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Does antibodies get produced in female body against sperms?

Does antibodies get produced in female body against sperms?


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Our immune system produces antibodies against any foreign particles entering our body.In female body sperms are a foreign particle .Does females produce antibodies or some sort of chemical resistance against these sperms?If so how come sperms able to survive this?Is it because there a huge number of sperms entering female body?


Boston University Medical CampusSexual Medicine

Female sexual dysfunction is defined as disorders of sexual desire, arousal, orgasm and/or sexual pain, which results in significant personal distress and may have an impact on the quality of life and interpersonal relationships. Although each specific condition can be separately defined in medical terms, clinically there is significant overlap in afflicted patients. The limited available data on female anatomy, physiology, biochemistry and molecular biology of the female sexual response makes this field particularly challenging to clinicians, psychologists and basic science researchers alike.

The sexual response cycle consists of desire, arousal, orgasm and resolution (both physiologic and psychologic). Desire is the mental state created by external and internal stimuli that induces a need or want to partake in sexual activity. Desire may be said to consist of: 1) biologic roots, which in part are based on hormones such as androgen and estrogen, 2) motivational roots, which are in part based on intimacy, pleasure and relationship issues and 3) cognitive issues such as risk and wish. Arousal is the state with specific feelings and physiologic changes usually associated with sexual activity involving the genitals. Arousal may be said to consist of: 1) central mechanisms including activation of thoughts, dreams and fantasies, 2) non-genital peripheral mechanisms such as salivation, sweating, cutaneous vasodilation and nipple erection and 3) genital mechanisms such as clitoral, labial and vaginal engorgement. Orgasm is the altered state of consciousness associated with primarily genital sensory input. Orgasm consists of multiple sensory afferent information from trigger points such as clitoris, labia, vagina, periurethral glans, etc., which pass centrally to supraspinal structures likely involving the thalamic septum. Following sufficient sensory stimulation, central neurotransmitter discharge during orgasm results in repeated 1-second motor contractions of the pelvic floor (3 – 8/orgasm) followed in 2 – 4 seconds by repeated uterine and vaginal smooth muscle contraction. Pleasurable sensory information is also carried to the cortical pleasure sites.

Epidemiology of Female Sexual Dysfunction

Well-designed, random-sample, community-based epidemiologic investigations of women with sexual dysfunction are limited. Current data reveals that up to 76% of women have some type of sexual dysfuntion. U.S. population census data suggest that approximately 10 million American women ages 50-74 self-report complaints of diminished vaginal lubrication, pain and discomfort with intercourse, decreased arousal, and difficulty achieving orgasm. Recently, Laumann and Rosen found that sexual dysfunction is more prevalent in women (43%) than in men (31%) and is associated with various psychodemographic characteristics such as age, education, and poor physical and emotional health. More importantly, female sexual dysfunction is associated with negative sexual relationship experiences.

Anatomy and physiology of genital sexual arousal

There is a paucity of data concerning the anatomy, physiology, pathophysiology of sexual function in women. The female external genitalia consist of various structures. The vagina is a midline cylindrical organ that connects the uterus with the external genitalia. The vaginal wall consists of three layers: a) an inner mucous type stratified squamous cell epithelium supported by a thick lamina propia, that undergoes hormone-related cyclical changes, b) the muscularis composed of outer longitudinal smooth muscle fibers and inner circular fibers, and c) an outer fibrous layer, rich in collagen and elastin, which provides structural support to the vagina. The vulva, bounded by the symphysis pubis, the anal sphincter and the ischial tuberosities, consists of labial formations, the interlabial space, and erectile tissue. The labial formations are two paired cutaneous structures: a) the labia majora are fatty folds covered by hair-bearing skin that fuses anteriorly with the mons veneris, or anterior prominence of the symphysis pubis, and posteriorly with the perineal body or posterior commissure b) The labia minora are smaller folds covered by non-hearing skin laterally and by vaginal mucosa medially, that fuses anteriorly to form the prepuce of the clitoris, and posteriorly in the fossa navicularis. The interlabial space is composed of the vestibule, the urinary meatus, and vaginal opening and is bounded by the space medial to the labia minora, the fossa navicularis and the clitoris. The clitoris is a 7-13 cm Y shaped organ comprised of glans, body, and crura. The body of the clitoris is surrounded by tunica albuginea and consists of two paired corpora cavernosa composed of trabecular smooth muscle and lacunar sinusoids. Finally, the vestibular bulb consists of paired structures located beneath the skin of the labia minora and represents the homologue of the corpus spongiosum in the male.

There is limited understanding of the precise location of autonomic neurovascular structures related to the uterus, cervix, and vagina. Uterine nerves arise from the inferior hypogastric plexus formed by the union of hypogastric nerves (sympathetic T10-L1) and the splanchnic fibers (parasympathetic S2-S4). This plexus has three portions: Vesical plexus, the rectal plexus, and the uterovaginal plexus (Frankenhauser’s ganglion), which lies at the base of the broad ligament, dorsal to the uterine vessels, and lateral to the uterosacral and cardinal ligament. This plexus provides innervation via the cardinal ligament and uterosacral ligaments to the cervix, upper vagina, urethra, vestibular bulbs and clitoris. At the cervix, sympathetic and parasympathetic nerves form the paracervical ganglia. The larger one is called the uterine cervical ganglion. It is at this level that injury to the autonomic fibers of the vagina, labia, cervix may occur during hysterectomy. The pudendal nerve (S2-S4) reaches the perineum through Alcock’s canal and provides sensory and motor innervation to the external genitalia.

Large gaps exist in our knowledge of how the central nervous system controls female sexual function. Limited data suggest that descending supraspinal modulation of female genital reflexes emanates from: 1) brainstem structures such as the nucleus paragigantocellularis (inhibitory via serotonin), locus ceruleus (norepinephrine, nocturnal engorgement during REM sleep) and midbrain periaqueductal gray, 2) hypothalamic structures such as the medial pre-optic area, ventromedial nucleus and paraventricular nucleus and 3) forebrain structure such as the amygdala. Multiple factors interact at the supraspinal levels to influence the excitability of spinal sexual reflexes such as: 1) gonadal hormones, 2) genital sensory information via the mylenated spinothalamic pathway and the unmyelinated spinoreticular pathway and 3) input from higher cortical centers of cognition.

The sexual arousal responses of the multiple genital and non-genital peripheral anatomic structures are largely the product of spinal cord reflex mechanisms. The spinal segments are under descending excitatory and inhibitory control from multiple supraspinal sites. The afferent reflex arm is primarily via the pudendal nerve. The efferent reflex arm consists of coordinated somatic and autonomic activity. One spinal sexual reflex is the bulbocavernosus reflex involving sacral cord segments S 2,3 and 4 in which pudendal nerve stimulation results in pelvic floor muscle contraction. Another spinal sexual reflex involves vaginal and clitoral cavernosal autonomic nerve stimulation resulting in clitoral, labial and vaginal engorgement.

In the basal state, clitoral corporal and vaginal smooth muscles are under contractile tone. Following sexual stimulation, neurogenic and endothelial release of nitric oxide (NO) plays an important role in clitoral cavernosal artery and helicine arteriolar smooth muscle relaxation. This leads to a rise in clitoral cavernosal artery inflow, an increase in clitoral intracavernosal pressure, and clitoral engorgement. The result is extrusion of the glans clitoris and enhanced sensitivity.

In the basal state, the vaginal epithelium reabsorbs sodium from the submucosal capillary plasma transudate. Following sexual stimulation, a number of neurotransmitters including NO and vasoactive intestinal peptide (VIP) are released modulating vaginal vascular and nonvascular smooth muscle relaxation. Dramatic increase in capillary inflow in the submucosa overwhelms Na-reabsorption leading to 3-5 ml of vaginal transudate, enhancing lubrication essential for pleasurable coitus. Vaginal smooth-muscle relaxation results in increased vaginal length and luminal diameter, especially in the distal two-thirds of the vagina (Fig. 1). Vasoactive intestinal polypeptide is a non-adrenergic non-cholinergic neurotransmitter that plays a role in enhancing vaginal blood flow, lubrication and secretions.

Experimental models for investigation of female sexual genital arousal

I Results from in vivo animal studies:
The absence of established animal models to investigate female sexual genital arousal has hampered progress in this field. Recently, Park et al., investigated vaginal and clitoral hemodynamics in female New Zealand White rabbits in response to pelvic nerve stimulation (PNS) in order to mimic genital arousal in response to sexual stimulation. This elegant study showed that pelvic nerve-stimulation caused an increase in vaginal blood flow, vaginal wall pressure, vaginal length, clitoral intracavernosal pressure and clitoral blood flow and a decrease in vaginal luminal pressure. This study represents an approach to study genital arousal in an animal model and paved the way for the investigation of genital arousal in a laboratory setting. Using a rat model, Vachon et al., confirmed genital hemodynamic changes reported by Park et al., in the rabbit model. More recently, Giuliano et al., further demonstrated that PNS induced an increase in vaginal wall tension and a decrease in vaginal vascular resistance in the rat model. In addition, this study showed that atropine did not significantly affect vaginal blood flow response to pelvic nerve stimulation despite the fact that cholinergic fibers innervate vascular smooth muscle in the rat vagina, suggesting that acetylcholine may not be the primary neurotransmitter responsible for the increase in vaginal engorgement during sexual arousal. These studies documented that genital arousal is a neurovascular event characterized by increase in genital blood flow and smooth muscle relaxation. These hemodynamic changes are mediated by neurotransmitters and vasoactive agents and modulated by the hormonal milieu. Park et al., investigated the effects of estrogen deprivation and replacement on genital hemodynamics. They reported that ovariectomy significantly reduced vaginal and clitoral blood flow in response to pelvic nerve stimulation. We also investigated the effects of ovariectomy and estrogen and androgen treatment on genital blood flow using a novel, non-invasive laser oximetry technique. In contrast to the observations made by Park et al. we found that ovariectomy did not significantly alter genital blood flow in the rabbit model. The discrepancy may be attributed to differences in methodologies. In our studies, we determined genital blood flow two-weeks post ovariectomy, while Park et al. performed their studies six weeks after ovariectomy. The longer period of estrogen deprivation may have produced tissue structural changes that altered the engorgement response. Since the female rabbit remains in continuous diestrus until mounted, serum estrogen levels are normally low (32-38 pg/ml), and ovariectomy does not produce a dramatic decrease in estrogen levels (22-25 pg/ml). As a consequence, genital hemodynamic changes before and after ovariectomy may be minimal. In addition, laser oximetry was used in our studies to assess changes in genital blood flow, whereas Park et al., used laser Doppler-flowmetry. Further studies using other animal models that undergo menstrual cycling (e.g. rat) are necessary to investigate this discrepancy.

Park et al., also reported that estrogen replacement normalized genital hemodynamics to control levels. In our studies, treatment of ovariectomized animals with estradiol significantly increased pelvic nerve-stimulated genital blood flow above control levels (Fig.2). Interestingly, treatment with testosterone did not restore blood flow to that observed in control animals. Park et al., also noted marked thinning of the vaginal epithelial layers, decreased vaginal submucosal microvasculature, and diffuse clitoral cavernosal fibrosis in ovariectomized animals. In addition, the percentage of clitoral cavernosal smooth muscle was significantly decreased in ovariectomized animals. These studies suggest that estrogens modulate genital hemodynamics and are critical for maintaining tissue structural integrity.

Vaginal lubrication, an estrogen-dependent physiological process, is one of the indicators of genital arousal and tissue integrity. Min et al., showed that vaginal lubrication in ovariectomized animals under basal conditions and after pelvic nerve stimulation was reduced and normalized with estrogen treatment (Fig 3 and 4). In contrast, androgen treatment of ovariectomized animals with testosterone alone or in combination with estradiol did not restore vaginal lubrication to that observed in control animals. Finally, it was noted that ovariectomy caused vaginal atrophy and reduced vaginal epithelial cell maturation, which was normalized by estrogen but not androgen treatment.

In summary, data derived from in vivo animal models indicates that estrogen but not androgens modulate genital blood flow, vaginal lubrication and vaginal tissue structural integrity. It should be noted that estradiol levels used in these studies were supra-physiological with potential pharmacologic effects different from those achieved physiologically. Although estrogen replacement increases vaginal lubrication and restores vaginal epithelial integrity, this therapy may not be appropriate for all patients, due to associated risk of breast and endometrial cancer. An alternative to hormonal treatment is the utilization of P2Y2 receptor agonists, which have been shown to increase mucin production and blood flow in other systems. We investigated the effects of P2Y2 receptor agonists as a feasible non-hormonal alternative for the treatment of vaginal dryness in an animal model. P2Y2 receptors are expressed in cervical and vaginal tissues, and these agonists increased vaginal lubrication under conditions of estrogen deprivation.

II. Effects of vasoactive substances on genital blood flow
Limited data are available on the effects of vasoactive substances on genital hemodynamics. Park et al., 1997 demonstrated that injection of papaverine hydrochloride and phentolamine mesylate into the vaginal spongy muscularis layer increased vaginal wall pressure and vaginal blood flow. Sildenafil, a PDE5-selective inhibitor, has been utilized in the treatment of women with sexual arousal disorders with mixed results and pre-clinical data supporting the use of this agent in the management of female sexual dysfunction remains equivocal. We have shown that sildenafil administration caused significant increase in genital blood flow and vaginal lubrication in intact and ovariectomized animals. However, this response was more pronounced in animals treated with estradiol. These data suggested that the NO-cGMP pathway is involved, at least in part, in the physiologic mechanism of female genital arousal and that sildenafil facilitates this response in an in vivo animal model.

The effects of apomorphine, a non-selective dopamine receptor agonist, on genital blood flow were investigated by Tarcan et al., who suggested that systemic administration of apomorphine improved clitoral and vaginal engorgement by increasing clitoral intracavernosal and vaginal wall arterial inflow.

In summary, data derived from in vivo animal models indicate that vasoactive agents play a role in genital arousal. Although sildenafil and apomorphine enhanced genital blood flow in the animal model, clinical use of vasoactive agents remains controversial.
Studies in organ baths:

Physiological studies of the arousal phase of the female sexual response involve, in part, an understanding of the various local regulatory mechanisms, which modulate tone in the clitoral erectile tissue and the vaginal muscularis. Immunohistochemical studies in human vaginal tissues have shown the presence of nerve fibers containing NPY, VIP, NOS, CGRP and substance P.10 Previous studies have suggested that VIP may be involved in the regulation of clitoral and vaginal smooth muscle tone but, as yet, no conclusive experimental evidence of its functional involvement has been forthcoming. There is physiological evidence supporting a role for the alpha-adrenergic system in female sexual arousal. The alpha-2 adrenergic agonist clonidine impaired both vaginal engorgement and lubrication when administered to healthy volunteers.

There is limited data on the functional activity of the inhibitory non-adrenergic non-cholinergic transmission in the clitoral corpus cavernosum. Cellek and Moncada have shown that electrical field stimulation induces NANC relaxation responses in the clitoral corpus cavernosum of the rabbit. These responses were inhibited by NG-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) or tetrodotoxin. In addition, the inhibitory effect of L-NAME was partially reversed by L-arginine but not by D-arginine. EFS-induced relaxations were enhanced by an inhibitor of type V cyclic GMP phosphodiesterase, zaprinast. It was concluded that nitrergic neurotransmission is responsible for the NANC relaxation responses in the clitoral corpus cavernosum of the rabbit. Furthermore, the role of phosphodiesterase type 5 inhibition in the modulation of female sexual dysfunction was investigated by Vemulapalli and Kurowski. Pretreatment of clitoral corpus cavernosum strips with sildenafil enhanced the electrical field stimulation-induced relaxations, both in magnitude and duration. Thus, the NO pathway is critical for smooth muscle relaxation in the clitoris. However, in the vagina, this pathway plays only a partial role, as demonstrated by Ziessen et al. These investigators showed that in the rat and rabbit vaginal wall, NANC relaxations were partly mediated by nitric oxide. The remaining part was neurogenic since it could be inhibited by tetrodotoxin. This non-nitrergic NANC response was not associated with any known neuropeptides or purines. Thus, the nature of the non-adrenergic, non-cholinergic neurotransmitter in the vagina remains elusive.

We have carried out preliminary experiments in organ bath chambers to assess clitoral and vaginal tissue responses to: a) electric field stimulation b) alpha-adrenergic agonists c) NO donors and d) VIP. Electrical field stimulation resulted in a biphasic (contraction/relaxation) response in clitoral and vaginal tissue strips. Bretylium (inhibitor of NE release) abolished the contractile response induced by EFS in both tissues. Exogenously added norepinephrine caused a dose-dependent contraction in vaginal and clitoral tissues. These observations suggest that adrenergic nerves mediate the contractile response. Sodium nitroprusside and papaverine caused dose dependent relaxation of vaginal and clitoral strips pre-contracted with norepinephrine. Alpha-1 (prazosin and tamsulosin) and alpha-2 (delequamine) selective antagonists inhibited contraction of vaginal tissue strips to exogenous norepinephrine. Further studies using specific molecular probes and RNase protection assays have detected mRNA for both alpha 1A and alpha 2A adrenergic receptors in human clitoral and vaginal smooth muscle cells (Traish et al., unpublished data). Thus, vaginal and clitoral smooth muscle contraction is the result of activation of alpha-adrenergic receptors by norepinephrine released from adrenergic nerves. It remains to be determined if other vasoconstrictor agents, such as endothelin, neuropeptide Y (NPY), angiotensin or eicosanoids may play a role in regulating smooth muscle tone in these tissues.

Giraldi et al., have characterized the effect of experimental diabetes on neurotransmission in rat vagina. It was suggested that diabetes interferes with adrenergic-, cholinergic- and NANC-neurotransmitter mechanisms in the smooth muscle of the rat vagina.8 The changes in the nitrergic neurotransmission were attributed to reduction in NOS-activity, but may also be attributed to inhibition of various reactions in the L-arginine/NO/guanylate cyclase/cGMP system.

We investigated the effects of hormonal manipulations on vaginal smooth muscle contractility in response to electrical field stimulation (EFS) and vasoactive substances. Ovariectomy reduced norepinephrine-induced contractile response and treatment with estradiol or testosterone normalized the contractile response. Ovariectomy also attenuated EFS-induced relaxation response and treatment with testosterone facilitated EFS-induced smooth muscle relaxation. Moreover, VIP induced a dose-dependent relaxation response that was attenuated in tissues from ovariectomized animals or in animals treated with estradiol. In contrast, VIP-induced relaxation was facilitated in tissues from ovariectomized animals treated with testosterone. These observations suggest that testosterone and estradiol produce distinct physiological responses in vaginal smooth muscle and that androgens facilitate vaginal smooth muscle relaxation.

In summary, the data reported from several laboratories suggest that NO is a key pathway in mediating clitoral smooth muscle relaxation. However, in the vagina, NO appears to play only a partial role in mediating smooth muscle relaxation. VIP also induces vaginal smooth muscle relaxation yet its exact functional role remains to be determined. Functional alpha-adrenergic receptors are expressed in the vagina and mediate norepinephrine induced contraction. Hyperglycemia affects vaginal smooth muscle response to neurotransmission affecting multiple physiological pathways. We have observed that androgens but not estrogens at pharmacological doses enhanced smooth muscle relaxation. Further studies with hormonal manipulations at physiological doses are necessary to establish the role of hormones on vaginal smooth muscle relaxation.

Studies in cell culture:

Park et al. and Traish et al.recently sub-cultured and characterized human and rabbit vaginal and clitoral smooth muscle cells and investigated the synthesis of second messenger cyclic nucleotides in response to vasodilators and determined the activity and kinetics of phosphodiesterase (PDE) type 5.32,37 Cultured vaginal and clitoral cells exhibited growth characteristics typical of smooth muscle cells and immunostained positively with antibodies against alpha smooth muscle actin. The cells retained functional prostaglandin E, VIP and b adrenergic receptors as demonstrated by increased intracellular cAMP synthesis in response to PGE1, VIP or isoproterenol. The response to these vasoactive substances was augmented with forskolin, suggesting stabilization of G-protein activated adenylyl cyclases. Treatment with the nitric oxide donor, sodium nitroprusside, in the presence of sildenafil, a PDE type 5 inhibitor, enhanced intracellular cGMP synthesis and accumulation. Incubation of rabbit vaginal tissue with sildenafil, sodium nitroprusside and PGE1 or forskolin produced a marked increase in intracellular cGMP. These observations were similar to those obtained with cultured cells and suggest that sub-cultured cells retained functional characteristics exhibited in intact tissue. The cells retained phosphodiesterase type 5 expression as shown by specific cGMP hydrolytic activity. Sildenafil and zaprinast inhibited cGMP hydrolysis competitively and bound with high affinity (inhibition constants Ki= 7 and 250 nM, respectively). These observations suggest that cultured human and rabbit vaginal smooth muscle cells retained their metabolic functional integrity and this experimental system should prove useful in investigating the signaling pathways that modulate vaginal smooth muscle tone.

Investigation of the distribution of NOS in the rat vagina in response to ovariectomy and estrogen replacement was recently performed using immunohistochemical analyses with n-NOS and e-NOS antibodies. In intact cycling animals, e-NOS and n-NOS expression were found to be highest during proestrous and lowest during metestrous while in ovariectomized animals n-NOS and e-NOS expression declined substantially. Estrogen replacement resulted in significant increase in e-NOS and n-NOS expression, when compared with NOS in intact animals. It was suggested that estrogen plays a critical role in regulating vaginal NOS expression of the rat vagina and that NO may modulate both vaginal blood supply and vaginal smooth musculature. More recent studies have shown the opposite observation. They found that rabbit vaginal NOS activity was considerably reduced by treatment with estradiol or estradiol and progesterone. They also noted that progesterone treatment alone up-regulated vaginal NOS. NOS-containing nerves could be demonstrated in vagina by immunohistochemistry. Vaginal smooth muscle responded with relaxation after EFS, which was inhibited by NG-nitro-L-arginine. A tissue specific role for NOS in vagina was suggested based on NO-dependent response of vaginal smooth muscle, expression of relatively high NOS, which is down-regulation by estradiol and up-regulation by progesterone.

This discrepancy in NOS regulation by estrogen in these studies may be due to species differences or to methods for assessment of NOS expression and activity. We have used both immunochemical (Western blots) and enzymatic activity assays to determine regulation of vaginal NOS in the rabbit model. In this study we demonstrated that nitric oxide synthase was predominantly expressed in the proximal vagina. The reason for this tissue distribution is yet to be determined. We further observed that ovariectomy enhanced NOS activity in the proximal vagina suggesting specific regulation of NOS by sex steroid hormones. Treatment of ovariectomized animals with estrogens resulted in decreased expression and activity of NOS in vaginal tissue, consistent with the research by Al-Hijji et al. In contrast, treatment of ovariectomized animals with androgens resulted in increased NOS expression and activity. These observations suggest that NOS in vaginal tissue is regulated by androgens and estrogens in an opposite manner.

Conclusions

The psychosocial and relationship aspects of female sexuality have been extensively investigated. However, studies concerning the anatomy, physiology and pathophysiology of female sexual function and dysfunction are limited. The paucity of biological data may be attributed to lack of reliable experimental models and tools for the investigation of female sexual function, and to limited funding, which is critical for the development of experimental approaches.
Research efforts by a number of investigators in different laboratories are establishing experimental models needed for the investigation of the physiological mechanisms involved in the genital arousal response of sexual function. These experimental models have permitted assessment of genital hemodynamics, vaginal lubrication, regulation of genital smooth muscle contractility and signaling pathways, providing preliminary information on the role of neurotransmitters and sex steroid hormones in sexual function. Further research is needed to define the neurotransmitters responsible for vaginal smooth muscle relaxation, the role of sex steroid hormones and their receptors in modulating genital hemodynamics, smooth muscle contractility and neurotransmitter receptor expression. Finally, a global and integral understanding of the biologic aspects of female sexual function requires investigation of the vascular, neurological (central and peripheral) and structural components of this extremely complex physiological process.


Talk to your doctor about any concerns you have regarding the need for the test, its risks, how it will be done, or what the results will mean.

Blood test

In general, there's nothing you have to do before this test, unless your doctor tells you to.

Semen sample

Your semen sample is collected after the blood and vaginal fluid samples are taken. You should not release your sperm (ejaculate) for 2 days before the test. It is important to not go longer than 5 days before the test without ejaculating.


Antibody Testing

Because immunoglobulins are matched to a specific pathogen, they can be used to diagnose some diseases based on their unique structure. Antibody tests are used to detect disease-specific antibodies in a blood sample.

Antibody tests are available to diagnose (or help diagnose) a wide variety of infectious and autoimmune diseases, including:

Antibody tests do not detect the actual pathogens that cause an infection—they detect the antibodies that are produced in response to the infection. A positive result means "yes," the test has detected the antibody or antigen. A negative result means "no," while borderline results are considered inconclusive.

Depending on the disease, it may take time for enough antibodies to be produced to reach detectable levels. If it's done too soon, during the early window period, the test may deliver a false negative result.

An antibody test can confirm that an infection has taken place, as with COVID-19 or HIV, although it cannot tell you when.

Sometimes, immunoglobulin levels can be used to characterize the stage of an infection. Because IgM levels usually increase before the IgG response kicks in, a disease-specific IgM and IgG test can help determine whether an infection has occurred recently. For example, herpes simplex is an infection for which IgM and IgG tests can help determine the timing of the infection.  

In people with allergies, IgE tests can be used to confirm that an allergic response has occurred. These tests can also be used as part of the diagnostic process to determine whether IgE levels increase when you are intentionally exposed to an allergen.


Contents

150 kDa) proteins of about 10 nm in size, [7] arranged in three globular regions that roughly form a Y shape.

In humans and most mammals, an antibody unit consists of four polypeptide chains two identical heavy chains and two identical light chains connected by disulfide bonds. [8] Each chain is a series of domains: somewhat similar sequences of about 110 amino acids each. These domains are usually represented in simplified schematics as rectangles. Light chains consist of one variable domain VL and one constant domain CL, while heavy chains contain one variable domain VH and three to four constant domains CH1, CH2, . [9]

Structurally an antibody is also partitioned into two antigen-binding fragments (Fab), containing one VL, VH, CL, and CH1 domain each, as well as the crystallisable fragment (Fc), forming the trunk of the Y shape. [10] In between them is a hinge region of the heavy chains, whose flexibility allows antibodies to bind to pairs of epitopes at various distances, to form complexes (dimers, trimers, etc.), and to bind effector molecules more easily. [11]

In an electrophoresis test of blood proteins, antibodies mostly migrate to the last, gamma globulin fraction. Conversely, most gamma-globulins are antibodies, which is why the two terms were historically used as synonyms, as were the symbols Ig and γ. This variant terminology fell out of use due to the correspondence being inexact and due to confusion with γ heavy chains which characterize the IgG class of antibodies. [12] [13]

Antigen-binding site Edit

The variable domains can also be referred to as the FV region. It is the subregion of Fab that binds to an antigen. More specifically, each variable domain contains three hypervariable regions – the amino acids seen there vary the most from antibody to antibody. When the protein folds, these regions give rise to three loops of β-strands, localised near one another on the surface of the antibody. These loops are referred to as the complementarity-determining regions (CDRs), since their shape complements that of an antigen. Three CDRs from each of the heavy and light chains together form an antibody-binding site whose shape can be anything from a pocket to which a smaller antigen binds, to a larger surface, to a protrusion that sticks out into a groove in an antigen. Typically however only a few residues contribute to most of the binding energy. [2]

The existence of two identical antibody-binding sites allows antibody molecules to bind strongly to multivalent antigen (repeating sites such as polysaccharides in bacterial cell walls, or other sites at some distance apart), as well as to form antibody complexes and larger antigen-antibody complexes. [2] The resulting cross-linking plays a role in activating other parts of the immune system.

The structures of CDRs have been clustered and classified by Chothia et al. [14] and more recently by North et al. [15] and Nikoloudis et al. [16] In the framework of the immune network theory, CDRs are also called idiotypes. According to immune network theory, the adaptive immune system is regulated by interactions between idiotypes.

Fc region Edit

The Fc region (the trunk of the Y shape) is composed of constant domains from the heavy chains. Its role is in modulating immune cell activity: it is where effector molecules bind to, triggering various effects after the antibody Fab region binds to an antigen. [2] [11] Effector cells (such as macrophages or natural killer cells) bind via their Fc receptors (FcR) to the Fc region of an antibody, while the complement system is activated by binding the C1q protein complex.

Another role of the Fc region is to selectively distribute different antibody classes across the body. In particular, the neonatal Fc receptor (FcRn) binds to the Fc region of IgG antibodies to transport it across the placenta, from the mother to the fetus.

Antibodies are glycoproteins, [17] that is, they have carbohydrates (glycans) added to conserved amino acid residues. [17] [18] These conserved glycosylation sites occur in the Fc region and influence interactions with effector molecules. [17] [19]

Protein structure Edit

The N-terminus of each chain is situated at the tip. Each immunoglobulin domain has a similar structure, characteristic of all the members of the immunoglobulin superfamily: it is composed of between 7 (for constant domains) and 9 (for variable domains) β-strands, forming two beta sheets in a Greek key motif. The sheets create a "sandwich" shape, the immunoglobulin fold, held together by a disulfide bond.

Antibody complexes Edit

Secreted antibodies can occur as a single Y-shaped unit, a monomer. However, some antibody classes also form dimers with two Ig units (as with IgA), tetramers with four Ig units (like teleost fish IgM), or pentamers with five Ig units (like mammalian IgM, which occasionally forms hexamers as well, with six units). [20]

Antibodies also form complexes by binding to antigen: this is called an antigen-antibody complex or immune complex. Small antigens can cross-link two antibodies, also leading to the formation of antibody dimers, trimers, tetramers, etc. Multivalent antigens (e.g., cells with multiple epitopes) can form larger complexes with antibodies. An extreme example is the clumping, or agglutination, of red blood cells with antibodies in the Coombs test to determine blood groups: the large clumps become insoluble, leading to visually apparent precipitation.

B cell receptors Edit

The membrane-bound form of an antibody may be called a surface immunoglobulin (sIg) or a membrane immunoglobulin (mIg). It is part of the B cell receptor (BCR), which allows a B cell to detect when a specific antigen is present in the body and triggers B cell activation. [21] The BCR is composed of surface-bound IgD or IgM antibodies and associated Ig-α and Ig-β heterodimers, which are capable of signal transduction. [22] A typical human B cell will have 50,000 to 100,000 antibodies bound to its surface. [22] Upon antigen binding, they cluster in large patches, which can exceed 1 micrometer in diameter, on lipid rafts that isolate the BCRs from most other cell signaling receptors. [22] These patches may improve the efficiency of the cellular immune response. [23] In humans, the cell surface is bare around the B cell receptors for several hundred nanometers, [22] which further isolates the BCRs from competing influences.

Antibodies can come in different varieties known as isotypes or classes. In placental mammals there are five antibody classes known as IgA, IgD, IgE, IgG, and IgM, which are further subdivided into subclasses such as IgA1, IgA2. The prefix "Ig" stands for immunoglobulin, while the suffix denotes the type of heavy chain the antibody contains: the heavy chain types α (alpha), γ (gamma), δ (delta), ε (epsilon), μ (mu) give rise to IgA, IgG, IgD, IgE, IgM, respectively. The distinctive features of each class are determined by the part of the heavy chain within the hinge and Fc region. [2]

The classes differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the table. [8] For example, IgE antibodies are responsible for an allergic response consisting of histamine release from mast cells, contributing to asthma. The antibody's variable region binds to allergic antigen, for example house dust mite particles, while its Fc region (in the ε heavy chains) binds to Fc receptor ε on a mast cell, triggering its degranulation: the release of molecules stored in its granules. [24]

Antibody isotypes of mammals
Class Subclasses Description
IgA 2 Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents colonization by pathogens. [25] Also found in saliva, tears, and breast milk.
IgD 1 Functions mainly as an antigen receptor on B cells that have not been exposed to antigens. [26] It has been shown to activate basophils and mast cells to produce antimicrobial factors. [27]
IgE 1 Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms. [5]
IgG 4 In its four forms, provides the majority of antibody-based immunity against invading pathogens. [5] The only antibody capable of crossing the placenta to give passive immunity to the fetus.
IgM 1 Expressed on the surface of B cells (monomer) and in a secreted form (pentamer) with very high avidity. Eliminates pathogens in the early stages of B cell-mediated (humoral) immunity before there is sufficient IgG. [5] [26]

The antibody isotype of a B cell changes during cell development and activation. Immature B cells, which have never been exposed to an antigen, express only the IgM isotype in a cell surface bound form. The B lymphocyte, in this ready-to-respond form, is known as a "naive B lymphocyte." The naive B lymphocyte expresses both surface IgM and IgD. The co-expression of both of these immunoglobulin isotypes renders the B cell ready to respond to antigen. [28] B cell activation follows engagement of the cell-bound antibody molecule with an antigen, causing the cell to divide and differentiate into an antibody-producing cell called a plasma cell. In this activated form, the B cell starts to produce antibody in a secreted form rather than a membrane-bound form. Some daughter cells of the activated B cells undergo isotype switching, a mechanism that causes the production of antibodies to change from IgM or IgD to the other antibody isotypes, IgE, IgA, or IgG, that have defined roles in the immune system.

Light chain types Edit

In mammals there are two types of immunoglobulin light chain, which are called lambda (λ) and kappa (κ). However, there is no known functional difference between them, and both can occur with any of the five major types of heavy chains. [2] Each antibody contains two identical light chains: both κ or both λ. Proportions of κ and λ types vary by species and can be used to detect abnormal proliferation of B cell clones. Other types of light chains, such as the iota (ι) chain, are found in other vertebrates like sharks (Chondrichthyes) and bony fishes (Teleostei).

In animals Edit

In most placental mammals the structure of antibodies is generally the same. Jawed fish appear to be the most primitive animals that are able to make antibodies similar to those of mammals, although many features of their adaptive immunity appeared somewhat earlier. [29] Cartilaginous fish (such as sharks) produce heavy-chain-only antibodies (lacking light chains) which moreover feature longer chains, with five constant domains each. Camelids (such as camels, llamas, alpacas) are also notable for producing heavy-chain-only antibodies. [2] [30]

Antibody classes not found in mammals
Class Types Description
IgY Found in birds and reptiles related to mammalian IgG. [31]
IgW Found in sharks and skates related to mammalian IgD. [32]

The antibody's paratope interacts with the antigen's epitope. An antigen usually contains different epitopes along its surface arranged discontinuously, and dominant epitopes on a given antigen are called determinants.

Antibody and antigen interact by spatial complementarity (lock and key). The molecular forces involved in the Fab-epitope interaction are weak and non-specific – for example electrostatic forces, hydrogen bonds, hydrophobic interactions, and van der Waals forces. This means binding between antibody and antigen is reversible, and the antibody's affinity towards an antigen is relative rather than absolute. Relatively weak binding also means it is possible for an antibody to cross-react with different antigens of different relative affinities.

The main categories of antibody action include the following:

    , in which neutralizing antibodies block parts of the surface of a bacterial cell or virion to render its attack ineffective , in which antibodies "glue together" foreign cells into clumps that are attractive targets for phagocytosis , in which antibodies "glue together" serum-soluble antigens, forcing them to precipitate out of solution in clumps that are attractive targets for phagocytosis (fixation), in which antibodies that are latched onto a foreign cell encourage complement to attack it with a membrane attack complex, which leads to the following:
      of the foreign cell
    • Encouragement of inflammation by chemotactically attracting inflammatory cells

    More indirectly, an antibody can signal immune cells to present antibody fragments to T cells, or downregulate other immune cells to avoid autoimmunity.

    Activated B cells differentiate into either antibody-producing cells called plasma cells that secrete soluble antibody or memory cells that survive in the body for years afterward in order to allow the immune system to remember an antigen and respond faster upon future exposures. [4]

    At the prenatal and neonatal stages of life, the presence of antibodies is provided by passive immunization from the mother. Early endogenous antibody production varies for different kinds of antibodies, and usually appear within the first years of life. Since antibodies exist freely in the bloodstream, they are said to be part of the humoral immune system. Circulating antibodies are produced by clonal B cells that specifically respond to only one antigen (an example is a virus capsid protein fragment). Antibodies contribute to immunity in three ways: They prevent pathogens from entering or damaging cells by binding to them they stimulate removal of pathogens by macrophages and other cells by coating the pathogen and they trigger destruction of pathogens by stimulating other immune responses such as the complement pathway. [33] Antibodies will also trigger vasoactive amine degranulation to contribute to immunity against certain types of antigens (helminths, allergens).

    Activation of complement Edit

    Antibodies that bind to surface antigens (for example, on bacteria) will attract the first component of the complement cascade with their Fc region and initiate activation of the "classical" complement system. [33] This results in the killing of bacteria in two ways. [5] First, the binding of the antibody and complement molecules marks the microbe for ingestion by phagocytes in a process called opsonization these phagocytes are attracted by certain complement molecules generated in the complement cascade. Second, some complement system components form a membrane attack complex to assist antibodies to kill the bacterium directly (bacteriolysis). [34]

    Activation of effector cells Edit

    To combat pathogens that replicate outside cells, antibodies bind to pathogens to link them together, causing them to agglutinate. Since an antibody has at least two paratopes, it can bind more than one antigen by binding identical epitopes carried on the surfaces of these antigens. By coating the pathogen, antibodies stimulate effector functions against the pathogen in cells that recognize their Fc region. [5]

    Those cells that recognize coated pathogens have Fc receptors, which, as the name suggests, interact with the Fc region of IgA, IgG, and IgE antibodies. The engagement of a particular antibody with the Fc receptor on a particular cell triggers an effector function of that cell phagocytes will phagocytose, mast cells and neutrophils will degranulate, natural killer cells will release cytokines and cytotoxic molecules that will ultimately result in destruction of the invading microbe. The activation of natural killer cells by antibodies initiates a cytotoxic mechanism known as antibody-dependent cell-mediated cytotoxicity (ADCC) – this process may explain the efficacy of monoclonal antibodies used in biological therapies against cancer. The Fc receptors are isotype-specific, which gives greater flexibility to the immune system, invoking only the appropriate immune mechanisms for distinct pathogens. [2]

    Natural antibodies Edit

    Humans and higher primates also produce "natural antibodies" that are present in serum before viral infection. Natural antibodies have been defined as antibodies that are produced without any previous infection, vaccination, other foreign antigen exposure or passive immunization. These antibodies can activate the classical complement pathway leading to lysis of enveloped virus particles long before the adaptive immune response is activated. Many natural antibodies are directed against the disaccharide galactose α(1,3)-galactose (α-Gal), which is found as a terminal sugar on glycosylated cell surface proteins, and generated in response to production of this sugar by bacteria contained in the human gut. [35] Rejection of xenotransplantated organs is thought to be, in part, the result of natural antibodies circulating in the serum of the recipient binding to α-Gal antigens expressed on the donor tissue. [36]

    Virtually all microbes can trigger an antibody response. Successful recognition and eradication of many different types of microbes requires diversity among antibodies their amino acid composition varies allowing them to interact with many different antigens. [37] It has been estimated that humans generate about 10 billion different antibodies, each capable of binding a distinct epitope of an antigen. [38] Although a huge repertoire of different antibodies is generated in a single individual, the number of genes available to make these proteins is limited by the size of the human genome. Several complex genetic mechanisms have evolved that allow vertebrate B cells to generate a diverse pool of antibodies from a relatively small number of antibody genes. [39]

    Domain variability Edit

    The chromosomal region that encodes an antibody is large and contains several distinct gene loci for each domain of the antibody—the chromosome region containing heavy chain genes ([email protected]) is found on chromosome 14, and the loci containing lambda and kappa light chain genes ([email protected] and [email protected]) are found on chromosomes 22 and 2 in humans. One of these domains is called the variable domain, which is present in each heavy and light chain of every antibody, but can differ in different antibodies generated from distinct B cells. Differences, between the variable domains, are located on three loops known as hypervariable regions (HV-1, HV-2 and HV-3) or complementarity-determining regions (CDR1, CDR2 and CDR3). CDRs are supported within the variable domains by conserved framework regions. The heavy chain locus contains about 65 different variable domain genes that all differ in their CDRs. Combining these genes with an array of genes for other domains of the antibody generates a large cavalry of antibodies with a high degree of variability. This combination is called V(D)J recombination discussed below. [40]

    V(D)J recombination Edit

    Somatic recombination of immunoglobulins, also known as V(D)J recombination, involves the generation of a unique immunoglobulin variable region. The variable region of each immunoglobulin heavy or light chain is encoded in several pieces—known as gene segments (subgenes). These segments are called variable (V), diversity (D) and joining (J) segments. [39] V, D and J segments are found in Ig heavy chains, but only V and J segments are found in Ig light chains. Multiple copies of the V, D and J gene segments exist, and are tandemly arranged in the genomes of mammals. In the bone marrow, each developing B cell will assemble an immunoglobulin variable region by randomly selecting and combining one V, one D and one J gene segment (or one V and one J segment in the light chain). As there are multiple copies of each type of gene segment, and different combinations of gene segments can be used to generate each immunoglobulin variable region, this process generates a huge number of antibodies, each with different paratopes, and thus different antigen specificities. [41] The rearrangement of several subgenes (i.e. V2 family) for lambda light chain immunoglobulin is coupled with the activation of microRNA miR-650, which further influences biology of B-cells.

    RAG proteins play an important role with V(D)J recombination in cutting DNA at a particular region. [41] Without the presence of these proteins, V(D)J recombination would not occur. [41]

    After a B cell produces a functional immunoglobulin gene during V(D)J recombination, it cannot express any other variable region (a process known as allelic exclusion) thus each B cell can produce antibodies containing only one kind of variable chain. [2] [42]

    Somatic hypermutation and affinity maturation Edit

    Following activation with antigen, B cells begin to proliferate rapidly. In these rapidly dividing cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate of point mutation, by a process called somatic hypermutation (SHM). SHM results in approximately one nucleotide change per variable gene, per cell division. [43] As a consequence, any daughter B cells will acquire slight amino acid differences in the variable domains of their antibody chains.

    This serves to increase the diversity of the antibody pool and impacts the antibody's antigen-binding affinity. [44] Some point mutations will result in the production of antibodies that have a weaker interaction (low affinity) with their antigen than the original antibody, and some mutations will generate antibodies with a stronger interaction (high affinity). [45] B cells that express high affinity antibodies on their surface will receive a strong survival signal during interactions with other cells, whereas those with low affinity antibodies will not, and will die by apoptosis. [45] Thus, B cells expressing antibodies with a higher affinity for the antigen will outcompete those with weaker affinities for function and survival allowing the average affinity of antibodies to increase over time. The process of generating antibodies with increased binding affinities is called affinity maturation. Affinity maturation occurs in mature B cells after V(D)J recombination, and is dependent on help from helper T cells. [46]

    Class switching Edit

    Isotype or class switching is a biological process occurring after activation of the B cell, which allows the cell to produce different classes of antibody (IgA, IgE, or IgG). [41] The different classes of antibody, and thus effector functions, are defined by the constant (C) regions of the immunoglobulin heavy chain. Initially, naive B cells express only cell-surface IgM and IgD with identical antigen binding regions. Each isotype is adapted for a distinct function therefore, after activation, an antibody with an IgG, IgA, or IgE effector function might be required to effectively eliminate an antigen. Class switching allows different daughter cells from the same activated B cell to produce antibodies of different isotypes. Only the constant region of the antibody heavy chain changes during class switching the variable regions, and therefore antigen specificity, remain unchanged. Thus the progeny of a single B cell can produce antibodies, all specific for the same antigen, but with the ability to produce the effector function appropriate for each antigenic challenge. Class switching is triggered by cytokines the isotype generated depends on which cytokines are present in the B cell environment. [47]

    Class switching occurs in the heavy chain gene locus by a mechanism called class switch recombination (CSR). This mechanism relies on conserved nucleotide motifs, called switch (S) regions, found in DNA upstream of each constant region gene (except in the δ-chain). The DNA strand is broken by the activity of a series of enzymes at two selected S-regions. [48] [49] The variable domain exon is rejoined through a process called non-homologous end joining (NHEJ) to the desired constant region (γ, α or ε). This process results in an immunoglobulin gene that encodes an antibody of a different isotype. [50]

    Specificity designations Edit

    An antibody can be called monospecific if it has specificity for the same antigen or epitope, [51] or bispecific if they have affinity for two different antigens or two different epitopes on the same antigen. [52] A group of antibodies can be called polyvalent (or unspecific) if they have affinity for various antigens [53] or microorganisms. [53] Intravenous immunoglobulin, if not otherwise noted, consists of a variety of different IgG (polyclonal IgG). In contrast, monoclonal antibodies are identical antibodies produced by a single B cell.

    Asymmetrical antibodies Edit

    Heterodimeric antibodies, which are also asymmetrical antibodies, allow for greater flexibility and new formats for attaching a variety of drugs to the antibody arms. One of the general formats for a heterodimeric antibody is the "knobs-into-holes" format. This format is specific to the heavy chain part of the constant region in antibodies. The "knobs" part is engineered by replacing a small amino acid with a larger one. It fits into the "hole", which is engineered by replacing a large amino acid with a smaller one. What connects the "knobs" to the "holes" are the disulfide bonds between each chain. The "knobs-into-holes" shape facilitates antibody dependent cell mediated cytotoxicity. Single chain variable fragments (scFv) are connected to the variable domain of the heavy and light chain via a short linker peptide. The linker is rich in glycine, which gives it more flexibility, and serine/threonine, which gives it specificity. Two different scFv fragments can be connected together, via a hinge region, to the constant domain of the heavy chain or the constant domain of the light chain. [54] This gives the antibody bispecificity, allowing for the binding specificities of two different antigens. [55] The "knobs-into-holes" format enhances heterodimer formation but doesn't suppress homodimer formation.

    To further improve the function of heterodimeric antibodies, many scientists are looking towards artificial constructs. Artificial antibodies are largely diverse protein motifs that use the functional strategy of the antibody molecule, but aren't limited by the loop and framework structural constraints of the natural antibody. [56] Being able to control the combinational design of the sequence and three-dimensional space could transcend the natural design and allow for the attachment of different combinations of drugs to the arms.

    Heterodimeric antibodies have a greater range in shapes they can take and the drugs that are attached to the arms don't have to be the same on each arm, allowing for different combinations of drugs to be used in cancer treatment. Pharmaceuticals are able to produce highly functional bispecific, and even multispecific, antibodies. The degree to which they can function is impressive given that such a change of shape from the natural form should lead to decreased functionality.

    The first use of the term "antibody" occurred in a text by Paul Ehrlich. The term Antikörper (the German word for antibody) appears in the conclusion of his article "Experimental Studies on Immunity", published in October 1891, which states that, "if two substances give rise to two different Antikörper, then they themselves must be different". [57] However, the term was not accepted immediately and several other terms for antibody were proposed these included Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin. [57] The word antibody has formal analogy to the word antitoxin and a similar concept to Immunkörper (immune body in English). [57] As such, the original construction of the word contains a logical flaw the antitoxin is something directed against a toxin, while the antibody is a body directed against something. [57]

    The study of antibodies began in 1890 when Emil von Behring and Kitasato Shibasaburō described antibody activity against diphtheria and tetanus toxins. Von Behring and Kitasato put forward the theory of humoral immunity, proposing that a mediator in serum could react with a foreign antigen. [61] [62] His idea prompted Paul Ehrlich to propose the side-chain theory for antibody and antigen interaction in 1897, when he hypothesized that receptors (described as "side-chains") on the surface of cells could bind specifically to toxins – in a "lock-and-key" interaction – and that this binding reaction is the trigger for the production of antibodies. [63] Other researchers believed that antibodies existed freely in the blood and, in 1904, Almroth Wright suggested that soluble antibodies coated bacteria to label them for phagocytosis and killing a process that he named opsoninization. [64]

    In the 1920s, Michael Heidelberger and Oswald Avery observed that antigens could be precipitated by antibodies and went on to show that antibodies are made of protein. [65] The biochemical properties of antigen-antibody-binding interactions were examined in more detail in the late 1930s by John Marrack. [66] The next major advance was in the 1940s, when Linus Pauling confirmed the lock-and-key theory proposed by Ehrlich by showing that the interactions between antibodies and antigens depend more on their shape than their chemical composition. [67] In 1948, Astrid Fagraeus discovered that B cells, in the form of plasma cells, were responsible for generating antibodies. [68]

    Further work concentrated on characterizing the structures of the antibody proteins. A major advance in these structural studies was the discovery in the early 1960s by Gerald Edelman and Joseph Gally of the antibody light chain, [69] and their realization that this protein is the same as the Bence-Jones protein described in 1845 by Henry Bence Jones. [70] Edelman went on to discover that antibodies are composed of disulfide bond-linked heavy and light chains. Around the same time, antibody-binding (Fab) and antibody tail (Fc) regions of IgG were characterized by Rodney Porter. [71] Together, these scientists deduced the structure and complete amino acid sequence of IgG, a feat for which they were jointly awarded the 1972 Nobel Prize in Physiology or Medicine. [71] The Fv fragment was prepared and characterized by David Givol. [72] While most of these early studies focused on IgM and IgG, other immunoglobulin isotypes were identified in the 1960s: Thomas Tomasi discovered secretory antibody (IgA) [73] David S. Rowe and John L. Fahey discovered IgD [74] and Kimishige Ishizaka and Teruko Ishizaka discovered IgE and showed it was a class of antibodies involved in allergic reactions. [75] In a landmark series of experiments beginning in 1976, Susumu Tonegawa showed that genetic material can rearrange itself to form the vast array of available antibodies. [76]

    Disease diagnosis Edit

    Detection of particular antibodies is a very common form of medical diagnostics, and applications such as serology depend on these methods. [77] For example, in biochemical assays for disease diagnosis, [78] a titer of antibodies directed against Epstein-Barr virus or Lyme disease is estimated from the blood. If those antibodies are not present, either the person is not infected or the infection occurred a very long time ago, and the B cells generating these specific antibodies have naturally decayed.

    In clinical immunology, levels of individual classes of immunoglobulins are measured by nephelometry (or turbidimetry) to characterize the antibody profile of patient. [79] Elevations in different classes of immunoglobulins are sometimes useful in determining the cause of liver damage in patients for whom the diagnosis is unclear. [1] For example, elevated IgA indicates alcoholic cirrhosis, elevated IgM indicates viral hepatitis and primary biliary cirrhosis, while IgG is elevated in viral hepatitis, autoimmune hepatitis and cirrhosis.

    Autoimmune disorders can often be traced to antibodies that bind the body's own epitopes many can be detected through blood tests. Antibodies directed against red blood cell surface antigens in immune mediated hemolytic anemia are detected with the Coombs test. [80] The Coombs test is also used for antibody screening in blood transfusion preparation and also for antibody screening in antenatal women. [80]

    Practically, several immunodiagnostic methods based on detection of complex antigen-antibody are used to diagnose infectious diseases, for example ELISA, immunofluorescence, Western blot, immunodiffusion, immunoelectrophoresis, and magnetic immunoassay. Antibodies raised against human chorionic gonadotropin are used in over the counter pregnancy tests.

    New dioxaborolane chemistry enables radioactive fluoride ( 18 F) labeling of antibodies, which allows for positron emission tomography (PET) imaging of cancer. [81]

    Disease therapy Edit

    Some immune deficiencies, such as X-linked agammaglobulinemia and hypogammaglobulinemia, result in partial or complete lack of antibodies. [87] These diseases are often treated by inducing a short term form of immunity called passive immunity. Passive immunity is achieved through the transfer of ready-made antibodies in the form of human or animal serum, pooled immunoglobulin or monoclonal antibodies, into the affected individual. [88]

    Prenatal therapy Edit

    Rh factor, also known as Rh D antigen, is an antigen found on red blood cells individuals that are Rh-positive (Rh+) have this antigen on their red blood cells and individuals that are Rh-negative (Rh–) do not. During normal childbirth, delivery trauma or complications during pregnancy, blood from a fetus can enter the mother's system. In the case of an Rh-incompatible mother and child, consequential blood mixing may sensitize an Rh- mother to the Rh antigen on the blood cells of the Rh+ child, putting the remainder of the pregnancy, and any subsequent pregnancies, at risk for hemolytic disease of the newborn. [89]

    Rho(D) immune globulin antibodies are specific for human RhD antigen. [90] Anti-RhD antibodies are administered as part of a prenatal treatment regimen to prevent sensitization that may occur when a Rh-negative mother has a Rh-positive fetus. Treatment of a mother with Anti-RhD antibodies prior to and immediately after trauma and delivery destroys Rh antigen in the mother's system from the fetus. It is important to note that this occurs before the antigen can stimulate maternal B cells to "remember" Rh antigen by generating memory B cells. Therefore, her humoral immune system will not make anti-Rh antibodies, and will not attack the Rh antigens of the current or subsequent babies. Rho(D) Immune Globulin treatment prevents sensitization that can lead to Rh disease, but does not prevent or treat the underlying disease itself. [90]

    Specific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat, rabbit, goat, sheep, or horse for large quantities of antibody. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen—in the serum, which can now be called antiserum. Antigens are also injected into chickens for generation of polyclonal antibodies in egg yolk. [91] To obtain antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody these antibodies are called monoclonal antibodies. [92] Polyclonal and monoclonal antibodies are often purified using Protein A/G or antigen-affinity chromatography. [93]

    In research, purified antibodies are used in many applications. Antibodies for research applications can be found directly from antibody suppliers, or through use of a specialist search engine. Research antibodies are most commonly used to identify and locate intracellular and extracellular proteins. Antibodies are used in flow cytometry to differentiate cell types by the proteins they express different types of cell express different combinations of cluster of differentiation molecules on their surface, and produce different intracellular and secretable proteins. [94] They are also used in immunoprecipitation to separate proteins and anything bound to them (co-immunoprecipitation) from other molecules in a cell lysate, [95] in Western blot analyses to identify proteins separated by electrophoresis, [96] and in immunohistochemistry or immunofluorescence to examine protein expression in tissue sections or to locate proteins within cells with the assistance of a microscope. [94] [97] Proteins can also be detected and quantified with antibodies, using ELISA and ELISpot techniques. [98] [99]

    Antibodies used in research are some of the most powerful, yet most problematic reagents with a tremendous number of factors that must be controlled in any experiment including cross reactivity, or the antibody recognizing multiple epitopes and affinity, which can vary widely depending on experimental conditions such as pH, solvent, state of tissue etc. Multiple attempts have been made to improve both the way that researchers validate antibodies [100] [101] and ways in which they report on antibodies. Researchers using antibodies in their work need to record them correctly in order to allow their research to be reproducible (and therefore tested, and qualified by other researchers). Less than half of research antibodies referenced in academic papers can be easily identified. [102] Papers published in F1000 in 2014 and 2015 provide researchers with a guide for reporting research antibody use. [103] [104] The RRID paper, is co-published in 4 journals that implemented the RRIDs Standard for research resource citation, which draws data from the antibodyregistry.org as the source of antibody identifiers [105] (see also group at Force11 [106] ).

    Production and testing Edit

    Traditionally, most antibodies are produced by hybridoma cell lines through immortalization of antibody-producing cells by chemically-induced fusion with myeloma cells. In some cases, additional fusions with other lines have created "triomas" and "quadromas". The manufacturing process should be appropriately described and validated. Validation studies should at least include:

    • The demonstration that the process is able to produce in good quality (the process should be validated)
    • The efficiency of the antibody purification (all impurities and virus must be eliminated)
    • The characterization of purified antibody (physicochemical characterization, immunological properties, biological activities, contaminants, . )
    • Determination of the virus clearance studies

    Before clinical trials Edit

    • Product safety testing: Sterility (bacteria and fungi), In vitro and in vivo testing for adventitious viruses, Murine retrovirus testing. Product safety data needed before the initiation of feasibility trials in serious or immediately life-threatening conditions, it serves to evaluate dangerous potential of the product.
    • Feasibility testing: These are pilot studies whose objectives include, among others, early characterization of safety and initial proof of concept in a small specific patient population (in vitro or in vivo testing).

    Preclinical studies Edit

    • Testing cross-reactivity of antibody: to highlight unwanted interactions (toxicity) of antibodies with previously characterized tissues. This study can be performed in vitro (Reactivity of the antibody or immunoconjugate should be determined with a quick-frozen adult tissues) or in vivo (with appropriates animal models).
    • Preclinical pharmacology and toxicity testing: preclinical safety testing of antibody is designed to identify possible toxicity in humans, to estimate the likelihood and severity of potential adverse events in humans, and to identify a safe starting dose and dose escalation, when possible.
    • Animal toxicity studies: Acute toxicity testing, Repeat-dose toxicity testing, Long-term toxicity testing
    • Pharmacokinetics and pharmacodynamics testing: Use for determinate clinical dosages, antibody activities, evaluation of the potential clinical effects

    The importance of antibodies in health care and the biotechnology industry demands knowledge of their structures at high resolution. This information is used for protein engineering, modifying the antigen binding affinity, and identifying an epitope, of a given antibody. X-ray crystallography is one commonly used method for determining antibody structures. However, crystallizing an antibody is often laborious and time-consuming. Computational approaches provide a cheaper and faster alternative to crystallography, but their results are more equivocal, since they do not produce empirical structures. Online web servers such as Web Antibody Modeling (WAM) [107] and Prediction of Immunoglobulin Structure (PIGS) [108] enables computational modeling of antibody variable regions. Rosetta Antibody is a novel antibody FV region structure prediction server, which incorporates sophisticated techniques to minimize CDR loops and optimize the relative orientation of the light and heavy chains, as well as homology models that predict successful docking of antibodies with their unique antigen. [109]

    The ability to describe the antibody through binding affinity to the antigen is supplemented by information on antibody structure and amino acid sequences for the purpose of patent claims. [110] Several methods have been presented for computational design of antibodies based on the structural bioinformatics studies of antibody CDRs. [111] [112] [113]

    There are a variety of methods used to sequence an antibody including Edman degradation, cDNA, etc. albeit one of the most common modern uses for peptide/protein identification is liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). [114] High volume antibody sequencing methods require computational approaches for the data analysis, including de novo sequencing directly from tandem mass spectra [115] and database search methods that use existing protein sequence databases. [116] [117] Many versions of shotgun protein sequencing are able to increase the coverage by utilizing CID/HCD/ETD [118] fragmentation methods and other techniques, and they have achieved substantial progress in attempt to fully sequence proteins, especially antibodies. Other methods have assumed the existence of similar proteins, [119] a known genome sequence, [120] or combined top-down and bottom up approaches. [121] Current technologies have the ability to assemble protein sequences with high accuracy by integrating de novo sequencing peptides, intensity, and positional confidence scores from database and homology searches. [122]

    Antibody mimetics are organic compounds, like antibodies, that can specifically bind antigens. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. Nucleic acids and small molecules are sometimes considered antibody mimetics, but not artificial antibodies, antibody fragments, and fusion proteins are composed from these. Common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. Antibody mimetics have being developed and commercialised as research, diagnostic and therapeutic agents. [123]


    The Starting Lineup

    One of the most important types of cells in the lymphatic and immune systems is the white blood cell. White blood cells are superstars. They include:

    • Natural killer cells (NK), which go after tumor cells and viruses and insert a protein into the cells that destroys them.
    • T cells, which destroy foreign or damaged cells. The helper T cells plan the attack and the killer T cells carry it out.
    • B cells, which create antibodies. Antibodies are specific to one antigen (a toxin or foreign substance). They attach to that antigen like a key in a lock. The B cells that create the antibodies “remember.” If an antigen enters your body and your B cells recognize it, either from having had the disease before or from being vaccinated against it, your B cells increase production of the appropriate antibody. When the antibody binds to the antigen, it is a signal for other cells and molecules of the immune system to come and destroy the antigen.

    Bone marrow, the spongy inside of most of your bones, is where your blood cells are made. Immature blood cells, called stem cells, can grow into different types of cells, including blood cells. When mature, most blood cells leave the bone marrow and move into your blood or lymph system.

    The immune system consists of more than just the lymphatic system. Your skin and mucus membranes act as the first line of defense. Skin presents a physical barrier. The mucus membranes that line your natural openings, such as your mouth, nose and anus, make and release substances that create a hostile environment for the invaders and/or attack and destroy the invaders directly.

    Vaccines work with your body’s natural defenses to create immunity to a specific disease. Long ago, people realized that survivors of a disease didn’t get that disease again. A British doctor is often credited with the first vaccine (for smallpox) in the 1790s, but a Chinese emperor who was a smallpox survivor himself started an inoculation program against the disease in the mid-1600s.

    Activating Your Immune System Against Cancer

    Now that you've learned a little bit about your immune system, find out how it's used to fight cancer.


    Placental Protection

    This article is part of Harvard Medical School’s continuing coverage of medicine, biomedical research, medical education, and policy related to the SARS-CoV-2 pandemic and the disease COVID-19.

    Pregnant women infected with SARS-CoV-2 appear to be at higher risk of developing severe cases of COVID-19 than infected women who are not pregnant.

    Yet newborns are mostly doing well. Reports indicate that only about 5 percent of babies born to COVID-19-infected mothers are themselves infected, and most newborns who test positive have mild or asymptomatic infections that rarely need mechanical ventilation.

    Three new studies from Harvard Medical School researchers at Massachusetts General Hospital and Boston Children's Hospital provide insight into what may be happening.

    The results could help researchers and doctors better understand how the body protects against COVID-19, improve care for pregnant women and newborns, and inform vaccine timing.

    Viral barrier

    The first study, published in October 2020 in the journal Placenta, examined how the placenta—the complex and little-understood organ linking parent to fetus—responds to the coronavirus itself.

    First author Elizabeth Taglauer, an HMS instructor in pediatrics and a placental immunologist at Boston Children’s, and senior author Elisha Wachman at Boston University School of Medicine and Boston Medical Center followed pregnant women with COVID-19 and studied a variety of maternal and fetal samples, including placentas discarded after delivery.

    The team's findings indicate that SARS-CoV-2 can only partially penetrate the placenta.

    Even when babies were uninfected, the researchers detected SARS-CoV-2 spike protein in the placental villi, specifically in the outermost layer, which directly contacts the mother’s blood and is the first barrier the virus must cross to get from mother to fetus.

    The researchers also found that ACE2, the primary receptor for SARS-CoV-2, was present in this placental layer at lower levels in infected than in uninfected mothers. Taglauer plans to investigate whether the placenta downregulates ACE2 as a protective measure against infection.

    The researchers found TMPRSS2, another protein the virus uses to enter cells, in placental tissues from infected and uninfected mothers at lower levels than ACE2. This suggests that the virus uses other receptors to penetrate the placenta—another question for further exploration.

    Taglauer and Wachman are now collaborating with Jeffrey Moffitt, HMS assistant professor of microbiology and of pediatrics at Boston Children's, to analyze every cell within the placental tissues of infected and uninfected mothers and profile the genes important to COVID-19. They will use a technology called MERFISH that directly images RNA.

    In the meantime, Taglauer and Wachman built a biorepository of almost 80 placentas from COVID-19-positive mothers and have made it available as a resource for others.

    The researchers hope the work will not only lead to better care for pregnant women but also reveal more about how the virus infects the lungs and other organs.

    “The anatomy of the placenta is very similar to that of the developing lungs and intestines at certain stages," said Taglauer. "And unlike lung or intestinal tissue from COVID-19 patients, we can get our hands on this tissue right away.”

    The Placenta study was funded by the Boston University Clinical and Translational Science Institute COVID-19 Pilot Grant Program (UL1TR001430), National Institutes of Health (1T32HD098061-01) and the BU School of Medicine Medical Student Summer Research Program.

    Antibody discrepancy

    The second study, published Dec. 22, 2020, in JAMA Network Open, similarly found that pregnant women with COVID-19 don’t pass the virus to newborns—but also found that mothers may pass along fewer than expected protective antibodies.

    A follow-up study by members of the same team, published the same day in Cell, expands on the antibody findings.

    The JAMA study included 127 pregnant women in their third trimester who received care at three Boston hospitals between April 2 and June 13, 2020. Among the 64 women who tested positive for SARS-CoV-2, investigators detected no virus in maternal or umbilical cord blood despite detection in the women’s respiratory system. They found no signs of the virus in the placenta, either, and no evidence of viral transmission to newborns.

    The team discovered that although most of the women who tested positive developed antibody responses against SARS-CoV-2 proteins, mother-to-newborn transfer of these antibodies through the placenta was significantly lower than transfer of antibodies against influenza.

    Transfer of antibodies across the placenta to the fetus is typically highest in the third trimester, so it was unexpected to see such a reduction compared with flu antibodies, said first author Andrea Edlow, HMS assistant professor of obstetrics, gynecology, and reproductive biology at Mass General.

    The Cell paper reveals that the cause of the lower than expected antibody transfer may be alterations to these antibodies after they’re produced.

    The team compared maternal antibodies against the flu, whooping cough, and SARS-CoV-2, and how these antibodies transferred across the placenta. They found not only that SARS-CoV-2-specific antibodies were transferred at significantly reduced rates during the third trimester compared with the flu and whooping cough but also that the coronavirus antibodies were less functional than those against influenza.

    The scientists found that alterations in glycosylation, a process of attaching carbohydrates to molecules, may be to blame for the reduced transfer. They observed that carbohydrates attached differently to SARS-CoV-2-specific antibodies in maternal blood than they did to influenza- and pertussis-specific antibodies. This abnormality may cause the coronavirus-specific antibodies to get stuck in the maternal circulation because they cannot bind as well to antibody receptors on the placenta.

    The team found that some functional antibodies were able to cross the placenta because of infection-induced increases in total maternal antibodies as well as higher placental expression of receptors that bind to the altered antibody carbohydrate pattern. Some of the antibodies that transferred the best were also the most functional, the researchers discovered.

    The findings from both papers have implications for designing and administering COVID-19 vaccines to pregnant women, the authors said.

    "Specifically, it highlights that pregnant women are a key population to consider in vaccine rollouts," said Edlow, who is co-senior author of the Cell paper. "It also raises questions regarding the optimal timing of vaccine administration to best support maternal and newborn immunity."

    “Vaccine regimens able to drive high levels of the COVID-specific antibodies with glycosylation patterns favored by the placenta for selective transfer to the fetus may lead to better neonatal and infant protection,” Edlow added.

    Understanding how antibody transfer varies by trimester may point to critical windows in pregnancy for vaccination to optimize parental and infant protection.

    “We are beginning to define the rules of placental antibody transfer of SARS-CoV-2 for the very first time—catalyzing our ability to rationally design vaccines to protect pregnant women and their newborns," said Galit Alter, HMS professor of medicine at Mass General, senior author of the JAMA Network paper and co-senior author of the Cell paper.

    Both studies were funded by the National Institutes of Health (grants 3R01HD100022-02S2 , K23HD097300, K08HL146963, K08HL143183, U19AI135995, R37AI80289 and R01AI146785)), with additional support from the March of Dimes, Mass General Department of Obstetrics and Gynecology, Massachusetts Consortium on Pathogen Readiness, Bill & Melinda Gates Foundation, U.S. Centers for Disease Control and Prevention (CK000490), and Harvard University Center for AIDS Research.

    Adapted from a post on Boston Children's Discoveries portal and from two Mass General news releases.


    Innate and adaptive immune system

    There are two subsystems within the immune system, known as the innate (non-specific) immune system and the adaptive (specific) immune system. Both of these subsystems are closely linked and work together whenever a germ or harmful substance triggers an immune response.

    The innate immune system provides a general defense against harmful germs and substances, so it’s also called the non-specific immune system. It mostly fights using immune cells such as natural killer cells and phagocytes (�ting cells”). The main job of the innate immune system is to fight harmful substances and germs that enter the body, for instance through the skin or digestive system.

    The adaptive (specific) immune system makes antibodies and uses them to specifically fight certain germs that the body has previously come into contact with. This is also known as an �quired” (learned) or specific immune response.

    Because the adaptive immune system is constantly learning and adapting, the body can also fight bacteria or viruses that change over time.


    Common mistakes that must be avoided

    Some of the common mistakes that candidates must avoid

    • Not adhering to the time table. Candidates must follow the timetable strictly so that they can get a good score in the exam.
    • There is no need to follow a hectic schedule or study for extended hours, as it might affect your retention power.
    • Most of the biology questions come from NCERT textbooks only, so ignoring the same can result in losing your scores. Check NEET 2020 Important Books
    • Don’t refer so many books, rather you must study from one book at a time. Else you will get confused and will mess up everything you learned so far.
    • Don’t ignore any chapter in human health and diseases, even a single page needs to be studied and revised properly.

    It is important to stick to the time table and follow the above-mentioned preparation tips that can help to get a good rank in NEET Exam. Also, at the same time, it is important to avoid all the mistakes that can lower your scores.

    Medical UG aspirants need to do NEET Biology Preparation by considering these tips and tricks that help them to achieve the best possible results. Stay focused and best of luck for the upcoming NEET exam.


    Materials and Methods

    Model of Selection on the CMAH(−) Allele.

    Homozygous CMAH(−/−) females have a nonself immune reaction to the Neu5Gc Sia on the sperm or embryos of CMAH(+/+) and CMAH(+/−) males. Fig. 5A shows a compatibility matrix that describes the reproductive output of all possible CMAH genotype combinations. In males, selection favors the CMAH(−) allele in females, selection acts against the CMAH(−) allele (Fig. 5A). The effect of selection and expected frequency change of the CMAH(−) allele over a single generation was calculated for each 0.01 frequency increment between 0 and 1 by substituting selection inferred from the compatibility matrix into standard equations for calculating frequency change by selection on dominant alleles (34).

    Males.

    The proportion of females compatible with a CMAH(−/−) male = 1. The proportion compatible with a CMAH(+/+) or CMAH(+/−) male was calculated as 1 − q 2 , where q is the frequency of the CMAH(−) allele. For completely incompatible crosses the disadvantage to CMAH(+/+) and CMAH(+/−) males, sm = q 2 . c is the compatibility, the fraction of the cross which is restored in cases of partial incompatibility. The strength of selection against CMAH(+/+) and CMAH(+/−) males is thus described by: sm = (1 − c) × q 2 . The expected frequency the CMAH(−) allele after a single generation of selection in males is given by the standard equation describing selection against a dominant allele: q1 = qsmq + smq 2 /1 − sm(1 − q 2 ).

    Females.

    The strength of selection in females depends on the probability of encountering a compatible mate. For completely incompatible crosses, homozygous CMAH(−/−) females can only be fertilized by CMAH(−/−) males, which are encountered in each mating attempt with a probability of q 2 . Partial compatibility raises the probability of encountering a compatible CMAH(−/−) male (ε) by a fraction proportional to the compatibility of a CMAH(−/−) female with CMAH(+/+) and CMAH(+/−) males: ε = q 2 + c(1 − q 2 ).

    The strength of selection against homozygous CMAH(−/−) females (sf) is the proportion of CMAH(−/−) females that did not successfully reproduce relative to CMAH(+/+) and CMAH(+/−) females. sf is calculated as the binomial probability that a CMAH(−/−) female will encounter zero (k = 0) compatible mates in a given number of mating attempts (n) with a given probability of encountering a compatible mate (ε). The expected frequency of the CMAH(−) allele after a single generation of selection in females is given by the standard equation describing selection favoring a dominant allele: q1 = qsfq/1 − sfq 2 .

    The combined effect of selection in males and females and the expected change in frequency of the CMAH(−) allele over a single generation was calculated by equally weighting the negative effect in females and the positive effect in males (assuming a 50:50 population sex ratio). Scripts were written in Perl by S. Springer (SI Appendix).

    The transgenic Cmah(−/−) mice have been described (19) and were used under protocol S01227. Age-matched female mice were immunized, and cohorts of females with similar spectra of anti-Neu5Gc titers were formed. Females were bred with a single male. Litters were removed at weaning.

    Mouse Sperm.

    Mouse sperm were harvested from the cauda epididymis of 12- to 20-wk-old males immediately after sacrificing the animals. Cauda epididymis tissue was minced and kept on a shaker at room temperature (RT) for 10 min, followed by 30 s of centrifugation at 500 × g and a swim up procedure in sperm storage buffer [SSB: 110 mM NaCl/27.2 mM KCl/0.36 mM NaH2PO4/0.49 mM MgCl2/2.40 mMCaCl2/25.00 mM Hepes/5.00 mM Mes/2-(N-morpholino)ethanesulfonic acid/25.00 mM lactic acid, pH 5.5 (35)]. Sperm were further subjected to a swim up procedure in 500 μL of SSB at 37 °C, 5% CO2 for 30 min before collection of the supernatant.

    Mouse Immunization and Determination of Anti-Neu5Gc Immune Response.

    Antibody titers in mouse serum and mouse uterine fluids were determined by ELISA using Neu5Gc- and Neu5Ac-polyacrylamide beads as plated targets (16). Sia dependence of antibody binding was confirmed by mild periodate treatment to destroy the side chain of Sia and controlled for by mock treatment without the premixed periodate and sodium borohydride (16). Uterine fluid was collected by immediate postsacrifice flushing of the uteri with PBS.

    Antibody–Sperm Binding Assays.

    Antibody–sperm binding by mouse immune sera was studied on plated and freshly fixed sperm of all three possible Cmah genotypes, which was collected immediately after sacrificing the male mice. Sperm were diluted to a concentration of 8 million/mL in Biggers Whitten Whittingham, 0.05% human serum albumin. A total of 100 μL of this suspension was added to each well of a COSTAR microplate. Plates were spun down at 250 × g for 5 min at RT. Supernatant was discarded. Cell densities were verified under microscope. The plate was allowed to air dry for 10 min. The plated sperm were fixed using freshly thawed formaldehyde adjusted to 1%. A total of 200 μL of 1% pfa in PBS was added to each well. After fixation, the plate was washed 3× with 200 μL of PBS (0.1%) per well. For sialidase treatment, 5 mU of A. ureafaciens sialidase (AUS) in buffer (50 mM NaPO4, pH 6) were added to each well, and the sample was incubated at 37 °C for 2 h. Controls were treated with buffer or heat-treated AUS. The plate was blocked with 1% ovalbumin at RT for 1 h, serum was added at a concentration of 1:100 in TBS ovalbumin (100 μL per well) and incubated at RT for 2 h. The plate was washed three times with 150 μL of TBS (1%) ovalbumin per well and then blotted. Secondary antibody (donkey anti-mouse IgG) was added at concentration of 1:500 in TBS and incubated for 30 min at RT. The plate was washed three times with TBS before adding alkaline phosphatase substrate, developing in the dark, and reading at 490 nm.

    Neu5Gc Antigens for ELISA.

    Antigen was prepared from homogenate of H. influenzae that was precultured with or without Neu5Gc, as described (17). The latter served as the control antigen.

    Hominid Sperm.

    Chimpanzee sperm were collected by a noninvasive method (36). The samples from a total of six different chimpanzees were shipped at RT and analyzed side-by-side with human samples that were collected the same day as the chimpanzee samples and kept at RT until arrival of the chimpanzee samples (UCSD protocol 040613).

    Sample Preparation for Fluorescence Microscopy.

    Human Sera.

    Human sera were collected by venipuncture from human volunteers under University of California, San Diego, IRB-approved protocol 080677x.

    Sialic Acid Analysis.

    Hominin Sperm Exposure to Anti-Neu5Gc Antibodies in Serum.

    Sperm were exposed to sera in a 1:2 dilution (20 million sperm in 50 μL of BWW medium plus 50 μL of neat serum) for 2 h at RT. Sera included human sera with high or low tiers of anti-Neu5Gc and chimpanzee sera (no anti-Neu5Gc antibodes). Anti-Neu5Gc content of human sera had been previously determined by ELISA using Neu5Gc target probes (16). Samples were then washed and stained for cell death with propidium iodine (PI, 15 min at RT) or for complement deposition with anti-C3 monoclonal antibody [1 h at RT, C3 (6C9), Santa Cruz Biotechnology]. Staining was quantified by flow cytometry. Sera were untreated or heat treated (30 min at 56 °C) to inactivate complement. Chimpanzee serum was added as an inhibitor due to its high content of Neu5Gc. The effect of serum exposure was measured by staining with PI followed by flow cytometry. Negative controls were incubated in BWW buffer.

    Statistics.

    Statistical analyses were performed using Prism v5.0a (GraphPad Software).


    Watch the video: Πως επιδρά η διατροφή στην ποιότητα του σπέρματος (May 2022).


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