Follow-Up and Comparative Assessment of SARS-CoV-2 IgA, IgG, Neutralizing, and Total Antibody Responses After BNT162b2 or mRNA-1273 Heterologous Booster Vaccination.

BACKGROUND: Priming with ChAdOx1 followed by heterologous boosting is considered in several countries. Nevertheless, analyses comparing the immunogenicity of heterologous booster to homologous primary vaccination regimens and natural infection are lacking. In this study, we aimed to conduct a comparative assessment of the immunogenicity between homologous primary vaccination regimens and heterologous prime-boost vaccination using BNT162b2 or mRNA-1273. METHODS: We matched vaccinated naïve (VN) individuals (n = 673) with partial vaccination (n = 64), primary vaccination (n = 590), and primary series plus mRNA vaccine heterologous booster (n = 19) with unvaccinated naturally infected (NI) individuals with a documented primary SARS-CoV-2 infection (n = 206). We measured the levels of neutralizing total antibodies (NTAbs), total antibodies (TAbs), anti-S-RBD IgG, and anti-S1 IgA titers. RESULTS: Homologous primary vaccination with ChAdOx1 not only showed less potent NTAb, TAb, anti-S-RBD IgG, and anti-S1 IgA immune responses compared to primary BNT162b2 or mRNA-1273 vaccination regimens (p < 0.05) but also showed ~3-fold less anti-S1 IgA response compared to infection-induced immunity (p < 0.001). Nevertheless, a heterologous booster led to an increase of ~12 times in the immune response when compared to two consecutive homologous ChAdOx1 immunizations. Furthermore, correlation analyses revealed that both anti-S-RBD IgG and anti-S1 IgA significantly contributed to virus neutralization among NI individuals, particularly in symptomatic and pauci-symptomatic individuals, whereas among VN individuals, anti-S-RBD IgG was the main contributor to virus neutralization. CONCLUSION: The results emphasize the potential benefit of using heterologous mRNA boosters to increase antibody levels and neutralizing capacity particularly in patients who received primary vaccination with ChAdOx1.

Nucleocapsid and Spike Protein-Based Anti-SARS-CoV-2 Assay Performance in the Minority and Rural Coronavirus Insights Study: Characteristics of Socioeconomically Disadvantaged Populations with Health Disparities.

BACKGROUND: COVID-19 has had a devastating impact on Black, Hispanic, and other underserved, disadvantaged populations. Here anti-SARS-CoV-2 tests are characterized in disadvantaged patients to examine equivalence in US populations. METHODS: Underserved participant adults (age > 18 years) were enrolled before the availability of SARS-CoV-2 vaccines in Federal Qualified Health Centers in California, Florida, Louisiana, Illinois, and Ohio and contributed samples to the Minority and Rural Coronavirus Insights Study (MRCIS). A subset coined the MRCIS SARS-CoV-2 Antibody Cohort of 2365 participants was tested with the Roche Anti-SARS-CoV-2 assay (Cobas e601). Five hundred ninety-five of these were also tested with the Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG assay (VITROS-5600); 1770 were also tested with the Abbott ARCHITECT SARS-CoV-2 IgG assay (ARCHITECT-2000). Assay-specific cutoffs classified negative/positive results. RESULTS: Eight point four percent (199/2365) of the MRCIS SARS-CoV-2 Antibody Cohort was SARS-CoV-2 RNA positive at enrollment. Agreement between the Ortho/Roche and the Abbott/Roche antibody testing did not vary by enrollment RNA status. The Ortho (anti-spike protein) vs Roche (anti-nucleocapsid protein) comparison agreed substantially: kappa = 0.63 (95% CI: 0.57-0.69); overall agreement, 83%. However, agreement was even better for the Abbott vs Roche assays (both anti-nucleocapsid protein tests): kappa = 0.85 (95% CI: 0.81-0.87); overall agreement, 95%. Anti-SARS-CoV-2 comparisons stratified by demographic criteria demonstrated no significant variability in agreement by sex, race/ethnicity, or age. CONCLUSIONS: Analytical agreement is 96.4% for anti-spike-protein vs anti-nucleocapsid-protein comparisons. Physiologically, seroreversion of anti-nucleocapsid reactivity after infection occurred in the disadvantaged population similarly to general populations. No anti-SARS-CoV-2 assays included demonstrated a clinically significant difference due to the demographics of the disadvantaged MRCIS SARS-CoV-2 Antibody Cohort.

Teste de antigliadina deaminada IgG para crianças com até 2 anos de idade e com suspeita de doença celíaca / IgG deaminated antigliadin test for children aged 2 years or older with suspected celiac disease

INTRODUÇÃO: A Doença Celíaca (DC) é uma doença autoimune crônica do intestino delgado caracterizada por intolerância permanente ao glúten. A sua prevalência global é de aproximadamente 1%. No Brasil, essa prevalência foi relatada em cerca de 0,54% em crianças (1-14 anos). O rastreamento por sorologia associado à confirmação por biópsia duodenal é padrão ouro para o diagnóstico em adultos e em crianças, mas a biópsia precisa ser bem indicada na prática pediátrica por ser um procedimento invasivo e potencialmente de alto risco. Os testes sorológicos para detectar anticorpos IgA são comumente utilizados, porém indivíduos com deficiência de IgA não podem ser diagnosticados/rastreados por esses testes, justamente porque apresentam déficit na síntese de todas as imunoglobulinas do tipo A. Uma alternativa de triagem para esses indivíduos, bem como para os menores de 2 anos, é a dosagem sérica dos anticorpos IgG, como o teste antigliadina deaminada IgG. PERGUNTA DE PESQUISA: O uso do teste sorológico antigliadina deaminada IgG para triagem é mais acurado e custo-efetivo em pacientes com deficiência de IgA e suspeita de doença celíaca e crianças menores de dois anos quando comparado à triagem com antitransglutaminase IgA e confirmação por biópsia de duodeno por via endoscópica (endoscopia digestiva alta + biópsia)? EVIDÊNCIAS CIENTÍFICAS: Este relatório incluiu oito estudos que avaliaram se o uso do teste sorológico antigliadina deaminada IgG é mais acurado em pacientes com deficiência de IgA e suspeita de doença celíaca de qualquer idade e crianças menores de dois anos quando comparado à biópsia de duodeno por via endoscópica para diagnóstico da doença celíaca. Segundo os resultados das metanálises apresentadas neste relatório, para as análises da acurácia obtidas por meio da sensibilidade e especificidades combinadas, destaca-se a boa especificidade do teste antigliadina deaminada IgG em crianças menores de dois anos, utilizando o ponto de corte determinado pelo fabricante (97,8%; IC95% 95,6% - 98,9%). Já a especificidade combinada foi máxima (100,0%; IC95%:0,0 - 100,0%), potencializando o valor preditivo negativo do teste antigliadina deaminada IgG neste grupo populacional. Estes achados mostram que a adição do teste antigliadina deaminada IgG pode melhorar a acurácia diagnóstica da detecção de DC em crianças menores de dois anos de idade. AVALIAÇÃO ECONÔMICA (AE): Conduziu-se análise de custo-efetividade para comparar os testes diagnósticos com base em suas efetividades e seus custos, por meio da razão de custoefetividade incremental (RCEI). Considerando-se as diferenças observadas no desempenho do teste para crianças menores de dois anos e indivíduos com deficiência de IgA e suspeita de doença celíaca, foram propostas duas árvores de decisão. No caso de indivíduos com deficiência de IgA, a realização de teste antigliadina deaminada associada à EDA com biópsia, comparada à antigliadina deaminada isolada resultaria em razão de custo-efetividade incremental (RCEI) de R$ 108,17 por biópsia evitada. Para a comparação entre antigliadina deaminada isolada e EDA com biópsia, a RCEI seria de R$ 2.063,16 por biópsia evitada. ANÁLISE DE IMPACTO ORÇAMENTÁRIO (AIO): Considerando a população elegível total e suspeita de doença celíaca, o cenário alternativo 01 (market share variando de 30% a 50%) provocaria uma economia de R$ 30.671.133,25, no caso da incorporação do teste antigliadina em substituição aos testes atuais. Já o cenário alternativo 02 (market share de 50% a 70%) de substituição dos testes atuais pelo teste da antigliadina provocaria também economia de R$ 46.018.687,48. Já no cenário em que o teste antigliadina seja adicionado aos testes atuais, ao invés de substituí-los, o impacto orçamentário acumulado em cinco anos seria de R$ 14.410.515,92. CONSIDERAÇÕES FINAIS: Segundo os resultados das metanálises apresentadas neste relatório, destaca-se a boa especificidade do teste de antigliadina deaminada IgG em crianças menores de dois anos, utilizando o ponto de corte determinado pelo fabricante (97,8%; IC95% 95,6% - 98,9%). Já a especificidade combinada foi máxima (100,0%; IC95%:0,0 ­ 100,0%), potencializando o valor preditivo negativo do teste de antigliadina deaminada IgG neste grupo populacional. Para a população de indivíduos com deficiência de IgA, a sensibilidade combinada foi de 76,7% (IC 95%: 54,7% a 90,0%) e a especificidade de 73,3% (IC 95%: 60,6% a 83,0%). A taxa de falsos positivos global correspondeu a 26,7% (IC 95%: 17,0% a 39,4%). Contudo, os achados devem ser interpretados com cautela, uma vez que os estudos primários incluídos foram considerados de qualidade metodológica baixa e muito baixa qualidade da evidência. Um dos estudos incluídos neste relatório é uma revisão sistemática cujo divergiu do encontrado na meta-análise feita pelo grupo elaborador. Enquanto a sensibilidade para o teste antigliadina deaminada IgG em relação à biópsia duodenal foram de 0,96 (IC 95% 0,91 a 0,98) na revisão sistemática, a metaanálise deste relatório teve como resultado 0,48 (IC de 95%: 0,23 a 0,97). Essa divergência está relacionada ao ponto de corte utilizado para a inclusão dos estudos nas metaanálises. Em relação à confiança nos resultados da revisão sistemática em questão, os resultados estão associados a uma baixa confiabilidade. RECOMENDAÇÃO PRELIMINAR DA CONITEC: O tema foi avaliado na 113ª Reunião Ordinária da Conitec em 5 de outubro de 2022. A recomendação inicial foi desfavorável à incorporação do teste de antigliadina deaminada IgG para pessoas com deficiência de IgA por considerar que os testes atualmente disponíveis no SUS já atendem satisfatoriamente a população. Ademais, a recomendação inicial foi favorável à incorporação do teste de antigliadina deaminada IgG para pessoas menores de 2 anos de idade por evitar as hospitalizações necessárias para realização de endoscopia digestiva alta nessa população. CONSULTA PÚBLICA: Foram recebidas 18 contribuições, sendo cinco pelo formulário para contribuições técnico-científicas e 13 pelo formulário para contribuições sobre experiência ou opinião de pacientes, familiares, amigos ou cuidadores de pacientes, profissionais de saúde ou pessoas interessadas no tema. Em relação às contribuições de cunho técnico-científico, quatro concordaram que o teste antigliadina deaminada IgG deve ser incorporado ao SUS e uma não concordou. A Federação Nacional das Associações de Celíacos do Brasil (FENACELBRA) se manifestou contrária à recomendação de incorporação do teste diagnóstico e a favor de seguir a Sociedade Europeia de Gastroenterologia, Hepatologia e Nutrição Pediátrica (ESPGHAN), que sugere que seja realizada a dosagem de IgA total e antitransglutaminase IgA (antitTG) enquanto o paciente estiver consumindo glúten diariamente por cerca de 2 a 3 meses. No caso de crianças com concentração de IgA baixa, a contribuição indicou a necessidade de uma das seguintes sorologias IgG: antigliadina deaminada IgG, antiendomísio IgG ou antitransglutaminase IgG. Um participante foi contrário à premissa de que o resultado para o teste antigliadina deaminada IgG positivo deveria substituir a endoscopia e biópsia, o que foi corroborado por um especialista que participou do grupo elaborador. Dessa forma, uma avaliação econômica e análise de impacto orçamentário adicional foram realizadas com o objetivo de avaliar o impacto dessa premissa. A AE resultou numa RCEI custo-efetiva (R$ 1.254 por QALY ganho) e a AIO resultou em impacto orçamentário positivo de R$ 17.460.094 ao longo de cinco anos. Quanto às contribuições referentes ao formulário de experiência ou opinião, todos foram favoráveis à incorporação. RECOMENDAÇÃO FINAL DA CONITEC: As contribuições da consulta pública foram apresentadas à Conitec por ocasião da 117ª Reunião Ordinária, realizada em 29 de março de 2023. Os membros presentes deliberaram, por unanimidade, recomendar a incorporação do teste de antigliadina deaminada IgG para crianças com até 2 anos de idade e com suspeita de doença celíaca, porque, para essa população, o teste anti-gliadina tem alta acurácia diagnóstica, enquanto para os deficientes de IgA a acurácia diagnóstica foi considerada moderada e de impacto clínico incerto. O teste anti-gliadina deaminada IgG para crianças de até 2 anos de idade deverá ser incorporado conforme Protocolo Clínico do Ministério da Saúde. Por fim, foi assinado o Registro de Deliberação Nº 811 / 2023. DECISÃO: ncorporar, no âmbito do Sistema Único de Saúde - SUS, o teste de antigliadina deaminada IgG para crianças com até 2 anos de idade e com suspeita de doença celíaca, publicada no Diário Oficial da União nº 74, seção 1, página 195, em 18 de abril de 2023.

Importance of anti-SARS-CoV-2 assay antigenic composition as revealed by the results of the Belgian external quality assessment (EQA) scheme.

We report on sample IS/17575 since it generated highly divergent results in the Belgian SARS-CoV-2 serology external quality assessment scheme. Sample IS/17575 was serum originating from a 30 years old male patient. 124 diagnostic laboratories analysed this sample. A total of 168 results was returned (including 5 doubles). Overall, 38 were positive. All tests against S1 were positive except the Euroimmun IgG ELISA and the Ortho clinical Diagnostics VITROS IgG CLIA. All tests against S1/S2 (Liaison, Diasorin) resulted in a signal above cutoff. Assays against RBD, mostly generate a negative result. An exception are the Wantai SARS-CoV-2 ELISA's. All tests targeting N protein were negative. The survey shows, when >6 months post-infection, assays targeting at least S1, and preferably S1 combined with S2, are the most sensitive. This finding accentuates the necessity of external quality assessment schedules and importance of antigenic composition of serologic SARS-CoV-2 assays.

Quality comparability assessment of a SARS-CoV-2-neutralizing antibody across transient, mini-pool-derived and single-clone CHO cells.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.

A homogeneous time-resolved fluorometric energy transfer assay for the binding assessment of FcRn with IgG antibodies.

We aimed to develop a homogeneous time-resolved fluorometric energy transfer assay for assessment of human neonatal Fc receptor binding activity with IgG-type antibodies. The assay was configured with FcRn-coupled with Eu cryptate via biotin and streptavidin interaction as donor and IgG1 labeled with d2 as acceptor. Only a single incubation step was involved and no wash step was required. The assay demonstrated good accuracy, precision, linearity and specificity. Our further investigation with a rat pharmacokinetics study revealed that the terminal t1/2 for Trastuzumab and its related three ADCs agreed with the EC50 data. The assay can be applied to various IgGs with modifications to identify antibodies with appropriate binding ability to human FcRn.

COVID-19 mRNA Vaccination in Lactation: Assessment of Adverse Events and Vaccine Related Antibodies in Mother-Infant Dyads.

Background: Data regarding symptoms in the lactating mother-infant dyad and their immune response to COVID-19 mRNA vaccination during lactation are needed to inform vaccination guidelines. Methods: From a prospective cohort of 50 lactating individuals who received mRNA-based vaccines for COVID-19 (mRNA-1273 and BNT162b2), blood and milk samples were collected prior to first vaccination dose, immediately prior to 2nd dose, and 4-10 weeks after 2nd dose. Symptoms in mother and infant were assessed by detailed questionnaires. Anti-SARS-CoV-2 antibody levels in blood and milk were measured by Pylon 3D automated immunoassay and ELISA. In addition, vaccine-related PEGylated proteins in milk were measured by ELISA. Blood samples were collected from a subset of infants whose mothers received the vaccine during lactation (4-15 weeks after mothers' 2nd dose). Results: No severe maternal or infant adverse events were reported in this cohort. Two mothers and two infants were diagnosed with COVID-19 during the study period before achieving full immune response. PEGylated proteins were not found at significant levels in milk after vaccination. After vaccination, levels of anti-SARS-CoV-2 IgG and IgM significantly increased in maternal plasma and there was significant transfer of anti-SARS-CoV-2-Receptor Binding Domain (anti-RBD) IgA and IgG antibodies to milk. Milk IgA levels after the 2nd dose were negatively associated with infant age. Anti-SARS-CoV-2 IgG antibodies were not detected in the plasma of infants whose mothers were vaccinated during lactation. Conclusions: COVID-19 mRNA vaccines generate robust immune responses in plasma and milk of lactating individuals without severe adverse events reported.

Immunological assessment of SARS-CoV-2 infection in pregnancy from diagnosis to delivery: A multicentre prospective study.

BACKGROUND: Background Population-based data on SARS-CoV-2 infection in pregnancy and assessment of passive immunity to the neonate, is lacking. We profiled the maternal and fetal response using a combination of viral RNA from naso-pharyngeal swabs and serological assessment of antibodies against SARS-CoV-2. METHODS: This multicentre prospective observational study was conducted between March 24th and August 31st 2020. Two independent cohorts were established, a symptomatic SARS-CoV-2 cohort and a cohort of asymptomatic pregnant women attending two of the largest maternity hospitals in Europe. Symptomatic women were invited to provide a serum sample to assess antibody responses. Asymptomatic pregnant women provided a nasopharyngeal swab and serum sample. RT-PCR for viral RNA was performed using the Cobas SARS-CoV-2 6800 platform (Roche). Umbilical cord bloods were obtained at delivery. Maternal and fetal serological response was measured using both the Elecsys® Anti-SARS-CoV-2 immunoassay (Roche), Abbott SARS-CoV-2 IgG Assay and the IgM Architect assay. Informed written consent was obtained from all participants. RESULTS: Ten of twenty three symptomatic women had SARS-CoV-2 RNA detected on nasopharyngeal swabs. Five (5/23, 21.7%) demonstrated serological evidence of anti-SARS-CoV-2 IgG antibodies and seven (30.4%, 7/23) were positive for IgM antibodies. In the asymptomatic cohort, the prevalence of SARS-CoV-2 infection in RNA was 0.16% (1/608). IgG SARS-CoV-2 antibodies were detected in 1·67% (10/598, 95% CI 0·8%-3·1%) and IgM in 3·51% (21/598, 95% CI 2·3-5·5%). Nine women had repeat testing post the baseline test. Four (4/9, 44%) remained IgM positive and one remained IgG positive. 3 IgG anti-SARS-CoV-2 antibodies were detectable in cord bloods from babies born to five seropositive women who delivered during the study. The mean gestation at serological test was 34 weeks. The mean time between maternal serologic positivity and detection in umbilical cord samples was 28 days. CONCLUSION: Using two independent serological assays, we present a comprehensive illustration of the antibody response to SARS-CoV-2 in pregnancy, and show a low prevalence of asymptomatic SARS-CoV2. Transplacental migration of anti-SARS-CoV-2 antibodies was identified in cord blood of women who demonstrated antenatal anti-SARS-CoV-2 antibodies, raising the possibility of passive immunity.

Longitudinal Assessment of Antisevere Acute Respiratory Syndrome Coronavirus 2 Immune Responses for Six Months Based on the Clinical Severity of Coronavirus Disease 2019.

BACKGROUND: There is insufficient data on the longevity of immunity acquired after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We aimed to evaluate the duration of SARS-CoV-2-specific humoral and cellular immunity according to the clinical severity of coronavirus disease 2019 (COVID-19). The study population comprised asymptomatic (n = 14), symptomatic/nonpneumonic (n = 42), and pneumonic (n = 41) patients. RESULTS: The anti-SARS-CoV-2 immunoglobulin class G and neutralizing antibody (NAb) titers lasted until 6 months after diagnosis, with positivity rates of 66.7% and 86.9%, respectively. Older age, prolonged viral shedding, and accompanying pneumonia were more frequently found in patients with sustained humoral immunity. Severe acute respiratory syndrome coronavirus 2-specific T-cell response was strongly observed in pneumonic patients and prominent in individuals with sustained humoral immunity. CONCLUSIONS: In conclusion, most (>85%) patients carry NAb until 6 months after diagnosis of SARS-CoV-2 infection, providing insights for establishing vaccination strategies against COVID-19.

Ultra-fast, high throughput and inexpensive detection of SARS-CoV-2 seroconversion using Ni2+ magnetic beads.

To monitor the levels of protecting antibodies raised in the population in response to infection and/or to immunization with SARS-CoV-2, we need a technique that allows high throughput and low-cost quantitative analysis of human IgG antibodies reactive against viral antigens. Here we describe an ultra-fast, high throughput and inexpensive assay to detect SARS-CoV-2 seroconversion in humans. The assay is based on Ni2+ magnetic particles coated with His tagged SARS-CoV-2 antigens. A simple and inexpensive 96 well plate magnetic extraction/homogenization process is described which allows the simultaneous analysis of 96 samples and delivers results in 7 min with high accuracy.

Assessment of avidity related to IgG subclasses in SARS-CoV-2 Brazilian infected patients.

SARS-CoV-2 is considered a global emergency, resulting in an exacerbated crisis in the health public in the world. Although there are advances in vaccine development, it is still limited for many countries. On the other hand, an immunological response that mediates protective immunity or indicates that predict disease outcome in SARS-CoV-2 infection remains undefined. This work aimed to assess the antibody levels, avidity, and subclasses of IgG to RBD protein, in symptomatic patients with severe and mild forms of COVID-19 in Brazil using an adapted in-house RBD-IgG ELISA. The RBD IgG-ELISA showed 100% of specificity and 94.3% of sensibility on detecting antibodies in the sera of hospitalized patients. Patients who presented severe COVID-19 had higher anti-RBD IgG levels compared to patients with mild disease. Additionally, most patients analyzed displayed low antibody avidity, with 64.4% of the samples of patients who recovered from the disease and 84.6% of those who died in this avidity range. Our data also reveals an increase of IgG1 and IgG3 levels since the 8th day after symptoms onset, while IgG4 levels maintained less detectable during the study period. Surprisingly, patients who died during 8-14 and 15-21 days also showed higher anti-RBD IgG4 levels in comparison with the recovered (P < 0.05), suggesting that some life-threatening patients can elicit IgG4 to RBD antibody response in the first weeks of symptoms onset. Our findings constitute the effort to clarify IgG antibodies' kinetics, avidity, and subclasses against SARS-CoV-2 RBD in symptomatic patients with COVID-19 in Brazil, highlighting the importance of IgG antibody avidity in association with IgG4 detection as tool laboratory in the follow-up of hospitalized patients with more significant potential for life-threatening.

Multiplex assessment of SARS-CoV-2 antibodies improves assay sensitivity and correlation with neutralizing antibodies.

OBJECTIVES: Detection of antibodies to multiple SARS-CoV-2 antigens in a single assay could increase diagnostic accuracy, differentiate vaccination from natural disease, and aid in retrospective exposure determination. Correlation of binding antibody assessment in clinical assays with neutralizing antibodies is needed to better understand the humoral response to SARS-CoV-2 infection and establish of correlates of protection. METHODS: A cohort of 752 samples was used to assess specificity, sensitivity, and comparison to 6 other Conformitè Europëenne serologic assays for the BioRad SARS-CoV-2 IgG multiplex assay which measures receptor binding domain IgG (RBD), spike-S1 IgG (S1), spike-S2 IgG (S2), and nucleocapsid IgG (N). A subset of serial specimens from 14 patients was also tested for neutralizing antibodies (n = 61). RESULTS: Specificity for RBD and S1 IgG was 99.4% (n = 170) and 100% for S2 and N IgG (n = 170) in a cohort selected for probable interference. Overall assay concordance with other assays was >93% for IgG and total antibody assays and reached 100% sensitivity for clinical concordance at >14 days as a multiplex assay. RBD and S1 binding antibody positivity demonstrated 79-95% agreement with the presence of neutralizing antibodies. CONCLUSIONS: The BioRad SARS-CoV-2 IgG assay is comparable to existing assays, and achieved 100% sensitivity when all markers were included. The ability to measure antibodies against spike and nucleocapsid proteins simultaneously may be advantageous for complex clinical presentations, epidemiologic research, and in decisions regarding infection prevention strategies. Additional independent validations are needed to further determine binding antibody and neutralizing antibody correlations.

Assessment of serological assays for identifying high titer convalescent plasma.

BACKGROUND: The COVID-19 pandemic has been accompanied by the largest mobilization of therapeutic convalescent plasma (CCP) in over a century. Initial identification of high titer units was based on dose-response data using the Ortho VITROS IgG assay. The proliferation of severe acute respiratory syndrome coronavirus 2 serological assays and non-uniform application has led to uncertainty about their interrelationships. The purpose of this study was to establish correlations and analogous cutoffs between multiple serological assays. METHODS: We compared the Ortho, Abbott, Roche, an anti-spike (S) ELISA, and a virus neutralization assay. Relationships relative to FDA-approved cutoffs under the CCP emergency use authorization were identified in convalescent plasma from a cohort of 79 donors from April 2020. RESULTS: Relative to the neutralization assay, the spearman r value of the Ortho Clinical, Abbott, Roche, anti-S ELISA assays was 0.65, 0.59, 0.45, and 0.76, respectively. The best correlative index for establishing high-titer units was 3.87 signal-to-cutoff (S/C) for the Abbott, 13.82 cutoff index for the Roche, 1:1412 for the anti-S ELISA, 1:219 by the neutralization assay, and 15.9 S/C by the Ortho Clinical assay. The overall agreement using derived cutoffs compared to a neutralizing titer of 1:250 was 78.5% for Abbott, 74.7% for Roche, 83.5% for the anti-S ELISA, and 78.5% for Ortho Clinical. DISCUSSION: Assays based on antibodies against the nucleoprotein were positively associated with neutralizing titers and the Ortho assay, although their ability to distinguish FDA high-titer specimens was imperfect. The resulting relationships help reconcile results from the large body of serological data generated during the COVID-19 pandemic.

Development of rapid and cost-effective top-loading device for the detection of anti-SARS-CoV-2 IgG/IgM antibodies.

Infection with SARS-CoV-2, the Betacoronavirus, caused a pandemic that affected the globe negatively. The gold method, RT-PCR, can detect SARS-CoV-2 but it is time-consuming and needs sophisticated equipment and professional personnel. On the other hand, rapid tests offer fast results and can detect anti-SARS-CoV-2 antibodies (Abs). The aim of this study is to develop a new rapid and cost-effective method for the detection of anti-SARS-CoV-2 IgG/IgM Abs. A new top-loading detection device was developed and composed of a small piece of plastic (25 × 25 × 0.5 mm) with an opening in the center, a piece of nitrocellulose (NC) membrane enough to block the opening from one side and adhesive tape to affix the NC to the plastic piece. The NC is blotted with anti-human IgG/IgM and rabbit serum. The device was evaluated against a commercially available IgG/IgM ELISA detection kit using normal, Covid-19-positive, HCV, HBV, and Cytomegalovirus-positive sera. Outcomes demonstrated simplicity, reproducibility, and accuracy of the new device and results can be obtained in less than 5 min. We anticipate our developed assay method to be used widely in point of care before deciding on the use of expensive nucleic acid assays.

A systematic assessment of structural heterogeneity and IgG/IgE-binding of ovalbumin.

Ovalbumin (OVA), one of the major allergens in hen egg, exhibits extensive structural heterogeneity due to a range of post-translational modifications (PTMs). However, analyzing the structural heterogeneity of native OVA is challenging, and the relationship between heterogeneity and IgG/IgE-binding of OVA remains unclear. In this work, ion exchange chromatography (IXC) with salt gradient elution and on-line detection by native electrospray ionization mass spectrometry (ESI MS) was used to assess the structural heterogeneity of OVA, while inhibition-ELISA was used to assess the IgG/IgE binding characteristics of OVA. Over 130 different OVA proteoforms (including glycan-free species and 32 pairs of isobaric species) were identified. Proteoforms with acetylation, phosphorylation, oxidation and succinimide modifications had reduced IgG/IgE binding capacities, whereas those with few structural modifications had higher IgG/IgE binding capacities. OVA isoforms with a sialic acid-containing glycan modification had the highest IgG/IgE binding capacity. Our results demonstrate that on-line native IXC/MS with salt gradient elution can be used for rapid assessment of the structural heterogeneity of proteins. An improved understanding of the relationship between IgG/IgE binding capacity and OVA structure provides a basis for developing biotechnology or food processing methods for reducing protein allergenicity reduction.

Performance assessment of seven SARS-CoV-2 IgG enzyme-linked immunosorbent assays.

The pandemic of COVID-19 has caused enormous fatalities worldwide. Serological assays are important for detection of asymptomatic or mild cases of COVID-19, and sero-prevalence and vaccine efficacy studies. Here, we evaluated and compared the performance of seven commercially available enzyme-linked immunosorbent assay (ELISA)s for detection of anti-severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) immunoglobulin G (IgG). The ELISAs were evaluated with a characterized panel of 100 serum samples from qRT-PCR confirmed COVID-19 patients, collected 14 days post onset disease, 100 SARS-CoV-2 negative samples and compared the results with that of neutralization assay. Results were analysed by creating the receiver operating characteristic curve of all the assays in reference to the neutralization assay. All kits, were found to be suitable for detection of IgG against SARS-CoV-2 with high accuracy. The DiaPro COVID-19 IgG ELISA showed the highest sensitivity (98%) among the kits. The assays demonstrated high sensitivity and specificity in detecting the IgG antibodies against SARS-CoV-2. However, the presence of IgG antibodies does not always correspond to neutralizing antibodies. Due to their good accuracy indices, these assays can also aid in tracing mild infections, in cohort studies and in pre-vaccine evaluations.

High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay.

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

Analytic comparison between three high-throughput commercial SARS-CoV-2 antibody assays reveals minor discrepancies in a high-incidence population.

Performance of three automated commercial serological IgG-based assays was investigated for assessing SARS-CoV-2 "ever" (past or current) infection in a population-based sample in a high exposure setting. PCR and serological testing was performed on 394 individuals. SARS-CoV-2-IgG seroprevalence was 42.9% (95% CI 38.1-47.8%), 40.6% (95% CI 35.9-45.5%), and 42.4% (95% CI 37.6-47.3%) using the CL-900i, VidasIII, and Elecsys assays, respectively. Between the three assays, overall, positive, and negative percent agreements ranged between 93.2-95.7%, 89.3-92.8%, and 93.8-97.8%, respectively; Cohen's kappa statistic ranged from 0.86 to 0.91; and 35 specimens (8.9%) showed discordant results. Among all individuals, 12.5% (95% CI 9.6-16.1%) had current infection, as assessed by PCR. Of these, only 34.7% (95% CI 22.9-48.7%) were seropositive by at least one assay. A total of 216 individuals (54.8%; 95% CI 49.9-59.7%) had evidence of ever infection using antibody testing and/or PCR during or prior to this study. Of these, only 78.2%, 74.1%, and 77.3% were seropositive in the CL-900i, VidasIII, and Elecsys assays, respectively. All three assays had comparable performance and excellent agreement, but missed at least 20% of individuals with past or current infection. Commercial antibody assays can substantially underestimate ever infection, more so when infection rates are high.

Evaluation of 11 SARS-CoV-2 antibody tests by using samples from patients with defined IgG antibody titers.

We evaluated the performance of 11 SARS-CoV-2 antibody tests using a reference set of heat-inactivated samples from 278 unexposed persons and 258 COVID-19 patients, some of whom contributed serial samples. The reference set included samples with a variation in SARS-CoV-2 IgG antibody titers, as determined by an in-house immunofluorescence assay (IFA). The five evaluated rapid diagnostic tests had a specificity of 99.0% and a sensitivity that ranged from 56.3 to 81.6% and decreased with low IFA IgG titers. The specificity was > 99% for five out of six platform-based tests, and when assessed using samples collected ≥ 22 days after symptom onset, two assays had a sensitivity of > 96%. These two assays also detected samples with low IFA titers more frequently than the other assays. In conclusion, the evaluated antibody tests showed a heterogeneity in their performances and only a few tests performed well with samples having low IFA IgG titers, an important aspect for diagnostics and epidemiological investigations.

Patient-blood management for COVID19 convalescent plasma therapy: relevance of affinity and donor-recipient differences in concentration of neutralizing antibodies.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) is being extensively investigated as a treatment, with mixed results to date. Overall, there has been a generalized lack of appropriateness in prescriptions, which, in the field of transfusion medicine, is termed patient-blood management. OBJECTIVES: We aimed to separate study design variables that could affect clinical outcome after CCP therapy. We focus here on variables such as pretransfusion antibody testing in recipients, dose adjustments and antibody affinity measurements. SOURCES: We searched PubMed and preprint servers for relevant preclinical and clinical studies discussing each of these variables in the field of CCP therapy. CONTENT: We show evidence that neglecting those variables has affected the outcomes of the vast majority of CCP clinical trials to date. IMPLICATIONS: A better understanding of such variables will improve the design of the next generation of CCP clinical trials. This will likely lead to better clinical outcomes and will minimize risks of immune evasion from subneutralizing doses of neutralizing antibodies.