Enzyme-linked Immunosorbent Assay For Detection Of Antibodies Research Articles

WITHDRAWN: Development of a double-antigen sandwich ELISA for African swine fever virus antibody detection based on K205R protein

African swine fever (ASF) is a highly fatal infectious disease that affects swine caused by the African swine fever virus (ASFV) and poses a significant threat to the swine industry, and its effective diagnosis is crucial due to the current lack of commercially available vaccine for disease prevention and control. In this regard, the K205R protein is a suitable diagnostic antigen for enzyme-linked immunosorbent assay (ELISA) owing to its continuous expression throughout all ASFV replication stages, high immunogenicity, and sequence conservation. The double-antigen sandwich ELISA (DAgS-ELISA) is capable of detecting all immunoglobulins present in the serum, and outperforms alternative methods by enabling earlier antibody detection. Therefore, in this study, the K205R protein was prokaryotically expressed, and the K205R protein digested His-tag was labeled with horseradish peroxidase (HRP) and named K205R-HRP. The DAgS-ELISA was conducted using a combination of digested His-tag proteins and K205R-HRP. This method exhibits exceptional properties, including high sensitivity and no cross-reactivity with other porcine antibodies, with both the intra- and inter-assay coefficients of variation (CV) below 10 %. Antibody kinetics experiments have demonstrated that this method is capable of detecting antibodies as early as 7 to 9 days post-infection, which is a significant improvement compared to the commercial kit that can only detect antibodies from 11 to 13 days post-infection. In 120 clinical serum tests, the Kappa value between the DAgS-ELISA and the commercial kit was 0.96 (almost perfect). Owing to its high sensitivity, impressive repeatability, and strong specificity, this method enables earlier antibody detection and is suitable for ASFV antibody testing.

A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

Development of in-house ELISA for detection of antibodies against lumpy skin disease virus in cattle and assessment of its performance using a bayesian approach

Lumpy skin disease (LSD) is a contagious disease among cattle and buffalo worldwide. Currently, an enzyme-linked immunosorbent assay (ELISA) has been recognized as an efficient diagnostic tool that is less time-consuming and easier than the viral neutralization test to measure the antibody levels. In the present study, an in-house method of indirect ELISA was developed to detect the bovine antibodies against Lumpy skin disease virus (LSDV) and its performance was assessed using field samples. This in-house method has been compared with the commercial ELISA test kit for detection of bovine antibodies against LSDV. The sensitivity (Se) and the specificity (Sp) of the test were estimated using a Bayesian latent class model. Checkerboard titration was performed using the naturally LSDV-infected bovine sera and colostrum-deprived calf sera. The LSDV antigen concentrations (1 TCID50/mL), the sample serum (1:500), and goat anti-bovine immunoglobulin G (IgG) labeled with horseradish peroxidase (HRP) (1:10,000) were determined to be optimal for this assay. The calculated cut-off value was 0.067, and there were no differences in the results of tests that utilized positive and negative sera (p < 0.05). The characteristics of two diagnostic tests were evaluated using a conditional dependent and one-population Bayesian model. The Se value of an in-house indirect ELISA were almost similar to ELISA test kit. On the other hand, the Sp value of the in-house ELISA test was lower than that of the commercial ELISA test with the median values of 89% (95% PPI = 75.9–99.3%) and 91.4% (95% PPI = 85.3–95.5%), respectively. A posterior estimate for the prevalence was 66.9% (95% PPI = 60.8–83.3%) and higher than initially expected.

Serological response to vaccination against coronavirus disease-19 in patients with inflammatory bowel disease.

Vaccination against coronavirus disease-19 (COVID-19) is effective in preventing the occurrence or reduction in the severity of the infection. Patients with inflammatory bowel disease (IBD) are on immunomodulators, which may alter serological response to vaccination against COVID-19. Accordingly, we studied (i) the serological response to vaccination against COVID-19 in IBD patients and (ii) a comparison of serological response in IBD patients with that in healthy controls. A prospective study was undertaken during a 6-month period (July 2021 to January 2022). Seroconversion was assessed among vaccinated, unvaccinated IBD patients and vaccinated healthy controls using anti-severe acute respiratory syndrome coronavirus 2 immunoglobulin G (anti-SARS-CoV-2 IgG) antibody detection enzyme-linked immunosorbent assay (ELISA) kit, and optical density (OD) was measured at 450nm. OD is directly proportional to the antibody concentration. One hundred andthirty-two blood samples were collected from 97 IBD patients (85 [87.6%] ulcerative colitis and 12 [12.4%] Crohn's disease). Forty-one of the seventy-one (57.7%) unvaccinated and 60/61 (98.4%) vaccinated IBD patients tested positive (OD > 0.3) for SARS-CoV-2 IgG antibodies. Fourteen of the sixteen (87.5%) healthy controls tested positive for SARS-CoV-2 IgG antibodies. Vaccinated IBD patients had higher ODs than unvaccinated IBD patients (1.31 [1.09-1.70] vs. 0.53 [0.19-1.32], p < 0.001) and 16 vaccinated healthy controls (1.31 [1.09-1.70] vs. 0.64 [0.43-0.78], p < 0.001). Three of the seventy-one (4.2%) unvaccinated IBD patients reported having recovered from COVID-19. Most IBD patients seroconvert after vaccination against SARS-CoV-2, similar to a healthy population. A large proportion of IBD patients had anti-SARS-CoV-2 antibodies even before vaccination, suggesting the occurrence of herd immunity.

Development and evaluation of recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (ELISA) and double antigen sandwich ELISA for detection of antibodies to Chikungunya virus

Chikungunya virus (CHIKV) reemerged and caused millions of human infections since 2004. The disease could be established, when the virus has been introduced to areas where the appropriate vectors are endemic. The differential diagnosis of CHIKV infection varies based on place of residence, travel history, and exposures. Serological tests are commonly used to diagnose CHIKV infection, but their availability and assessments of the performance of the diagnostics have been limited. To develop and evaluate antibodies detection methods for chikungunya diagnosis and serological investigation. Recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (Mac-ELISA) and double antigen sandwich ELISA (Das-ELISA) for detection of antibodies to Chikungunya virus were developed and evaluated. The repeatability was evaluated by testing of three reference sera at single dilutions in triplicated for 5 times. The sensitivity, specificity, accuracy, and agreement of the MAC-ELISA and Das-ELISA were obtained by comparing the detection results of 225 serum samples (45 positive; 180 negative) with a real-time RT-PCR assay and an IFA commercial tests manufactured by Euroimmun. The established ELISA assays were standardized by determining the optimal concentrations of the key reagents. The coefficient values of repeat testing were within 10% and 20% for intraassay and interassay precision, respectively. A sensitivity of 60.0% and 52.5%, a specificity of 96.2% and 96.8%, and an accuracy of 89.8% and 88.9% were obtained for the Mac-ELISA and Das-ELISA, respectively, when compared to a CHIKV qRT-PCR method. And a sensitivity of 100%, a specificity of 97.5% and 99.5%, and an accuracy of 97.8% and 99.6% were yielded respectively when using the IIFT as a reference method, which showed a highly consistence to the commercial IIFT assay with a Kappa value greater than 0.90. The Mac-ELISA and Das-ELISA based on recombinant E2 protein of CHIKV were developed and standardized, which could detect IgM or total antibodies against CHIKV in 2-3 hours with acceptable sensitivities and specificities. These assays can be used for laboratory diagnosis and serological investigation of CHIKV infections to evaluate the risk of CHIKV transmission.

Plasmodium relictum MSP-1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus).

Avian malaria, caused by Plasmodium spp. and transmitted by mosquitos, is a leading cause of mortality of captive penguins. Antimalarial drugs are currently used to control infections in penguins. However, the effectiveness of treatment reduces significantly by the time the clinical signs appear, while early and unnecessary treatment interferes with development of protective immunity. Therefore, for suppressing parasitemia without affecting the development of immunity in captive penguins, antimalaria drugs need to be administered at the right time, which requires reliable diagnostic tools that can determine the levels of circulating antimalaria antibodies. In the present study, we have developed an enzyme-linked immunosorbent assay (ELISA) diagnostic assay based on the merozoite surface protein 1 (MSP-1) of P. relictum isolate SGS1 to specifically detect and relatively quantify antimalaria antibodies in penguins. We expressed and purified a truncated P. relictum isolate SGS1 MSP-1 and optimized its biotinylation and subsequent conjugation to streptavidin alkaline phosphatase for signal generation in ELISA. We tested the assay by analyzing sera obtained from penguins at the Baltimore Zoo, from Spring through Fall, and found that levels of detectable antibodies against MSP-1 varied seasonally for individual penguins, consistent with the expected seasonal variations in avian malaria prevalence. Corroboratively, we analyzed the sensitivity of the assay by titrating positive sera and found that the signal intensity generated was serum concentration-dependent, thus validating the ability of the assay to detect and relatively quantify the levels of antimalaria antibodies in penguin sera.

Development of robust, indigenous ELISA for detection of IgG antibodies against CoV-2N and S proteins: mass screening.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has adversely affected humankind and caused millions of deaths globally since January 2020. Robust and quick serological tests such as antibody detection assays for SARS-CoV-2 provide relevant information and aid in the process of vaccine development and diagnostics, as well as in sero-epidemiological monitoring of antibody response to the virus. The receptor-binding domain (RBD) of spike and nucleocapsid protein are specific targets for detecting SARS-CoV-2 antibodies. Here, we present the development of a stable spike (S) and nucleocapsid (N) protein-based ELISA antibody detection test “CoroSuchak,” with 99% sensitivity, 98% specificity, cost-effective, and detection in a minimum time for serodiagnosis and mass screening of the population for antibodies against SARS-CoV-2. Blood samples were analyzed from 374 SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) positive, 772 negative and asymptomatic, and 874 random groups of subjects. We found that the antibody titer was significantly higher (p < 0.0001) in infected and vaccinated group compared to the only vaccinated and only infected group. Using enzyme-linked immunosorbent assay (ELISA), we detected SARS-CoV-2 immunoglobulin G (IgG) antibodies in 118/123 (96%) infected individuals, 570/653 (87%) non-infected but vaccinated individuals, 231/237 (97%) individuals who were both infected and vaccinated, and 499/874 (57%) from randomly selected individuals from the first and second waves of the pandemic. Similarly in the third wave, 14/14 (100%) infected and 16/20 (80%) RT-PCR-negative but symptomatic subjects were detected. Thus, the highly sensitive and specific in-house developed ELISA antibody detection kit “CoroSuchak” is extremely useful to determine the seroprevalence of SARS-CoV-2 antibodies in the coronavirus-exposed population.Key points•Indigenous kit using a combination of spike and nucleocapsid proteins and peptide sequences.•High sensitivity and specificity to detect variants.•Highly sensitive for mass screening.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-12113-8.

Prokaryotic expression and purification of nucleoprotein of Guertu virus and its establishment of ELISA detection method

Objective: To obtain purified protein antigen of guertu virus (GTV) nucleoprotein (NP) and establish a rapid and accurate enzyme-linked immunosorbent assay (ELISA) method for detection of GTV antibody. Methods: Codon optimized GTV NP encoding genes were synthesized, cloned into the pet32a (+) vector, and recombinant expression plasmids were constructed and transformed into BL21 (DE3). Recombinant protein (rNP) obtained from the optimized expression were purified over a Ni column and identified by SDS-PAGE and Western blot. The purified protein was used as the antigen to optimize the reaction conditions, and an indirect ELISA assay for GTV IgG antibody was developed and optimized, which was evaluated and initially applied. Results: The prokaryotic expression plasmid pet32a-NP was successfully constructed, the recombinant protein was highly expressed in E. coli in the form of inclusion bodies, the size was about 44 kD, and the results of Western blot indicated that the recombinant protein had good antigenicity with GTV positive serum. The optimized ELISA (GTV-rNP-iELISA) established in this study showed strong specificity, high sensitivity, and the coefficient of variation within and between batches is less than 10%, and has good repeatability; the detection results are consistent with the IFA detection results. Using the established ELISA method to detect 162 sheep sera from some regions of Xinjiang in 2017-2019, the total positive rate of antibodies was 39.8%. Conclusions: The GTV NP antibody detection ELISA method has good sensitivity, reproducibility, and specificity and has the potential to be a powerful tool for the diagnosis and serological investigation of GTV infection.

Seroprevalence of antiSARS-CoV-2 IgG antibodies in children with household exposure to confirm cases of COVID-19 during the second wave of COVID-19 pandemic in Kota, Rajasthan

Background: Weather and the susceptibility of children to SARS-CoV-2 infection is still a debated question particularly in view of important decisions regarding opening schools. Therefore, we planned this cross-sectional analysis of antiSARS-CoV-2 Immunoglobulin-G (IgG) antibodies in children with known household exposure to SARS-CoV-2. This serosurveillance, to find the presence of IgG antibodies among children of probable household exposure of confirmed COVID-19 cases will help in the better understanding of the immune response after COVID-19 infection/exposure. Aims and Objectives: The aim of the study was to investigate the prevalence of anti-SARS-CoV-2 antibodies in children among the families who had confirm case/cases of COVID 19 during the second wave of the COVID-19 pandemic. This study is designed as a cross-sectional serosurveillance study in Kota, Rajasthan India. Materials and Methods: Population-based serosurveillance among children with probable household exposure to confirmed COVID-19 cases who was admitted in Govt. Medical College attached COVID 19 dedicated hospital was carried out during October 2021 to December 2021 using the COVID IgG Antibody Detection Enzyme-Linked Immunosorbent Assay kits. Seropositivity among children was measured and with various other factors to understand the immunity status among COVID-19 cases. Descriptive statistical analysis done. Results: 156/1285 eligible candidates (19.53%) having a total of 251 household contacts of &lt;17 years age group agreed to participate in study while rest of them refused. Anti-SARS-CoV-2 IgG antibodies were present in 127/251 household contacts (50.59%). Among them maximum 28.68% of seropositive were from age group 11–17 years age, symptoms were observed in 29.13% IgG positive contacts, maximum seropositivity was seen when only one family member was hospitalized, 63% seropositivity was seen when primary relative of child was hospitalized as compared to secondary. Maximum seroreactivity (55.9%) was seen when the Chest CT score of index case was high, that is, 14–25. max reactivity was observed 76/127 (59.84%) when hospital duration of index case is &lt;1 week. 118/127 (92.91%) of seroreactive children were found when the index case was unvaccinated or partially vaccinated. Conclusion: Seropositivity of 50.59% suggests that all the cases may not have IgG antibodies. Among the seronegative cases, the antibodies either not developed, or are undetectable, or have disappeared during the post COVID period. Seropositivity based on gender difference is statistically not significant. Proportion of positivity shows an increasing trend with increasing age among pediatric household contacts. Complete COVID-19 vaccination is an effective tool to minimize the disease frequency/severity. Severe disease patients with high CT score transmits the infection to household contacts more efficiently.

Performance of a Mycoplasma hyopneumoniae serum ELISA for antibody detection in processing fluids

The diagnostic performance of a commercial Mycoplasma hyopneumoniae (MHP) serum enzyme-linked immunosorbent assay (ELISA) was evaluated for MHP antibody detection in processing fluids (n = 494) using samples from three commercial swine farms. Based on historical monitoring, one farm was considered MHP positive and two were considered MHP negative. Samples were tested at a 1:10 dilution and diagnostic sensitivities and specificities estimated for specific ELISA sample-to-positive (S:P) cutoffs. At S:P ≥ 0.40, diagnostic sensitivity and specificity were estimated as 97.6% and 100.0%, respectively. Overall, the results suggest that processing fluids can be used for MHP antibody surveillance in breeding herds.

Detection of antibodies against domestic cat hepadnavirus using baculovirus-expressed core protein.

A novel orthohepadnavirus (domestic cat hepadnavirus [DCH]) similar to human hepatitis B virus has been recently detected in serum and liver samples from domestic cats with chronic hepatitis and hepatocellular carcinoma. Molecular investigations by independent research groups around the world have revealed positivity rates ranging from 6.5% to 12.5% in blood samples and up to 14.0% in liver tissue. In this study, we screened an age-stratified collection of feline sera (n=256) by using an antibody detection enzyme-linked immunosorbent assay based on the recombinant core antigen of DCH (DCHc). Specific antibodies (DCHc Abs) were detected with a prevalence of 25.0%. The DNA of DCH was detected in 35.9% (23/64) of seropositive cats and only in 1.0% (2/192) of seronegative animals. Based on the serological (IgG and IgM anti-DCHc) and virological status, the possible stages of DCH infection were predicted.

Novel Synthetic Peptide-based Enzyme Linked Immunosorbent Assay for Antibody Detection in Viral Infections of the Central Nervous System

Introduction: Viral infections of Central Nervous System (CNS) are associated with severe neurological sequelae and can lead to significant morbidity if not adequately diagnosed and treated. An immunological assay detecting antibodies in the Cerebrospinal Fluid (CSF) of the patients is widely used for diagnosis of viral infections of the CNS. In the present study, diagnostic efficacy of in-house designed synthetic peptide based Enzyme Linked Immunosorbent Assay (ELISA) was evaluated for detection of antibodies against neurotropic viruses panel such as Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella Zoster Virus (VZV), Japanese Encephalitis Virus (JEV), Dengue (DENV), West Nile Virus (WNV) and Chandipura Virus (CHPV) in CSF of suspected cases. Aim: To determine the diagnostic efficacy of in-house developed synthetic peptide-based ELISA in viral infections of the CNS. Materials and Methods: A retrospective study was conducted in Central India Institute of Medical Science, Nagpur, Maharashtra, India. The suspected case of CNS viral infections of the patients were enrolled in this study from January 2013 to December 2015. Total 150 suspected cases of viral CNS infections and 135 control cases were recruited. Total 32 synthetic peptides of highly immunogenic proteins of respective seven neurotropic viruses were designed and synthesised. The designed peptides were evaluated in CSF samples of both the viral CNS infections and control cases for detection of Imunoglobulin G (IgG) and IgM antibodies using in-house developed antibody detection method. The developed tests were further compared with commercially available antibody detection ELISA kit. The sensitivity and specificity of peptide-ELISA were determined by Receiver Operating Curve (ROC) analysis and p-value was calculated by comparison of means by t-test. Results: Out of total 150 cases of CNS viral infections, a total of 31 CSF samples including 15 for CMV and 16 of JEV were positive by in-house peptide ELISA. The synthetic peptides STGDVVDISP, KQKSLVEL, RTLEVFKE, RSSNVED of CMV and ITYECPK, RRSVSVQT, GESSLVN of JEV showed a significant difference in mean absorbance value for IgM and IgG antibodies in CSF of suspected cases of viral CNS infection. Conclusion: The results demonstrated that the synthetic peptide-based ELISA is rapid, cost-effective, simple and efficient immunodiagnostic assay for initial screening of viral CNS infection.

Investigation of Canine Distemper Virus Infection in Dogs in the Antalya Province

Canine distemper virus is one of the most prevalent infectious agents causing severe clinical symptoms among canids. Communal life-habitat for example clinics, dog-shelters, or rural areas is critical in terms of the transmission dynamics of CDV. In this study, blood samples from 92 dogs, aged 2 to 12 months, that were brought to the Rehabilitation Center and private veterinary clinics in Antalya with various internal medical problems were examined for Canine Distemper Virus (CDV) infection. Samples were tested using commercial immunochromatographic rapid test for detection of CDV-antigens and Enzyme Linked Immunosorbent Assay (ELISA) for detection of CDV-specific IgG and IgM antibodies. &#x0D; The most common clinical findings in the 92 dogs sampled were mucopurulent discharge in the eye (45.65%), nasal hyperkeratosis (35.87%), nasal mucopurulent discharge (25%), cough (13.04%), diarrhea (8.70%), and fatigue (6.52%). It was observed that clinical findings were more intense in the early reconvalescent period. 5.43% (5/92) of the samples examined by immunochromatographic rapid test were positive. The positivity rates of IgG and IgM antibodies by ELISA were 19.56% (18/92) and 94.56% (87/92), respectively. Of the 92 dogs tested by ELISA for detection of IgG and IgM antibodies, 91 (98.91%) were positive for one or both IgG or IgM antibodies, and 1 (1.09%) was negative for both antibodies. &#x0D; In conclusion, it was determined that CDV infection is actively circulating in the Antalya province and poses a risk for unvaccinated dogs in the region.

Bacterial coloration immunofluorescence strip for ultrasensitive rapid detection of bacterial antibodies and targeted antibody-secreting hybridomas

The indirect enzyme-linked immunosorbent assay (ELISA) is the gold standard method for monoclonal antibody (McAb) detection and plays a unique role in the preparation of bacterial antibodies. To solve the laborious issues associated with indirect ELISA, a novel bacterial coloration immunofluorescence strip (BCIFS) for antibody detection using colored bacteria instead of a labeled antibody as the antigen and tracer simultaneously and goat anti-mouse IgG as the test line was developed. The affinity range survey of BCIFS indicated that hybridoma cell cultures of E. coli O157:H7 (D3, E7) and Vibrio parahemolyticus (H7, C9) were detected, which complied with the results of indirect ELISA. Compared with the traditional indirect ELISA, the BCIFS sensitivity for E7 cell cultures, ascites, and purified antibodies was at least 4-fold more sensitive, and the BCIFS cross-reactivity for E7 cell cultures was almost consistent with that of indirect ELISA. In addition, the BCIFS isotypes for E. coli O157:H7 cell cultures and Vibrio parahemolyticus were IgG2a and IgG1, respectively, which were identical to the indirect ELISA. Furthermore, the BCIFS method was confirmed by McAb preparation, effective antibody use, and targeted antibody-secreted hybridoma preparation and screening, which showed excellent performance and substitution of the indirect ELISA method. Combined with methylcellulose semisolid medium, BCIFS offers a novel, easy to operate, rapid preparation method for antigen-specific hybridomas. This is the first report using BCIFS instead of indirect ELISA for bacterial antibody detection and application in different samples, which demonstrates a rapid and powerful tool for antibody engineering.

Seroprevalence of Immunoglobulin-G Antibody Among Confirm Cases of COVID-19.

BackgroundSero-surveillance to find the presence of IgG antibodies among COVID-19 cases helps in the better understanding of the immune response after COVID-19 infection.ObjectivesTo estimate seropositivity among confirmed COVID-19 cases and to correlate the seropositivity with various factors affecting seropositivity.MethodsPopulation-based sero-surveillance among COVID-19 cases was carried out during the second half of August 2020 in Ahmedabad using the COVID KAVACH, Immunoglobulin-G (IgG) Antibody Detection Enzyme-Linked Immunosorbent Assay (ELISA) kits. Seropositivity among cases was measured and compared with various other factors to understand the immunity status among COVID-19 cases.ResultsWith 1073 positive for IgG antibodies from 1720 samples, the seropositivity among COVID-19 cases is 62.38% [95%CI 60.07-64.64%]. The difference in seropositivity based on gender was statistically not significant (Z=0.26, P=0.79). Children have the highest seropositivity (94.44%) and from young adults, to the elderly, the proportion of positivity among cases shows an increasing trend. Time gap analysis from the date of diagnosis shows that the proportion of cases with IgG antibodies increases gradually reaching its peak at around 10 weeks (third month) and then declines gradually.ConclusionSeropositivity among COVID-19 cases is 62.38%. The proportion of cases with IgG antibodies reaches its peak at around 10 weeks (third month) after diagnosis and then declines gradually. This fall indicates that the detected antibodies may not be long-lasting and may become undetectable/absent over a period of time. The reason for seronegative results in COVID-19 cases needs further in-depth scientific research.

Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer–antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

Effect of pig serum storage conditions on detection of anti-ASFV antibodies by ELISA

One of the measures used to control and prevent African swine fever spread in the Russian Federation involves testing pig and boar sera using inter alia serological tools based on enzyme-linked immunosorbent assay (ELISA) for anti-viral antibody detection. However, there is no unified regulatory document specifying storage conditions for sera used in the ELISA for anti-ASFV antibody detection. There are also lack of published data on the maximum admissible shelf life of the pig sera, and the effect of storage conditions on the serological status of the pig sera as for ASF is understudied. The paper demonstrates results of the experiment aimed at the determination of the effect of storage temperatures and shelf life on the serological status of ASFV seropositive and seronegative pig sera when tested by INgezim PPA Compac (Ingenasa, Spain) ELISA as well as on the possibility of false results. During the experiment and analysis of its results, the new data were obtained, and they indicated from none to non-significant effect of the simulated storage conditions on the serological status of sera used for ASFV detection, while hemolyzed sera demonstrated more significant changes proportional to hemolysis degree and storage duration. Although the results of detection of antibodies against the agents of some diseases cannot be used in case of other pathogens, this study has a substantial applied significance as it allows to specify the dependence of the valid results of ASF serodiagnosis on the storage conditions of the samples.

Serosurvey for Health-Care Workers Provides Supportive Evidence for the Effectiveness of Hydroxychloroquine Prophylaxis against SARS-CoV-2 Infection

BackgroundThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has resulted in occupational exposure among Healthcare Workers (HCWs) and a high risk of nosocomial transmission. Asymptomatic infection and transmission of infection before the development of symptoms are well-recognized factors contributing to the spread of infection. We conducted a cross-sectional observational study to understand the seroprevalence of SARS-CoV-2 infection among HCWs and to verify the appropriateness of infection control measures, particularly Hydroxychloroquine (HCQ) prophylaxis.MethodsA cross-sectional sero-surveillance study was conducted among 500 HCWs in Dombivli and surrounding Mumbai Metropolitan area (Maharashtra, India) between 21st July and 3rd August 2020. The vulnerability of the study participants to SARS-CoV-2 infection was ascertained through a history of (i) involvement in direct care, (ii) exposure to aerosol-generating procedures, (iii) co-morbidities, (iv) Personal Protective Equipment (PPE) use, and (v) HCQ prophylaxis. SARS-CoV-2 IgG antibodies were tested using COVID KAVACH anti-SARS-CoV-2 IgG antibody detection enzyme-linked immunosorbent assay (ELISA) from Zydus Cadila. A systematic analysis of the correlation between the development of antibodies and factors affecting vulnerability to infection was performed.ResultsThe overall SARS-CoV-2 seroprevalence in the study population was 11%. Providing direct care to COVID-19 patients (Adjusted OR 16.4, 95% CI 3.3–126.9, p = 0.002) for long hours and irregular use of PPE (Adjusted OR 3.78, 95% CI 1.1–11.9, p = 0.02) were associated with an increased incidence of seropositivity. Prophylaxis with HCQ may have a role in reducing the vulnerability to infection as depicted by univariate and multivariate analysis (Adjusted OR 0.55, 95% CI 0.3–0.9, p = 0.047). It was also noted that those not on HCQ prophylaxis were threefold more prone to infection and developed severe disease as compared to those on HCQ prophylaxis.ConclusionProphylaxis with HCQ may have a role in mitigating the incidence and severity of SARS-CoV-2 infection. Although vaccination is the most robust strategy to safeguard against COVID-19, it will be months before vaccination percolates to the masses. In the face of the second wave of COVID-19, the use of HCQ prophylaxis in combination with use of face-masks regularly may be considered as a cost-effective measure for population dense areas like urban slums where social distancing is not possible.

Seroprevalence of antibodies to SARS-CoV-2 and predictors of seropositivity among employees of a teaching hospital in New Delhi, India.

ObjectivesHealthcare workers (HCWs) are at a high risk of contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to the increased likelihood of clinical exposure during patient management. The study objective was to determine the seroprevalence of antibodies to SARS-CoV-2 and its predictors among hospital employees.MethodsThe cross-sectional study was conducted at a teaching hospital from August 2020 to September 2020 among 1,401 employees, including 1,217 HCWs, in New Delhi, India. The serum samples were examined for immunoglobulin G (IgG) antibodies to SARS-CoV-2 using the COVID Kavach-Anti-SARS-CoV-2 IgG Antibody Detection enzyme-linked immunosorbent assay kit. Data were collected electronically using the EpiCollect mobile platform. A p < 0.05 was considered to indicate statistical significance.ResultsA total of 169 participants (12.1%) had detectable IgG antibodies to SARS-CoV-2. The highest seropositivity rate was observed in the administrative staff (20.1%), while it was lowest among medical doctors (5.5%, p < 0.001). Male sex and ever having lived in a containment zone were independently associated with past infection with SARS-CoV-2.ConclusionThe seroprevalence of SARS-CoV-2 infection in health workers may be lower than in the general population in New Delhi. However, nonpharmaceutical interventions were not associated with a reduction in the risk of acquisition of SARS-CoV-2.

Detection of SARS-CoV-2 Antibodies in Oral Fluid Obtained Using a Rapid Collection Device.

Current commercially available methods for reliably detecting antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain expensive and inaccessible due to the need for whole-blood collection by highly trained phlebotomists using personal protective equipment (PPE). We have evaluated an antibody detection approach using the OraSure Technologies oral antibody collection device (OACD) and their proprietary SARS-CoV-2 total antibody detection enzyme-linked immunosorbent assay (ELISA). We found that the OraSure test for total antibody detection in oral fluid had comparable sensitivity and specificity to commercially available serum-based ELISAs for SARS-CoV-2 antibody detection while allowing for a more accessible form of specimen collection with the potential for self-collection.