Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Development of an Indirect ELISA Antibodies Detection Method on Tandem-epitope Peptide of African Swine Fever Virus p30 and p54 Proteins.

Here, we report the development of an indirect ELISA antibodies detection method for African swine fever virus (ASFV). Two purified monoclonal antibodies (mAbs) against ASFV p30 and p54 protein were used as targets and a phage-displayed 12-mer peptide library was used to conduct four rounds of biopanning to screen peptide epitopes, then amino acids GGG was used as a linker to synthesize tandem-epitope peptide of ASFV p30 and p54 protein which was used as coating antigen. The optimum reaction conditions of indirect ELISA were determined by chessboard titration, and clinical serum samples were used to evaluate the specificity, sensitivity, stability and conformity of this method. The biopanning experiment indicated that 146PAEPYTT152 was a core domain of the B cell linear epitope of p54 protein. The optimization results of ELISA reaction conditions showed that the tandem-epitope peptide coupled with ovalbumin (OVA) at N-terminal had low background of non-specific serum reaction. And the optimum reaction effect was obtained when the polypeptide antigen was coated with carbonate buffer in 2 mug.mL-1, the serum was diluted 100-fold with blocking solution (1% gelatin solution), and the HRP-antibody was diluted 5 000 times with 0.05% PBST solution. The cut-off value was determined to be 0.339. Furthermore, the results of specificity, sensitivity and stability tests showed that there is no cross-reaction in positive serum samples of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV), the detection limit of ASFV positive sera is 1:1 600, and the method had high repeatability. Finally, Total 320 swine serum samples were detected simultaneously by the present established method and commercial ASFV antibody detection kit. The results showed that the relative specificity and sensitivity of the two methods were 97.6% and 97.3%, respectively. And the coincidence rate was 97.5%. In conclusion, this method showed good specificity, sensitivity, repeatability and coincidence rate, that had the potential value of developing clinical diagnostic kit.Copyright © 2022 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.

Seroincidencia de anticuerpos IgG del SARS-CoV-2 y factores de riesgo en trabajadores sanitarios asintomáticos del Hospital Departamental de Villavicencio

Objetivo: Estimar la seroincidencia acumulada de inmunoglobulinas (Ig) clase G (IgG) anti-SARS-CoV-2 en trabajadores de la salud asintomáticos y su asociación epidemiológica dentro de las áreas funcionales del Hospital Departamental de Villavicencio (HDV). Metodología: Se llevó a cabo un estudio observacional analítico longitudinal de una cohorte de trabajadores, donde cada 21 días, en tres oportunidades, se midieron IgG anti-SARS-CoV-2 en suero sanguíneo, a través de ELISA indirecto, en una muestra representativa aleatoria (n = 105) de trabajadores sanitarios del hospital (N = 756). Como instrumento de recolección de datos se utilizó una encuesta, donde cada trabajador sanitario declaró no haber sido diagnosticado con COVID-19, e igualmente registró la información sobre las variables independientes: sexo, edad, condición laboral, área funcional y comorbilidades. Resultados: La prevalencia inicial para SARS-CoV-2 entre los trabajadores sanitarios asintomáticos del HDV fue de 9,52 % (IC 95 % 5,25-16,65). La seroincidencia acumulada durante 42 días fue de 12,38 % (IC 95 % 7,38-20,04). El riesgo relativo (RR) se utilizó para establecer los factores de riesgo asociados a las variables independientes. El sexo masculino (RR ajustado = 3,34, IC 95 % 1,98-5,86), obesidad (RR ajustado = 10,98, IC 95 % 1,41-85,98) y sexo femenino (RR ajustado = 2,15, IC 95 % 1,12-4,31) en las áreas funcionales de Hospitalización, Medicina Crítica y Urgencias, respectivamente, son factores de riesgo en el HDV. Conclusión: Un total de 13 de 105 trabajadores sanitarios del hospital seroconvirtieron positivamente para SARS-CoV-2 y fueron asintomáticos durante 42 días de seguimiento epidemiológico. Además, existen factores de riesgo importantes en su exposición a este virus en el HDV.Alternate : Objetivo: Estimar a incidência zero acumulada de imunoglobulinas (Ig) classe G (IgG) anti-SARS-CoV-2 em profissionais de saúde assintomáticos e sua associação epidemiológica dentro das áreas funcionais do Hospital Estadual de Villavicencio (HDV). Metodologia: Foi realizado um estudo observacional analítico longitudinal de uma coorte de profissionais, no qual a cada 21 dias, em três ocasiões mediram-se IgG anti-SARS-CoV-2 em soro sanguíneo, através de ELISA indireto, em uma amostra representativa aleatória (n = 105) de profissionais de saúde do hospital (N =756). Como instrumento de recolecção de dados foi usada uma pesquisa, onde cada profissional de saúde declarou não ter sido diagnosticado com COVID-19, e igualmente registrou a informação sobre as variáveis independentes: sexo, idade, condições de trabalho, área de atuação e comorbidades. Resultados: A prevalência inicial para SARS-CoV-2 entre os profissionais de saúde assintomáticos do HDV foi de 9,52% (IC 95% 5,25-16,65). A incidência zero acumulada durante 42 dias foi de 12,38% (IC 95% 7,38-20,04). O risco relativo (RR) foi utilizado para estabelecer os fatores de risco associados às variáveis independentes. O sexo masculino (RR ajustado 3,34, IC 95% 1,98-5,86), obesidade (RR ajustado 10,98, IC 95% 1,41-85,98) e sexo feminino (RR ajustado 2,15, IC 95% 1,12-4,31) nas áreas funcionais de Internação, Unidade de Terapia Intensiva e Urgências, respectivamente, são fatores de risco no HDV. Conclusão: Um total de 13 de 105 profissionais de saúde do hospital foram detectados positivamente para SARS-CoV-2 e foram assintomáticos durante 42 dias de seguimento epidemiológico. Além disso, existem importantes fatores de risco na sua exposição a este vírus no HDV.Alternate : Objective: To estimate the cumulative seroincidence of anti-sars-CoV-2 immunoglobulin (Ig) class G (IgG) in asymptomatic health care workers and its epidemiological association within the functional areas of the Villavicencio Departmental Hospital (HDV). Methodology: A longitudinal analytical observational study of a cohort of workers was conducted in which anti-SARS-CoV-2 IgG levels in blood serum were measured every 21 days on three occasions using an in irect ELISA in a random representative sample (n = 105) of hospital health workers (N = 756). The data collection tool was a survey in which each healthcare worker indicated that they had not been diagnosed with COVID-19 and provided information on the independent variables: sex, age, job status, functional area, and comorbidities. Results: The baseline prevalence for SARS-CoV-2 among asymptomatic HDV healthcare workers was 9.52% (CI 95% 5.25-16.65). Cumulative seroincidence over 42 days was 12.38% (CI 95% 7.38-20.04). Relative risk (RR) was used to establish the risk factors associated with the independent variables. Male sex (adjusted RR 3.34, CI 95% 1.98-5.86), obesity (adjusted RR 10.98, CI 95% 1.41-85.98) and female sex (adjusted RR 2.15, CI 95% 1.12-4.31) in the functional areas of Hospitalization, Critical Medicine and Emergency, respectively, are risk factors in the HDV. Conclusion: During 42 days of epidemiological follow-up, 13 out of 105 hospital healthcare workers seroconverted positively for SARS-CoV-2 and remained asymptomatic. Additionally, significant risk factors are associated with their exposure to this virus in the HDV.

Enzyme-Linked Immunosorbent Assay: Types and Applications.

Enzyme-linked immunosorbent assay (ELISA) is an immunological assay widely used in basic science research, clinical application studies, and diagnostics. The ELISA technique relies on the interaction between the antigen (i.e., the target protein) versus the primary antibody against the antigen of interest. The presence of the antigen is confirmed through the enzyme-linked antibody catalysis of the added substrate, the products of which are either qualitatively detected by visual inspection or quantitatively using readouts from either a luminometer or a spectrophotometer. ELISA techniques are broadly classified into direct, indirect, sandwich, and competitive ELISA-all of which vary based on the antigens, antibodies, substrates, and experimental conditions. Direct ELISA relies on the binding of the enzyme-conjugated primary antibodies to the antigen-coated plates. Indirect ELISA introduces enzyme-linked secondary antibodies specific to the primary antibodies bound to the antigen-coated plates. Competitive ELISA involves a competition between the sample antigen and the plate-coated antigen for the primary antibody, followed by the binding of enzyme-linked secondary antibodies. Sandwich ELISA technique includes a sample antigen introduced to the antibody-precoated plate, followed by sequential binding of detection and enzyme-linked secondary antibodies to the recognition sites on the antigen. This review describes ELISA methodology, the types of ELISA, their advantages and disadvantages, and a listing of some multifaceted applications both in clinical and research settings, including screening for drug use, pregnancy testing, diagnosing disease, detecting biomarkers, blood typing, and detecting SARS-CoV-2 that causes coronavirus disease 2019 (COVID-19).

Preparation of the Monoclonal Antibody against the African Swine Fever Virus p54 Protein and Identification of the Antigenic Epitope.

The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times' subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all kappa chains. The lowest titer of mAb was 1:25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine. Copyright © 2023 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.

Serological response to SARS-CoV- 2 in patients under immunomodulatory treatment for rheumatoid arthritis

Background/Purpose: To determine and compare the magnitude of humoral immune response after the first, second, and third and overall exposure to SARS-CoV- 2, either by natural infection or vaccination, between rheumatoid arthritis (RA) patients under various immunosuppressive treatments and healthy controls (HCs). Method(s): Blood samples from various time points before and after the COVID-19 pandemic and longitudinal data on exposure history were obtained from RA patients and HCs recruited for this study. Antibody levels were then determined using indirect ELISA and statistically analysed in relation to exposure history. Result(s): A significant rise in antibody levels after overall exposure to SARS-CoV- 2 in RA patients and HCs was found, with no significant difference between the two groups. Amongst RA patients, a progressive rise in antibodies following each exposure to SARS-CoV- 2 was found, with antibody levels rising above the cut-off for seropositivity following the second and third exposures. Incidentally, HCs, despite having high antibody levels following the first and second exposure, were found to have antibody levels below the cut-off for seropositivity upon their third exposure to SARS-CoV- 2. Conclusion(s): Contrary to popular belief, RA patients, despite under immunosuppressive treatments, are capable of eliciting a humoral response that is comparable to that of healthy individuals. The lower antibody levels found in HCs by third exposure could signify that more vaccine boosters may be required to increase the public's immunity against SARS-CoV- 2 for herd immunity and avoid creating a reservoir for the emergence of novel variants with persistent infections.

Kinetics of Humoral Immune Response in Patients with Asymptomatic or Mild Covid-19: A Longitudinal Study Based in an in-House Indirect Elisa Method

Background: In the COVID-19 pandemic, individuals may be asymptomatic, with mild or severe symptoms associated with respiratory tract infections. Patients with COVID-19 have IgG antibodies on average two weeks after infection and persist at stable levels for a few months. The influence of the levels of these antibodies and the time in circulation, protection, and severity of the disease in reinfections, has not yet been completely elucidated. The objective of this work was to evaluate the titers of anti-SARS-CoV-2 IgG antibodies in asymptomatic patients or with mild symptoms in a period of six months. Method(s): In this longitudinal study, we selected 62 individuals (median age 42.5 years;IQR 33.3-52.0;59.7% female) tested positive for SARS-CoV-2 Immunoglobulin G (IgG) antibodies using a Fluorescence Immunoassay (FIA) (iChroma II, BioSys + Kovalent) from July 2020 (peak of the first wave) to March 2021 (begging of the second wave associated with transmission of the Gamma variant) in the state of Sergipe, Northeast Brazil. All participants included in the present study had a history of asymptomatic or mild COVID-19, were not vaccinated against the disease, and were selected from the EpiSERGIPE Project conducted in the state of Sergipe. An in-house indirect Enzyme-Linked Immunosorbent assay (ELISA)-based method was used to evaluate the serum titers of anti-nucleocapsid SARS-CoV-2 antibodies in three moments: baseline (D0), 90 days (D90), and 180 days (D180). Briefly, 96-microwell plates (Nunc, Thermo Fisher Scientific). Result(s): In D0, 79% (49 of 62) of individuals had a positive result for the presence of anti-nucleocapsid SARS-CoV-2 antibodies. In D90 and D180, the percentage of positive results was 69.3% (43 of 62) and 53.2% (33 of 62), respectively. We found a progressive decline in the levels of anti-nucleocapsid SARS-CoV-2 antibodies over time, ranging from 26.2 (Interquartile Range [IQR] 12.4-37.7) in D0 to 11.7 (IQR 5.6-18.2) in D180 (p<0.001). In addition, we found that individuals over 40 years old had higher levels of IgG antibodies to the SARS-CoV-2 nucleocapsid in D90, regardless of sex. However, an influence of sex and age was not observed on D0 and D180. Discussion(s): Studies describe that IgG levels remain for only 3-4 months in the body of individuals who have had previous contact with SARS-CoV-2, however in this study it was observed that most individuals had a significant and gradual decrease in anti-nucleocapsid SARS-CoV-2 antibodies circulating, even before completing three months after exposure to the virus. Conclusion(s): This study showed a progressive decline in the levels of anti-nucleocapsid SARS-CoV-2 antibodies during the first six months after SARS-CoV-2 infection among individuals with asymptomatic or mild COVID-19. Approximately 50% of individuals have no detectable antibodies six months after infection. Moreover, we found a potential influence of age on the humoral response against SARS-CoV-2. Copyright © 2022

Design and optimization of an IgG human ELISA assay reactive to recombinant RBD SARS-CoV-2 protein.

Serology assays are essential tools to mitigate the effect of COVID-19, help to identify previous SARS-CoV-2 infections or vaccination, and provide data for surveillance and epidemiologic studies. In this study, we report the production and purification process of the receptor-binding domain (RBD) of SARS-CoV-2 in HEK293 cells, which allowed the design, optimization, and validation of an indirect ELISA (iELISA) for the detection of human anti-RBD antibodies. To find the optimal conditions of this iELISA, a multivariate strategy was performed throughout design of experiments (DoE) and response surface methodology (RSM), one of the main tools of quality by design (QbD) approach. The adoption of this strategy helped to reduce the time and cost during the method development stage and to define an optimum condition within the analyzed design region. The assay was then validated, exhibiting a sensitivity of 94.24 (86.01-98.42%; 95% CI) and a specificity of 95.96% (89.98-98.89%; 95% CI). Besides, the degree of agreement between quality results assessed using kappa's value was 0.92. Hence, this iELISA represents a high-throughput technique, simple to perform, reliable, and feasible to be scaled up to satisfy the current demands. Since RBD is proposed as the coating antigen, the intended use of this iELISA is not only the detection of previous exposure to the virus, but also the possibility of detecting protective immunity. KEY POINTS: • RBD was produced in 1-L bioreactor and highly purified. • An iELISA assay was optimized applying QbD concepts. • The validation procedure demonstrated that this iELISA is accurate and precise.

Lab Parameter among Corona Patients

Introduction: The COVID-19 pandemic has been raging across the globe since early January 2020. Various geographical regions have been passing multiple swells of upsurge of cases which aren't matched temporally as well as in severity. The diapason of the complaint ranges from asymptomatic to severe life-hanging complaint. Advanced age and the presence of comorbidities similar as cardiovascular complaint, diabetes mellitus, hypertension, chronic lung complaint, chronic kidney complaint, cancer, and obesity are among the major threat factors for severe disease. Aims and objectives: Significance of lab parameter among Corona Patients. Materials and methods: The covid- 19 opinion was verified by reverse transcription- polymerase chain reaction (RT- PCR) assay of nasopharyngeal swab sample. Hematology blood samples were used to analyze by flow cytometry. Biochemical samples were used to analyze by completely auto analyzer diagnostic outfit. Serology tests were carried out the styles based on indirect ELISA technique, immune plates are coated with a admixture of purified viral antigen and probe using the patient serum. Results: It is found that there is statistically significant (p-value<0.05) mean difference within the lab parameters (IL-6, LDH and Ferritin) in Covid patients using the Post Hoc Analysis. It is also found that there statistically significant (p-value<0.05) mean difference between RBC, Hb level, Hematocrit, MCV, MCH, MCHC, Platelet, RDW, PCT and NL ratio while Age, WBC, MPV, M(Monocyte), E(Eosinophil), B(Basophil), D-dimer and PDW were found to be statistically insignificant (p-value>0.05) with respect to gender. Discussion: CBC, D- dimer, IL-6, LDH and Ferritin were analysed and found associated with adverse outcomes. There is significant association of age, gender, comorbidity. Conclusion: High NLR at admission associated with a higher mortality. Laboratory features (e.g., IL-6, LDH, Ferritin D-dimer etc.) were associated with poor outcomes.

Development of an indirect ELISA to detect PEDV specific IgA antibody based on a PEDV epidemic strain.

BACKGROUND: Porcine epidemic diarrhea (PED), a swine epidemic disease caused by porcine epidemic diarrhea virus (PEDV), is characterized by severe watery diarrhea, vomiting, dehydration and high mortality in piglets, and has caused serious economic losses to the global porcine industry. The level of PEDV IgA antibody is a key marker to assess the extent of passive immunity of the resistance against PEDV infection. However, current commercial structure proteins-based kits for detection of PEDV antibody are not affordable, and those kits require complicated antigen preparation procedures, which cannot meet the scope of economic benefits of many large-scale pig companies in China. Therefore, there is an urgent need to develop an accurate, simple, and economical method for IgA detection in clinical samples. In this study, an indirect ELISA (i-ELISA) method was developed based on a purified PEDV epidemic strain (NH-TA2020). RESULTS: The results show that optimal working dilution ratios of PEDV antigen and HRP anti-swine IgA are at 1: 1000 and 1:15000 respectively. The sensitivity of this method is high with the maximum dilution of samples up to 1:160, and coefficients of variation (CV) of both the intra assays and inter assays were no more than 15%. In addition, the relative sensitivities of the i-ELISA were above 90% compared with values from commercial kits in both serum and oral fluid samples. CONCLUSIONS: Our results suggested that the i-ELISA developed in this study was an accurate, simple, and economical method for PEDV-IgA detection in clinical samples.

Improvement of the Detection Method for the Serum Titer of the Inactivated SARS-CoV-2 Vaccine

A mixed antigen coating method was designed to optimize the method for detecting the titer of the immune serum to inactivated SARS-CoV-2 vaccine, which based on the traditional single antigen coating.The authors determined the optimal coating concentration of the single antigen first by the checkerboard method, then combined two kinds of antigen and obtained coating concentration of the mixed antigen.The best combination of mixed coating antigen contains the whole-coronavirus antigen with total protein concentration of 25 ng/well and the recombinant new coro-navirus S1 protein antigen with protein concentration of 50 ng/well.This method can be used to detect all antibody-specific titers in serum effectively, especially serum containing high-level S protein-specific antibodies.

Seroprevalence of Anti Sars Cov 2 Igg Antibodies in Healthcare Workers Working In Covid Isolation Wards at a Tertiary Care Centre in Hyderabad

Background:Healthcare workers are at a high risk of contracting SARS CoV 2 infection due to their close contact with COVID19 confirmed and suspected cases. Preventing infection amongst healthcare workers is crucial, not only for main- taining a healthy and functional workforce during the pandemic, but also to reduce secondary transmission to collegues and other patients. Prevalence of IgG antibodies against the infection provides essential information regarding unde- tected infection and transmission. The present study is being conducted to estimate the seroprevalence of SARS CoV 2 antibodies among healthcare workers working in COVID19 isolation wards. Methods:90 healthcare workers working in covid isolation wards were recruited into the study. A questionnaire was administered for risk assessment and history of previous RT-PCR confirmed COVID-19 infection,if any. Serum sample collected from the participants were tested for anti SARS CoV 2 IgG antibodies by Indirect ELISA (Covid Kawach IgG Mi- crolisa by J Mitra). Results:Out of 48 samples processed so far, 16(33.3%) samples were positive for SARS CoV 2 IgG antibody.of the 16 pos- itive samples, 14 samples were negative by RT-PCR previously. The remaining results will be produced at the time of the presentation. Conclusions:Presence of anti SARS CoV 2 IgG antibodies among healthcare workers at high risk, who tested negative by RT-PCR previously can indicate a previou asymptomatic infection, which calls for further evaluation

Sero-Positivity of SARS-COV-2 Antibodies among Healthy Blood Donors in Delhi-National Capital Region

Introduction: A novel Coronavirus (2019-nCoV) was first reportedfrom Wuhan, China, in December 2019. This 2019-nCoV was foundto be an enveloped RNA beta coronavirus which contains fourstructural proteins: spike (S), envelope (E), membrane (M) andnucleocapsid (N). During infections, neutralizing antibodies aremainly targeted against the spike protein, which has been the mainfocus of vaccine development. Severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) is disease implicated in the infection bythis novel pathogen.The actual percentage of asymptomatic cases is unclear and how longthey continue to be carriers is unknown. Sero-prevalence data cantherefore throw light on the exposure, immune status and diseaseseverity in a population.Aims &Objectives: To determine the sero-positivity of SARS-CoV-2 Antibodies among healthy blood donors attending a tertiary carehospital and voluntary blood donor camps, in Delhi-National CapitalRegion.Materials &Methods: After obtaining written informed consent, 2 ml of venous bloodwas collected in plain sterile vials, during phlebotomy of thevoluntary blood donors. Separated serum samples were used fortesting. Serum samples were tested for the presence of IgG antibodiesagainst COVID-19 using a commercially available ELISA kit. ErbaLisa COVID-19 IgG, developed and manufactured byCalbiotech. Inc USA will be used. The immunoassay is based on "Indirect ELISA." Statistical analysis: Prevalence of healthy blood donors positivefor SARS-CoV-2 IgG Antibodies is expressed as a percentage. Outcome measures: Percentage of healthy blood donors positivefor SARS-CoV-2 IgG Antibodies in Delhi-National CapitalRegion.Results: 119/182 (65.38%) of healthy blood donors were positive for Spikeprotein of SARS-CoV-2 IgG antibodies. Majority of donors were male (97.8%) in the age group18-52 years with a mean age of 30.38 years. Antibody titers ranged from 0.265 to 2.039 None were positive for transfusion transmitted infections alloantibodies. Blood group wise percentage of sero-positivity was as follows:A: 64.28% (27/42))A-: 0/0B: 69.01% (49/71)B-: 60% (03/05)O: 60.78% (31/51)O-: 100% (1/1)AB: 66.66% (08/12)AB-: 0/0 Conclusions: Results obtained from our study will be useful to gaugethe current immunological status, which will allow further policymaking to be guided by more reliable evidence-based data.

Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Real-time RT-PCR is the most commonly used method for COVID-19 diagnosis. However, serological assays are urgently needed as complementary tools to RT-PCR. Hachim et al. 2020 and Burbelo et al. 2020 demonstrated that anti-nucleocapsid(N) SARS-CoV-2 antibodies are higher and appear earlier than the spike antibodies. Additionally, cross-reactive antibodies against N protein are more prevalent than those against spike protein. We developed a less cross-reactive immunoglobulin G (IgG) indirect ELISA by using a truncated recombinant SARS-CoV-2 N protein as assay antigen. A highly conserved region of coronaviruses N protein was deleted and the protein was prepared using an E. coli protein expression system. A total of 177 samples collected from COVID-19 suspected cases and 155 negative control sera collected during the pre-COVID-19 period were applied to evaluate the assay's performance, with the plaque reduction neutralization test and the commercial SARS-CoV-2 spike protein IgG ELISA as gold standards. The SARS-CoV-2 N truncated protein-based ELISA showed similar sensitivity (91.1% vs. 91.9%) and specificity (93.8% vs. 93.8%) between the PRNT and spike IgG ELISA, as well as also higher specificity compared to the full-length N protein (93.8% vs. 89.9%). Our ELISA can be used for the diagnosis and surveillance of COVID-19.

Predictive Analysis of the Neutralization Activity in Convalescent Plasmas From COVID-19 Recovered Patients in Zhejiang Province, China, January-March, 2020.

Background: Convalescent plasma (CP) transfusion is considered to be the priority therapeutic option for COVID-19 inpatients when no specific drugs are available for emerging infections. An alternative, simple, and sensitive method is urgently needed for clinical use to detect neutralization activity of the CP to avoid the use of inconvenient micro-neutralization assay. Method: This study aims to explore optimal index in predicting the COVID-19 CP neutralization activity (neutralizing antibody titers, NAb titers) in an indirect ELISA format. Fifty-seven COVID-19-recovered patients plasma samples were subjected to anti-SARS-CoV-2 RBD, S1, and N protein IgG antibody by indirect ELISA. Results: ELISA-RBD exhibited high specificity (96.2%) and ELISA-N had high sensitivity (100%); while ELISA-S1 had low sensitivity (86.0%) and specificity (73.1%). Furthermore, ELISA-RBD IgG titers and pseudovirus-based NAb titers correlated significantly, with R2 of 0.2564 (P < 0.0001). Conclusion: ELISA-RBD could be a substitute for the neutralization assay in resource-limited situations to screen potential plasma donors for further plasma infusion therapy.