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Antibodies have the ability of recognising highly specific peptide sequences and bind it at their antigen-binding site.
This ability is harnessed as a tool in research to purify target structures in the cell (e.g. in chromatin immunoprecipitation, ChIP).
Now let's say that I've identified an interesting target structure (such as a particular transcription factor) and I want to design an antibody to use in ChIP against said target. How would I go about producing such an antibody, taking into account that it should be both highly sensitive and specific?
I worked for a long time at a leading high-quality antibody company, so I'll try and share some of my experiences with you. The process of making a highly specific antibody (I'll focus on monoclonals) has three important parts - antigen design and immunization, cloning and subcloning, and screening/validation. Each part is crucial on its own, and the better one is performed, the higher your chances of success downstream become.
In order for an antibody to work, it needs to bind specifically to its target. I won't get into all the specifics here, as there is a good open literature on antigen design, but basically you need something that will promote a strong immune response in the host, give you a variety of clones to choose from, and be specific - it shouldn't bind other targets besides your protein of interest. There are lots of factors to consider, including antigenicity, solubility, ease of production (for immunizing and screening), steric considerations (does another protein or nucleic acid obscure the target site in vivo?), and others. Antigens can be roughly divided into two types - peptides (typically less than 20 or 30 amino acids) and proteins or protein fragments. You will most likely want to try several antigens to maximize your probability of success. After the antigen is synthesized and purified, you need to come up with an immunization schedule for priming and boosting the animal(s), then determine when and how to test them (screening), and at what time and how to harvest it for the monoclonal process.
The cloning process then comes next. Clones can be generated by a variety of methods, some widely available, such as the various hybridoma methods, and some proprietary to individual companies. My former company employed a mix of both, depending on the species of animal, using some really cool in-house tech that was constantly being improved and expanded upon. These processes are very dependent on the quality of materials being used and the expertise of the cloners. The input B cells (which make the antibodies) need to have been harvested, treated, and stored according to a set of precise steps to allow for the highest number of viable clones. Once the immortalized cells have been obtained (after fusion with the myeloma partner cells in the hybridoma process, for example) they are diluted to a pre-determined cell count and plated in 96-well plates and allowed to recover and grow for a time, during which they are secreting antibodies into the medium. This media is then tested quickly (before the cells overgrow the well), and positive wells are diluted out to (hopefully) single-cell suspensions and subcloned, or frozen down for later. The first testing is frequently by ELISA using the immunizing antigen, although depending on your application of interest it may be by high-throughput screening via flow cytometry, immunohistochemistry, or immunofluorescence. After the initial testing, subsequent steps of subcloning and re-testing can occur at a more measured pace, as the cells can be frozen indefinitely after generating sufficient quantities of antibodies in the supernatant.
This is where the quality of your screening program comes in. Your assays need to be well-designed, robust, well-documented and performed, and have appropriate positive and negative controls so that you can truly tell whether a certain clone is of interest, or performs better than an existing product. This means controlled conditions, standardized reagents and protocols, and a high degree of repeatability from assay to assay. Also, the appropriateness of your controls cannot be emphasized enough. Do you have a knockout or non-expressing sample for comparison, or a way of looking at baseline vs. induced or inhibited activity? For ChIP, unless you have a good control antibody and primer pair for your gene of interest, how will you know if the assay works? You may get a great result doing a straight immunoprecipitation-Western blot, but still not pull down DNA-bound protein. If your ChIP method doesn't produce clean DNA, you may never get binding. And if you don't have appropriate data analysis protocols in place with multiple replicate wells, you may waste time chasing noise instead of specific signal. Additionally, along with testing all your clones, you also need to titrate them to determine the optimal working concentration. I've seen a lot of antibodies that look like crap when you first test the supe, but diluted 100 or 1000X they are clean, specific, and still strong. Be patient, testing is a lot of work and requires a ton of repetition to confirm your results, but it'll be well worth it in the end.
Finally, after all this is done, you hopefully have a great clone that does what you want it to do, and better than anything else out there. You now need to decide what you're going to do with it, because if you're an academic lab and publish with it, the world will come knocking at your door. Don't slack off at the end and decide 100 ml of supernatant will be enough to last forever - save the clones and put them in a cell bank, or try to get a commercialization deal with a manufacturing company so you don't have to do all the work!
I hope this helps, please let me know if you have any additional questions or concerns.
Answering Konrad's comment, you produce the peptide or protein of interest using bacteria or chemically synthesize them (chemical synthesis generally only work for short peptide chains. After which, you get a lot of side products). This peptide or protein of your interest serves as your antigen. You inject this antigen into mouse or rabbit. The animal's immune system will naturally mount an immune response to produce antibodies against it. The B cells which are responsible for producing those antibodies can then be harvested. After which, you basically follow the protocol Matt stated.
You could, of course, also choose to just directly isolate the serum of the animal and purify it. However, this leads to the production of polyclonal antibodies which can be less specific than a monoclonal antibody.
An antibody (Ab), also known as an immunoglobulin (Ig),  is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen.   Each tip of the "Y" of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly (for example, by blocking a part of a virus that is essential for its invasion).
To allow the immune system to recognize millions of different antigens, the antigen-binding sites at both tips of the antibody come in an equally wide variety. In contrast, the remainder of the antibody is relatively constant. It only occurs in a few variants, which define the antibody's class or isotype: IgA, IgD, IgE, IgG, or IgM. The constant region at the trunk of the antibody includes sites involved in interactions with other components of the immune system. The class hence determines the function triggered by an antibody after binding to an antigen, in addition to some structural features. Antibodies from different classes also differ in where they are released in the body and at what stage of an immune response.
Together with B and T cells, antibodies comprise the most important part of the adaptive immune system. They occur in two forms: one that is attached to a B cell, and the other, a soluble form, that is unattached and found in extracellular fluids such as blood plasma. Initially, all antibodies are of the first form, attached to the surface of a B cell – these are then referred to as B-cell receptors (BCR). After an antigen binds to a BCR, the B cell activates to proliferate and differentiate into either plasma cells, which secrete soluble antibodies with the same paratope, or memory B cells, which survive in the body to enable long-lasting immunity to the antigen.  Soluble antibodies are released into the blood and tissue fluids, as well as many secretions. Because these fluids were traditionally known as humors, antibody-mediated immunity is sometimes known as, or considered a part of, humoral immunity.  The soluble Y-shaped units can occur individually as monomers, or in complexes of two to five units.
Antibodies are glycoproteins belonging to the immunoglobulin superfamily. The terms antibody and immunoglobulin are often used interchangeably,  though the term 'antibody' is sometimes reserved for the secreted, soluble form, i.e. excluding B-cell receptors. 
1. Tell us your goal
We at Precision Antibody want to learn about your goals so that we can custom design a project that will cover all your requirements. We have expertise in a wide range of antibody development from straight-forward projects like an antibody against a peptide, to more “challenging” projects developing an antibody that recognizes a single amino acid mutation, or a specific cleavage site. For a full description of types of antibodies we develop, please visit Our Services.
2. Finalize the Plan
Precision Antibody will design your project through in-depth communication with you. We will discuss multiple approaches to meet your goals. At the end of the discussion, we will put together a Statement of Work. Based on the SOW, PA will issue a quote. All the discussions whether we have executed CDA or not will be kept confidential. Once all steps have been approved and finalized, it is time to get started! We understand how important your project is, so we will keep you informed every step along the progression of your project. Our project manager will send you updates after every step and guide you through the next steps. We will work together closely to help you achieve your goal.
3. Optimize Your Antigen to Meet Your Goals
Every successful project starts from well-thought out optimized antigen design. Our team of experienced and knowledgeable immunologists will optimize your target for immunization. Our services include but not limited to goal-specific antigen design using our proprietary TED technology that transforms non- or weak immunogens to become highly immunogenic, designing appropriate immunogen targeting multi-span transmembrane proteins, and design strategies for small molecule targets.
4. Find and Execute the Best Immunization Strategy Precision Antibody’s proprietary immunization technology is designed to be tailored to your project. All variables are considered to assure we can obtain the best titer in a short amount of time (1:100,000 in 2 ½ weeks) which guarantees the best antibody. PA also offers specialized immunization strategies for cell based immunizations and other challenging targets.
5. Optimized Process for Hybridoma Generation and Screening
At the end of the initial immunization cycle, serum titer check will be performed. PA will not fuse unless a sufficient titer (higher than 3-times the control OD at serum dilution of 1:100,000) is obtained. PA uses electrofusion to ensure a high fusion rate and viability. Utilization of well-designed screening method is of a critical importance in the selection of hybridoma clones producing mAb of interest. Precision Antibody utilizes screening method most compatible with the intended application of your antibodies to ensure that the selected clones generate antibodies that meet your acceptance criteria.
PA will send you data at each milestone (Phase) to keep you up-to-date. we will guide you through the next phases that will lead you to the selection of best hybridoma(s) for your application. This may include Octet based binding affinity measurements, Protein Simple WES assay, and flow-based functional assays. Please view our full list of our services.
Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces
Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.
Conflict of interest statement
The authors have declared that no competing interests exist.
Fig 1. Deep mutational scanning of an…
Fig 1. Deep mutational scanning of an antibody variable fragment (Fv).
Fig 2. Gains in affinity, stability, and…
Fig 2. Gains in affinity, stability, and aggregation resistance through vL-vH interface design guided by…
Fig 3. Mutational-tolerance mapping by Rosetta atomistic…
Fig 3. Mutational-tolerance mapping by Rosetta atomistic energy calculations (ΔΔ G ) and evolutionary-conservation scores…
Fig 4. Fully automated antibody stability and…
Fig 4. Fully automated antibody stability and affinity optimization using AbLIFT.
Fig 5. Substantial increase in antibody expression…
Fig 5. Substantial increase in antibody expression yields following AbLIFT design.
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We have 10,000+ antibody products with validated images available for applications like ELISA, WB, IHC, FC..etc, including Rabbit polyclonal & Mouse monoclonal primary antibody and tagged secondary antibody such as HRP, FITC, Biotin…etc.
Not Just About Antibodies: Why mRNA COVID Vaccines May Shield From Variants
TUESDAY, May 4, 2021 (HealthDay News) -- Two widely used COVID-19 vaccines — Pfizer and Moderna — will likely remain powerfully protective against developing serious illness even if coronavirus variants somehow manage to infect vaccinated patients, new research suggests.
Both vaccines are based on messenger RNA (mRNA) technology. And investigators say that, at least in theory, such technology can deploy multiple levels of defense to keep ever-changing coronavirus variants in check.
"It is important to note that only time will tell whether the vaccines do in fact protect against other human coronaviruses," cautioned study author Dr. Joel Blankson, a professor of medicine at Johns Hopkins Medicine, in Baltimore. "But this could be a good news story."
The reasoning, said Blankson, is that both the Pfizer and Moderna vaccines actually do two things at once.
On the one hand, the vaccines trigger the immune system into producing protective antibodies. "In terms of COVID-19, antibodies would work to prevent virus from infecting cells" in the first place, he explained.
At the same time, the vaccines also cause the immune system to generate so-called "helper T-cells," known as CD4+ T lymphocytes. "And T-cells would kill cells that are infected, to help prevent the infection from spreading," Blankson said.
CD4+ T-cells do this by setting in motion a chain of immune-bolstering events, including the activation of a particularly powerful "killer" T-cell known as CD8+, the study team noted.
After conducting an in-depth blood sample analysis, Blankson's team found that such killer T-cells seem to keep the coronavirus firmly in the immune system's crosshairs, even if antibodies designed to combat an early iteration of the virus fail to prevent infection with a newer variant.
The blood analysis involved 30 men and women, ranging in age from 20 to 59. All had been vaccinated with two doses of one of the two mRNA vaccines, and none had tested positive for COVID-19 either prior to vaccination or afterwards. Both the Pfizer and the Moderna vaccine are approved for emergency use in the United States. Another approved vaccine produced by Johnson & Johnson is based on a different technology, and was not included in the study.
As intended, the mRNA vaccines first triggered the production of a harmless version of a "spike protein" found on the surface of the coronavirus. That, in turn, launched each recipient's immune system into action, generating antibodies to fight off infection.
But beyond that, initial lab tests revealed that the mRNA vaccine also produced a strong T-cell response following exposure to the original strain of the coronavirus.
That initial response generated 23 different types of T-cell protein building blocks (or peptides). And subsequent blood analyses further showed that fewer than one-fifth of those peptides seemed to be impeded by newer coronavirus strains, such as the ones that recently took hold in the United Kingdom and South Africa.
According to Blankson, "T-cells and antibodies recognize different parts of the spike protein, and the parts of the variant proteins that allowed them to partially evade the antibody response are not important for T-cell recognition."
And that, he added, means that "even if the variants are able to escape the antibody response and infect cells, T-cells should be able to kill infected cells before the virus replicates to a high level that would cause severe disease."
A number of researchers offered upbeat reactions to the findings.
"Yes, it is a good story," said Rustom Antia, a professor in the department of biology at Emory University and affiliate faculty with Emory Vaccine Center, in Atlanta.
"The implications are that having a coordinated immune response with multiple components is better than one that is focused on a single component. Much like waging a successful military campaign requires Army, Navy and Air Force," said Antia.
Suzanne Judd, an epidemiologist and professor in the school of public health at the University of Alabama at Birmingham, agreed. "From a disease control and prevention standpoint, these research findings are as good as it gets," she said.
Blankson and colleagues "demonstrate that the vaccine can stop the spread of variants, which if true means we can be more comfortable returning to normal," Judd added.
At the same time, both Blankson and Judd said that other non-mRNA vaccines might end up proving equally adept at staving off serious illness.
"We don't have enough information to know if the J&J and AstraZeneca vaccines produce the same type of T-cell response yet," Judd noted. "The results of this study don't mean the same response is not true for other types of vaccines, because they did not examine other types of vaccines."
Indeed, Dr. David Hirschwerk, an attending infectious disease and internal medicine physician with Northwell Health in Manhasset, N.Y., said that "while this study looked only at mRNA vaccines, it does seem likely that the T-cell responses will be similar" in other vaccines like J&J, though he stressed that more research will be needed to know for sure.
He agreed that the latest finding "does add a layer of reasoning to why the vaccines appear protective against most of the important variants currently circulating." But, Hirschwerk stressed that "we still primarily need to rely upon the clinical observations of how patients infected with variants continue to fare over time after vaccination."
The report by Blankson's team was published recently in the Journal of Clinical Investigation.
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Antibodies: Meaning, Structure and Mechanism
Antibodies, the magic bullets of the immune system, are glycoproteins formed in response to antigenic stimulation and counteract with antigens with great specificity.
The antibodies are found in the serum fraction of the blood and are also known as immunoglobulins (Ig). The chemical composition and structure of antibodies was revealed by G.M. Edelman and R.M. Porter who received in 1972 Nobel Prize in Physiology and Medicine for this contribution.
Structure of Antibodies:
Antibodies (immunoglobulins) have molecular weights ranging from 150,000 to 900,000 daltons. Electron microscopic viewing reveals that the antibody molecules resemble the letter “T” before they combine with antigens (Fig. 41.6A) while they resemble the letter “Y” after antigens combine with them (Fig. 41.6B).
It is considered that the antigen-antibody binding causes a rearrangement in the T-shape structure of the antibody molecule resulting in Y-shape thus providing more exposure to complement binding site of heavy-chain for further reactions.
An immunoglobulin (antibody) molecule is composed of four polypeptide chains (Fig. 41.7). Two of the four chains are identical to each other and are called heavy (H) chains because of greater number of amino acids (approximately 440 amino acids in each chain) and thus high molecular weight (approximately 50,000 daltons).
The remaining two chains, also identical to each other, are termed light (L) chains because of lesser number of amino acids, (approximately 440 amino acids in each chain) and thus low molecular weight (approximately 23,000 daltons). Each antibody molecule consists of a stem-part and two arms.
The stem-part of the antibody molecule is formed by approximately one-half of each heavy-chain, and the two chains are joined together by sulphur to sulphur (disulphide) bonds. Each arm of the antibody molecule consists of approximately one-half of a heavy-chain and one light-chain, again joined by a disulphide bond.
Both, light and heavy-chains also possess intrachain disulphide bonds that create “loops”, each loop called a domain. Each light-chain contains a single variable-domain (VL) and a single constant-domain (CL) whereas each heavy-chain contains a singly variable-domain (VH) and three, sometimes, four constant-domains (CH1, CH2, CH3 and in some cases CH4).
The variable-domains form the variable (V) region while the constant-domains form the constant (C) region of each of the light and heavy-chains. Variable (V) regions of both the chains lie opposite to each other at the top of the two arms of the antibody molecule and represent the site where the antigen-antibody-binding takes place.
Remaining part of each of the chains represents the constant (C) region which varies in different class of antibodies with respect to its amino acid sequence, and thus determines the type of antigen-antibody reaction.
Classes of Antibodies:
All immunoglobulins (antibodies) physicochemical properties vary considerably because of variations in amino acid sequence in their heavy polypeptide chain. Therefore, according to their physicochemical properties, the immunoglobulins are subdivided into five different classes, namely, IgG, IgA, IgM, IgD, and IgE (Table 41.6).
Mechanism of Antibody Formation: Clonal Selection Theory:
These are the B-lymphocytes that produce antibodies in the body. Various theories have been proposed regarding the formation of antibodies, it is the clonal selection theory which has gained wide support. This theory states that there are a variety of B- lymphocytes present in the immune system.
These lymphocytes produce a small number of antibody molecules without any antigenic stimulation, and these antibody molecules integrate into the cytoplasmic membrane of their producer lymphocyte to serve as receptor site for specific antigen. When specific antigens enter the immune system, they interact only with complementary antibody- receptor site of B-lymphocyte.
In this way, such B-lymphocyte is “selected out” or “differentiated” by the union of specific antigen and its complementary antibody. This “selected out” or “differentiated” B-lymphocyte is stimulated to undergo multiplication leading to clones of plasma cells which synthesize and secrete a crop of antibodies complementary’ to the specific antigens that have entered the immune system.
In fact, the “selected out” or “differentiated” B-lymphocytes initiate multiplication forming two different cell populations: the primary B-lymphocytes and the secondary B-lymphocytes.
The primary, in response to antigenic stimulation, divide and transform into plasma cells which enter into the process of antibody formation, but the secondary B- lymphocytes do not. The latter circulate actively from blood to lymph and liver, and constitute memory cells.
These memory cells, however, transform into plasma cells when they are exposed to subsequent antigenic stimulation, perhaps years later, and enter into the process of antibody formation (Fig. 41.8A).
The B-lymphocytes get transformed into antibody-synthesizing mature plasma cells through a prolonged process (Fig. 41.8B). It has been revealed that a transformation period of about five days involving at-least eight successive cell generations is required for the formation of mature plasma cells from the B-lymphocyte cells.
With each cell generation, there is a progressive development of ribosomes and endoplasmic reticulum. By the fifth day, the RNA content of the plasma cell is very much increased and its entire protein synthesizing machinery is so activated that the endoplasmic reticulum cisternae are filled with antibody molecules, and the plasma cells rapidly secrete them.
It is estimated that about 90-95% of the total protein produced in plasma cells gives rise to antibodies, and about 10,000 antibody molecules are secreted per plasma cell per second. However, obviously thousands of plasma cells throughout the circulatory system are in operation for the production of antibodies at any given time.
Advanced Photon Source helps reveal how antibodies bind a molecule linked to cancer
Scientists are harnessing hard X-rays in the fight against cancer. A team of researchers, in conjunction with the U.S. Department of Energy’s ( DOE ) Argonne National Laboratory, has used ultrabright X-ray light to determine how specific types of antibodies can tell the difference between different forms of a cancer-linked molecule. These new insights will help scientists design better antibodies for potential treatments.
Tony Hunter, professor at the Salk Institute for Biological Studies, led this new research, building on years of study at his lab into amino acids, the building blocks of proteins. Hunter and his team were the first to show that adding phosphate to tyrosine, one of 20 amino acids in the human body, contributes to the progression of cancer. Their discovery not only led to the development of anticancer drugs, but also inspired researchers to start examining phosphate in combination with other amino acids.
“ Our antibodies are going to be key to studying this relatively understudied process of histidine phosphorylation, and thanks to X-ray crystallography, we now know how they work. This means we can potentially improve them for specific purposes and even perhaps for use in the clinical arena where we see evidence that histidine phosphorylation plays a role in disease.” — Tony Hunter, professor, Salk Institute for Biological Studies
Histidine, an amino acid the body uses to synthesize proteins, is the new target under study at the Hunter Lab. When phosphate is added, it forms phosphohistidine, an unstable molecule that has been linked to liver and breast cancer and neuroblastoma, a type of cancer often found in the adrenal glands.
To better understand phosphohistidine’s potential role in cancer, the Hunter research team has, for the past eight years, been developing and studying antibodies that can bind to it. But to discern exactly how these antibodies work, they needed a more specialized set of tools.
The Advanced Photon Source ( APS ), a DOE Office of Science User Facility at Argonne, was one of three light source facilities the research team used to gain more insight into this problem. At the facilities, they used a technique known as X-ray crystallography to determine the crystal structures of their antibodies bound to peptides (short amino acid sequences) containing phosphohistidine. Their work and findings were recently published in the Proceedings of the National Academy of Sciences ( PNAS ).
“ Our antibodies are going to be key to studying this relatively understudied process of histidine phosphorylation, and thanks to X-ray crystallography, we now know how they work,” said Hunter “ This means we can potentially improve them for specific purposes and even perhaps for use in the clinical arena where we see evidence that histidine phosphorylation is connected to cancer.”
To make use of this technique, Hunter’s team worked alongside researchers at The Scripps Research Institute and used three different light sources — the APS , the Advanced Light Source at DOE ’s Lawrence Berkeley National Laboratory, and the Stanford Synchrotron Radiation Lightsource at DOE ’s SLAC National Accelerator Laboratory. All three are DOE Office of Science User Facilities that provide extremely intense, small X-ray beams that are particularly useful for this type of technique.
Researchers first grew crystals of their antibodies bound to phosphohistidine peptides. These were then sent to the light sources, which had capabilities that allowed the researchers to place their crystals in an X-ray beam remotely. Upon contact with the crystals, the beams scattered, creating diffraction patterns that were collected and used to determine the 3 -D atomic structure of the antibodies combined with the phosphohistidine peptides.
“ X-rays have wavelengths that are about the size of atoms, and they scatter strongly. But it’s not so easy to make a lens that can recombine these rays to form an image near atomic resolution,” said protein crystallographer Michael Becker of Argonne’s X-ray Science Division. “ So instead, researchers collect diffraction data on detectors and use mathematics, physics and chemistry in the computer to essentially calculate an image of the molecule in the crystal.”
X-ray crystallography allows scientists to determine the molecular and atomic structure of these tiny crystals. By measuring these diffracted beams, scientists can reconstruct an image of the atoms and their position in the sample, as well as a host of other information.
“ What crystallography did was enable us to look at atomic interactions between the antibody and the antigen, which in this case was the phosphohistidine,” said Ian Wilson, a structural biology professor at The Scripps Research Institute and a co-author on the paper.
The resulting insights not only advance our understanding of phosphohistidine’s potential role in cancer, but can also help other scientists looking to design better antibodies to suit their own research purposes.
“ From the data, we learned how small differences in atomic interactions help the antibodies to differentiate the two different isoforms of phosphohistidine, and also how these antibodies are able to recognize different peptides which undergo histidine phosphorylation,” said Rajasree Kalagiri, a Salk postdoctoral researcher and lead author of the study.
Additional authors of the study include Jill Meisenhelder and Stephen R. Fuhs of the Salk Institute Robyn Stanfield of The Scripps Research Institute and James J. La Clair of the University of California San Diego.
The Advanced Photon Source is operated for the DOE Office of Science by Argonne National Laboratory. X-ray crystallography at the APS was performed at the General Medical Sciences and Cancer Institutes Structural Biology Facility which has been funded by the National Cancer Institute ( ACB- 12002 ) and the National Institute of General Medical Sciences ( AGM- 12006 , P 30 GM 138396 ).
Use of the Stanford Synchrotron Radiation Lightsource was supported by DOE Office of Science’s Office of Basic Energy Sciences, under Contract DE-AC 02 - 76 SF 00515 . The Advanced Light Source is a DOE Office of Science User Facility under Contract DE-AC 02 - 05 CH 11231 . This project was also funded by the Leona M. and Harry B. Helmsley Charitable Trust and the Skaggs Institute for Chemical Biology at The Scripps Research Institute.
About the Advanced Photon Source
The U. S. Department of Energy Office of Science’s Advanced Photon Source ( APS ) at Argonne National Laboratory is one of the world’s most productive X-ray light source facilities. The APS provides high-brightness X-ray beams to a diverse community of researchers in materials science, chemistry, condensed matter physics, the life and environmental sciences, and applied research. These X-rays are ideally suited for explorations of materials and biological structures elemental distribution chemical, magnetic, electronic states and a wide range of technologically important engineering systems from batteries to fuel injector sprays, all of which are the foundations of our nation’s economic, technological, and physical well-being. Each year, more than 5 , 000 researchers use the APS to produce over 2 , 000 publications detailing impactful discoveries, and solve more vital biological protein structures than users of any other X-ray light source research facility. APS scientists and engineers innovate technology that is at the heart of advancing accelerator and light-source operations. This includes the insertion devices that produce extreme-brightness X-rays prized by researchers, lenses that focus the X-rays down to a few nanometers, instrumentation that maximizes the way the X-rays interact with samples being studied, and software that gathers and manages the massive quantity of data resulting from discovery research at the APS .
1. Extract potential epitopes based on the interface between target and ligand protein.
2. Construct antibody library based on 3D structure.
3. Place antibodies based on surface complementary with target.
4. Refine “hitting” antibody- target to increase its stability according to amino acids bias at different locations in antibody.
5. Select the best antibody hits according to evaluation, optimize DNA and then synthesize gene/library using Syno ® 3.0 synthesis.
100 antibody sequences for the antigen, finding hits with affinity greater than 10 -10 M