scholarly journals A "Made-in-Canada" serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination

2021 ◽  
Author(s):  
Karen Colwill ◽  
Yannick Galipeau ◽  
Matthew Stuible ◽  
Christian Gervais ◽  
Corey Arnold ◽  
...  
Keyword(s):  
Immune Responses ◽  
High Throughput ◽  
Natural Infection ◽  
National Research Council ◽  
World Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Assay ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Made In

BACKGROUND: Testing for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable "Made-in-Canada" solution that can detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. METHODS: We developed a set of methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD), and the nucleocapsid (N) protein. These antigens are complemented by a detection antibody (human anti-IgG fused to horseradish peroxidase (HRP)) and a positive control reference antibody (recombinant IgG against the RBD), permitting intra- and inter-laboratory comparisons. Using this toolkit and commercial reagents, we optimized automated ELISAs on two different high throughput platforms to measure antibody responses to SARS-CoV-2 antigens. The assays were calibrated to a reference standard from the World Health Organization. We also automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-Spike or -RBD interactions by antibodies using biotinylated ACE2. RESULTS: Our individual IgG-based ELISAs measure antibody levels in single-point measurements in reference to a standard antibody curve to accurately distinguish non-infected and infected individuals (area under the curve > 0.96 for each assay). Positivity thresholds can be established in individual assays using precision-recall analysis (e.g., by fixing the false positive rate), or more stringently, by scoring against the distribution of the means of negative samples across multiple assays performed over several months. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for at least 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. Here, we present detailed protocols to perform these assays using either serum/plasma or dried blood spots both manually and on two automated platforms, and to express the results in international units to facilitate data harmonization and inter-study comparisons. We also demonstrate that the snELISA can be performed automatically at single points, increasing the scalability of this functional assay for large seroprevalence studies. INTERPRETATION: The ability to measure antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput assays enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The "Made-in-Canada" set of protein reagents, produced at the National Research Council of Canada are publicly available to enable the up-scaling of standardized serological assays, permitting nationwide data comparison and aggregation.

scholarly journals Idiotypes of anti-Ia antibodies. I. Expression of the 14-4-4S idiotype in humoral immune responses.

1981 ◽  
Vol 154 (2) ◽  
pp. 397-409 ◽  
Author(s):  
S L Epstein ◽  
K Ozato ◽  
J A Bluestone ◽  
D H Sachs
Keyword(s):  
Immune Responses ◽  
Immunosorbent Assay ◽  
Heavy Chain ◽  
Antigenic Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Absorption Analysis ◽  
Specific Enzyme ◽  
Cellular Receptors ◽  
Humoral Immune ◽  
Humoral Immune Responses

The idiotype of a mouse monoclonal anti-I-E antibody, 14-4-4S, has been studied using a heterologous anti-idiotypic reagent. This antibody recognizes Ia. 7, an antigenic specificity present in all strains expressing a product of the I-E subregion. Expression of the 14-4-4S idiotype in humoral immune responses was analyzed by an idiotype-specific enzyme-linked immunosorbent assay system. The idiotype was readily detectable in C3H.SW anti-C3H alloantisera, the same immunization combination from which the hybridoma was derived. Absorption analysis demonstrated the anti-I-E specificity of the idiotype-positive molecules in these alloantisera. Penetrance of idiotype expression was high among individual C3H.SW immune mice (9 of 10 tested). To examine genetic requirements for idiotype expression, an immunization was performed using as responders CWB mice, congenic with C3H.SW but differing at the heavy chain allotype loci. Immune sera of individual CWB mice contained very little or no idiotype, demonstrating that levels of idiotype expression are influenced by allotype-linked genes, although the influence of other genes has not been ruled. The 14-4-4S idiotype therefore represents a shared idiotype of anti-Ia antibodies and provides opportunities for analysis of the idiotypes of cellular receptors for the corresponding Ia antigen.

scholarly journals Limited Local and Systemic Antibody Responses toNeisseria gonorrhoeae during Uncomplicated Genital Infections

1999 ◽  
Vol 67 (8) ◽  
pp. 3937-3946 ◽  
Author(s):  
Spencer R. Hedges ◽  
Matthew S. Mayo ◽  
Jiri Mestecky ◽  
Edward W. Hook ◽  
Michael W. Russell
Keyword(s):  
Immune Responses ◽  
Transmitted Disease ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Genital Infections ◽  
Sexually Transmitted ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Repeated Infections ◽  
Antibody Levels

ABSTRACT Repeated infections with Neisseria gonorrhoeae are common among patients attending sexually transmitted disease clinics. We examined whether previous infections or site of infection altered the local and systemic antigonococcal antibody levels in males and females. Antibodies against N. gonorrhoeae MS11 and the patients’ homologous infecting isolates were measured by enzyme-linked immunosorbent assay. In general, the local and systemic immune responses to gonococci were extremely modest. There was a slight increase in serum immunoglobulin G (IgG) against the MS11 strain and the homologous isolates in infected males. Levels of serum IgA1 antibodies against MS11 were slightly higher in infected than in uninfected females. A history of previous infections with N. gonorrhoeae did not alter the antibody levels in patients with a current infection, suggesting that immunological memory is not induced by uncomplicated gonococcal infections. Antibody responses to infected subjects’ homologous isolates were observed in cervical mucus; IgA1 levels increased while IgG levels decreased. The decline in mucosal IgG against the homologous isolates was less common in subjects having both rectal and cervical infections; otherwise, no effect of rectal involvement was observed. The absence of substantially higher antibody levels to gonococci where there is infection at a site known to contain organized lymphoid tissue suggests that the low levels of responses to uncomplicated infections may not be due simply to an absence of inductive sites in the genital tract. We propose that in addition to its potential ability to avoid the effects of an immune response,N. gonorrhoeae does not elicit strong humoral immune responses during uncomplicated genital infections.

scholarly journals Humoral Immune Responses against the Mycobacteriumtuberculosis 38-Kilodalton, MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis

2010 ◽  
Vol 17 (3) ◽  
pp. 372-375 ◽  
Author(s):  
Xueqiong Wu ◽  
Yourong Yang ◽  
Junxian Zhang ◽  
Bangying Li ◽  
Yan Liang ◽  
...  
Keyword(s):  
Immune Responses ◽  
Healthy Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Single Antigen ◽  
Human Sera ◽  
Low Sensitivity ◽  
Humoral Responses ◽  
Culture Negative

ABSTRACT The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging. The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in 250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and 73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.

scholarly journals Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

2003 ◽  
Vol 10 (3) ◽  
pp. 383-387 ◽  
Author(s):  
Masahiro Yoneda ◽  
Takao Hirofuji ◽  
Noriko Motooka ◽  
Koji Nozoe ◽  
Kayoko Shigenaga ◽  
...  
Keyword(s):  
Immune Responses ◽  
Gel Electrophoresis ◽  
Early Onset ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Healthy Control ◽  
Specific Antigens

ABSTRACT Bacteroides forsythus is one of the important periodontopathic bacteria, and this microorganism is known to have an S-layer outside the outer membrane. The S-layer-like antigens were recently isolated from B. forsythus, and they were found to be 270- and 230-kDa proteins in the envelope fraction. In this study, these proteins were confirmed to be specific to B. forsythus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they were clearly recognized by sera from patients with adult and early-onset periodontitis in Western immmunoblot analysis. We compared the immunoglobulin G (IgG) responses against the purified S-layer-like antigen by enzyme-linked immunosorbent assay. IgG responses against this antigen were low in healthy control subjects, but they were significantly higher in subjects with adult and early-onset periodontitis. Together with the fact that the IgG responses against the crude extract of B. forsythus did not rise significantly in patients with periodontitis, S-layer-like proteins are considered to be specific antigens of B. forsythus and may play an important role in the progression of periodontitis.

scholarly journals Humoral Immune Responses to Neisseria meningitidis in Children

1999 ◽  
Vol 67 (5) ◽  
pp. 2441-2451 ◽  
Author(s):  
Andrew J. Pollard ◽  
Rachel Galassini ◽  
Eileene M. Rouppe van der Voort ◽  
Robert Booy ◽  
Paul Langford ◽  
...  
Keyword(s):  
Immune Responses ◽  
Neisseria Meningitidis ◽  
Bactericidal Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Older Children ◽  
Recent Infection ◽  
Humoral Immune ◽  
Serum Bactericidal Activity ◽  
Humoral Immune Responses ◽  
Bactericidal Antibody

ABSTRACT An understanding of the nature of immunity to serogroup B meningococci in childhood is necessary in order to establish the reasons for poor responses to candidate vaccines in infancy. We sought to examine the nature of humoral immune responses following infection in relation to age. Serum bactericidal activity was poor in children under 12 months of age despite recent infection with Neisseria meningitidis. The highest levels of bactericidal activity were seen in children over 10 years of age. However, infants produced levels of total immunoglobulin G (IgG) and IgG subclass antibodies similar to those in older children in a meningococcal enzyme-linked immunosorbent assay. Most antibody was of the IgG1 and IgG3 subclasses. This striking age dependency of bactericidal antibody response following infection is not apparently due to failure of class switching in infants but might be due to qualitative differences in antibody specificity or affinity.

scholarly journals Serum but not mucosal antibody responses are predicted by pre-existing SARS-CoV-2 spike cross-reactive CD4+ T cells following BNT162b2 vaccination in the elderly

2021 ◽  
Author(s):  
Lil Meyer-Arndt ◽  
Tatjana Schwarz ◽  
Lucie Loyal ◽  
Larissa Henze ◽  
Beate Kruse ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Natural Infection ◽  
The Elderly ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Mucosal Antibody ◽  
Age Related ◽  
Neutralizing Capacity ◽  
Efficient Induction

AbstractAdvanced age is a main risk factor for severe COVID-19 and thus elderly were often prioritized for vaccination. However, low vaccination efficacy and accelerated waning immunity have been reported in this age group. To elucidate age-related differences in immunogenicity, we analysed cellular, serological and salivary SARS-CoV-2 spike glycoprotein-specific immune responses to BNT162b2 COVID-19 vaccine in old (69-92 years) and middle-aged (24-57 years) vaccinees compared to natural infection (COVID-19 convalescents of 21-55 years). Serological humoral responses to vaccination exceeded those of convalescents but salivary anti-spike subunit 1 (S1) IgA and neutralizing capacity were less durable in vaccinees. In old vaccinees, we observed that pre-existing spike-specific CD4+ T cells correlated with efficient induction of serological anti-S1 IgG and neutralizing capacity after vaccination. Our results highlight the role of pre-existing cross-reactive CD4+ T cells with respect to SARS-CoV-2 vaccination particularly in old individuals, in whom their presence predicted efficient COVID-19-vaccine-induced humoral immune responses.

scholarly journals Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

2015 ◽  
Vol 22 (9) ◽  
pp. 1033-1039 ◽  
Author(s):  
Ola H. Negm ◽  
Mohamed R. Hamed ◽  
Elizabeth M. Dilnot ◽  
Clifford C. Shone ◽  
Izabela Marszalowska ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Immune Responses ◽  
Protein Microarray ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Antigens ◽  
Igg Antibody ◽  
Content Type ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Specific Antigens

ABSTRACTClostridium difficileis an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course ofC. difficileinfection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purifiedC. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid andCandida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses toC. difficileprotein antigens and may have potential advantages in throughput, convenience, and cost.

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