Preparation Of Antibodies Research Articles

Histone derived antimicrobial peptides identified from Mytilus coruscus serum by peptidomics

Histones and their N-terminal or C-terminal derived peptides have been studied in vertebrates and presented as potential antimicrobial agents playing important roles in the innate immune defenses. Although histones and their derived peptides had been reported as components of innate immunity in invertebrates, the knowledge about the histone derived antimicrobial peptides (HDAPs) in invertebrates are still limited. Using a peptidomic technique, a set of peptide fragments derived from the histones was identified in this study from the serum of microbes challenged Mytilus coruscus. Among the 85 identified histone-derived-peptides with high confidence, 5 HDAPs were chemically synthesized and the antimicrobial activities were verified, showing strong growth inhibition against Gram-positive bacteria, Gram-negative bacteria, and fungus. The gene expression level of the precursor histones matched by representative HDAPs were further tested using q-PCR, and the results showed a significant upregulation of the histone gene expression levels in hemocytes, gill, and mantle of the mussel after immune stress. In addition, three identified HDAPs were selected for preparation of specific antibodies, and the corresponding histones and their derived C-terminal fragments were detected by Western blotting in the blood cell and serum of immune challenged mussel, respectively, indicating the existence of HDAPs in M. coruscus. Our findings revealed the immune function of histones in Mytilus, and confirmed the existence of HDAPs in the mussel. The identified Mytilus HDAPs represent a new source of immune effector with antimicrobial function in the innate immune system, and thus provide promising candidates for the treatment of microbial infections in aquaculture and medicine.

Preparation of ultra-sensitive anti-carbendazim antibodies based on new haptens and their application in lateral flow immunoassay

To overcome limitations with respect to the low sensitivity of rapid immunoassay methods due to dilution of the extract in extraction solution, ultra-sensitive antibodies are needed. The linker-tethering site between the target and carrier protein, the length of the spacer arm, and the atomic charge of the linker-tethering site between the target and spacer arm in haptens design play a pivotal role in the preparation of ultra-sensitive antibodies. In this study, carbendazim (CBZ), a systemic broad-spectrum fungicidal pesticide, was used as model compound. 2-Aminobenzimidazole was chosen as the common skeletal structure and reacted with different groups, such as alkanes and urine groups, to form haptens H1 (previously reported), H2 (novel), and H3 (novel), respectively. Subsequently, eight monoclonal antibodies (mAbs) were generated using these three haptens, which exhibited notable variation in sensitivity. The half maximal inhibitory concentration (IC50) value of mAb 4B11 based on hapten H3 against CBZ was 0.04 ng/mL, which was seven times better than that of mAb 5B10 (IC50 value of 0.28 ng/mL) prepared using hapten H2 and 129 times better than that of mAb 6D5 (IC50 value of 5.15 ng/mL) prepared using hapten H1. Finally, the optimal antigen–antibody combination was employed to establish a sensitive colloidal gold lateral flow immunoassay (CG-LFA) for CBZ detection in vegetables and fruits with convenient sample pretreatment. The developed CG-LFA shows promising practical utility and prospects for application due to its low limit of detection (0.10–0.18 ng/mL) and high recovery ratios (76.0%–96.1%) in six different samples.

Activates B lymphocytes and enhanced immune response: A promising adjuvant based on PLGA nanoparticle to improve the sensitivity of ZEN monoclonal antibody

In preparing monoclonal antibodies by hybridoma cell technology, the quality of B lymphocytes used for cell fusion directly affects the sensitivity of monoclonal antibodies. To obtain B-lymphocytes producing high-quality specific antibodies for cell fusion during the immunization phase of the antigen, we prepared a TH2-Cell stimulatory delivery system as a novel adjuvant. Astragalus polysaccharide has a good ability to enhance antigenic immune response, and it was encapsulated in biocompatible materials PLGA as an immunostimulatory factor to form the delivery system (APS-PLGA). The preparation conditions of APSP were optimized using RSM to attain the highest utilization of APS. Immunization against ZEN-BSA antigen using APSP as an adjuvant to obtain B lymphocytes producing ZEN-specific antibodies for cell fusion. As results present, APSP could induce a stronger TH2 immune response through differentiating CD4 T cells and promoting IL-4 and IL-6 cytokines. Moreover, it could slow down the release efficiency of ZEN-BSA and enhance the targeting of ZEN-BSA to lymph nodes in vivo experiments. Ultimately, the sensitivity of mouse serum ZEN-specific antibodies was enhanced upon completion of immunization, indicating a significant upregulation of high-quality B lymphocyte expression. In the preparation of monoclonal antibodies, the proportion of positive wells for the first screening was 60%, and the inhibition rates of the antibodies were all similar (>50%). Then we obtained the ZEN monoclonal antibody with IC50 of 0.049 ng/mL, which was more sensitive than most antibodies prepared under conventional adjuvants. Finally, a TRFIAS strip assay was preliminarily established with a LOD value of 0.246 ng/mL.

Advancement in the development of single chain antibodies using phage display technology.

Phage display technology has become an important research tool in biological research, fundamentally changing the traditional monoclonal antibody preparation process, and has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigenic epitope research, and cellular signal transduction research.The phage display is a powerful platform for technology development. Using phage display technology, single chain fragment variable (scFv) can be screened, replacing the disadvantage of the large size of traditional antibodies. Phage display single chain antibody libraries have significant biological implications. Here we describe the types of antibodies, including chimeric antibodies, bispecific antibodies, and scFvs. In addition, we describe the phage display system, phage display single chain antibody libraries, screening of specific antibodies by phage libraries and the application of phage libraries.

Highly selective and sensitive immunoassays for flurogestone acetate analysis in goat milk: From rational hapten design and antibody production to assay development

The preparation of high specificity and affinity antibodies is challenging due to limited information on characteristic groups of haptens in traditional design strategy. In this study, we first predicted characteristic groups of flurogestone acetate (FGA) using quantitative analysis of molecular surface combined with atomic charge distribution. Subsequently, FGA haptens were rationally designed to expose these identified characteristic groups fully. As a result, seven monoclonal antibodies were obtained with satisfactory performance, exhibiting IC50 values from 0.17 to 0.45 μg/L and negligible cross-reactivities below 1% to other 18 hormones. The antibody recognition mechanism further confirmed hydrogen bonds and hydrophobic interactions involving predicted FGA characteristic groups and specific amino acids in the antibodies contributed to their high specificity and affinity. Finally, one selective and sensitive ic-ELISA was developed for FGA determination with a detection limit as low as 0.12 μg/L, providing an efficient tool for timely monitoring of FGA in goat milk samples.

Preparation of a dual-specific antibody targeting human CD123 and exploration of its anti-acute myeloid leukemia effects

Objective: To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) . Methods: Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified. Results: ① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123(+) tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group (P<0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10(5)/ml to 3.2×10(6)/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8(+) PD-1(+) LAG-3(+) T cells was 10.90%, and the proportion of propidium iodide (PI) (-) Annexin Ⅴ(+) T cells and PI(+) Annexin Ⅴ(+) T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group (P<0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group (P<0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group (P<0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123(+) tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123(+) MV4-11 cells, CD123(+) Molm13 cells, and CD123(+) THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group (P<0.05) . Conclusion: In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123(+) tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

Preparation of limonin monoclonal antibody and establishment of a sensitive icELISA for analyzing limonin in citrus and herbal samples

Limonin is an intensely bitter and highly oxidized tetracyclic triterpenoid secondary metabolite, which is abundant in the Rutaceae and Meliaceae, especially in Citrus. In order to detect limonin content in complex substrates such as citrus and traditional Chinese medicine, monoclonal antibodies specifically recognizing limonin were prepared and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established. The median inhibition concentration (IC50) was 5.40 ng/mL and the linear range was 1.25–23.84 ng/mL. The average recoveries from citrus peel and pulp samples were 95.9%–118.8% and 77.5%–113.1%, respectively. Moreover, the contents of limonin in 6 citrus samples and 4 herbal samples were analyzed by icELISA and UPLC-MS, and the results of the two methods were consistent. This validation is sufficient to demonstrate that the developed immunoassay is applicable for the detection of limonin in citrus and herbal samples and has the advantage of high efficiency, sensitivity, and convenience.

Reverse design of haptens based on antigen spatial conformation to prepare anti-capsaicinoids&gingerols antibodies for monitoring of gutter cooking oil

Rapid simultaneous detection of multi-component adulteration markers can improve the accuracy of identification of gutter cooking oil in edible oil, which is made possible by broad-spectrum antibody (bs-mAb). This study used capsaicinoids (CPCs) and gingerol derivatives (GDs) as adulteration markers, and two broad-spectrum haptens (bs-haptens) were designed and synthesized based on a reverse design strategy of molecular docking. Electrostatic potential (ESP) and monoclonal antibodies (mAbs) preparation verified the strategy's feasibility. To further investigate the recognition mechanism, five other reported antigens and mAbs were also used. Finally, the optimal combination (Hapten 5-OVA/1-F12) and key functional groups (f-groups) were determined. The half maximal inhibitory concentration (IC50) for CPCs-GDs was between 88.13 and 499.16 ng/mL. Meanwhile, a preliminary lateral flow immunoassay (LFIA) study made practical monitoring possible. The study provided a theoretical basis for the virtual screening of bs-haptens and simultaneous immunoassay of multiple exogenous markers to monitor gutter oil rapidly and accurately.

Enhanced targeting: Production of filamentous bacteriophage monoclonal antibodies using the major coat protein pVIII as anchor.

Filamentous bacteriophage display technology has been employed in antibody discovery, drug screening, and protein-protein interaction study across various fields, including food safety, agricultural pollution, and environmental monitoring. Antifilamentous bacteriophage antibodies for identifying filamentous bacteriophage are playing a pivotal role in this technology. However, the existing antifilamentous bacteriophage antibodies lack sensitivity and specificity, and the antibodies preparation methods are cumbersome and hyposensitive. The major coat protein pVIII of filamentous bacteriophage has an advantage in quantification, which is benefit for detecting signal amplification but its full potential remains underutilized. In this study, the partial polypeptide CT21 of the major coat protein pVIII of filamentous bacteriophage was intercepted as the targeted immunogen or coating antigen to prepare antifilamentous bacteriophage antibodies. Six filamentous bacteriophage-specific monoclonal antibodies (mAbs) M5G8, M9A2, P6B5, P6D2, P8E4, and P10D4 were obtained. The limit of detections of the prepared six mAbs for detecting filamentous bacteriophage was 1.0×107 pfumL-1 . These mAbs stayed stable under different pH, temperature, and exhibited high specificity in real application. This study not only provides a new idea for simplifying the preparation of antifilamentous bacteriophage antibodies which could apply in filamentous bacteriophage display, but it also presents a novel strategy for preparing antibodies against protein-specific epitopes with high sensitivity.

Biological therapy of severe bronchial asthma in a child

Currently, bronchial asthma is a global health problem, due to its high prevalence, economic component, as well as a violation of the social adaptation of children suffering from this disease. This article is devoted to a detailed analysis of the clinical case of a patient born in 2009 with an established diagnosis of Bronchial asthma, atopic form, severe persistent partially controlled course, which initiated therapy with a genetically engineered biological preparation of a humanized monoclonal antibody against IgE – omalizumab. However, in subsequent years, the patient’s condition was unstable, severe seizures were repeated, she was repeatedly hospitalized in the department where infusion therapy was performed and basic therapy was reviewed, dose adjustments and administration regimens of monoclonal antibodies to IgE (omalizumab) were carried out. A retrospective analysis of the results of objective, instrumental and laboratory examinations of the patient was also carried out over the past three years, when the child received the genetically engineered drug omalizumab without interruption. Taking into account the severe course of AD, the lack of control over the disease against the background of basic therapy with combined drugs in combination with therapy with monoclonal antibodies to IgE (omalizumab), it was decided to correct treatment and initiate therapy with a genetically engineered drug, recombinant human monoclonal antibody IgG4 (dupilumab). This clinical example once again shows how personalized the approach should be when prescribing therapy to patients with severe asthma, and also dictates the need to develop new diagnostic methods and management tactics for patients with uncontrolled forms of this disease.

The characteristics of BCR-CDR3 repertoire in COVID-19 patients and SARS-CoV-2 vaccinated volunteers.

The global COVID-19 pandemic has caused more than 1 billion infections, and numerous SARS-CoV-2 vaccines developed rapidly have been administered over 10 billion doses. The world is continuously concerned about the cytokine storms induced by the interaction between SARS-CoV-2 and host, long COVID, breakthrough infections postvaccination, and the impact of SARS-CoV-2 variants. BCR-CDR3 repertoire serves as a molecular target for monitoring the antiviral response "trace" of B cells, evaluating the effects, mechanisms, and memory abilities of individual responses to B cells, and has been successfully applied in analyzing the infection mechanisms, vaccine improvement, and neutralizing antibodies preparation of influenza virus, HIV, MERS, and Ebola virus. Based on research on BCR-CDR3 repertoire of COVID-19 patients and volunteers who received different SARS-CoV-2 vaccines in multiple laboratories worldwide, we focus on analyzing the characteristics and changes of BCR-CDR3 repertoire, such as diversity, clonality, V&J genes usage and pairing, SHM, CSR, shared CDR3 clones, as well as the summary on BCR sequences targeting virus-specific epitopes in the preparation and application research of SARS-CoV-2 potential therapeutic monoclonal antibodies. This review provides comparative data and new research schemes for studying the possible mechanisms of differences in B cell response between SARS-CoV-2 infection or vaccination, and supplies a foundation for improving vaccines after SARS-CoV-2 mutations and potential antibody therapy for infected individuals.

Method development for 14C-labeling of IgG antibodies in preparation for clinical trials

Abstract Objectives Carbon-14 (14C) labeling is a standard technology for tracing molecules and providing their pharmacokinetic profiles. However, its primary focus has been on small molecules, with limited application to biomacromolecules. Particularly in the development of new biological entities (NBE), the utilization of microdosing with a 14C-labeled biomacromolecule proves beneficial in the early stages of drug development, contributing to significant time and cost savings. This study investigates the 14C-labeling of antibody and explores the stability of 14C-labeled antibody under various storage conditions. Methods and results In this study, the utilization of 14C-formaldehyde for labeling target antibodies at various molar ratios revealed a direct correlation between labeling efficiency and the quantity of 14C-formaldehyde applied: 1.5 mol/mol for 14C-labeled antibody with the use of 10 equivalents of 14C-formaldehyde, 3.8 mol/mol for 14C-labeled antibody with the use of 10 equivalents of 14C-formaldehyde, and 10.5 mol/mol for 14C-labeled antibody with the use of 60 equivalents of 14C-formaldehyde. All the reaction conditions exhibited no antibody degradation, as evidenced by the absence of a significant change in HPLC purity compared to the unlabeled antibody. Stability tests revealed that all groups maintained their purities over a 4-week period at both − 75 ± 10 °C and 5 ± 3 °C. Given safety concerns related to internal radiation exposure in potential human subjects during microdosing, this study established optimal conditions for employing 14C-labeled antibodies. Therefore, it is optimized that 10 equivalents of 14C-formaldehyde can be used for 14C-antibody labeling through reductive amination, storing the antibodies at 5 ± 3 °C, and assigning a storage period of 4 weeks. Conclusion The findings from this study offer valuable insights into the effective application of 14C-labeling in microdosing studies, especially for larger molecules such as antibodies.

Development of monoclonal antibodies targeting the conserved fragment of hexon protein to detect different serotypes of human adenovirus.

In this study, we selected the three most conserved antigenic fragments of human adenovirus to prepare a murine monoclonal antibody for the first time, and human adenovirus antigenic fragments with heretofore unheard of degrees of conservatism were isolated. The three monoclonal antibodies with the ability to recognize human respiratory adenovirus over a broad spectrum were screened by hybridoma and monoclonal antibody preparation. Human adenovirus infections are serious; however, therapeutic drugs and diagnostic reagents are scarce. Thus, to reduce the serious consequences of human viral infections and adenovirus pneumonitis, early diagnosis of infection is required. The present study provides three monoclonal antibodies capable of recognizing a wide range of human adenoviruses, thereby offering guidance for subsequent research and development.

Preparation and characterization of monoclonal antibodies against porcine gasdermin D protein

Pyroptosis is a newly discovered type of pro-inflammatory programmed cell death that plays a vital role in various processes such as inflammations, immune responses, and pathogen infections. As one of the main executioners of pyroptosis, gasdermin D (GSDMD) is a membrane pore-forming protein that typically exists in a self-inhibitory state. Once activated, GSDMD will be cleaved into an N-terminal fragment with pore-forming activity, becoming the key indicator of pyroptosis activation, and a C-terminal fragment. Although commercial antibodies against human and murine GSDMD proteins are currently available, their reactivity with porcine GSDMD (pGSDMD) is poor, which limits research on the biological functions of pGSDMD and pyroptosis in pigs in vivo and in vitro. Here, five monoclonal antibodies (mAbs) were prepared by immunizing BALB/c mice with procaryotically expressed full-length pGSDMD, all of which did not cross react with human and murine GSDMD proteins. Epitope mapping demonstrated that 15H6 recognizes amino acids (aa) at positions 28–34 of pGSDMD (LQTSDRF), 19H3 recognizes 257–260aa (PPQF), 23H10 and 27A10 recognize 78–82aa (GPFYF), and 25E2 recognizes 429–435aa (PPTLLGS). The affinity constant and isotype of 15H6, 19H3, 23H10, 27A10, and 25E2 mAbs were determined to be 1.32 × 10−9, 3.66 × 10−9, 9.04 × 10−9, 1.83 × 10−9, and 8.00 × 10−8 mol/L and IgG1/κ, IgG2a/κ, IgG2a/κ, IgG1/κ, and IgG1/κ, respectively. Heavy- and light-chain variable regions sequencing showed that the heavy-chain complementarity-determining region (CDR) sequences of all five mAbs are completely different, while the light-chain CDR sequences of the four mAbs that recognize the N-terminus of pGSDMD are identical. Our prepared mAbs provide valuable materials for studying pGSDMD function and pyroptosis.Key points• A total of five mouse anti-pGSDMD mAbs were prepared, of which four recognize the N-terminus of pGSDMD and one recognize its C-terminus.• The main performance parameters of the five mAbs, including epitope, antibody titer, affinity constant, isotype, and heavy- and light-chain CDR, were characterized.• All five mAbs specifically recognize pGSDMD protein and do not cross react with human and murine GSDMD proteins.

P546 Endoscopic findings related to the long-term response to anti-TNF-α antibody treatment in patients with Crohn's disease

Abstract Background Anti-TNF-α antibody treatment in Crohn's disease patients has been established, but loss of treatment response has become a problem in recent years. No studies have investigated the endoscopic findings related to loss of response particularly in the long-term treatment. In this study, we investigated the endoscopic findings associated with diminished efficacy of anti-TNF-α antibodies in Crohn's disease patients treated over a long period of time. Methods A total of 79 patients with Crohn's disease who were observed for at least 1 year after the anti-TNF-α antibody treatment was initiated at our hospital were included in this study. These patients were stratified into cases with and without loss of response to the anti-TNF-α antibody treatment, and the endoscopic findings were compared between the two groups. A case with loss of response was defined as one in which the dose was increased or the duration of treatment was shortened during the anti-TNF-α antibody treatment or the anti-TNF-α antibody preparation was changed to another due to lack of response to the initial preparation. The findings examined were aphtha, erosion, cobblestone-like mucosa, fistula, multiple ulcer, longitudinal ulcer, and anal lesion. Mann-Whitney's U test, χ2 test, and logistic regression analysis were used for statistical analysis, and differences with p &amp;lt; 0.05 were considered significant. Results The mean observation period for all patients was 85.5 months, and the longest period was 188 months. Of the 79 patients included, 29 met the criteria with loss of response, and 50 did not (loss of response rate 37%). Comparisons between the loss of response group and the no loss of response group by univariate analysis showed that anal lesions (66% vs 42%, p = 0.028), stenosis (75% vs 48%, p = 0.021), and multiple ulcers (82% vs 53%, p = 0.011) were significantly more frequent in the loss of response group. The multivariate analysis on these items identified anal lesions (OR: 3.06, 95% CI 1.04-9.04, p = 0.042) and multiple ulcers (OR: 5.33, 95% CI 1.59-17.9, p = 0.0068) as independent risk factors for loss of response. Conclusion In this study, Crohn's disease patients with endoscopic findings of anal lesions and multiple ulcers were prone to loss of response to the anti-TNF-alpha antibody treatment after long-term administration. This result is considered to be very useful for developing a treatment strategy for patients requiring long-term anti-TNF-α antibody treatment.

Improved detection sensitivity of anti-PRV variant antibodies through preparation of anti-gB and anti-gE monoclonal antibodies and development of blocking ELISAs

Since 2011, PRV has resurged in China and is characterized by a mutated strain with significant alterations in antigenicity and virulence. Therefore, we hypothesized that antibody detection kits based on classic PRV strains may have limitations in detecting PRV variants. For more sensitive antibody detection of PRV variants, two MABs targeting the gB and gE proteins were developed. IFA revealed that these MABs exhibited strong reactivity toward both classic and variant PRV strains. MAB-gE recognizes a novel conserved linear B-cell epitope (41PSAEVWD47), while MAB-gB recognizes a conformational B-cell epitope. The binding of both MABs was effectively inhibited in the PRV-positive pig blood samples. Accordingly, we established blocking-ELISAs to detect anti-PRV gB and gE antibodies, which achieved higher sensitivity than commercial kits. Moreover, the clinical serum samples results of our method and that of IFA were in high agreement, and our test results had a higher coincidence rate than that of a commercial kit. Assessing antibody levels by our methods at various times following immunization and challenge accurately reflected the trend of antibody-level changes and revealed the conversion to positive antibody status before the commercial kit. Our method is crucial for monitoring PRV infections, assessing immune responses, and controlling disease.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Humanized Antibodies. Modern Developments and Prospects for the Creation of Medical Protectors Against Biological Threads and Hazards

Passive immunization is the variant of the immunization, in which antibodies are introduced into the body in quantities, sufficient to provide a therapeutic effect. One of the «windows of opportunity» for passive immunization is an urgent post-exposure prophylaxis of infectious diseases and their preventive therapy, especially in the absence of chemotherapy drugs suitable for these purposes or their insufficient effectiveness. The existing heterologous drugs based on hyperimmune sera are highly reactogenic, and obtaining donor human immunoglobulins is associated with a number of ethical and technical restrictions. Therefore, biotechnologies that make it possible to obtain, on an industrial scale, low-reactogenic preparations of chimeric monoclonal antibodies with partially human specificity, as well as recombinant antibodies with fully human specificity, have enormous prospects. This trend is called «humanization of antibodies.» The purpose of this article is to analyze modern developments and to show the prospects for creating humanized antibodies specific to antigens of pathogens of especially dangerous infections and toxins as medical biological protection agents. The sources of the research are English-language studies and the scientific literature available via the Internet. The research method is an analysis of scientific sources on the topic being studied from the general to the specific. Results and discussion. The history of the creation of specific prevention drugs based on heterologous and homologous sera/immunoglobulins and monoclonal antibodies is presented. It has been shown in the article, that humanized specific monoclonal antibodies are widely used currently for the treatment of a number of severe chronic diseases (for example, rheumatoid arthritis, psoriasis, immunoinflammatory bowel diseases, malignant tumors). Conclusion. Recently, there has been an increase in numbers of both scientific research and developments, and drugs of recombinant antibodies already approved for use in clinical practice, specific to antigens of pathogens of especially dangerous infections and toxins - potential agents of bioterrorism, such as the anthrax microbe, botulinum toxins of various types, plant toxins ricin and abrin, ebolaviruses, coronaviruses. Such drugs can also be used as medical protectors against biological threats and hazards.

Standardization of flow cytometric detection of antigen expression.

Since response to antigen-based immunotherapy relies upon the level of tumor antigen expression we developed an antigen quantification assay using ABC values. Antigen quantification as a clinical assay requires methods for quality control and for interlaboratory and inter-cytometer platform standardization. A single lot of Cytotrol™ Lyophilized Control Cells (Beckman Coulter) used for all studies. The variability in antigen quantification across 4 different instrument platforms in 2 separate laboratories was evaluated. The effect of the antibody clone utilized, importance of custom 1:1 molar ratio (fluorophore to protein, F/P) verses off-the-shelf antibodies, and QuantiBrite PE calibration verses linearity calibration combined with a single point scale transformation with CD4 as reference were determined. Use of single lot control cells allowed validation of reproducibility between flow cytometer platforms and laboratories and allowed assessment of different antibody lots, cocktail preparation, and different antibody clones. Off the shelf antibody preparations provide reproducible estimates of antigen density, however custom 1:1 unimolar antibody preparations should be utilized for definitive measurement of antigen expression.Geometric Mean fluorescent Intensity (GeoMFI) was not comparable across instruments and inter-laboratory. The use of CD4 as the reference marker can minimize variability in ABC values. Comparable antigen quantification is vital in managing patients receiving antigen-based immunotherapy. If this assay is to be utilized in a clinical setting, quality control methods have to be instituted to assure reproducibility and allow validation across laboratories. We have demonstrated that use of a lyophilized cell control is highly valuable in achieveing these goals.

TWO CASES OF α-GAL SYNDROME CAUSED BY INGESTION OF WILD BOAR MEAT

α-Gal syndrome (AGS) is an allergic reaction to galactose-α-1,3-galactose (α-gal) found in the salivary glands of ticks, mammalian meat excluding primates, and some antibody preparations, such as cetuximab. We report two cases of AGS diagnosed after ingestion of wild boar meat. Patient 1, a male in his 70s, developed anaphylactic shock about 3 h after eating wild boar meat. He was transported to our acute and critical care center in Nagasaki University Hospital because he had difficulty in moving. Patient 2, a female in her 60s, developed a skin rash about 2.5 h after ingesting wild boar meat. After visiting our department to investigate the cause of the disease, the sera of both patients were found to be positive for α-gal specific IgE antibody and were diagnosed with AGS caused by ingestion of wild boar meat. Reports of AGS diagnosed after ingestion of wild boar meat are rare in Japan. Compared with other prefectures, the consumption of wild boar meat in Nagasaki is relatively high in Japan. In the past 10 years, four cases of AGS were diagnosed at our department, half of which were caused by the ingestion of wild boar meat, the ratio is possibly higher than that in other prefectures in Japan.