Purification Of Antigen Research Articles

Development of a double-antibody sandwich ELISA for rapidly quantitative detection of residual non-structural proteins in inactivated foot-and-mouth disease virus vaccines

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals. Vaccination and surveillance against non-structure protein (NSP) are the most efficacious and cost-effective strategy to control this disease. Therefore, vaccine purity control is vital for successful prevention. Currently, vaccine purity is tested by an in-vivo test that recommended in the World Organization for Animal Health (WOAH), but it is time consuming and costly. Herein, we develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for quantitative detection of residual NSPs in inactivated FMD virus (FMDV) vaccines. In this assay, the monoclonal antibody 3A24 was selected as capture antibody and biotinylated 3B4B1 (Biotin-3B4B1) as detection antibody. A standard curve was developed using the NSP 3AB concentration versus OD value with the linear range of concentration of 2.5–160 ng/mL. The lowest limit of detection was 2.5 ng/mL. In addition, we determined 2.5 ng/mL of NSP as an acceptable threshold value of FMD vaccine purity using a dose-response experiment in cattle. The DAS-ELISA combined with the threshold value of FMD vaccine purity could provide a quick and simple tool for evaluation the antigenic purity of FMD vaccine during the manufacturing process.

Immunogenicity of a secreted, C-terminally truncated, form of bovine viral diarrhea virus E2 glycoprotein as a potential candidate in subunit vaccine development

Both current live, attenuated, and killed virus vaccines for bovine viral diarrhea virus (BVDV) have their limitations. Here, we report the development of a BVDV subunit vaccine by (i) the expression of a secreted form of a recombinant E2 glycoprotein using BHK21 cells and (ii) determination of the immune responses in mice. The E2 glycoprotein was modified by deletion of the C-terminal transmembrane anchor domain and fusion to a V5 epitope tag. This allowed detection using anti-V5 monoclonal antibodies together with simple purification of the expressed, secreted, form of E2 from the cell media. Furthermore, we genetically fused green fluorescent protein (GFP) linked to E2 via a Thosea asigna virus 2A (T2A) ribosome skipping sequence thereby creating a self-processing polyprotein [GFP-T2A-BVDV-E2trunk-V5], producing discrete [GFP-T2A] and [E2trunk-V5] translation products: GFP fluorescence acts, therefore, as a surrogate marker of E2 expression, BALB/c mice were inoculated with [E2trunk-V5] purified from cell media and both humoral and cellular immune responses were observed. Our antigen expression system provides, therefore, both (i) a simple antigen purification protocol together with (ii) a feasible strategy for further, large-scale, production of vaccines.

Preparation, Purification and Performance Evaluation of Polyclonal Antibody Against SARS-CoV-2 Produced in Rat.

Among all known human coronaviruses, some viruses (e.g., SARS-CoV-2) cause severe pneumonia or even death. With the regard to its spread and the importance of its rapid identification/treatment, and because pAbs are relatively cheap, able to bind to more sites on antigens and even neutralize them, this study was done for the production and purification of anti-SARS-CoV-2 polyclonal antibodies (pAb) in rats. Viral antigen purification was performed by PEG/NaCl precipitation. The efficiency of the sucrose cushion method was also investigated to produce a purer antigen. Immunization was done and antibody purification was performed by ammonium sulfate precipitation (33%), dialysis, and ion-exchange chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to verify the antibody specificity. All data were analyzed by SPSS software. The results showed that the amount of concentrated virus increased with the increase of PEG concentration. Moreover, the sucrose cushion method increased its purity. Besides, induction of immune response in rats for pAb production with high titers was reached via these antigens and ELISA/western blot results indicated a suitable antibody-antigen interaction. Additionally, it was shown that ion-exchange chromatography could be a suitable technique for IgG purification. Herein, we presented a simple and cheap method for the purification of whole viral particles with relatively high quality. The results verified that these antigens could elicit a good immune response in the rat. The obtained pAbs showed a good specificity toward SARS-CoV-2 antigens. Accordingly, this study proposes a promising method for viral vaccine development.

Suppression of Coffee-Ring Effect on Nitrocellulose Membrane: Effect of Polyethylene Glycol

In the development of the diagnostic kit, it was favorable to have a low antigen concentration due to the difficulty of antigen preparedness and purification. However, it can cause the coffee-ring effect, producing different pattern formations on the selected membrane. It can lead to a false interpretation of the result. Thus, the immobilization of protein solution (lysozyme) as a model protein for antigen, with the addition of hydrosoluble polymer additive onto a membrane, was evaluated to suppress the coffee-ring effect. This research aims to evaluate the effect of polyethylene glycol on the protein solution for coffee-ring effect suppression and to analyze the image of the coffee-ring effect. From the experimental studies, 5 different concentrations (v/v%) of PEG which are 3.0, 2.0, 1.0, 0.1 and 0.01 v/v% is added at 4.0 mg/mL of lysozyme solution before being spotted onto nitrocellulose membrane. The color intensity of the dried spot, together with the formation of the coffee-ring effect, is analyzed by Image-J software. It is the approach to measure the suppression of the ring effect, in which 0.01 v/v% concentration portrays the most faded ring effect on nitrocellulose membrane. This effect occurs due to a surface tension gradient that causes the solute particles to accumulate at the edge of the droplet. As Marangoni flow has been altered, the coffee-ring effect is successfully suppressed; thus, uniform pattern deposition is achieved.

A Comparative Evaluation of Four Different Immunoassays in the Diagnosis of Cystic Echinococcosis Using a Crude and Purified Hydatid Cyst Fluid Antigen.

Cystic echinococcosis (CE) is one of the most neglected tropical diseases as per WHO which has an immense public health significance. Diagnosis of CE is difficult as specific clinical signs are manifested only after the hydatid cyst attains a considerable size. Immunodiagnosis is a reliable method of diagnosing CE. SDS-PAGE was performed for the hydatid cyst fluid antigens. The antigen purity was tested by Western blotting and four different immunoassays were evaluated using these two antigens in sheep and buffalo in diagnosis of CE. SDS-PAGE revealed four bands of 72, 64, 48 and 24kDa for crude antigen and a single 72kDa band for purified antigen. Among sheep sera, ELISA was most sensitive (70%) using crude antigen and also while using the purified antigen (80%). In case of buffaloes, ELISA, DID and CIEP were more sensitive (83.3%) using crude antigen, whereas DID and CIEP were more sensitive (83.3%) using purified antigen. In sheep, while using the crude antigen ELISA was the most sensitive assay and IHA was the least sensitive assay. While using the purified antigen also, ELISA was the most sensitive and others were absolutely specific except for IHA being less sensitive. In buffaloes, using crude antigen, all the immunoassays CIEP, DID and ELISA were highly sensitive in diagnosing CE infection except IHA, whereas using the purified antigen, both CIEP and DID were more sensitive than ELISA and IHA which were comparatively less sensitive in detecting CE in buffalo sera.

Protective effect of two new nanovaccines against Pseudomonas aeruginosa based on LPS and OPS: A comparison study

Pseudomonas aeruginosa is one of the most important infectious pathogens in medicine. This bacterium causes various infections, especially in patients with severe burns and people with defective immune systems. The purpose of this study was to develop a nanovaccine based on PLGA nanoparticles and lipopolysaccharide and oligopolysaccharide antigens for appropriate stimulation of the humoral and cellular immune systems against P. aeruginosa. LPS-PLGA and OPS-PLGA conjugates were synthesized using the carbodiimide reaction. The prepared conjugates of as well as the pure antigens of LPS and OPS were injected to BALB/c mice in three periods at 2 week intervals. The ELISA test showed that the IgM, IgA, IgG, IgG1, IgG2b, IgG2a and IgG3 antibodies produced against LPS-PLGA or OPS-PLGA conjugates were tens of times higher than the pure antigens. Also, the opsonophagocytosis test showed that the performance and the effect of produced anti-LPS-PLGA antibodies were higher than other groups. In addition, the mice treated with LPS-PLGA conjugate were more resistant to P. aeruginosa infection than other groups. These findings indicated that LPS and OPS antigens in conjugation with PLGA nanoparticles have the ability to create and effective immunity against P. aeruginosa and LPS-PLGA is more effective than OPS-PLGA.

Genetically detoxified tetanus toxin as a vaccine and conjugate carrier protein

Tetanus toxoid (TTxd), developed over 100 years ago, is a clinically effective, legacy vaccine against tetanus. Due to the extreme potency of native tetanus toxin, manufacturing and regulatory efforts often focus on TTxd production, standardization, and safety, rather than product modernization. Recently, a genetically detoxified, full-length tetanus toxin protein (8MTT) was reported as a tetanus vaccine alternative to TTxd (Przedpelski et al. mBio, 2020). Here we describe the production of 8MTT in Gor/MetTM E. coli, a strain engineered to have an oxidative cytoplasm, allowing for the expression of soluble, disulfide-bonded proteins. The strain was also designed to efficiently cleave N-terminal methionine, the obligatory start amino acid for E. coli expressed proteins. 8MTT was purified as a soluble protein from the cytoplasm in a two-column protocol to > 99 % purity, yielding 0.5 g of purified 8MTT/liter of fermentation broth with low endotoxin contamination, and antigenic purity of 3500 Lf/mg protein nitrogen. Mouse immunizations showed 8MTT to be an immunogenic vaccine and effective as a carrier protein for peptide and polysaccharide conjugates. These studies validate 8MTT as commercially viable and, unlike the heterogenous tetanus toxoid, a uniform carrier protein for conjugate vaccines. The development of a recombinant, genetically detoxified toxin produced in E. coli aligns the tetanus vaccine with modern manufacturing, regulatory, standardization, and safety requirements.

Evaluation of PLGA nanoparticles containing outer membrane proteins of Acinetobacter baumannii bacterium in stimulating the immune system in mice.

Background and purpose:Acinetobacter baumannii (A. baumannii) is known as a pathogen with antibiotic resistance, causing respiratory infections. PLGA has been approved for use in vaccines as well as drug delivery. This study was performed to evaluate PLGA nanoparticles containing the outer membrane proteins (OMPs) of A. baumannii in stimulating the mice’s immune system and improving pneumonia.Experimental approach:Double emulsion solvent evaporation technique was used. The properties of the obtained nanospheres were determined using a zetasizer, FTIR, and AFM devices. Nanoparticles were administered to mice BALB/c by applying the intramuscular route. ELISA was used to measure the amounts of immunoglobulins produced; also, an opsonophagocytic killing assay was used to measure the effectiveness of immunoglobulins. Immunized mice were then challenged with live A. baumannii through the lungs; their internal organs were also removed for bacteriological studies.Findings/Results:The prepared particles were 550 nm in diameter with a negative surface charge. The production of the OMPs specific IgG was much higher in the group receiving nanoparticles containing antigen as compared to those getting pure antigen. The immunoglobulins produced against nanoparticles were superior to those developed against pure antigens. Mice that received the new nanovaccine were more resistant to pneumonia caused by this bacterium than those that received pure antigen.Conclusion and implication:Overall, it can be said that PLGA nanoparticles could deliver their internal antigens (OMPs) well to the immune system of mice and stimulate humoral immunity in these animals, thus protecting them against pneumonia caused by A. baumannii.

Preprint Highlight: Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models.

Developing cancer immunotherapies relies on an accurate understanding of the diversity of cancer antigen–MHC complexes presented by immune cells, yet popular in vitro cell culture systems do not fully replicate the native tumor microenvironment. To characterize the cancer antigen fragments presented by immune cells in vivo, the authors engineered a mouse model with an affinity tag linked to the MHC heavy chain protein H2-Kb, enabling cell type-specific purification of cancer antigen–MHC complexes. In a mouse model of lung adenocarcinoma, this system revealed context-dependent changes in the peptide signatures obtained at different stages of tumorigenesis, including potential immunotherapeutic targets. Overall, this model offers a new way for the scientific community to evaluate in vivo immune cell interactions that is applicable across multiple cancer and disease settings.

Methodology of Purification of Inactivated Cell-Culture-Grown SARS-CoV-2 Using Size-Exclusion Chromatography.

Various types of COVID-19 vaccines, including adenovirus, mRNA, and inactivated ones, have been developed and approved for clinical use worldwide. Inactivated vaccines are produced using a proven technology that is widely used for the production of vaccines for the prevention and control of infectious diseases, including influenza and poliomyelitis. The development of inactivated whole-virion vaccines commonly includes several stages: the production of cellular and viral biomass in cell culture; inactivation of the virus; filtration and ultrafiltration; chromatographic purification of the viral antigen; and formulation with stabilizers and adjuvants. In this study, the suitability of four resins for Size-Exclusion Chromatography was investigated for the purification of a viral antigen for the human COVID-19 vaccine.

Development of antibacterial Type III secretion system binding antibodies

Common agricultural crops such as wild rice and tomatoes are infected by the bacterial pathogens Xanthomonas Oryzae pv. oryzae and Pseudomonas Syringae pv. syringae respectively. These species use the same cellular structure to infect their host cells called a type three secretion system or T3SS. T3SSs are composed of multiple protein subunits used to insert effector proteins into the host cell's cytoplasm. These effectors in turn begin a cascade of events that ultimately results in invasion of the host cell. Once infected, the pathogens can take over the cell’s system and ultimately kill it. Given the importance of tomatoes and rice as food sources, the spread of these infections could potentially become disastrous for the populations that depend on them. To combat this infection, biological antibiotics will be developed to bind to a critical subunit in each of the respective pathogen’s T3SS, preventing proper assembly and function, and rendering the bacteria non‐pathogenic.A combination of yeast surface display and directed evolution are being used to find nanobodies specific to the T3SS in Xanthomonas Oryzae pv. oryzae and Pseudomonas Syringae pv. syringae. Nanobodies are inexpensive and stable antibody fragments that hold substantial potential as antimicrobials in human and agriscience applications. To do this, an antigen mimicking that of the bacterial secretion systems must be created. The antigens herein were chosen for their relative conservation and role in T3SS pathogenicity. This antigen selection strategy is expected to both increase the applicability of the method across bacterial species, and reduce the potential for the development of antibiotic resistance. This poster outlines the selection, cloning, and purification of a T3SS antigen for directed evolution of nanobodies as T3SS‐targeting antimicrobials.

Induced antigen-binding polyreactivity in human serum IgA

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.

Engineering His-Tagged Senecavirus A for One-Step Purification of Viral Antigens.

Senecavirus A (SVA) is a picornavirus that causes vesicular disease in swine, and the inactivated vaccine is used to prevent and control SVA infection. To develop a new chromatography strategy for the purification and concentration of SVA vaccine antigens, we inserted a 6×His-tag at the VP1 C-terminal of the SVA/HLJ/CHA/2016 in an infectious clone to rescue a His-tagged SVA. The constructed and rescued recombinant virus, named as rSVA-His, exhibited similar growth kinetics to that of its parental virus. In addition, the expression of a 6×His-tag on the surface of SVA showed genetic stability in cell passages in vitro, which allowed one-step purification of SVA antigens by Ni2+ affinity columns. Furthermore, the immunogenicity of the inactivated rSVA-His was evaluated by inoculating rabbits and detecting neutralizing antibodies. The animals receiving two doses of the inactivated rSVA-His emulsified with oil adjuvant developed a high titer of neutralizing antibodies, indicating that SVA VP1 is tolerant to His-tag insertion without detriment to its antigenicity. In summary, the constructed 6×His-tagged SVA may offer a feasible approach to the affinity purification and concentration of antigens in the process of SVA inactivated vaccine production.

Development of the ELISA Test System for Quantitative Determination of IgG to the Varizella zoster virus in Human Serum and Assessment of its Diagnostic Efficiency

Relevance. The introduction of Varicella vaccine prophylaxis explains the need to develop a methodology for monitoring the vaccination effectiveness and the intensity of population immunity. This problem can be solved using quantitative immunoassay methods. Aim. Development of an enzyme-linked immunosorbent assay for the concentration of class G immunoglobulins (AB) to Varicella zoster virus (VZV) determining and assessing its functional characteristics and diagnostic efficiency. Materials and methods. Recombinant antigen GE VZV. WHO International Standard for Antibodies to VZV W1044. Blood serum samples from healthy people and patients with Chickenpox and Herpes zoster, blood serum samples containing IgG antibodies to herpes simplex viruses of the first and second types, cytomegalovirus, Epstein-Barr virus. Anti-VZV ELISA (IgG) reagent kit (Euroimmun, Germany). Indirect enzyme-linked immunosorbent assay. Immunization of animals with recombinant antigen GE, isolation, and purification of specific antibodies. Conjugation of monoclonal antibodies to human IgG with antibodies to antigen GE and with horseradish peroxidase. Results. An enzyme-linked immunosorbent assay in «an indirect» format has been developed to determine the specific antibodies to VZV concentration (IU/ml) in human serum/plasma. An artificial calibrator for determining the concentration of AB-VZV had been synthesized and standardized according to the International WHO-standard W1044. The main functional characteristics of the developed enzyme-linked immunosorbent assay are determined in accordance with GOST 51352-2013. The diagnostic kit was tested on blood serum samples from children with chickenpox (n = 43), adults with Herpes zoster (n = 158), healthy individuals (n = 781). The diagnostic sensitivity of the test system was 85%, the diagnostic specificity was 87% according to the ROC analysis. The absence of cross-reactivity of the test system was shown on samples with serological markers of other herpesvirus infections (n = 94). Comparative trials of the developed test system and its commercial analog, the Anti-VZV ELISA (IgG) reagent kit, did not reveal statistically significant differences between their functional characteristics. Conclusions. The developed test system for determining of the AB-VZV concentration in human serum/plasma in terms of its functional characteristics meets the GOST requirements, is characterized by high diagnostic efficiency, can be used to monitor the effectiveness of vaccine prophylaxis and strength of population immunity, as well as to assess the immune response in chickenpox and Herpes zoster.

Improved Efficiency of Bluetongue Viral Antigen Isolation for Successful Immunization

Bluetongue (BT) is a viral disease transmitted by Culicoides spp. The clinical presentation of BT varies widely among susceptible sheep, and in most cases results in severe illness and death in the infected animals. The mortality among susceptible sheep ranges from 2-30% but can occasionally be as high as 70%. Therefore, we investigated a new method to increase the purified BTV-antigen. BTV viral suspensions were purified using Freon-113 and ultracentrifugation through 40% sucrose. We obtained 94.5-95.8% purification of the BT-16 viral antigen. Sheep and cows were immunized with the isolated BTV antigen obtained from this method to confirm antibody specificity to BTV. The antibody activity measured by enzyme-linked immunosorbent assay (ELISA) from goat serum was 7.0log2 to 13.0log2 (on average 10.8±2.28log2) relative to that of sheep (P<0.05 to P<0.0001). We show here that this method can successfully purify BTV-16 antigen and could be used for large-scale production and other BTV serotypes.

X-ray Crystal Structure Analysis of VHH-Protein Antigen Complexes.

VHHs are antigen-binding domains cloned from heavy-chain antibodies found in camelids. These proteins have generated considerable interest in a variety of applications as research reagents, crystallization chaperones, and therapeutics. The evolutionary adaptations of VHHs have resulted in biophysical properties and antigen-binding modalities which are distinct from those of conventional antibodies. A detailed molecular analysis of VHH interactions with their cognate protein antigens is valuable for understanding structure-function relationships and for protein engineering. The majority of VHHs bind to folded proteins and thus recognize discontinuous three-dimensional epitopes. While multiple approaches exist for dissecting the interaction between a protein antigen and a VHH, X-ray crystallography remains the highest resolution method available. Here, we provide an updated procedure for determining and analyzing the X-ray structure of a VHH in complex with a protein antigen. We describe the recombinant expression and purification of VHHs and protein antigens, purification and analysis of protein complexes, crystallization, and optimization, X-ray structure determination by molecular replacement, and analysis of the complex.

Using crystallography tools to improve vaccine formulations.

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Validation of Pretreatment Methods for the In-Process Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is controlled by vaccine policy in many countries. For vaccine potency, the content of intact virus particles (146S antigens) is critical, and the sucrose density gradient (SDG) fractionation is the gold standard for the quantification of 146S antigens. However, this method has several drawbacks. Although size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace the classic method, its application is generally confined to purified samples owing to the interfering signals. Therefore, we aimed to develop optimal pretreatment methods for SE-HPLC quantification in less purified samples. Crude virus infection supernatant (CVIS) and semi-purified samples with PEG precipitation (PEG-P) were used. Chloroform pretreatment was essential to remove a high level of non-specific signals in CVIS, whereas it caused loss of 146S antigens without the distinctive removal of non-specific signals in PEG-P. Benzonase pretreatment was required to improve the resolution of the target peak in the chromatogram for both CVIS and PEG-P. Through spiking tests with pure 146S antigens, it was verified that the combined pretreatment with chloroform and benzonase was optimal for the CVIS, while the sole pretreatment of benzonase was beneficial for PEG-P.

Purification of Prospective Vaccine Antigens from Gram-Positive Pathogens by Immunoprecipitation.

Immunoprecipitation is an affinity purification technique that exploits the highly specific interactions formed between antibodies and their cognate antigens to purify molecules of interest from complex biological solutions. The generation of an effective humoral response provides protection against a wide range of gram-positive pathogens, and thus immunoprecipitation using antibodies purified from immune humans or animals provides a simple but effective means of isolating prospective vaccine antigens from fractionated bacterial cells for downstream identification. The commercial availability of antibody preparations from donated human plasma, containing antibodies against many common gram-positive pathogens, allows the protocol to be performed in the absence of bespoke vaccination experiments. Thus, immunoprecipitation has the potential to reduce the number of animals used in vaccine studies by allowing an initial screen for promising antigens to be conducted in vitro.

Purification and concentration of antigen for ELISA using epizootic isolates of Infectious laryngotrachitis virus isolated in Ukraine

Infectious laryngotracheitis of chickens is one of the most dangerous viral respiratory diseases of chickens, which causes significant economic losses to poultry farms. A key component in this disease control is timely rapid serological diagnosis. To date, the basic method of serological diagnosis and monitoring is enzyme-linked immunosorbent assay (ELISA). The main components of ELISA test systems are purified and concentrated infectious laryngotracheitis virus antigens. Our research aimed to develop a technology for the production of purified and concentrated antigens of infectious laryngotracheitis virus, as well as to test the suitability of epizootic isolates for the production of antigens for ELISA. Based on the results of research, an improved scheme for obtaining purified infectious laryngotracheitis virus antigens using epizootic isolates has been developed. The scheme consists of accumulation of virus raw material, its inactivation, verification of inactivation completeness, concentration of infectious laryngotracheitis virus by PEG-6000 precipitation followed by ultracentrifugation at 14,000 rpm through a 30% sucrose pad. Samples of purified concentrated infectious laryngotracheitis virus antigens from isolates “В 59-11”, “Б 2-10”, “ЧП 96-10”, and “A 4-12” with protein content 1,520–3,720 μg/cm3 have been obtained. The ratio of protein concentration before and after purification ranged from 4.17 to 7.24. ELISA found that all these antigens were suitable for use as antigens. When testing for specificity, it was found that all antigens did not react with heterologous sera to other poultry viral diseases, but reacted only with homologous sera positive for infectious laryngotracheitis, which proves their specificity