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Neutralizing TCA washes

Neutralizing TCA washes


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Trichloroacetic Acid (TCA) is commonly used for protein precipitation but the wash waste needs to be neutralized prior to disposal. What exactly is required to effectively neutralize TCA waste? Do I just add caustic?


A neutralization reaction occurs between Brönsted acids and bases to form a salt.

In this case, you have to add a Brönsted base to neutralize your acid. If the final pH is important, you should work out the exact quantity of base to add. Otherwise, just use litmus paper to check the pH of your solution, and add base until it's neutral.

EDIT

In view of the other answer proposed, I want to stress the following:

Do NOT dispose of your solution in the sink/sewer. Leave it in its plastic bin until a lab technician takes it to the furnace.

If the base you use is sodium bicarbonate (NaHCO3), there will be bubbling, caused by the decomposition of carbonic acid (H2CO3) into water (H2O) and carbon dioxide (CO2). You've successfully neutralized the TCA once there's no bubbling upon addition of NaHCO3.

It is indeed best to proceed slowly while adding your base.


I've been fortunate to find a protocol hidden away in my lab archives.

  • The acid waste can be accumulated for 10 days before it needs to be neutralized. The waste should be kept in a carboy in a fume hood.
  • 50 mL 50% w/v Sodium Bicarbonate slurry should be added per liter of TCA waste. During neutralization fumes will be generated which may include chlorine gas.
  • When the pH reaches 6-8, dispose the waste into the sewer.

Trichloroacetic Acid

Like DCA, TCA exists almost exclusively in salt form at pH found in drinking water.

Toxicokinetics

TCA is readily absorbed from the gastrointestinal tract in experimental animals and humans and its clearance from blood is relatively slow relative to other HAAs. Approximately half of the administered dose was eliminated unchanged. There are substantial differences in this clearance by different species. Clearance is much faster in mice than in rats and human clearance is very slow. TCA produces same metabolites as DCA with or without being converted to DCA.

Toxicity

The oral LD50 of TCA (neutralized to pH 6–7) is found to be 3.32 g kg −1 in rats and 4.97 g kg −1 in mice when administered in aqueous solution. The most obvious target organ for TCA is the liver. Repeated administration of TCA in drinking water only produced minimal evidence of liver toxicity. TCA administration resulted in body weight reductions, soft tissue malformations, and interventricular septal defect. TCA (neutralized) induces hepatic tumors in male and female mice, but not in rats when administered via drinking water. After short-term exposures, TCA induces basophilic liver foci in mice, similar to those caused by several peroxisome proliferators.

TCA is a strong acid. It is widely recognized that skin contact of TCA has the potential to produce acid burns, and ingestion of TCA has the potential to damage tissues of the gastrointestinal tract or produce systemic acidosis, even though specific studies of these effects do not appear in the literature. TCA is frequently utilized for chemical peeling by physicians practicing dermatologic surgery. TCA is a major metabolite of commonly used solvents such as TCE and PERC and occupational exposures to these solvents have been quite high in the past, but few, if any, effects of the solvents in humans have been attributed to TCA. So it is reasonable to presume that TCA is relatively nontoxic to humans under circumstances of low exposures such as those encountered in chlorinated drinking water. In addition, the mode of tumor induction – peroxisomal proliferation – in animals is not relevant for humans. IARC has classified TCA as a group 3 compound for its carcinogenicity.

Mode of Action

TCA causes carcinogenicity mainly by peroxisomal proliferation mechanism. Additional mechanisms suggested are increased oncogene expression via DNA hypomethylation and promotion of spontaneous liver tumors.


Which of these peels must be neutralized: glycolic, salicylic, TCA, lactic acid, Jessner?

Thank you for your question. From the peels listed that must be neutralized is the Glycolic Acid peel, the other peels are self neutralizing. Peels are easily applied, but can cause many issues/problems if not done correctly. I would recommend you find an experienced dermatologist or cosmetic surgeon to do your peels. - Dr. Dickerson

Answer: Do not try this at home.

Thank you for your question. From the peels listed that must be neutralized is the Glycolic Acid peel, the other peels are self neutralizing. Peels are easily applied, but can cause many issues/problems if not done correctly. I would recommend you find an experienced dermatologist or cosmetic surgeon to do your peels. - Dr. Dickerson

Answer: It is a terrible mistake to perform these peels on yourself.

The peeling agents are available online. That does not mean you should order them and apply them to yourself. A great deal of judgement is required to safely perform a facial peel. Do yourself a favor and find an experience dermatologist or cosmetic surgeon to do your peels.

Answer: It is a terrible mistake to perform these peels on yourself.

The peeling agents are available online. That does not mean you should order them and apply them to yourself. A great deal of judgement is required to safely perform a facial peel. Do yourself a favor and find an experience dermatologist or cosmetic surgeon to do your peels.


How do you neutralize skin after a chemical peel?

After a chemical peel, your skin may feel tight, inflamed and sensitive, depending on the strength of the acid. The purpose of a neutralizer is to stop the acid and comfort the skin. You may have heard that baking soda and water can be used in place of a ready-made after peel neutralizer.

Additionally, can you neutralize a chemical peel with water? &ldquoA chemical peel is an acid,&rdquo says Dr. Jhin. &ldquoAcids have a very low pH level, so you want to neutralize it by making it alkaline and not acidic. I recommend that they apply this to a clean face, leave it on for five minutes, and then splash it off with water to neutralize it.

Accordingly, how long does it take to recover from a chemical peel?

Treated areas take about one to seven days to heal after a light chemical peel.

Do I wash off glycolic acid?

One of the main benefits of the glycolic acid serum is that while most products must be washed off, you can apply a serum and leave it on. Your skin benefits over a longer period of time from the serum.


Introduction

The rapid international spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is associated with numerous mutations that alter viral fitness. Mutations have been documented in all 4 structural proteins encoded by the viral genome including the small envelope glycoprotein (E), membrane glycoprotein (M), nucleocapsid (N) protein, and the spike (S) protein. The most prominent mutations are in the spike protein, which mediates entry of the virus into cells by engaging with the angiotensin converting enzyme 2 (ACE2) receptor. Several structures of SARS-CoV-2 spike protein variants in pre- and post-fusion conformations have been reported, including complexes with ACE2 and a variety of antibodies [1–13]. Mutations that emerge in the receptor binding domain (RBD) of the spike protein are especially of interest given their high potential to alter the kinetics and strength of the interaction of the virus with target cells. These mutations could also affect the binding of antibodies capable of binding and blocking engagement of the virus with ACE2.

In December 2020, new variants of SARS-CoV-2 carrying several mutations in the spike protein were documented in the UK (SARS-CoV-2 VOC202012/01) and South Africa (501Y.V2) [14,15]. Early epidemiological and clinical findings have indicated that these variants show increased transmissibility in the population [16]. Despite being phylogenetically distinct, a common feature of both the UK and South African variants is the mutation of residue 501 in the RBD from Asn to Tyr (N501Y). X-ray crystallography and cryo-electron microscopy (cryo-EM) structural studies have identified N501 as a key residue in the spike protein at the interface between RBD and ACE2 that is involved in critical contacts with several ACE2 residues [5,6,10,13]. Studies carried out in a mouse model before the identification of the new UK variant suggested that mutations of residue 501 could be linked to increased receptor binding and infectivity [17,18]. Understanding the impact of N501Y on antibody neutralization, ACE2 binding, and viral entry is therefore of fundamental interest in the efforts to prevent the spread of COVID-19.


COMMENTARY

Background Information

Antibodies perform several key functions during viral infections, including complement recruitment, opsonization, and neutralization. Pathogen-specific antibodies can form immune complexes that inactivate the virus and are cleared by phagocytic cells (Corti & Lanzavecchia, 2013 ). However, this neutralizing ability can have a wide range of effectivity based on the pathogen or even the patient. The plaque reduction neutralization test (PRNT) is considered to be a classic method for measuring neutralization against Betacoronaviruses (Suthar et al., 2010 Zhu et al., 2007 ). In PRNT, sera containing the antibodies of interest are serially diluted and incubated with the target virus to form immune complexes. These complexes are then overlaid on a monolayer of susceptible cells, and virus diffusion is limited by overlaying agar. Over a period of several days, the monolayer is examined for cytopathic effect, which will result in plaque formation. The neutralization ability of the antibodies is quantified by comparing the number of plaques between cells treated with sera and an untreated control. This method is reliable and well established, and requires few specific reagents. However, PRNTs take several days to perform and to provide adequate plaque resolution. A PRNT must be performed in a 6- or 12-well plate format, which results in the need for large quantities of cells, plates, and reagents to analyze a large number of samples. Thus, while highly sensitive, PRNTs are not ideal for a high-throughput setting.

Focus reduction neutralization (FRNT) assays have been developed for several viruses as an alternative to PRNTs (Friedrich & Beasley, 2016 Quicke et al., 2016 Vaidya et al., 2010 ). This assay uses a similar principle to the PRNT however, instead of visualizing viral growth with plaques, virus is detected using antibodies. In an FRNT assay, the immune complexes are overlaid on a cell monolayer, and virus diffusion is immobilized using methylcellulose. The cells are fixed, and virus-specific antibodies conjugated to HRP are used to visualize foci of infected cells. This method provides several advantages over a traditional PRNT assay: (1) FRNTs can be performed in a 96-well plate format, making sample processing much quicker and requiring fewer reagents and cells (2) FRNTs take 3 days to perform instead of the 5-6 days that a PRNT requires (3) fixation of the cells allows for more flexibility in processing, as samples can be stored at 4°C for a few days and visualized in a BSL-2 setting. The major drawback of a FRNT assay compared to the PRNT assay is the requirement for a virus-specific antibody to visualize the foci.

In Basic Protocol 2, we describe a second method to detect virus neutralization, termed the FRNT-mNG assay, which utilizes an mNeonGreen-expressing SARS-CoV-2 virus (Xie et al., 2020 ). Here, antibodies are complexed with SARS-CoV-2-mNG and then overlaid on a monolayer of cells in a manner similar to the FRNT. Infected foci are directly visualized using a fluorescent ELISPOT reader. This method is advantageous for high-throughput applications, as it reduces bench time compared to the FRNT assay by the omission of the permeabilization, primary and secondary antibody incubations, and washing steps. However, a drawback for the FRNT-mNG is that it requires a virus that expresses an mNeonGreen protein and a method to visualize the foci, such as an ELISPOT reader with fluorescence capability.

Critical Parameters and Troubleshooting

For both the Basic and Alternate Protocol, the following considerations are applicable. It is important to use a low-passage sequence-verified SARS-CoV-2 isolate, as cell culture adaptations may influence neutralization titers. It is also essential to specifically use the E6 Vero clone to obtain robust infection. The multiplicity of infection (MOI) may need to be carefully titrated for optimal foci resolution. If the MOI is too low, there will not be enough foci to accurately quantify neutralization activity if the MOI is too high, there will be too many foci to count. When optimizing the size of the foci, the incubation time post infection is the most important factor. SARS-CoV-2 needs at least 18 hr to develop visible foci in Vero E6 cells. We have observed that longer incubation times lead to larger-sized foci, making it difficult to distinguish individual foci (Panel A in Figs. 3 and 4). In this method, the optimal incubation time was found to be 24-30 hr post-infection however, longer or shorter incubation times may work better in different environments. For the Basic Protocol, in order to obtain good staining of the foci, it is important to perform the washes precisely as described. We have found that reducing the number of washes or volume of PBS per wash can significantly increase the background signal. Additionally, overnight incubation with the primary antibody generally increases the robustness of the staining. For the Alternate Protocol, if the cells are fixed, it is important to fix cells with paraformaldehyde, not a methanol-based fixation method, which will quench the GFP fluorescence.

Understanding Results

The Basic and Alternate Protocol describe methods to measure the neutralization ability of SARS-CoV-2-specific antibodies in a given biological sample. To correctly interpret the results of each assay, it is important to include the following controls: (1) uninfected, untreated mock samples (2) SARS-CoV-2 infected, untreated controls and (3) serum from healthy, SARS-CoV-2-naïve individuals (Fig. 2). Representative images of the foci for the experimental and control samples for a FRNT and FRNT-mNG are shown in Figures 3 and 4. To quantify antibody neutralization, the number of foci are counted for each sample using the Viridot program (Katzelnick et al., 2018 ). To calculate percentage neutralization, foci numbers are compared between an untreated, infected control and serum-treated, infected wells (Figs. 3 and 4). The neutralization titers were calculated as follows: ratio of the mean number of foci in the presence of sera and foci at the highest dilution of respective sera sample. Each specimen is tested in two independent assays performed at different times. The FRNT50 titer, the dilution at which the serum or plasma sample reaches 50% neutralization capability, is interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3. The FRNT50 provides a quantitative measure of neutralization capacity and can be used to compare antibody effectiveness between groups or patients. Overall, both the FRNT and FRNT-mNG assays provide methods to quickly and accurately quantify the neutralizing antibodies in a sample and compare between groups.

Time Considerations

The Basic and Alternate Protocol require the establishment of a Vero E6 culture, which is not taken into account for the time considerations. It will take several days to revive a stock from liquid nitrogen and to reach confluency. Both the Basic and Alternate Protocols require 3 days to complete, with equal time required for both on day 1 and 2. However, on day 3, the Basic Protocol takes about 6 hr to complete, while the Alternate Protocol requires only 1-2 hr of work.

Acknowledgments

Thanks to Kathy Stephens and Laila Hussaini for their assistance in recruiting the patients. The research reported in this publication was supported in part by an Emory EVPHA Synergy Fund award (M.S.S. and J.W.), COVID-Catalyst-I 3 Funds from the Woodruff Health Sciences Center (M.S.S), Center for Childhood Infections and Vaccines (M.S.S), Children's Healthcare of Atlanta (M.S.S), Woodruff Health Sciences Center 2020 COVID-19 CURE Award (M.S.S), by the National Institutes of Health (NIH) through the National Institute for Allergy and Infectious Diseases under award numbers ORIP/OD P51OD011132 (M.S.S), 3U19AI057266-17S1 (M.S.S.), HIPC COVID-19 Supplement U19AI090023 (M.S.S), R01AI127799 (M.S.S.), R01AI148378 (M.S.S.) and by the Infectious Diseases Clinical Research Consortium UM1AI148684 (E.A., N.R., J.W., M.S.S.), R00AG049092 (V.D.M), and the World Reference Center for Emerging Viruses and Arboviruses R24AI120942 (V.D.M). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflicts of Interest Statement

The authors declare no competing interests.

Author Contributions

Abigail Vanderheiden: Conceptualization data curation formal analysis methodology writing-original draft writing-review & editing. Venkata Viswanadh Edara: Conceptualization data curation formal analysis methodology writing-original draft writing-review & editing. Katharine Floyd: Conceptualization data curation methodology writing-review & editing. Robert C. Kauffman: Conceptualization resources. Grace Mantus: Conceptualization data curation resources. Evan Anderson: Conceptualization data curation resources writing-review & editing. Nadine Rouphael: Conceptualization resources. Sri Edupuganti: Resources. Pei-Yong Shi: Methodology resources. Vineet D. Menachery: Methodology resources. Mehul S. Suthar: Conceptualization data curation formal analysis funding acquisition investigation supervision visualization writing-review & editing.


PRESERVATIVES | Traditional Preservatives – Organic Acids

Citric Acid

Citric acid is one of the most versatile, inexpensive, and widely used organic acidulants, and it commonly is applied to the production of fruit-flavored beverages. It is contained in all fruits listed in Table 1 and represents one of two major acid constituents contained in most of these fruits. In addition, citric acid is also used in jams, confectioneries, candy, cheeses, juices, wine, canned vegetables, and sauces. Owing to its widespread usage, citric acid has become the gold standard against which other acidulants are measured, including such parameters as taste, titratable acidity, and acidification. In particular, citric acid is highly favored by the food industry on account of its light fruity taste, solubility, low cost, and abundant supply.


This work was supported by the Initiative and Networking Fund of the Helmholtz Association of German Research Centers (grant number SO-96), the European Union’s Horizon 2020 research and innovation program under grant agreement No 101003480—CORESMA. This work has further received funding from State Ministry of Baden-Württemberg for Economic Affairs, Labour and Housing Construction (FKZ 3-4332.62-NMI/68). We thank Florian Krammer for providing expression constructs for SARS-CoV-2 homotrimeric Spike and RBD. Open Access funding enabled and organized by Projekt DEAL.

Study design: NSM, TRW, MB, UR Nb selection and biochemical characterization: PDK, BT, TRW Immunization of the animal: HS, SN, AS Multiplex binding assay: JH, DJ, MB HDX-MS experiments: MG, AZ Organization and providing patient samples: MoS, AN, JSW, KSL Designing and performing crystallization studies: EO, GZ, TS Virus neutralization assays: NR, MiS Data analysis and statistical analysis: TRW, MB, JH, MG, AZ, NR, MiS, UR Manuscript drafting: TRW, AD, UR Study supervision: NSM, UR Manuscript reading: All authors.


2. Materials and methods

2.1. Viruses and cells

All SARS-CoV-2 virus strains used in this work were isolated from hospitalized patients including domestic and foreign patients with confirmed COVID-19 in Yunnan Hospital of Infectious Diseases from January to May 2020. A strain with a D614G mutation in the S protein was isolated from a pediatric patient who had returned from the U.S. to their hometown and was identified as being infected with SARS-CoV-2 through clinical diagnosis and q-RT-PCR in March 2020. The virus was proliferated in Vero cells (WHO) and was titrated with a microtitration assay. Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM Corning, NY, USA) containing 5% fetal bovine serum (FCS HyClone, Logan, USA).

2.2. Inactivated vaccine

The SARS-CoV-2 inactivated vaccine was developed by the Institute of Medical Biology (IMB), Chinese Academy of Medical Sciences (CAMS) in its facility in accordance with GMP requirements. Briefly, the virus strain, named KMS-1 (GenBank No: MT226610.1), was inoculated into Vero cells for production in an environment that met the BSL requirements. The harvested viruses were inactivated by formaldehyde (v:v =ਁ:4000) for 48 h to lyse the viral membrane, purified via chromatography and concentrated. A second inactivation with beta-propiolactone (v:v =ਁ:2000) was performed to destroy the viral genomic structure, followed by a second purification and concentration using the same protocol. The vaccine stock was evaluated using various quality indexes, including antigen content, immunogenicity, sterility and residue testing. The viral antigen content was measured via ELISA. The vaccine contained 50, 100 or 150 EU of inactivated viral antigen adsorbed to 0.25 mg of Al(OH)3 adjuvant and suspended in 0.5 ml of buffered saline for each dose. Before this vaccine was released for clinical study by the CFDA under No. 2020L00020, the protective efficacy of the vaccine was tested in the macaque challenge test [11] , [12] , and various toxicity studies, including an acute toxicity test, a long-term toxicity test and an allergic test, demonstrated its safety in nonhuman primates and rodents. The placebo contained only the same amount of Al(OH)3 in buffer.

2.3. Study design and participants

The trial was designed based upon the principles of randomization, double-blinding and placebo control. The study protocol was reviewed and approved by the Ethics Committee of the West China Second University Hospital, Sichuan University (Approval Number: Y2020008). An independent data safety monitoring board was established to oversee the safety data during the study, specifically during the 7ꃚys after each inoculation (p.i.). The trial was conducted according to the principles of the Declaration of Helsinki and Good Clinical Practice at the West China Second University Hospital, Sichuan University. Healthy volunteers 18� years of age were eligible for enrollment after providing informed consent. The inclusion and exclusion criteria are listed in the supplementary appendix. The enrolled participants were randomly assigned at a ratio of 1:1 to receive two inoculations at an interval of 14ꃚys or 28ꃚys, and the subjects in each schedule were assigned at a ratio of 1:1:1:1 to receive one of the three vaccine doses (50 EU, 100 EU and 150 EU) or the placebo. All the enrolled participants were asked to record solicited and unsolicited adverse events, if any, for a period of 28ꃚys. Study staff visited participants on site to track their health status and determine whether they needed medical care. Blood samples were taken from the enrolled participants on the day before immunization (baseline) and days 7, 14 and 28 (0, 14 schedule) or days 7 and 28 (0, 28 schedule) after the booster immunization to evaluate the immunogenicity of the vaccine at different time points, determine the level of 48 cytokines in the serum and analyze mRNA gene expression in peripheral blood mononuclear cells (PBMCs).

2.4. Randomization and masking

The randomization number for each vaccination schedule was generated by SAS software (version 9.4), and stratified block randomization (block size 8) by subgroups, generated by an independent statistician, was used. Within each randomization block, the ratio of vaccine to placebo was 3:1. A randomization number was sequentially assigned to each participant, and then the participant was injected with a vaccine or placebo with the same number. All participants, investigators, and laboratory staff were blinded to the treatment allocation.

2.5. End points of the clinical trial

The primary end point was the total rate of adverse reactions from 0 to 28ꃚys post-immunization. The secondary end points were serological evidence of the immunogenicity of the vaccine.

2.6. Laboratory detection

2.6.1. Neutralizing antibody test

The neutralizing antibody assay was performed via microtitration, and the neutralizing titer in the sera was determined by CPE observation. Briefly, heat-inactivated serum was diluted and incubated with live virus (100 lgCCID50/well) for 2 h at 37 ଌ followed by the addition of Vero cells (10 5 /mL), and the mixture was incubated at 37 ଌ in 5% CO2 for 7ꃚys. The CPEs were observed and assessed to determine the neutralizing antibody titer of the serum. The geometric mean titers (GMTs) of neutralizing antibodies were measured. Antibody titers ≥਄ were considered positive. Seroconversion was defined as seropositivity after immunization in previously seronegative subjects.

2.6.2. ELISA

ELISAs were conducted with antibodies against the S protein, the N protein and virions that were developed by this institute. S/N protein (Sanyou Biopharmaceuticals Co., Ltd., Shanghai, China) and purified viral antigen were used to coat 96-well ELISA plates (Corning, NY, USA) at a concentration of 5 μg/well, and the plates were incubated at 4 ଌ overnight. The plates were then blocked with 5% BSA and incubated with serum samples, and immune complexes were visualized using an HRP-conjugated antibody (Abcam, MA, USA) and TMB substrate (Solarbio, Beijing, China) as described in a previous report [13] . The absorbance of each well at 450 nm was measured using an ELISA plate reader (Gene Company, Beijing, China). The antibody serum samples that yielded OD values at least 2.1-fold higher than that of the negative control at a test sample dilution of 1:400 were defined as positive. The endpoint titer (ET) was defined as the highest serum dilution that yielded a positive OD value. The GMT was calculated as the geometric mean of the ETs of the positive serum samples in each group. For neutralizing antibodies, seroconversion was defined as seropositivity after immunization in previously seronegative subjects.

2.6.3. ELISpot assay

An ELISpot assay was performed with a Human IFN-γ ELISpot Kit (Mabtech, Cincinnati, OH, USA) according to the manufacturer’s protocol. PBMCs were isolated from blood obtained from 50% of the subjects in the immunization and placebo groups via lymphocyte isolation (Ficoll-Paque PREMIUM GE Healthcare, Piscataway, NJ, USA) and plated in duplicate wells. Purified virions, recombinant S protein and recombinant N protein (Sanyou Biopharmaceuticals Co., Ltd.) were added to the wells to stimulate the cells. The positive control was phytohemagglutinin (PHA), and the negative controls included a cell-only control and a medium-only control. The plates were incubated at 37 ଌ for 24 h, the cells were removed, and the spots were developed. The colored spots were counted using an ELISPOT reader (CTL, Shaker Heights, OH, USA).

2.6.4. Immune cell populations

The isolation of immune cell populations was performed according to a standard protocol. PBMCs were isolated via lymphocyte isolation (Ficoll-Paque PREMIUM GE Healthcare). Anti-CD3, anti-CD20 and anti-CD16 antibodies (BD558639 BD, USA) were added to the PBMCs. The mixtures were incubated at room temperature (RT) for 30 min in the dark. Reagents for red blood cell lysis (BD, USA) and membrane permeabilization (BD, USA) were added in sequence. After two washes with PBS, the cells were resuspended in PBS and subjected to flow cytometry (BD, USA). T cells (CD3 + anti-human CD3, BD555332), T helper cells (CD3 + /CD4 + anti-human CD4, BD566319) and CD8 (+) T cells (CD3 + /CD8 + anti-human CD8, BD555368) were evaluated. The T cells were further categorized as T helper (Th)1 and Th2 cells using anti-CD4 (BD550631), anti-IL-4 (BD500824) and anti-IFN-γ (BD506507) antibodies (BD, USA). The percentages of immune cells of each type were determined using a flow cytometer (Beckman Coulter Cytoflex BD, USA). Th1 cells (CD4 + /IFN-γ + ) and Th2 cells (CD4 + /IL-4 + ) were assessed.

2.6.5. Cytokine assay

The levels of 48 cytokines in the serum of the subjects were measured using the Bio-Plex Pro Human Cytokine 48-Plex (Bio-Rad, Hercules, California, USA) according to the manufacturer’s protocol. Briefly, 4-fold dilutions of serum samples were added to tubes containing detection beads. The antibodies used for detection were then added, and the mixtures were incubated at RT for 0.5 h in the dark. The beads were then washed, resuspended in wash buffer and measured using a flow cytometer (BD, USA). The concentration of each cytokine was determined based on a calibration curve that was constructed independently for each cytokine. The samples were analyzed in duplicate.

2.6.6. Transcriptome assay

Transcriptome assays were performed by Novogene Co., Ltd., China. To exclude individual differences, each group included two samples (A and B) at each time point, and each sample was mixed with PBMCs from five individuals. Total RNA was extracted from PBMCs using an RNeasy Mini Kit (QIAGEN, GmBH, Germany). The RNA was checked for quality and quantified. Double- and single-stranded DNA and ribosomal RNA (rRNA) were removed. Magnetic beads were then used to purify and recover the reaction products, and sequencing libraries were generated according to the manufacturer’s recommendations. In brief, the reaction products were heated, denatured and circularized using a splint oligo sequence. The RNA-seq libraries were sequenced on an Illumina HiSeq X TEN platform (2 ×򠅐-bp paired-end reads).

The read pairs were filtered using software to remove those with low-quality bases (Phred quality <ਅ) or with more than 10% uncertain bases. The RNA-seq reads were aligned to the human genome (http://ftp.ensembl.org/pub/release-84/fasta/homo_sapiens/dna/) using Bowtie V2.0.6. The raw count was used by DESeq2 to quantify the gene expression level. Cuffdiff was used to identify genes that were differentially expressed in the vaccine and control samples. Significantly differentially expressed genes were identified at P <਀.05. The raw microarray data are available from the National Genomics Data Center of the China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (CNCB-NGDC), under accession number HRA000347.

2.6.7. qRT-PCR

RNA was extracted from samples using TRIzol reagent (Invitrogen Tiangen Biotech, China). Real-time RT-PCR assays were performed using the One-Step PrimeScript RT-PCR Kit (Takara, Shuzo, Japan) and a Real-Time PCR System (Bio-Rad, USA). Primers and probes for SARS-CoV-2 were used to measure mRNA levels for N, these were forward: 5′-GGGGAACTTCTCCTGCTAGAAT-3′, reverse: 5′-CAGACATTTTGCTCTCAAGCTG-3′, and probe: 5′-TTGCTGCTGCTTGACAGATT-3′, and for ORF 1ab, they were forward: 5′-CCCTGTGGGTTTTACACTTAA-3′, reverse: 5′-ACGATTGTGCATCAGCTGA-3′, and probe: 5′-CCGTCTGCGGTATGTGGAAAGGTTATGG-3′. Primers and probes targeting the 3′ untranslated region (UTR) of DENV serotypes 1𠄳 were used according to previously described methods [14] . An RT-PCR assay with a universal single probe for the diagnosis of dengue virus infections was performed [14] .

2.6.8. Statistical analysis

The sample size was determined based on the requirement of the National Medical Products Administration of China, which requires a minimum sample size of 20� participants for each vaccine dose in an immunization schedule in phase 1 clinical trials. Means and standard deviations are used to describe normally distributed continuous variables, and frequencies and proportions are used to describe categorical variables. The safety analysis was performed with the data from the participants who had received at least one dose of the vaccine and for whom safety data were available. The numbers and proportions of participants with adverse reactions or events were summarized. The immunogenicity analysis was conducted with the data from the full cohort, including all participants who received injections and had results for the antibody test. The antibodies against SARS-CoV-2 are summarized as geometric mean titers with 95% CIs, and the cellular responses are presented as the proportion of positive responders. The chi-square test or Fisher’s exact test was used to analyze the seroconversion rate of antibodies against SARS-CoV-2, and the rank-sum test was used to compare antibody titers. When the comparison among all four groups of each schedule showed a significant difference, pairwise comparisons were performed. A P value lower than 0.05 (two-sided) was considered to be significant. The statistical analysis was performed by an independent statistician using GraphPad Prism software (San Diego, CA, USA) and STATA software (Version 15.0 STATA Corp., College Station, TX, USA).


SARS-CoV-2 (COVID-19) Spike RBD antibody

SARS-CoV-2 (COVID-19) Spike RBD antibody detects SARS-CoV-2 (COVID-19) Spike RBD protein at cytoplasm by immunohistochemical analysis. Sample: Paraffin-embedded human SARS-CoV-2 (COVID-19) Spike transfected 293T cell FFPE Cell Pellet Block (GTX435640). Green: SARS-CoV-2 (COVID-19) Spike RBD stained by SARS-CoV-2 (COVID-19) Spike RBD antibody (GTX135709) diluted at 1:1000. Blue: Fluoroshield with DAPI (GTX30920). Antigen Retrieval: Citrate buffer, pH 6.0, 15 min

GTX135709 ICC/IF Image

SARS-CoV-2 (COVID-19) Spike RBD antibody detects SARS-CoV-2 (COVID-19) Spike RBD protein by immunofluorescent analysis. Sample: Mock and transfected 293T cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: SARS-CoV-2 (COVID-19) Spike RBD stained by SARS-CoV-2 (COVID-19) Spike RBD antibody (GTX135709) diluted at 1:1000. Blue: Fluoroshield with DAPI (GTX30920).

GTX135709 ELISA Image

Sandwich ELISA detection of recombinant full-length SARS-CoV-2 spike (trimer) protein using SARS-CoV / SARS-CoV-2 (COVID-19) spike antibody [1A9] (GTX632604) as capture antibody at concentration of 5 μg/mL and SARS-CoV-2 (COVID-19) Spike RBD antibody (GTX135709) as detection antibody at concentration of 1 μg/mL. Rabbit IgG antibody (HRP) (GTX213110-01) was diluted at 1:10000 and used to detect the primary antibody.

GTX135709 WB Image

Non-transfected (–) and transfected (+) 293T whole cell extracts (30 μg) were separated by 5% SDS-PAGE, and the membrane was blotted with SARS-CoV-2 (COVID-19) Spike RBD antibody (GTX135709) diluted at 1:5000. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody.

GTX135709 FACS Image

SARS-CoV-2 (COVID-19) Spike RBD antibody (GTX135709) detects SARS-CoV-2 (COVID-19) Spike RBD protein by flow cytometry analysis.
Sample: 293T cells transfected SARS-CoV-2 (2019-nCoV) Spike.
Blue: Unlabelled sample was used as a control.
Red: SARS-CoV-2 (COVID-19) Spike RBD antibody (GTX135709) dilution: 1:50.
Acquisition of 20,000 events were collected for FACS analysis.

GTX135709 Neutralizing/Inhibition Image

Inhibition analysis of immobilized recombinant SARS-CoV-2 (COVID-19) Spike RBD (N501Y Mutant) protein, His tag (active) (UK variant) (GTX136014-pro), SARS-CoV-2 (COVID-19) Spike RBD (K417N, E484K, N501Y Mutant) protein, His tag (active) (South Africa variant) (GTX136022-pro), SARS-CoV-2 (COVID-19) Spike RBD (K417T, E484K, N501Y Mutant) protein, His tag (active) (Brazil variant) (GTX136043-pro), SARS-CoV-2 (COVID-19) Spike RBD (E484K, N501Y Mutant) protein, His tag (active) (UK variant) (GTX136058-pro) and SARS-CoV-2 (COVID-19) Spike RBD protein, His tag (active) (GTX136090-pro) (coated at 2 μg/mL) binding to soluble recombinant Human ACE2 (ECD) protein, mouse IgG Fc tag (active) (GTX135683-pro) (1000 ng/mL). ACE2 binding was inhibited by increasing concentrations of SARS-CoV-2 (COVID-19) Spike RBD antibody (GTX135709) (13.72-10000 ng/mL). Bound ACE2 was detected by Goat Anti-Mouse IgG antibody (HRP) (GTX213111-01) (1:10000).

GTX135709 WB Image

Non-transfected (–) and transfected (+) Vero E6 whole cell extracts (30 μg) were separated by 5% SDS-PAGE, and the membrane was blotted with SARS-CoV-2 (COVID-19) Spike RBD antibody (GTX135709) diluted at 1:5000. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody.