Assay For IgG Antibodies Research Articles

A Unique Presentation of Excitatory Catatonia Alongside AntiNmda Receptor Encephalitis: A Case Report

Introduction: Anti-NMDA receptor encephalitis, first described in 2007, is a prominent cause of autoimmune encephalitis, predominantly affecting females. Its clinical presentation includes a prodromal phase with flu-like symptoms, followed by psychosis, unresponsiveness, hyperkinesis, and recovery [1]. Catatonia can coexist sounds better than cooccur in patients with anti-NMDA receptor encephalitis, complicating diagnosis and effective treatment. Case Summary: This case report discusses a 41-year-old African American female with a history of seizure disorder, schizoaffective disorder with catatonia, developmental delay, Catatonia in itself is not the diagnosis as per dsm 5. We could use it as a specifier with other diagnosis, be it schizoaffective disorder or seizure disorder here. So we could mention as schizoaffective disorder with catatonia. and recent bereavement. Her symptoms included variable motor hyperactivity, alternating between restlessness and sedation, with an initial Busch Francis Rating Scale score of 18. Initially diagnosed with excitatory catatonia secondary to schizoaffective disorder and treated with benzodiazepines, her condition failed to improve, prompting further investigation. Despite additional treatment for a urinary tract infection and medication adjustment, which included adding valproic acid and amantadine, her condition remained uncontrolled. Subsequent lumbar puncture revealed inflammatory markers, and her anti-NMDA receptor IgG antibody assay was positive at 1:1280, confirming the diagnosis of anti-NMDA receptor encephalitis. Treatment was escalated to include methylprednisolone and intravenous immunoglobulin, alongside six sessions of ECT for management of her catatonia. The patient showed significant improvement, achieving functional recovery and independence in daily activities before discharge. Conclusion: This case emphasizes the importance of considering anti-NMDA receptor encephalitis in patients with unexplained, refractory neuropsychiatric symptoms, such as catatonia, and highlights the value of a multidisciplinary approach in managing and rehabilitating affected individuals. Prompt diagnosis and treatment are crucial to improving outcomes and reducing the risk of persistent cognitive deficits.

Diagnostic value of IgG antibody and stool antigen tests for chronic Helicobacter pylori infections in Ibb Governorate, Yemen

The stool antigen test (SAT) and the serum Helicobacter pylori (H. pylori) IgG antibody assays exhibit significant utility in the clinical diagnosis of H. pylori infection and in distinguishing between acute and chronic infections. The main objective of the current study was to identify the diagnostic value of serum H. pylori IgG antibody and SAT in the detection of H. pylori infections among chronic H. pylori-infected patients residing in Ibb Governorate, Yemen. 200 patients with H. pylori infection, confirmed through positive results in the serum immunochromatographic antibody test, were selected for H. pylori infection confirmation using serum H. pylori IgG antibodies and SAT across diverse hospitals, gastroenterology, and Hepatology clinics in Ibb Governorate. After the selection of patients, blood and stool specimens were obtained from all participants and underwent analysis via the Statistical Package for the Social Sciences (SPSS). The prevalence of H. pylori infection demonstrated variability based on the confirmatory tests, with rates of 54% for SAT and 78.5% for serum H. pylori IgG antibody, contrasting with a 100% prevalence observed in the screening serum immunochromatographic antibody test. Clinically, the study categorized H. pylori infections into four stages, whereby a significant proportion of patients (40.5%) exhibited positivity for both serum H. pylori IgG antibody and SAT, indicative of active chronic infections. The majority of positive cases only manifested serum H. pylori IgG antibody presence (chronic infections) at 38%, whereas 13.5% exclusively tested positive for SAT, corresponding to acute infections. Moreover, 88% of patients did not have either serum H. pylori IgG antibody or SAT (absence of infections) during confirmatory tests. Noteworthy is the study's approach employing multiple tests for H. pylori infection detection, focusing predominantly on chronic infections-prevailing types caused by H. pylori. The results revealed a significant association between serum levels of H. pylori IgG antibody and SAT results with the presence of diverse gastrointestinal symptoms among patients, which increased with long H. pylori infection durations.

Epidemiology and enzyme-linked immunosorbent assay of Leptospira spp. in humans in the Republic of Guinea

Introduction: Leptospirosis is a zooanthroponosis caused by spirochete bacteria called leptospire of the genus Leptospira. Objective: To contribute to the knowledge and circulation of the Leptospira germ in humans in the Republic of Guinea. Methods: This prospective and descriptive cross-sectional study lasted 18 months, from June 2019 to December 2020. Results: Of 2346 human blood samples analysed in the laboratory using the enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against leptospirosis, the largest number of samples was taken in Lower Guinea (31.58%), followed by Forest Guinea (27.66%), Middle Guinea (20.97%) and Upper Guinea (19.77%). More samples were taken from females (50.89%) than males (49.10%), for a sex ratio of 1.03 in favour of females. In terms of natural regions, Basse Guinée recorded the highest number of female samples (36.85%). On the other hand, the highest number of male samples was observed in Guinée Forestière with 28.55%. The results of anti-leptospira class G immunoglobulin tests identified the germ in 93 patients, representing a prevalence of 4%. The average age of the patients was 45, with extremes ranging from 10 to 90 years. Of the 93 positive cases, the prevalence of Leptospira infection was highest in subjects aged 31 to 40 (46.23%), followed by individuals in the 41 to 50 age group (18.27%). Prevalence was lower in the 11-20 age group (9.67%), the 51-60 age group (7.52%) and the 61-70 age group (3.22%). No cases of Leptospire infection were recorded in the 0-10, 71-80 and 81-90 age groups. Males accounted for 53.76% of Leptospire infections, compared with 46.23% for females, with a 95% confidence interval (CI) ranging from [13.08-20.82]. Lower Guinea recorded 25.80%, followed by Middle Guinea with 32.25%, Upper Guinea with 20.43% and Forest Guinea with a prevalence of 21.51%. The prevalence of Leptospira infection is higher among women in Middle Guinea (21.50%) than in the other natural regions. We also note that Lower Guinea recorded the highest rate of positivity among males at 18.28% compared with the other regions of Guinea. Farmers are the most affected by infections with 46.23%, followed by veterinarians with 25.80%, health workers with 18.28%, retired people with 4.30% and commercial workers with 3.22% (3/93). Students and workers were the least represented in this study, with 1 case each, representing a prevalence of 1.07%. Conclusion: The results obtained prove that the leptospirosis pathogen does exist in the Republic of Guinea in the entire human population.

Diagnostic probability classification in suspected borreliosis by a novel Borrelia C6-peptide IgG1- subclass antibody test.

The tick-borne multisystemic infection caused by Borrelia burgdorferi sensu lato, Lyme borreliosis, or Lyme disease, occurring in temperate regions of the northern hemisphere, continues to spread geographically with the expanding tick population. Despite the rising perceived risk of infection in the population, the clinical diagnosis of Borrelia infection is not always obvious and the most important laboratory test, antibody detection, has limited accuracy in diagnosing active disease. According to international guidelines, the primary serology test, which has a high sensitivity-low specificity, should, be verified using a high specificity confirmation test to improve the specificity. However, this enhancement in specificity comes at the cost of lower sensitivity. This two-step procedure is often omitted in everyday clinical practice. An optimal primary test would be one where no secondary tests for confirmation would be necessary. In the present study, the performance of a novel assay for quantitating IgG1-subclass antibodies to Borrelia C6-peptide was compared to a commercial reference assay of total IgG and IgM antibodies to Borrelia C6-peptide in the setting of a high endemic area for borreliosis. A derivation study on a retrospective clinical material was performed to compare the performance parameters and assess the discriminatory properties of the assays, followed by a prospective validation study. The IgG1-antibody assay achieved comparable summary performance parameters to those of the reference assay. The sensitivity was almost 100% while the specificity was about 50%. In a high-endemic setting, characterized by high background seropositivity of about 50% and disease prevalence of approximately 10%, antibody tests are unable to rule-in active Borrelia infection. The rule-out assessment of the methods revealed that of 1000 patients, 7 - 54 with negative results based on the reference method could have an active Borrelia infection. Such uncertainty was not found for the index test and may help improve the risk classification of patients.

Riding the Omicron BA.5 Wave: Improved Humoral Response after Vaccination with Bivalent Omicron BA.4-5-Adapted mRNA SARS-CoV-2 Vaccine in Chronic Hemodialysis Patients.

Hemodialysis patients faced an excess morbidity and mortality during the COVID-19 pandemic. We evaluated the effect of second-generation mRNA vaccines against Omicron BA.4 and BA.5 variants of SARS-CoV-2 on humoral immunity. The study population comprised 66 adult hemodialysis patients who have encountered four SARS-CoV-2 antigen contacts through vaccination or infection. We assessed their humoral response using an anti-SARS-CoV-2 spike receptor binding domain IgG antibody assay (S-RBD-ab), measuring neutralizing antibodies against ancestral strain of SARS-CoV-2, Delta, and Omicron in a surrogate virus neutralization test (SVNT), and specifically against BA.5 in a plaque reduction neutralization test (PRNT) before and four weeks after vaccination with Comirnaty Original/Omicron BA.4-5. During the following six months, SARS-CoV-2 infections and symptom severity were documented. The bivalent mRNA vaccine led to a 7.6-fold increase in S-RBD-ab levels and an augmented inhibition of the Omicron variant in SVNT by 35% (median). Seroconversion in the Omicron BA.5-specific PRNT was attained by in 78.4% of previously negative patients (29/37). Levels of S-RBD-ab correlated with inhibition in the Omicron-specific SVNT and neutralization titers in the BA.5-PRNT. Eleven SARS-CoV-2 infections occurred in the six-month follow-up, none of which took a life-threatening course. The bivalent mRNA vaccine improved the SARS-CoV-2 virus variant-specific humoral immunity in chronic hemodialysis patients. Measurement of S-RBD-ab can be used in hemodialysis patients to estimate their humoral immunity status against Omicron BA.5.

Seroprevalence of IgG Antibodies Directed against Dengue, Chikungunya and West Nile Viruses and Associated Risk Factors in Madagascar, 2011 to 2013.

Arboviruses have been shown to circulate in Madagascar, including West Nile, dengue, and chikungunya viruses, though the extent of their circulation remains poorly documented. We estimated the seroprevalence of these three arboviruses in Madagascar and determined risk factors associated with seropositivity. Serum samples obtained from 1680 individuals surrounding the Sentinel Health Centers network in all regions of the country were analyzed using ELISA and hemagglutination inhibition assays for dengue, chikungunya, and West Nile viruses IgG antibodies, and multivariate logistic regression models were run. Overall, 6.5% [IC 95% 3.2-9.9] were seropositive for dengue virus, predominantly of Dengue serotype 1, 13.7% [IC 95% 6.5-20.9] for chikungunya virus, and 12.7% [IC 95% 9.0-16.5] for West Nile virus. There was no association with age, showing that dengue and chikungunya viruses were likely recently introduced. Eastern and Northern parts were more affected by dengue and chikungunya viruses, while West Nile virus seemed to circulate in all parts of the country. Dengue and chikungunya seropositivity were notably associated with high levels of vegetation, as well as frequent work in the forest, and West Nile seropositivity with the presence of cultivated areas, as well as standard of living. This analysis gives a new insight into arboviruses circulation and transmission patterns in Madagascar.

Development of a cost-effective quantitative in-house ELISA assay for screening anti-S1 IgG antibodies targeting SARS-CoV-2.

The RBD, S, and N proteins, the three main antigens of the SARS-CoV-2 virus, activate the host immune system and cause the formation of IgM and IgG antibodies. While IgM indicates an early, acute infection stage, IgG shows a past infection or persistent sickness. This study used an indirect ELISA assay that targets the S1 subunit of the SARS-CoV-2 S protein to create an in-house, qualitative serological test specific to COVID-19. A total of 60 serum samples were examined using ELISA for anti-SARS-CoV-2 IgG, and 50 of those results were positive. An additional 20 samples were taken from cases that occurred before the pandemic. For the in-house ELISA assay, a plasmid containing the gene coding for the S1 subunit was transformed into E. coli DH5ɑ bacterial cells and the protein was synthesized and purified. The purified protein was utilized to coat the ELISA plate, which was subsequently used to assess the levels of IgG among individuals with SARS-CoV-2 infection. The study found a significant association (p-value=0.01) between the in-house and the commercial anti-S1 subunit IgG antibodies kits. The in-house ELISA responded well, with a sensitivity and specificity of 75.0% and 88.89%, respectively. Furthermore, a library of SARS-CoV-2 recombinant S1 subunits was created by competent bacteria and may be employed for various tasks, such as creating diagnostic tools and scientific investigation. Overall, the in-house anti-SARS-CoV-2 human IgG-ELISA proved to be sensitive and specific for identifying IgG antibodies in patients exposed to SARS-CoV-2.

The incidence of infection in tumor and eye fluid system, and specific humoral immunity to herpes viruses in patients with uveal melanoma

Studies aimed at a direct research of human herpes viruses (HHVs) in the tumor material and eye media have not been carried out so far. Research goal to establish the frequency of detection HHVs DNA in the biomaterial of the eye and blood and to assess the specific humoral immunity to the causative agents of herpes virus infections in patients with uveal melanoma. 38 patients with the uveal tract tumor were examined for the presence of DNA of HHV types 1 and 2 (HSV-1, 2), Cytomegalovirus (CMV), Varicella Zoster virus (VZV), EpsteinBarr virus (EBV) and herpes viruses 6 and 8 types (HHV-6, HHV-8) in tumor tissue, vitreous body, aqueous humour and blood plasma by real-time polymerase chain reaction; blood serum was studied by enzyme-linked immunosorbent assay (ELISA) for IgG and IgM antibodies to HHVs. EBV DNA was present in tumor tissue in 20.6% of cases, in vitreous body in 4.2%, in blood plasma in 2.7%, and was not found in aqueous humor. Ig G antibodies to HSV-1, 2 and CMV were detected in 97.3% of cases, VZV 94.6%, HHV-6 32.4%, antibodies to HHV-8 were not detected. 20 patients (55.6%) had reactivation of chronic HSV-1, 2 infection, and 14 (38.9%) patients had reactivation of CMV infection. Markers of chronic EBV infection were found in all patients, its atypical reactivation was observed in 2 cases (5.4%). Our findings suggest the possible participation of EBV in the oncogenesis of the uveal tract and emphasize the need for further in-depth study of this problem.

Comprehensive Comparison of Seven SARS-CoV-2-Specific Surrogate Virus Neutralization and Anti-Spike IgG Antibody Assays Using a Live-Virus Neutralization Assay as a Reference.

Neutralizing antibodies (nAbs) are considered a valuable marker for measuring humoral immunity against SARS-CoV-2. However, live-virus neutralization tests (NTs) require high-biosafety-level laboratories and are time-consuming. Therefore, surrogate virus neutralization tests (sVNTs) have been widely applied, but unlike most anti-spike (S) antibody assays, NTs and sVNTs are not harmonized, requiring further evaluation and comparative analyses. This study compared seven commercial sVNTs and anti-S-antibody assays with a live-virus NT as a reference, using a panel of 720 single and longitudinal serum samples from 666 convalescent patients after SARS-CoV-2 infection. The sensitivity of these assays for detecting antibodies ranged from 48 to 94% after PCR-confirmed infection and from 56% to 100% relative to positivity in the in-house live-virus NT. Furthermore, we performed receiver operating characteristic (ROC) curve analyses to determine which immunoassays were most suitable for assessing nAb titers exceeding a specific cutoff (NT titer, ≥80) and found that the NeutraLISA and the cPass assays reached the highest area under the curve (AUC), exceeding 0.91. In addition, when the assays were compared for their correlation with nAb kinetics over time in a set of longitudinal samples, the extent of the measured decrease of nAbs after infection varied widely among the evaluated immunoassays. Finally, in vaccinated convalescent patients, high titers of nAbs exceeded the upper limit of the evaluated assays' quantification ranges. Based on data from this study, we conclude that commercial immunoassays are acceptable substitutes for live-virus NTs, particularly when additional adapted cutoffs are employed to detect nAbs beyond a specific threshold titer. IMPORTANCE While the measurement of neutralizing antibodies is considered a valuable tool in assessing protection against SARS-CoV-2, neutralization tests employ live-virus isolates and cell culture, requiring advanced laboratory biosafety levels. Including a large sample panel (over 700 samples), this study provides adapted cutoff values calculated for seven commercial immunoassays (including four surrogate neutralization assays and a protein-based microarray) that robustly correlate with specific titers of neutralizing antibodies.

LB747. Immune Responses of the Recombinant MVA-BN-RSV Vaccine against Respiratory Syncytial Virus Infection in an RSV Human Challenge Trial (HCT) in Healthy Adult Participants

Abstract Background Respiratory Syncytial Virus (RSV) causes considerable morbidity and mortality in infants and older adults. Even though throughout life multiple infections occur, there is no natural protection. The MVA-BN vector-based RSV vaccine was designed to induce humoral and cellular RSV-specific immune responses against 5 RSV proteins (F, G [A and B subtype], M2-1 and N). We present immune response data after vaccination and challenge with an RSV-A strain in a human challenge trial conducted in healthy, young adults. Methods In a double-blinded, placebo controlled, HCT (EudraCT-no. 2020-004814-36) subjects (N=61) were randomly vaccinated with MVA-BN-RSV or placebo (30 MVA-BN-RSV and 31 placebo), intranasally challenged with RSV-A (Memphis-37b strain) after 28 days and quarantined for 12 days. Exploratory endpoints included RSV specific humoral and cellular immune responses after vaccination and after challenge throughout follow up until Day 155 assessed by treatment group and infected vs non infected in subjects receiving either MVA-BN-RSV or placebo. Results For all serum-based RSV specific antibody assays including ELISA IgG, IgA and neutralizing antibody assays PRNT A and B, antibody titers increased after vaccination in the MVA-BN-RSV group and stayed above baseline level over the entire study without being influenced by the challenge with Memphis-37b. In the placebo group, the antibody titers increased after challenge and were not affected by vaccination. The geometric mean spot forming units (GMSFUs) based on IFN-γ producing PBMCs for the stimulating agents pool F, G(A), G(B), N, M2-1 and RSV increased after vaccination in the MVA-BN-RSV group, peaking at Day 7 post vaccination for most peptide pools without any effect after challenge. In the placebo group, there was no increase after vaccination, but an increase with a peak on Day 10 post challenge was observed for all pools. Conclusion The results confirmed the broad humoral and cellular immune response elicited by the vaccination with MVA-BN-RSV which was not further boosted by the challenge virus. Disclosures Darja Schmidt, PhD, Bavarian Nordic: Employed|Bavarian Nordic: Warrants Günter Silbernagl, MSc, Bavarian Nordic: Employed|Bavarian Nordic GmbH: employee Employee and received warrants Elke Jordan, PhD, Bavarian Nordic: Employed Victoria A. JENKINS, PhD MBA, Bavarian Nordic: Employed Stephanie Schultz, Dipl Biol, Bavarian Nordic: Employed Heinz Weidenthaler, Dr med, Bavarian Nordic: Employed.

Low seroprevalence of hepatitis delta virus co-infection in hepatitis B virus-infected blood donors in China: A multicenter study.

Hepatitis delta virus (HDV) coinfected with HBV causes severe viral hepatitis, however, the number of HDV infection may be underestimated. In the present study, we enrolled 1,141,331 blood donations, routinely tested for HBsAg and/or HBV DNA, from 21 blood establishments in China. 2,690 donors were HBsAg and/or HBV DNA positive after screening tests. After verification of HBsAg and HBV DNA, 1,490 samples were HBsAg confirmed-positive, including 1,459 HBV DNA-positive samples, and 825 samples were seronegative but HBV DNA positive. We first analyzed demographic characteristics of involved 2,690 donors with different HBV infection status and found the proportions of males, the older donors, workers and farmers were higher in HBsAg-/HBV DNA+ group. Then we evaluated specificity of HDV IgG and IgM antibody assays with 375 HBsAg and HBV DNA confirmed-negative samples, and 374 were tested negative using the two assays, respectively, suggesting a specificity of 99.73% for both assays (374/375, 95% Cl: 98.51-99.95%). Subsequently, we tested for HDV IgG and IgM of 2,315 HBsAg and/or HBV DNA confirmed-positive samples, and nine showed reactivity for IgG, while two were reactive for IgM. All these 11 reactive samples were tested again with another HDV pan-Ig and IgM testing assays and HDV RNA, and only one donor was identified as HDV IgG positive and HDV RNA negative, showing an HDV seroprevalence of 0.067% (95%CI: 0.012-0.38%) among HBsAg-positive blood donors in China. The positive donor was followed up for 2 years after the donation date, and decreased antibody titer of HDV IgG and HBsAg conversion were observed, and the infection status of the donor was HDV infection with recovery and occult hepatitis B virus infection with genotype C2. These results indicated a low seroprevalence of HDV infection among blood donors and a low risk of HDV transmission through blood transfusion in China.

Construction and protective efficacy of a novel Streptococcus pneumoniae fusion protein vaccine NanAT1-TufT1-PlyD4.

During the past decades, with the implementation of pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccines (PCVs), a dramatic reduction in vaccine type diseases and transmissions has occurred. However, it is necessary to develop a less expensive, serotype-independent pneumococcal vaccine due to the emergence of nonvaccine-type pneumococcal diseases and the limited effect of vaccines on colonization. As next-generation vaccines, conserved proteins, such as neuraminidase A (NanA), elongation factor Tu (Tuf), and pneumolysin (Ply), are promising targets against pneumococcal infections. Here, we designed and constructed a novel fusion protein, NanAT1-TufT1-PlyD4, using the structural and functional domains of full-length NanA, Tuf and Ply proteins with suitable linkers based on bioinformatics analysis and molecular cloning technology. Then, we tested whether the protein protected against focal and lethal pneumococcal infections and examined its potential protective mechanisms. The fusion protein NanAT1-TufT1-PlyD4 consists of 627 amino acids, which exhibits a relatively high level of thermostability, high stability, solubility and a high antigenic index without allergenicity. The purified fusion protein was used to subcutaneously immunize C57BL/6 mice, and NanAT1-TufT1-PlyD4 induced a strong and significant humoral immune response. The anti-NanAT1-TufT1-PlyD4 specific IgG antibody assays increased after the first immunization and reached the highest value at the 35th day. The results from in vitro experiments showed that anti-NanAT1-TufT1-PlyD4 antisera could inhibit the adhesion of Streptococcus pneumoniae (S. pneumoniae) to A549 cells. In addition, immunization with NanAT1-TufT1-PlyD4 significantly reduced S. pneumoniae colonization in the lung and decreased the damage to the lung tissues induced by S. pneumoniae infection. After challenge with a lethal dose of serotype 3 (NC_WCSUH32403), a better protection effect was observed with NanAT1-TufT1-PlyD4-immunized mice than with the separate full-length proteins and the adjuvant control; the survival rate was 50%, which met the standard of the marketed vaccine. Moreover, we showed that the humoral immune response and the Th1, Th2 and Th17-cellular immune pathways are involved in the immune protection of NanAT1-TufT1-PlyD4 to the host. Collectively, our results support that the novel fusion protein NanAT1-TufT1-PlyD4 exhibits extensive immune stimulation and is effective against pneumococcal challenges, and these properties are partially attributed to humoral and cellular-mediated immune responses.

Evidence of co-exposure with Brucella spp, Coxiella burnetii, and Rift Valley fever virus among various species of wildlife in Kenya.

BackgroundCo-infection, especially with pathogens of dissimilar genetic makeup, may result in a more devastating impact on the host. Investigations on co-infection with neglected zoonotic pathogens in wildlife are necessary to inform appropriate prevention and control strategies to reduce disease burden in wildlife and the potential transmission of these pathogens between wildlife, livestock and humans. This study assessed co-exposure of various Kenyan wildflife species with Brucella spp, Coxiella burnetii and Rift Valley fever virus (RVFV).MethodologyA total of 363 sera from 16 different wildlife species, most of them (92.6%) herbivores, were analysed by Enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against Brucella spp, C. burnetii and RVFV. Further, 280 of these were tested by PCR to identify Brucella species.ResultsOf the 16 wildlife species tested, 15 (93.8%) were seropositive for at least one of the pathogens. Mean seropositivities were 18.9% (95% CI: 15.0–23.3) for RVFV, 13.7% (95% CI: 10.3–17.7) for Brucella spp and 9.1% (95% CI: 6.3–12.5) for C. burnetii. Buffaloes (n = 269) had higher seropositivity for Brucella spp. (17.1%, 95% CI: 13.0–21.7%) and RVFV (23.4%, 95% CI: 18.6–28.6%), while giraffes (n = 36) had the highest seropositivity for C. burnetii (44.4%, 95% CI: 27.9–61.9%). Importantly, 23 of the 93 (24.7%) animals positive for at least one pathogen were co-exposed, with 25.4% (18/71) of the positive buffaloes positive for brucellosis and RVFV. On molecular analysis, Brucella DNA was detected in 46 (19.5%, CI: 14.9–24.7) samples, with 4 (8.6%, 95% CI: 2.2–15.8) being identified as B. melitensis. The Fisher’s Exact test indicated that seropositivity varied significantly within the different animal families, with Brucella (p = 0.013), C. burnetii (p = <0.001) and RVFV (p = 0.007). Location was also significantly associated (p = <0.001) with Brucella spp. and C. burnetii seropositivities.ConclusionOf ~20% of Kenyan wildlife that are seropositive for Brucella spp, C. burnetii and RVFV, almost 25% indicate co-infections with the three pathogens, particularly with Brucella spp and RVFV.

The development and validation of a high-capacity serological assay for celiac disease

BackgroundThe aim of the present study was to develop and clinically validate a high-throughput assay for serum IgA and IgG antibodies against transglutaminase-2 (TG2) and to determine appropriate assay cut-offs for large-scale population screening for celiac disease. MethodAn automated method was developed using dual label time-resolved fluorometry on the AutoDELFIA platform. Individuals (n = 1920) from the general population were screened. Subjects with serum anti-TG2 concentrations above a preliminary cut-off (>0.3 mg*/L anti-TG2 IgA or >0.5 mg*/L anti-TG2 IgG) were offered endoscopic examination and biopsy. A diagnosis of celiac disease was given if villous atrophy (Marsh grade 3) was found. ResultsThe assay had a limit of quantification of 0.25 mg*/L (anti-TG2 IgA) and 0.60 mg*/L (anti-TG2 IgG) with imprecision (CV) < 16% and <18% respectively. A total of 66 individuals were above the preliminary cut-off, and 56 underwent endoscopy. Of these, 26 were diagnosed with celiac disease. Sixty-eight percent of subjects with anti-TG2 IgA ≥ 0.7 mg*/L or anti-TG2 IgG ≥ 1.0 mg*/L had biopsy-proven celiac disease, and utilization of these higher cut-offs identified 96% of biopsy-positive patients. At the time of endoscopy, all individuals with anti-TG2 IgA > 2.0 mg*/L had celiac disease, and this cut-off identified 88% of newly diagnosed celiac patients. Eight percent (2/26) of the newly diagnosed patients had primarily anti-TG2 IgG. ConclusionsIn this study we developed and clinically validated a robust and automated assay suitable for celiac disease screening in the general population.

Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay.

The establishment of an approach for detecting the anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-receptor-binding domain (RBD) neutralizing antibodies (nAbs) by a safe, easy, and rapid technique without requiring the use of live viruses is essential for facing the coronavirus disease 2019 (COVID-19) pandemic. Depending on competitive enzyme-linked immunosorbent assay (ELISA) methodology, the current study assay was designed to simulate the virus-host interaction using purified SARS-COV-2-RBD from the spike protein and the host cell receptor human angiotensin-converting enzyme 2 protein. The performance of this in-house neutralizing ELISA assay was validated using freshly prepared standards with different known concentrations of the assay. In this regard, a cohort of 50 serum samples from convalescent COVID-19 individuals with different disease severity at different time points post-recovery and a cohort of 50 serum samples from healthy individuals were processed by the in-house developed assay for detecting SARS-CoV-2 nAbs, in comparison with a commercial total anti-SARS-CoV-2 IgG antibody assay as a gold standard. The assay obtained a sensitivity of 88% (95% CI: 75.69-95.47) and a specificity of 92% (95% CI: 80.77- 97.78%). A negative strong correlation was demonstrated in the standard curve between the optical density absorbance and log concentration of the nAbs with a statistical measure of r2 (coefficient of determination) = 0.9539. The SARS-COV-2-RBD neutralizing ELISA assay serves as a high throughput qualitative and quantitative tool that can be applied in most laboratory settings without special biosafety requirements to detect anti-RBD nAbs for seroprevalence, pre-clinical, and clinical evaluation of COVID-19 vaccines efficiency and the rapid selection of convalescent plasma donors for the treatment of COVID-19 patients.

A new testing platform using fingerstick blood for quantitative antibody response evaluation after SARS-CoV-2 vaccination

ABSTRACT Testing and vaccination have been major components of the strategy for combating the ongoing COVID-19 pandemic. In this study, we have developed a quantitative anti-SARS-CoV-2 spike (S1) IgG antibody assay using a fingerstick dried blood sample. We evaluated the feasibility of using this high-throughput and quantitative anti-SARS-CoV-2 spike (S1) IgG antibody testing assay in vaccinated individuals. Fingerstick blood samples were collected and analyzed from 137 volunteers before and after receiving the Moderna or Pfizer mRNA vaccine. Anti-SARS-CoV-2 S1 IgG antibody could not be detected within the first 7 days after receiving the first vaccine dose, however, the assay reliably detected antibodies from day 14 onwards. In addition, no anti-SARS-CoV-2 nucleocapsid (N) protein IgG antibody was detected in any of the vaccinated or healthy participants, indicating that the anti-SARS-CoV-2 S1 IgG assay is specific for the mRNA vaccine-induced antibodies. The S1 IgG levels detected in fingerstick samples correlated with the levels found in venous blood plasma samples and with the efficacy of venous blood plasma samples in the plaque reduction neutralization test (PRNT). The assay displayed a limit of quantification (LOQ) of 0.59 μg/mL and was found to be linear in the range of 0.51-1000 μg/mL. Finally, its clinical performance displayed a Positive Percent Agreement (PPA) of 100% (95% CI: 0.89-1.00) and a Negative Percent Agreement (NPA) of 100% (95% CI: 0.93-1.00). In summary, the assay described here represents a sensitive, precise, accurate, and simple method for the quantitative detection and monitoring of post-vaccination anti-SARS-CoV-2 spike IgG responses.

CROSS-REACTIVITY OF COVID-19 IGG ASSAY WITH KNOWN IMMUNE-MEDIATED AND INFECTIOUS DISORDERS

Objective: To assess the cross-reactivity of COVID-19 IgG assay with known immune-mediated and infectious disorders and evaluate for any false-positive reactions to determine the specificity of the serological assay.&#x0D; Study Design: Cross-sectional analytical study.&#x0D; Place and Duration of Study: Department of Chemical Pathology, Chughtai Institute of Pathology, Lahore Pakistan, from Sep to Oct 2020.&#x0D; Methodology: A total of 116 samples were included in the study of both males and females. Diagnosed cases of typhoid fever, viral hepatitis, systemic lupus erythematosus (SLE), syphilis, multiple connective tissue disorders (MCTD), varicella-zoster infection, rabies, toxoplasmosis, epstein-barr virus (EBV) infection, rubella, rheumatoid arthritis, AIDS, cytomegalovirus (CMV) infection and dengue fever were included in the study. Three samples of multiparous women aged more than 40 years were also included in the study. IgG antibody levels were measured against SARS-CoV-2 with a cut-off index of 1.4.&#x0D; Results: Out of 116, only 3 (2%) samples were reactive for IgG against SARS-CoV-2. The categories showing cross-reactivity were typhoid, hepatitis C, and CMV. All specimens showing cross-reactivity were of females. Assay under consideration showed a specificity of 97.4%.&#x0D; Conclusion: Cross-reactivity was seen in pre-pandemic cases of infectious diseases with COVID-19 IgG antibody assay. Medical lab professionals must verify the serological assays before use in the clinical laboratory to avoid false-positive results.

Evaluation of five widely used serologic assays for antibodies to SARS-CoV-2

Reliable diagnostic technologies are pivotal to the fight against COVID-19. While real-time reverse transcription-polymerase chain reaction (rRT-PCR) remains the gold standard, commercial assays for antibodies against (SARS-CoV-2) have emerged. We sought to examine 5 widely used commercial methods. We measured antibodies against SARS-CoV-2 with assays, Abbott-IgG, Roche-IgT (total antibodies, isotype-unspecific), EUROIMMUN-IgG, EUROIMMUN-IgA, DiaSorin-IgG, in 191 serum samples from patients with rRT-PCR proven COVID-19 between days 0 and 47 after the onset of clinical symptoms and in biobank samples collected in 2018. The assays were calibrated using the manufacturers’ instructions; results are in multiples of the assay specific cut-offs (Abbott, Roche, EUROIMMUN) or in arbitrary units (AU/mL, DiaSorin). The assays for IgG and IgT have approximately the same sensitivity and specificity for detecting seroconversion which starts at approximately day 3 after symptom onset, sensitivity reached 93% on day 16 and was 100% for each assay on day 20. The assay for IgA antibodies was superior in sensitivity and had a lower specificity than the others. Bivariate non–parametric correlation coefficients ranged between 0.738 and 0.991. Commercial assays for IgG or total antibodies against SARS-CoV-2 are largely equivalent for establishing seroconversion but differ at high antibody titres. Increased sensitivity to detect seroconversion is afforded by including IgA antibodies. Further international efforts to harmonise assays for antibodies against SARS-CoV-2 are urgently needed.

A Systematic Evaluation of IgM and IgG Antibody Assay Accuracy in Diagnosing Acute Zika Virus Infection in Brazil: Lessons Relevant to Emerging Infections.

Accurate diagnostics underpin effective public health responses to emerging viruses. For viruses, such as Zika virus (ZIKV), where the viremia clears quickly, antibody-based (IgM or IgG) diagnostics are recommended for patients who present 7 days after symptom onset. However, cross-reactive antibody responses can complicate test interpretation among populations where closely related viruses circulate. We examined the accuracy (proportion of samples correctly categorized as Zika positive or negative) for antibody-based diagnostics among Brazilian residents (Rio de Janeiro) during the ZIKV outbreak. Four ZIKV enzyme-linked immunosorbent assays (ELISAs; IgM and IgG Euroimmun, IgM Novagnost, and CDC MAC), two dengue ELISAs (IgM and IgG Panbio), and the ZIKV plaque reduction neutralization test (PRNT) were evaluated. Positive samples were ZIKV PCR confirmed clinical cases collected in 2015-2016 (n = 169); negative samples (n = 236) were collected before ZIKV was present in Brazil (≤2013). Among serum samples collected ≥7 days from symptom onset, PRNT exhibited the highest accuracy (93.7%), followed by the Euroimmun IgG ELISA (77.9%). All IgM assays exhibited lower accuracy (<75%). IgG was detected more consistently than IgM among ZIKV cases using Euroimmun ELISAs (68% versus 22%). Anti-dengue virus IgM ELISA was positive in 41.1% of confirmed ZIKV samples tested. The Euroimmun IgG assay, although misdiagnosing 22% of samples, provided the most accurate ELISA. Anti-ZIKV IgG was detected more reliably than IgM among ZIKV patients, suggesting a secondary antibody response to assay antigens following ZIKV infection. Antibody ELISAs need careful evaluation in their target population to optimize use and minimize misdiagnosis, prior to widespread deployment, particularly where related viruses cocirculate.

Multicentric evaluation of a novel point of care electrochemical ELISA platform for SARS-CoV-2 specific IgG and IgM antibody assay

New diagnostics technologies for the efficient detection and quantification of SARS-CoV-2 antibodies are very crucial to manage the COVID-19 pandemic, especially in the context of emerging vaccination paradigms. Herein, we report on a novel point-of-care Electrochemical ELISA platform with disposable screen printed electrodes functionalized with SARS-CoV-2 Spike Glycoprotein S1, to enable fast and accurate quantitative estimation of total antibody concentration (IgG and IgM) in clinical samples. The quantification is performed with a comparison of electrochemical redox current against the current produced by the spiked monoclonal antibodies with known concentration. The assay is validated through multicentric evaluation against 3 different FDA authorized Laboratory standard techniques, using both EDTA whole blood and serum samples. We demonstrate that the proposed assay has excellent sensitivity and specificity, making it a suitable candidate for epidemiological surveys and quantification of antibodies in COVID-19 vaccination programs.