In-house ELISA Research Articles

SARS-CoV-2 Circulation during the First Year of the Pandemic: A Seroprevalence Study from January to December 2020 in Tuscany, Italy.

Italy was the second country affected by the SARS-CoV-2 pandemic; the virus spread mainly in Northern Italy with a subsequent diffusion to the center and southern part of the country. In this study, we aimed to assess the prevalence of antibodies against SARS-CoV-2 in the general population of the Siena province in the Tuscany region (Central Italy) during 2020. A total of 2480 serum samples collected from January to December 2020 were tested for IgM and IgG antibodies against SARS-CoV-2 by a commercial ELISA. Positive and borderline samples were further tested for the presence of anti-receptor-binding domain (RBD) IgM and IgG antibodies by an in-house ELISA and by a micro-neutralization assay. Out of the 2480 samples tested by the commercial ELISA, 81 (3.3%) were found to be positive or borderline for IgG and 58 (2.3%) for IgM in a total of 133 samples (5.4%) found to be positive or borderline for at least one antibody class. When the commercial ELISA and in-house ELISA/micro-neutralization assay results were combined, 26 samples (1.0%) were positive for RBD IgG, 11 (0.4%) for RBD IgM, and 23 (0.9%) for a neutralizing antibody. An increase in seroprevalence was observed during the year 2020, especially from the end of summer, consistent with the routine epidemiological surveillance of COVID-19 cases.

The Occluded Epitope Residing in Spike Receptor-Binding Motif Is Essential for Cross-Neutralization of SARS-CoV-2 Delta Variant.

Concerns over vaccine efficacy after the emergence of the SARS-CoV-2 Delta variant prompted revisiting the vaccine design concepts. Monoclonal antibodies (mAbs) have been developed to identify the neutralizing epitopes on spike protein. It has been confirmed that the key amino acid residues in epitopes that induce the formation of neutralizing antibodies do not have to be on the receptor-binding domain (RBD)- angiotensin-converting enzyme 2 (ACE2) contact surface, and may be conformationally hidden. In addition, this epitope is tolerant to amino acid mutations of the Delta variant. The antibody titers against RBD in health care workers in Thailand receiving two doses of CoronaVac, followed by a booster dose of BNT162b2, were significantly increased. The neutralizing antibodies against the Delta variant suggest that the overall neutralizing antibody level against the Wuhan strain, using the NeutraLISA, was consistent with the levels of anti-RBD antibodies. However, individuals with moderate anti-RBD antibody responses have different levels of a unique antibody population competing with a cross-neutralizing mAb clone, 40591-MM43, determined by in-house competitive ELISA. Since 40591-MM43 mAb indicates cross-neutralizing activity against the Delta variant, this evidence implies that the efficiency of the vaccination regimen should be improved to facilitate cross-protective antibodies against Delta variant infections. The RBD epitope recognized by 40591-MM43 mAb is hidden in the close state.

Cross-Reactivity of IgG Antibodies and Virus Neutralization in mRNA-Vaccinated People Against Wild-Type SARS-CoV-2 and the Five Most Common SARS-CoV-2 Variants of Concern.

The rapid development, approval, and production of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than 1 year after the first reports of a new infectious disease was a real game changer, providing 80%–90% efficacy in preventing severe etiopathologies of the coronavirus disease 2019 (COVID-19). These vaccines induce an immune response against the SARS-CoV-2 spike (S) protein located on the surface of the virus particle. Antibodies (Abs) recognizing the S-protein can inhibit binding of the virus via the S-protein to the angiotensin-converting enzyme-2 (ACE-2) receptor expressed on different human cells, especially when these Abs bind to the interaction site, the so-called receptor-binding domain (RBD). We have expressed the RBDs of wild-type SARS-CoV-2 and five variants of concern (VOCs) to test the immune response in people before vaccination with mRNA vaccines BNT162b2 and mRNA-1273 and after up to three vaccinations using in-house ELISA and inhibition assays. The methods of both assays are provided. Both vaccines initiated similarly high IgG titers after two vaccinations against the wild-type and even two VOC-RBDs (alpha and delta) and strongly inhibited the corresponding RBD-ACE-2 binding. The IgG titers and inhibition of ACE-2 binding were lower for beta and gamma RBDs and much lower for omicron RBD. The third vaccination after 6 months strongly increased both the IgG titers and the neutralizing effect against all variants, especially for omicron, leading to 63% ± 13% neutralization potential. Importantly, neutralization linearly increased with the IgG titers.

Abstract 654: CD28 shedding is a novel resistance mechanism to anti PD-1 therapy

Abstract Background: Resistance mechanisms to anti-PD1 therapy remain poorly understood. Studies have implicated that efficient anti-PD1 therapy relies specifically on intact CD28/B7 signaling. We have previously shown that membranal CD28 is actively shed by MMP-2 and MMP-13 upon T cell stimulation in humans, and that soluble CD28 can counteract the efficacy of anti-PD1 blocking antibodies in-vitro. Here we describe that CD28-shedding process is a potential novel resistance mechanism to PD-1 therapies in cancer patients, by using several ex-vivo and in-vivo methodologies. The prominent unfavorable effect of CD28-shedding on PD-1 blockade prompted the generation of BND-67, an agent that can selectively and efficiently block CD28 shedding. Methods: Soluble CD28 detection in plasma of cancer patients and in the culture media of stimulated T cells was achieved using a proprietary in-house ELISA. Cancer patient processed tumors and PBMC were used ex-vivo to identify membrane/soluble CD28 by ELISA and flow-cytometry. MC-38 challenged human CD28 transgenic mice (CD28-shedding permissive model), and WT mice (non-permissive CD28-shedding model) were compared for efficacy to anti-PD1 blocking antibody. BND-67 was isolated by screening a VHH library for binding to the CD28-shedding prone region and blocking of CD28 shedding. The lead clone was further affinity-matured and reformatted into a half-life extended construct. Results: CD28 receptor density was found to be lower on tumor-resident T cells then their matched counterparts in the periphery. Evaluation of plasma samples from patients undergoing anti-PD-1 therapy demonstrated an association of diseases relapse with elevated plasma levels of soluble CD28. Correspondingly, relapses from response to anti-PD-1 therapy were significantly higher in CD28-shedding permissive mice vs non-permissive mice. To overcome this novel regulatory mechanism, we generated BND-67, which is an affinity-matured VHH that can effectively bind CD28, block CD28 shedding and has no agonistic activity. In addition, an optimal approach for increasing the half-life of the agent was identified by fusing the VHH to human Fc. Conclusions: We report here the discovery of a potential novel resistance mechanism to anti-PD1 blockade involving the shedding of the CD28 receptor. This process occurs in the setting of cancer and was further reinforced by results observed in transgenic mice. Biond has generated BND-67, a therapeutic drug candidate for the inhibition of this novel regulatory mechanism. Citation Format: Motti Hakim, Anna Fridman Dror, Tal Shahar Gabay, Dror Alishekevitz, Edna Meilin, Ilana Mandel, Yair Sapir, Avidor Shulman, Tehila Ben Moshe. CD28 shedding is a novel resistance mechanism to anti PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 654.

Diagnosis of Human Leptospirosis: Comparison of Microscopic Agglutination Test with Recombinant LigA/B Antigen-Based In-House IgM Dot ELISA Dipstick Test and Latex Agglutination Test Using Bayesian Latent Class Model and MAT as Gold Standard.

Leptospirosis is a spirochaetal infection that possesses a broad host range affecting almost all mammals. In the present study, the microscopic agglutination test (MAT) was compared with recombinant LigA/B antigen-based point-of-care diagnostics such as the in-house IgM dot ELISA dipstick test (IgM- DEDT) and the latex agglutination test (LAT) for the serodiagnosis of human leptospirosis. The comparison of the MAT with these two point–of-care diagnostics was performed using the MAT as the gold standard test and using Bayesian latent class modelling (BLCM), which considers all diagnostic tests as imperfect. The N-terminal conserved region of the LigA/B protein spanning the first to fifth big tandem repeat domains (rLigA/BCon1-5) was employed as a serodiagnostic marker in both of the bedside assays. A total of 340 serum samples collected from humans involved in high risk occupations were screened using the MAT, IgM DEDT and LAT. During the early phase of leptospirosis, BLCM analysis showed that the IgM DEDT and LAT had similar sensitivities (99.6 (96.0–100)) and (99.5 (95.2–100)), respectively, while the single acute phase MAT had the lowest sensitivity (83.3 (72.8–91.3)). Both the IgM DEDT and the LAT may be superior to the single acute phase MAT in terms of sensitivity during the early phase of infection and may be suitable for the early diagnosis of leptospirosis. However, BLCM analysis revealed that the use of both acute and convalescent samples substantially increased the sensitivity of the final MAT (98.2% (93.0–99.8%)) as a test to diagnose human leptospirosis. Both the IgM DEDT and LAT can be employed as bedside spot tests in remote locations where the MAT is not easily accessible.

Risk factors for SARS-CoV-2 seroprevalence following the first pandemic wave in UK healthcare workers in a large NHS Foundation Trust.

Background: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. Methods:HCWs at Sheffield Teaching Hospitals NHS Foundation Trust were prospectively enrolled and sampled at two time points. We developed an in-house ELISA for testing participant serum for SARS-CoV-2 IgG and IgA reactivity against Spike and Nucleoprotein. Data were analysed using three statistical models: a seroprevalence model, an antibody kinetics model, and a heterogeneous sensitivity model. Results:Our in-house assay had a sensitivity of 99·47% and specificity of 99·56%. We found that 24·4% (n=311/1275) of HCWs were seropositive as of 12th June 2020. Of these, 39·2% (n=122/311) were asymptomatic. The highest adjusted seroprevalence was measured in HCWs on the Acute Medical Unit (41·1%, 95% CrI 30·0-52·9) and in Physiotherapists and Occupational Therapists (39·2%, 95% CrI 24·4-56·5).Older age groups showed overall higher median antibody titres. Further modelling suggests that, for a serological assay with an overall sensitivity of 80%, antibody titres may be markedly affected by differences in age, with sensitivity estimates of 89% in those over 60 years but 61% in those ≤30 years. Conclusions:HCWs in acute medical units and those working closely with COVID-19 patients were at highest risk of infection, though whether these are infections acquired from patients or other staff is unknown. Current serological assays may underestimate seroprevalence in younger age groups if validated using sera from older and/or more severe COVID-19 cases.

Seroprevalence of Anti-S1-RBD Antibodies in Pre-pandemic and Pandemic Subjects From Hail Region, KSA.

BackgroundTwo years into the pandemic, yet the threat of new SARS-CoV-2 variants continues to loom large. Sustained efforts are required to fully understand the infection in asymptomatic individuals and those with complications. Identification, containment, care, and preventative strategies rely on understanding the varied humoral immune responses.MethodsAn in-house ELISA was developed and standardized to screen for serum IgG antibodies against the SARS-CoV-2 S1-RBD protein as an antigen. This study aims to investigate the seroprevalence of serum antibodies against S1-RBD antigen in pre-pandemic (n = 120) and during the early pandemic period (n = 120) in subjects from the Hail region, KSA and to correlate it with clinical and demographic factors.ResultsSamples collected from both male (n = 60) and female (n = 60) subjects during the pandemic in the age groups of 20–40 (0.31 ± 0.029 and 0.29 ± 0.024, respectively) and 41–60 years (0.35 ± 0.026 and 0.30 ± 0.025, respectively) showed significantly higher levels of serum antibodies against S-RBD antigen than the age-matched pre-pandemic samples [male (n = 60) and female (n = 60)]. Pandemic subjects exhibited significantly (p < 0.01) higher inhibition (80–88%) than age-matched pre-pandemic subjects (32–39%). Antibodies against S1-RBD antigen were detected in approximately 10% of the total pre-pandemic population (males and females). However, subjects > 60 years did not show antibodies.ConclusionAntibody levels increased in samples collected during the pandemic, even though these subjects were not clinically COVID-19 positive. A small number of pre-pandemic subjects showed serum antibodies, suggesting prior exposure to other coronaviruses in the region. With dwindling neutralizing antibody levels and reduced vaccine efficacy against newer variants, it remains crucial to develop better assays for surveillance, management, and future research.

Age and gender associated changes in immunoglobulin subclass levels specific to S. pneumoniae, serotype 1

S. pneumoniae is an important human pathogen which has a polysaccharide capsule with virulent properties. This work aims to estimate the titres of S. pneumoniae specific IgG and IgA isotypes, with respect to age and sex.An in-house whole bacterial cell ELISA was used for the determination of relative levels and endpoint titres of IgG subclasses and IgA1 subclass specific for S. pneumoniae serogroup 1, and to quantify specific IgG1 and IgG2 levels.Significantly lower anti-pneumococcus IgG1 titres were found in older individuals, which was more pronounced in men. Lower IgG2 titres were detected in men over 50 years of age, in comparison to women under 50 years of age. The levels of IgG3 and IgG4 did not differ between different sex and age groups. Lower IgA1 levels were detected in male individuals in both age groups in comparison to females under 50 years of age. The levels of IgG1 showed a moderate correlation with IgG4 in younger individuals of both sexes (r = 0.61 in men and 0.63 in women) which was not noted in the older age group.We highlight the deficiency in humoral immunity in older people, especially male and suggest immunization of this population with pneumococcal vaccines.

Quantification of infliximab and adalimumab in human plasma by aliquid chromatography tandem mass spectrometrykitand comparison with two ELISA methods.

Background: This study compared the performance of plasma infliximab and adalimumab quantification using a commercially available kit (mAbXmise kit) and mass spectrometry readout to that of two ELISA methods in patients treated for inflammatorybowel disease. Methods & results: The mAbXmise method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) was linear from 2 to 100μg/ml. It was validated according to international guidelines. Regarding cross-validation for infliximab (n=70), the mean bias with LC-MS/MS assay was approximately threefold higher with the commercial ELISA assay compared with thein-house ELISA (-6.1 vs -1.8μg/ml, respectively). The mean bias between theLC-MS/MS assay and in-house ELISA was -1.2μg/ml for adalimumab (n=35). Conclusion: The LC-MS/MS method is a powerful alternative to immunoassays to monitor concentrations of infliximab and adalimumab.

Characterization of recombinant Ybgf protein for the detection of Coxiella antibodies in ruminants.

Q fever remains a One Health problem, posing a zoonotic threat and causing significant economic losses to the livestock industry. The advancement of detection tools is critical to the effective control of infection. In humans, laboratory investigations depend largely on the immunofluorescence assay, considered the gold standard. In contrast, serologic tools routinely used for veterinary screening have several gaps, resulting in interpretations that are frequently misleading. We investigated the potential application of recombinant Ybgf antigen (r-Ybgf), a periplasmic protein described as one of the most immunodominant antigens in humans, in an indirect ELISA. Following successful expression in the prokaryotic system and the preliminary evaluation of immunoreactivity in western blot, we used r-Ybgf to develop an in-house ELISA using serum samples from sheep, goats, and cattle, which were tested in parallel with an Idexx ELISA kit. The results obtained with the 2 tests were compared, and r-Ybgf performed favorably, with 81.8% sensitivity and 90.1% specificity and substantial agreement, as revealed by receiver operating characteristic analysis. Moreover, we evaluated the serologic response against phase I (PhI) and phase II (PhII) antigens, and r-Ybgf antigen induced by vaccination, using phase-specific ELISAs. The dynamics of antibody response showed a significant increase in reactivity against PhI and PhII, but not against r-Ybgf, antigens. This property may be very useful given the absence of a protocol for the differentiation of infected from vaccinated animals.

ELISA, protein immunoprecipitation and line blot assays for anti-TIF1-gamma autoantibody detection in cancer-associated dermatomyositis.

Anti-TIF1-gamma autoantibodies can be detected with immunoprecipitation (IP), line blot (LB) and ELISA. We compared assay performance in patients with DM and the potential of these assays to detect anti-TIF1-gamma positive cancer-associated DM (CADM). We included sera from 131 patients with DM followed at Karolinska University Hospital, Stockholm, Sweden and 82 healthy controls. Serum samples taken at DM diagnosis were tested for anti-TIF1-gamma autoantibodies with IP, two ELISAs (in-house and commercial) and LB. Cancer diagnosis and dates were obtained from the Swedish national cancer register. CADM was defined as a malignancy that developed within 3 years of DM diagnosis. Anti-TIF1-gamma autoantibodies were detected in 19/101 (18.8%), 15/113 (13.2%), 34/131 (26%) and 45/131 (34.4%) of the patients with IP, LB, in-house and commercial ELISA, respectively. The anti-TIF1-gamma results from the in-house ELISA were confirmed with IP in 93 of 101 (92%) cases, κ = 0.76, with a commercial ELISA in 110 of 131 (84%) cases, κ = 0.63, and with LB in 101 of 113 (89.3%) cases, κ = 0.67. Anti-TIF1-gamma results with IP were confirmed with LB in 85 of 92 (92.4%) cases, κ = 0.73. For detecting CADM, the anti-TIF1-gamma in-house ELISA had a sensitivity of 58% and specificity of 86%, the commercial ELISA had a sensitivity of 63% and specificity of 82%, IP had a sensitivity of 52% and specificity of 92%, LB had a sensitivity of 40% and specificity of 96%. The two anti-TIF1-gamma ELISA assays had advantages both for autoantibody detection and to identify anti-TIF1-gamma-positive CADM.

Assessment of specific human antibodies against SARS-CoV-2 receptor binding domain by rapid in-house ELISA.

The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. The urgent need to control the spread of the virus and rapid application of the suitable health measures raised the importance of available, rapid, and accurate diagnostic approaches. The purpose of this study is to describe a rapid in-house optimized ELISA based on the expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in a prokaryotic system. We show the expression of the 30 kDa recombinant SARS-CoV-2 RBD-6×His in four different E. coli strains (at 28∘C using 0.25mM IPTG) including the expression strain E. coli BL21 (DE3) Rosetta Gami. SARS-CoV-2 rRBD-6×His protein was purified, refolded, and used as an antigen coat to assess antibody response in human sera against SARS-CoV-2 infection. The assessment was carried out using a total of 155 human sero-positive and negative SARS-CoV-2 antibodies. The ELISA showed 69.5% sensitivity, 88% specificity, 78.5% agreement, a positive predictive value (PPV) of 92.3%, and a negative predictive value of 56.5%. Moreover, the optical density (OD) values of positive samples significantly correlated with the commercial kit titers. Specific human antibodies against SARS-CoV-2 spike protein were detected by rapid in-house ELISA in sera of human COVID-19-infected patients. The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications, particularly in developing countries. Future studies are planned for the use of the generated SARS-CoV-2 rRBD-6×His protein in vaccine development and other diagnostic applications.

Decadal Change in Seroprevalence of Chikungunya Virus Infection in Pune City, India.

Chikungunya virus (CHIKV) is an arthropod-borne virus capable of causing large outbreaks. We aimed to determine the decadal change in the extent of chikungunya virus infection from 2009 to 2019. We implemented a prospective cross-sectional survey in Pune City using a 30-cluster approach with probability-proportion-to-size (PPS) sampling, with blood samples collected from 1654 participants in early 2019. The study also included an additional 799 blood samples from an earlier serosurvey in late 2009. The samples were tested by an in-house anti-CHIKV IgG ELISA assay. The overall seroprevalence in 2019 was 53.2% (95% CI 50.7–55.6) as against 8.5% (95% CI 6.5–10.4) in 2009. A fivefold increase in seroprevalence was observed in a decade (p < 0.00001). The seroprevalence increased significantly with age; however, it did not differ between genders. Modeling of age-stratified seroprevalence data from 2019 coincided with a recent outbreak in 2016 followed by the low-level circulation. The mean estimated force of infection during the outbreak was 35.8% (95% CI 2.9–41.2), and it was 1.2% after the outbreak. To conclude, the study reports a fivefold increase in the seroprevalence of chikungunya infection over a decade in Pune City. The modeling approach considering intermittent outbreaks with continuous low-level circulation was a better fit and coincided with a recent outbreak reported in 2016. Community engagement and effective vector control measures are needed to avert future chikungunya outbreaks.

Rapid Generation of In-House Serological Assays Is Comparable to Commercial Kits Critical for Early Response to Pandemics: A Case With SARS-CoV-2.

IntroductionAccurate and sensitive measurement of antibodies is critical to assess the prevalence of infection, especially asymptomatic infection, and to analyze the immune response to vaccination during outbreaks and pandemics. A broad variety of commercial and in-house serological assays are available to cater to different laboratory requirements; however direct comparison is necessary to understand utility.Materials and MethodsWe investigate the performance of six serological methods against SARS-CoV-2 to determine the antibody profile of 250 serum samples, including 234 RT-PCR-confirmed SARS-CoV-2 cases, the majority with asymptomatic presentation (87.2%) at 1–51 days post laboratory diagnosis. First, we compare to the performance of two in-house antibody assays: (i) an in-house IgG ELISA, utilizing UV-inactivated virus, and (ii) a live-virus neutralization assay (PRNT) using the same Cambodian isolate as the ELISA. In-house assays are then compared to standardized commercial anti-SARS-CoV-2 electrochemiluminescence immunoassays (Elecsys ECLIAs, Roche Diagnostics; targeting anti-N and anti-S antibodies) along with a flow cytometry based assay (FACS) that measures IgM and IgG against spike (S) protein and a multiplex microsphere-based immunoassay (MIA) determining the antibodies against various spike and nucleoprotein (N) antigens of SARS-CoV-2 and other coronaviruses (SARS-CoV-1, MERS-CoV, hCoVs 229E, NL63, HKU1).ResultsOverall, specificity of assays was 100%, except for the anti-S IgM flow cytometry based assay (96.2%), and the in-house IgG ELISA (94.2%). Sensitivity ranged from 97.3% for the anti-S ECLIA down to 76.3% for the anti-S IgG flow cytometry based assay. PRNT and in-house IgG ELISA performed similarly well when compared to the commercial ECLIA: sensitivity of ELISA and PRNT was 94.7 and 91.1%, respectively, compared to S- and N-targeting ECLIA with 97.3 and 96.8%, respectively. The MIA revealed cross-reactivity of antibodies from SARS-CoV-2-infected patients to the nucleocapsid of SARS-CoV-1, and the spike S1 domain of HKU1.ConclusionIn-house serological assays, especially ELISA and PRNT, perform similarly to commercial assays, a critical factor in pandemic response. Selection of suitable immunoassays should be made based on available resources and diagnostic needs.

Effectiveness of in-house developed Sandwich ELISA for antigen detection of tubercular antigen in resource constraint setting

Background: Around 26% of World’s tuberculosis (TB) cases are in India. In resource constrained settings, it is difficult to use diagnostic tests such as nucleic acid amplification technique and mycobacterial culture due to their complexity and expensiveness. Developing an assay using polyclonal antibody may help to solve this problem. Aims and Objectives: On the basis of this, we worked to detect tubercular antigen by developing Sandwich ELISA from the serum of TB patients. Materials and Methods: For this study, 50 cases, 50 disease controls, and 30 healthy control subjects were taken. Commercially available reagents were used for the development of Sandwich ELISA. The data were statistically analyzed with the help of software SPSS version 16.0. Results: Our effectively developed test had 82% area under the ROC curve. About 81% (n=35) patients of pulmonary TB cases with non-HIV infection detected by our Sandwich ELISA and 57% cases with the coinfection of HIV-TB. Developed test had 70.91% positive predictive value and 75.56% negative predictive value. Conclusion: We successfully developed Sandwich ELISA for tubercular antigen detection and to increase the specificity of the Sandwich ELISA, as per the WHO guidelines, further work is needed for the detection of tubercular antigen.

Development of an ELISA to distinguish between foot-and-mouth disease virus infected and vaccinated animals utilising the viral non-structural protein 3ABC.

Introduction. Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of livestock and is endemic in much of Asia, including Pakistan. Vaccination is used to control disease outbreaks and sensitive diagnostic methods which can differentiate infected animals from vaccinated animals (DIVA) are essential for monitoring the effectiveness of disease control programmes. Tests based on the detection of the non-structural protein (NSP) 3ABC are reliable indicators of virus replication in infected and vaccinated populations.Hypothesis/Gap statement. Diagnosis of FMD is expensive using commercial ELISA kits, yet is essential for controlling this economically-important disease.Aim. The development of a low-cost diagnostic ELISA, using protein made in Escherichia coli.Methodology. In this study, the viral precursor protein 3ABC (r3ABC) was expressed in E. coli, solubilised using detergent and purified using nickel affinity chromatography. The fusion protein contained an attenuating mutation in the protease and a SUMO tag. It was characterised by immunoblotting and immunoprecipitation, which revealed antigenicity against virus-specific polyclonal sera. Using r3ABC, an indirect ELISA was developed and evaluated using field sera from healthy/naïve, vaccinated and infected animals.Results. The diagnostic sensitivity and specificity of the r3ABC in-house ELISA were 95.3 and 96.3% respectively. The ELISA was validated through comparison with the commercially available ID Screen FMD NSP competition kit. Results indicated good concordance rates on tested samples and high agreement between the two tests.Conclusion. The ELISA described here can effectively differentiate between infected and vaccinated animals and represents an important low cost tool for sero-surveillance and control of FMD in endemic settings.

Validation of an In-House ELISA Method in the Diagnosis of Cutaneous Leishmaniasis Caused by Leishmania donovani in Hambantota District, Sri Lanka.

Clinical diagnosis has become a challenge amidst a surge of cutaneous leishmaniasis in Southern Sri Lanka. The routine diagnostic method, slit-skin smear (SSS), has variable sensitivity, leading to undiagnosed cases. Improved diagnostics are urgently needed. We assessed a new in-house ELISA method for its diagnostic capabilities against ITS-1 nested PCR (gold standard—Gs). A cohort of 190 clinical CL cases was examined by SSS microscopy, anti-rKRP42 IgG ELISA (serum- and urine-based), and rK39-Immunochromatographic strip test. Validation was done using non-endemic sera, and cutoffs were developed using the receiver operating curve. The sensitivity of SSS for case detection was 77.9% (authors) and 76.3% (technicians). ELISA vs. Gs demonstrated sensitivity (Sn) = 94.4%; specificity (Sp) = 50.0%; positive predictive value (PPV) = 97.1%; negative predictive value (NPV) = 33.3%; Kappa agreement (Kp) = 0.39/p < 0.01. Comparison of the combination method (SSS by technicians and ELISA) vs. Gs showed: Sn = 98.9%; Sp = 30.0; PPV = 96.2; NPV 60.0%; Kp = 0.378/p < 0.01. All methods performed better compared to SSS (29.4%) where the clinical diagnosis was doubtful (PCR = 94.15%; serum ELISA = 88.2%; combination = 94.1%; p < 0.01 for all). High serum anti-rKRP42 titers were seen in those with multiple lesions. Anti-rKRP42 urine ELISA was suboptimal as a diagnostic test. A 9% rate of positivity was seen for rk39-ICT, and positives recorded high anti-rKRP42 titers. The diagnostic accuracy can be increased above the level of the Gs by combining SSS and ELISA. Advanced studies are required to understand the association between rk39-ICT positivity and high anti-rKRP42 titers.

COVID-19 in Tunisia (North Africa): Seroprevalence of SARS-CoV-2 in the General Population of the Capital City Tunis.

Seroprevalence studies are essential to get an accurate estimate of the actual SARS-CoV-2 diffusion within populations. We report on the findings of the first serosurvey conducted in Tunis prior to the implementation of mass vaccination and analyzed factors associated with seropositivity. A household cross sectional survey was conducted (March–April 2021) in Tunis, spanning the end of the second wave and the beginning of the third wave of COVID-19. SARS-CoV-2 specific immunoglobulin G (IgG) antibodies to the spike (S-RBD) or the nucleocapsid (N) proteins were detected by in-house ELISA tests. The survey included 1676 individuals from 431 households. The mean age and sex ratio were 43.3 ± 20.9 years and 0.6, respectively. The weighted seroprevalence of anti-N and/or anti-S-RBD IgG antibodies was equal to 38.0% (34.6–41.5). In multivariate analysis, age under 10, no tobacco use, previous diagnosis of COVID-19, a history of COVID-19 related symptoms and contact with a COVID-19 case within the household, were independently associated with higher SARS-CoV-2 seroprevalence. More than one third of people living in Tunis obtained antibodies to SARS-CoV-2. Further studies are needed to monitor changes in these figures as Tunisian population is confronted to the subsequent epidemic waves and to guide the vaccine strategy.

Anti-HMGB1 antibody is a potential characteristic autoantibody for Sj\xf6gren's syndrome

Sjögren's syndrome (SS) is a common chronic inflammatory autoimmune disease that affects about 0.33–0.77% population in China. The positive for antinuclear antibodies (ANA) is one of the key features of SS, which shows a nuclear fine speckled (AC-4) pattern in an indirect immunofluorescent antibody test (IIFT). About 70% of ANA-positive SS patients have detectable anti-SS-A and/or SS-B antibodies, which indicates that other autoantibodies may present in SS patients. The anti-HMGB1 antibodies in 93 SS patients and 96 healthy controls were investigated with in-house developed ELISA and immunoblotting, and the locations of HMGB1 and fluorescent pattern of anti-HMGB1 antibody were investigated with IIFT. The contribution of anti-HMGB1 antibody in ANA-IF was evaluated with Cas9-induce HMGB1 knockout B16 cells. The anti-HMGB1 antibody level is higher in SS patients (9.96 ± 5.55 RU/ml) than in healthy controls (4.9 ± 1.4 RU/ml). With ROC curve analysis, when taking 8 RU/ml as the cutoff value, the sensitivity, specificity, and the area under the curve were 64.5%, 96.9%, and 0.83, respectively. A total of 18 patients (20.7%) with nuclear fine speckled (AC-4) pattern in ANA-IF test were anti-HMGB1 antibody positive only. With commercial antibody, anti-HMGB1 antibody showed the same nuclear fine speckled (AC-4) pattern. The serum from ANA-IF (+), SS-A (−), and SS-B (−) SS patients showed nuclear fine speckled (AC-4) pattern in wildtype B16 cells, but no fluorescence in HMGB1 knockout B16 cells. Anti-HMGB1 antibody may be one of the characteristic autoantibodies of SS in addition to anti-SS-A and SS-B. The detection of anti-HMGB1 antibody can provide more laboratory evidence for clinical diagnosis of SS.

Smartphone multiplex microcapillary diagnostics using Cygnus: Development andevaluation of rapid serotype-specific NS1 detection with dengue patient samples.

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device–termed Cygnus–with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-μl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58–0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63–0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.