Development of Monoclonal Antibody Based IgG and IgM ELISA for Diagnosis of Severe Fever with Thrombocytopenia Syndrome Virus Infection

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that possesses a great threat to human health because of the high fatality rate. Method: To develop sensitive and specific sero-diagnosis systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patient serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. Results: The MAb based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA with a sensitivity and specificity of 100%. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while was more sensitive comparing with the indirect IgM ELISA. Conclusions: The MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Comparison of IgA, IgG and neutralizing antibody responses following immunization with Moderna, BioNTech, AstraZeneca, Sputnik-V, Johnson and Johnson, and Sinopharm’s COVID-19 vaccines.

BACKGROUNDSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the resulting coronavirus disease 2019 (COVID-19) have afflicted millions of people in a worldwide pandemic. Several vaccines have been developed to prevent infection and illness. However, the safety and efficacy of most of the vaccines currently available are still being questioned by part of the public opinion. Even if vaccine-resistant individuals represent a minority in most countries, their hesitancy is sufficient to delay the highly desired ‘herd-immunity threshold.’ METHODSIn an ongoing multinational trial, we collected blood samples from 365 adults, 18 years of age or older, vaccinated with mRNA vaccines (Moderna, BioNTech), viral DNA-vectored vaccines (AstraZeneca, Sputnik-V, and Johnson and Johnson), or the attenuated virus vaccine from Sinopharm. Out of the 365 vaccinated individuals included in the study, 41 received two doses of Moderna Biotech's Spikevax, 92 received two doses of BioNTech’s Comirnaty, 52 were vaccinated with two doses of Oxford-AstraZeneca’s Vaxzevria, 34 received one dose of Johnson and Johnson’s Jansen, 35 two doses of Gamaleja’s Sputnik-V and 28 two doses of Sinopharm’s BBIBP-CorV. In addition, 40 received a prime dose of AstraZeneca followed by BioNTech as a booster, whereas 43 received Moderna’s vaccine as a booster after a prime dose of AstraZeneca. After collecting reactogenicity data, the expression of S-Protein binding IgG and IgA were analyzed before and after full vaccination in each group using an automated sandwich ELISA system. In addition, the neutralizing capacity of sera from individuals from all groups was investigated using an ACE-2-RBD neutralizing assay. RESULTSThe main side effects reported included short-term mild-to-moderate pain at the injection site, fatigue, and headache. More severe side effects were reported by vaccinees in the Moderna (10%), AstraZeneca (11%), Johnson and Johnson (5.9%), and Sputnik-V (7.2%) groups. No severe adverse reaction was reported in the BioNTech group, and the Sinopharm vaccinees presented the mildest reactogenicity profile, with 93.8% of the vaccinees declaring no adverse reactions. Moderna’s vaccine induced the highest amounts of SARS-CoV-2 specific IgG, IgA, and serum neutralization activity compared to the other groups. In contrast, people vaccinated with Sinopharm and Johnson and Johnson’s vaccines have the lowest SARS-CoV-2-specific antibody titers. Vaccinees from the Johnson and Johnson group presented significant levels of SARS-CoV-2 specific IgA but not IgG compared to the controls before vaccination. In the Sinopharm group, neither IgG nor IgA expression was significant. In addition, sera from vaccinees of these two groups presented no significant neutralization potential compared to the unvaccinated controls. Significant negative correlations between age and SARS-CoV-2- specific IgG expression were observed in the Johnson and Johnson (r=-0.4414, p=0.009) and Sinopharm (r=-0.6108, p=0.0006) groups. Remarkably, younger vaccinees (18-60 years old) in both Sinopharm and Johnson and Johnson groups produced substantial SARS-CoV-2 specific antibody expression and exhibited significant neutralization potential. While the AstraZeneca vaccine alone induced moderate IgG and IgA expression, the combination with Moderna or BioNTech mRNA vaccines induced higher antibody levels than a double dose of AstraZeneca and similar IgG expression and neutralization potential compared to Moderna, or BioNTech used alone. CONCLUSIONThe results suggest that the Moderna vaccine is the most immunogenic after two doses. AstraZeneca and Sputnik-V presented moderate but significant antibody expression and virus neutralizing properties. Low antibody and neutralization potential was observed in the elderly vaccinated with Sinopharm or Johnson and Johnson vaccines. The data also suggest that heterologous vaccination strategies combining the AstraZeneca DNA vectored vaccines and mRNA vaccines Moderna or BioNTech booster induced more robust antibody and virus neutralization potential compared to their homologous counterparts.

IgG antibody titers against SARS-CoV-2 nucleocapsid protein correlate with the severity of COVID-19 patients

Background The 2019 novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) is a current worldwide threat for which the immunological features after infection need to be investigated. The aim of this study was to establish a highly sensitive and quantitative detection method for SARS-CoV-2 IgG antibody and to compare the antibody reaction difference in patients with different disease severity. Results Recombinant SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to establish an indirect IgG ELISA detection system. The sensitivity of the ELISA was 100% with a specificity of 96.8% and a 98.3% concordance when compared to a colloidal gold kit, in addition, the sensitivity of the ELISA was 100% with a specificity of 98.9% and a 99.4% concordance when compared to a SARS-CoV-2 spike S1 protein IgG antibody ELISA kit. The increased sensitivity resulted in a higher rate of IgG antibody detection for COVID-19 patients. Moreover, the quantitative detection can be conducted with a much higher serum dilution (1:400 vs 1:10, 1:400 vs 1:100). The antibody titers of 88 patients with differing COVID-19 severity at their early convalescence ranged from 800 to 102,400, and the geometric mean titer for severe and critical cases, moderate cases, asymptomatic and mild cases was 51,203, 20,912, and 9590 respectively. Conclusion The development of a highly sensitive ELISA system for the detection of SARS-CoV-2 IgG antibodies is described herein. This system enabled a quantitative study of rSARS-CoV-2-N IgG antibody titers in COVID-19 patients, the occurrence of higher IgG antibody titers were found to be correlated with more severe cases.

Point-of-Need Disposable ELISA System for COVID-19 Serology Testing

A disposable enzyme-linked immunosorbent assay (dELISA) device for ate-home or doctor’s office use was developed to detect SARS-CoV-2 antibodies. Serology testing for SARS-CoV-2 antibodies is currently run using well-plate ELISAs in centralized laboratories. However, the scale of serology testing needed for epidemiological and clinical screening studies will overwhelm existing clinical laboratory resources. Instead, a point-of-need device that can be used at home or in doctor’s offices for COVID-19 serology testing must be developed and is one of four target products prioritized by the World Health Organization. Lateral flow assays are common and easy to use, but lack the sensitivity needed to reliably detect SARS-CoV-2 antibodies in clinical samples. This work describes a disposable ELISA device that is as simple to use as a lateral flow assay, but as sensitive as a well-plate ELISA. The device utilizes capillary-driven flow channels made of transparency films and double-sided adhesive combined with paper pumps to drive flow. The geometry of the channels and storage pads enables automated sequential washing and reagent addition steps with two simple end-user steps. An enzyme label is used to produce a colorimetric signal instead of a nanoparticle label in order to amplify signal and increase sensitivity, while the integrated washing steps decrease false positives and increase reproducibility. Naked-eye detection can be used for qualitative results or a smartphone camera for quantitative analysis. The device can detect antibodies at 2.8 ng/mL from whole blood, which was very close the concentration of detectable target in a well-plate ELISA (1.2 ng/mL). In this study the dELISA system was used to detect SARS-CoV-2 antibodies, but we believe that the device represents a fundamental step forward in point-of-care technology that will enable sensitive detection of many other analytes outside of a centralized laboratory.

Assessment of the diagnostic accuracy and relevance of a novel ELISA system developed for seroepidemiologic surveys of Helicobacter pylori infection in African settings

Beside diagnostic uncertainties due to the lack of a perfect gold standard test for Helicobacter pylori infection, the diagnosis and the prevalence estimation for this infection encounter particular challenges in Africa including limited diagnostic tools and specific genetic background. We developed and evaluated the accuracy of an enzyme-linked immunosorbent assay (ELISA) system tailored for H. pylori genetics in Africa (HpAfr-ELISA). Strains belonging to main genetic populations infecting Africans were exploited as sources for whole-cell antigens to establish in-house the ELISA system. A phase II unmatched case-control study explored the diagnostic accuracy of the HpAfr-ELISA using a training set of samples collected from dyspeptic patients from Kinshasa, the Democratic Republic of Congo (DRC) who had been tested with invasive standard tests (i.e., histology, culture, and rapid urease test) in 2017. Then the assay was cross-validated through a community-based survey assessing the prevalence of H. pylori and associated factors in 425 adults from Mbujimayi, DRC in 2018. Bayesian inferences were used to deal with statistical uncertainties of estimates (true prevalence, sensitivity, and specificity) in the study population. At its optimal cut-off-value 20.2 U/mL, the assay achieved an estimated sensitivity of 97.6% (95% credible interval [95%CrI]: 89.2; 99.9%) and specificity of 90.5% (95%CrI: 78.6; 98.5). Consistent outcomes obtained at repeated tests attested the robustness of the assay (negative and positive agreements always > 70%). The true prevalence of H. pylori was estimated 53.8% [95%CrI: 42.8; 62.7%]. Increasing age (adjusted odds ratio [aOR] = 1.0 [95% confidence interval (CI): 1.0; 1.1]; p<0.001), overcrowding households (aOR = 3.2 [95%CI: 2.0; 5.1]; p<0.001), and non-optimal hand hygiene (aOR = 4.5 [95%CI: 2.0; 11.4]; p = 0.001) were independently associated with the H. pylori-seropositivity. The novel ELISA system has demonstrated good diagnostic accuracy and potential usefulness for management and mitigation strategies for H. pylori infection in African settings.

Different Profiles of Antibodies and Cytokines Were Found Between Severe and Moderate COVID-19 Patients

ObjectivesOur objective was to determine the antibody and cytokine profiles in different COVID-19 patients.MethodsCOVID-19 patients with different clinical classifications were enrolled in this study. The level of IgG antibodies, IgA, IgM, IgE, and IgG subclasses targeting N and S proteins were tested using ELISA. Neutralizing antibody titers were determined by using a toxin neutralization assay (TNA) with live SARS-CoV-2. The concentrations of 8 cytokines, including IL-2, IL-4, IL-6, IL-10, CCL2, CXCL10, IFN-γ, and TNF-α, were measured using the Protein Sample Ella-Simple ELISA system. The differences in antibodies and cytokines between severe and moderate patients were compared by t-tests or Mann-Whitney tests.ResultsA total of 79 COVID-19 patients, including 49 moderate patients and 30 severe patients, were enrolled. Compared with those in moderate patients, neutralizing antibody and IgG-S antibody titers in severe patients were significantly higher. The concentration of IgG-N antibody was significantly higher than that of IgG-S antibody in COVID-19 patients. There was a significant difference in the distribution of IgG subclass antibodies between moderate patients and severe patients. The positive ratio of anti-S protein IgG3 is significantly more than anti-N protein IgG3, while the anti-S protein IgG4 positive rate is significantly less than the anti-N protein IgG4 positive rate. IL-2 was lower in COVID-19 patients than in healthy individuals, while IL-4, IL-6, CCL2, IFN-γ, and TNF-α were higher in COVID-19 patients than in healthy individuals. IL-6 was significantly higher in severe patients than in moderate patients. The antibody level of anti-S protein was positively correlated with the titer of neutralizing antibody, but there was no relationship between cytokines and neutralizing antibody.ConclusionsOur findings show the severe COVID-19 patients’ antibody levels were stronger than those of moderate patients, and a cytokine storm is associated with COVID-19 severity. There was a difference in immunoglobulin type between anti-S protein antibodies and anti-N protein antibodies in COVID-19 patients. And clarified the value of the profile in critical prevention.

Expression and Purification of Botulinum Neurotoxin Type ‘A’ Toxin Domain in E. coli and its Application in Detection

Botulism is a neuroparalytic disease caused by botulinum toxins type A-G. It has been listed as category ‘A’ biowarfare agent by CDC due to its extreme toxicity.. In the present study, we developed a simple method for the pro-duction and purification of recombinant toxin domain of BoNT type ‘A’ from Escherichia coli and established its application in BoNT detection. The BoNT/A LC gene was cloned in pET28b+ vector and expressed in E.coli. The recom-binant BoNT/A LC protein expression was achieved at 25°C with the induc-tion of 0.5mM IPTG at 16 h. The recombinant protein was purified under native conditions with more than 98% purity using simple affinity chroma-tography and confirmed by Mass spectrometry and Western blot analysis. Generated polyclonal antibodies and the toxin domain elicited a strong im-mune response in mice and rabbit. Optimized the Sandwich ELISA system resulted with the sensitivity of 7.5 ng/ml for the detection of BoNT/A. The limit of detection (LOD) in different food matrices were studied through spik-ing studies using rBoNT/A LC protein as antigen and achieved a detection limit ranging from7.5 ng/ml to 250 ng/ml. The developed method has the potential for the industrial production towards its application in detection, development of candidate vaccine molecule and production of neutralizing antibodies. Current study explored for detection of BoNT/A.

Periostin: A Biomarker Useful in Treating Patients with Progressive Fibrosing Interstitial Lung Diseases

Background: The concept of progressive fibrosing interstitial lung diseases (PF-ILD) has been recently proposed to describe a progressive phenotype in fibrosing ILDs, separate from the classical differential diagnosis of ILDs. However, we still do not know enough about how we can differentiate PF-ILD patients from non–PF-ILD patients in advance or how we can predict decline of lung function in PF-ILD patients. Periostin is a matricellular protein playing a critical role in the pathogenesis of pulmonary fibrosis significantly high in idiopathic pulmonary fibrosis patients. Methods: In this study, we applied three periostin ELISA systems—an original ELISA system recognizing total periostin, an ELISA system recognizing only the monomeric periostin, and a modified ELISA system that can measure periostin independently of the IgA complex established in this study—to serum from idiopathic interstitial pneumonia (IIP) patients to address whether periostin can differentiate PF-ILD from non–PF-ILD patients in advance, whether periostin can predict the decline of lung function in PF-ILD patients, and which ELISA system for periostin is the most suitable for these purposes.Results: We established the modified ELISA system using SS16A recognizing the R3 domain instead of SS18A recognizing the R1 domain as the primary mAb that can measure periostin independently of the IgA complex. All periostin values found by the three systems were significantly higher in PF-ILD patients than in healthy subjects in addition to SP-D and KL-6, whereas all the periostin values, but not SP-D or KL-6, could differentiate PF-ILD patients from patients with IIP other than PF-ILD, in the order of the monomer system, the modified system, and the original system. Moreover, the periostin values by all three systems, but not SP-D or KL-6, showed significant correlations with decline of either %VC or DL, CO, in order of the monomer system, the modified system, and the original system.Conclusions: Serum periostin, but not SP-D and KL-6, differentiates PF-ILD from non–PF-ILD and predicts decline of lung function in PF-ILD patients. The monomer periostin ELISA system is the best at doing so. These results demonstrate that periostin, particularly the monomeric form, is useful in treating PF-ILD patients.

Identification of small compounds regulating the secretion of extracellular vesicles via a TIM4-affinity ELISA

Extracellular vesicles (EVs) are secreted from most cells and play important roles in cell–cell communication by transporting proteins, lipids, and nucleic acids. As the involvement of EVs in diseases has become apparent, druggable regulators of EV secretion are required. However, the lack of a highly sensitive EV detection system has made the development of EV regulators difficult. We developed an ELISA system using a high-affinity phosphatidylserine-binder TIM4 to capture EVs and screened a 1567-compound library. Consequently, we identified one inhibitor and three activators of EV secretion in a variety of cells. The inhibitor, apoptosis activator 2, suppressed EV secretion via a different mechanism and had a broader cellular specificity than GW4869. Moreover, the three activators, namely cucurbitacin B, gossypol, and obatoclax, had broad cellular specificity, including HEK293T cells and human mesenchymal stem cells (hMSCs). In vitro bioactivity assays revealed that some regulators control EV secretion from glioblastoma and hMSCs, which induces angiogenesis and protects cardiomyocytes against apoptosis, respectively. In conclusion, we developed a high-throughput method to detect EVs with high sensitivity and versatility, and identified four compounds that can regulate the bioactivity of EVs.

Identification of an Alternative Glycyrrhizin Metabolite Causing Liquorice-Induced Pseudohyperaldosteronism and the Development of ELISA System to Detect the Predictive Biomarker

Liquorice is usually used as crude drug in traditional Japanese Kampo medicine and traditional Chinese medicine. Liquorice-containing glycyrrhizin (GL) can cause pseudohyperaldosteronism as a side effect. Previously, we identified 18β-glycyrrhetyl-3-O-sulfate (3) as a GL metabolite in Eisai hyperbilirubinuria rats (EHBRs) with the dysfunction of multidrug resistance-related protein (Mrp2). We speculated that 3 was associated with the onset of liquorice-induced pseudohyperaldosteronism, because it was mainly detected in serum of patients with suspected to have this condition. However, it is predicted that other metabolites might exist in the urine of EHBRs orally treated with glycyrrhetinic acid (GA). We explored other metabolites in the urine of EHBRs, and investigated the pharmacokinetic profiles of the new metabolite in EHBRs and normal Sprague-Dawley rats. We further analyzed the serum concentrations of the new metabolite in the patients of pseudohyperaldosteronism. Finally, we developed the analyzing method of these metabolites as a preventive biomarker for the onset of pseudohyperaldosteronism using an enzyme-linked immunosorbent assay (ELISA). We isolated a new GL metabolite, 18β-glycyrrhetyl-3-O-sulfate-30-O-glucuronide (4). Compound 4 significantly inhibited rat type-2 11β-hydroxysteroid dehydrogenase (11β-HSD2) and was a substrate of both organic anion transporter (OAT) 1 and OAT3. Compound 4 was also detected in the serum of patients with suspected pseudohyperaldosteronism at an approximately 10-fold lower concentrations than 3, and these concentrations were positively correlated. Compound 4 showed a lower serum concentration and weaker inhibitory titer on 11β-HSD2 than 3. We developed an enzyme-linked immunosorbent assay system using an anti-18β-glycyrrhetyl-3-O-glucuronide (3MGA) monoclonal antibody to measure the serum concentration of 3 to facilitate the measurement of biomarkers to predict the onset of pseudohyperaldosteronism. Although we found 4 as the secondary candidate causative agent, 3 could be the main potent preventive biomarker of liquorice-induced pseudohyperaldosteronism. Compound 3 was detected in serum at a higher concentration than GA and 4, implying that 3 may be a pharmacologically active ingredient mediating not only the development of pseudohyperaldosteronism but anti-inflammatory effects in humans administered GL or other liquorice-containing preparations.