scholarly journals A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity

2020 ◽  
Author(s):  
Eric D. Laing ◽  
Spencer L. Sterling ◽  
Stephanie A. Richard ◽  
Shreshta Phogat ◽  
Emily C. Samuels ◽  
...  
Keyword(s):  
The United States ◽  
Cross Reactivity ◽  
Spike Glycoprotein ◽  
Serum Samples ◽  
Igg Antibody ◽  
Military Hospitals ◽  
Before And After ◽  
Antibody Assays ◽  
Human Sera ◽  
Human Coronaviruses

ABSTRACTWith growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 µL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.

scholarly journals Longitudinal Analysis of Cryptosporidium Species-Specific Immunoglobulin G Antibody Responses in Peruvian Children

2006 ◽  
Vol 13 (1) ◽  
pp. 123-131 ◽  
Author(s):  
Jeffrey W. Priest ◽  
Caryn Bern ◽  
Lihua Xiao ◽  
Jacquelin M. Roberts ◽  
James P. Kwon ◽  
...  
Keyword(s):  
Immunoglobulin G ◽  
Antibody Responses ◽  
Health Interventions ◽  
Older Children ◽  
Serum Samples ◽  
Igg Antibody ◽  
Specific Immunoglobulin ◽  
Antibody Assays ◽  
Species Specific ◽  
Antibody Levels

ABSTRACT Cryptosporidium species are ubiquitous in the environment and are frequently detected in the stools of children who live where sanitation conditions are poor. To better characterize the immune response to these parasites, we monitored immunoglobulin G (IgG) antibody levels in a cohort of children from Lima, Peru. Two new enzyme-linked immunosorbent assays based on the C. parvum (bovine, subtype IIa) Iowa strain 17-kDa and 27-kDa antigens were used to measure IgG antibody levels in longitudinal serum samples. Antibody responses were detected during infections with C. parvum, C. felis, and C. meleagridis and with four different subtypes of C. hominis. We also noted that the magnitude of the antibody response was related to the number of previous infections and that older children generally had higher levels of antibodies to the two C. parvum antigens. Antibody responses were not associated with infections with either Cyclospora sp. or Giardia sp. We believe the antibody assays will be important tools for monitoring the success of future public health interventions.

The Significance of Hepatitis G Virus in Serum of Patients With Sporadic Fulminant and Subfulminant Hepatitis of Unknown Etiology

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1460-1464
Author(s):  
Santiago J. Muñoz ◽  
Harvey J. Alter ◽  
Yoshiyuki Nakatsuji ◽  
James W.-K. Shih ◽  
Rajender K. Reddy ◽  
...  
Keyword(s):  
United States ◽  
Fulminant Hepatitis ◽  
Acute Hepatic Failure ◽  
The United States ◽  
Unknown Etiology ◽  
Blood Product Transfusion ◽  
Serum Samples ◽  
Hepatitis G Virus ◽  
Before And After ◽  
Hepatitis G

Excluding acute hepatic failure caused by drugs, the etiology of fulminant hepatitis (FH) remains unknown in many patients. There are conflicting data about a possible pathogenic role for the hepatitis G virus (HGV) in patients with cryptogenic fulminant hepatitis (non–A-E FH). We investigated the presence of circulating HGV in 36 patients with well-documented non–A-E fulminant and 5 patients with subfulminant hepatitis from 3 geographic locations in the United States. Serum HGV RNA was determined by reverse transcriptase-polymerase chain reaction using primers from the NS5 region of the HGV genome. HGV RNA was also measured before and after liver transplantation in 5 patients and at different time points in 7 patients. Serum samples were recoded and reanalyzed for HGV RNA using different primer sets to assess the validity of the HGV RNA assay. HGV was present in serum of 14 of the 36 patients (38.8%) with non–A-E fulminant hepatitis. Twenty percent of patients from the Northeast, 11% of the patients from the Southeast, and 50% from the Mid-Atlantic regions of the United States had circulating HGV RNA. The use of therapeutic blood products was significantly associated with the presence of serum HGV RNA (P < .02). Retesting for HGV RNA with different primers was positive in all but 1 case. HGV RNA is not causally related to non–A-E fulminant hepatitis. The finding of HGV RNA in serum from these patients is likely related to the administration of blood product transfusion after the onset of fulminant hepatitis.

scholarly journals Characterization of Anaplasma phagocytophilum Major Surface Protein 5 and the Extent of Its Cross-Reactivity with A. marginale

2007 ◽  
Vol 14 (3) ◽  
pp. 262-268 ◽  
Author(s):  
N. I. Strik ◽  
A. R. Alleman ◽  
A. F. Barbet ◽  
H. L. Sorenson ◽  
H. L. Wamsley ◽  
...  
Keyword(s):  
United States ◽  
Anaplasma Phagocytophilum ◽  
Indirect Elisa ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
The United States ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Major Surface Protein ◽  
Major Surface

ABSTRACT Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

scholarly journals Development of a Dual Monoclonal Antibody Immunoassay for Total Human Kallikrein 2

2001 ◽  
Vol 47 (7) ◽  
pp. 1218-1224 ◽  
Author(s):  
Judith A Finlay ◽  
John R Day ◽  
Cindy L Evans ◽  
Robert Carlson ◽  
Kristine Kuus-Reichel ◽  
...  
Keyword(s):  
Specific Antigen ◽  
High Specificity ◽  
Cross Reactivity ◽  
Minimum Detectable Concentration ◽  
Serum Samples ◽  
Bodily Fluids ◽  
Before And After ◽  
Detectable Concentration ◽  
Human Kallikrein 2 ◽  
Kallikrein 2

Abstract Background: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-α1-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. Methods: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. Results: The minimum detectable concentration in the thK2 assay was 0.008 μg/L, and PSA cross-reactivity was &lt;0.001%. The assay detected prohK2 and three different hK2–serum protease complexes. The median serum concentration of thK2 in control samples (0.013 μg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 μg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at −70 °C. Conclusions: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.

scholarly journals Multi-peptide ELISAs overcome cross-reactivity and inadequate sensitivity of conventional Chlamydia pneumoniae serology

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kh Shamsur Rahman ◽  
Bernhard Kaltenboeck
Keyword(s):  
Chlamydia Pneumoniae ◽  
High Specificity ◽  
Cross Reactivity ◽  
Peptide Antigens ◽  
Specificity And Sensitivity ◽  
Peptide Antibody ◽  
Antibody Assays ◽  
Human Sera ◽  
Composite Reference Standard ◽  
Individual Peptide

Abstract Cross-reactivity of classical chlamydial antigens compromises Chlamydia (C.) pneumoniae serology. By testing with 185 human antisera, we expanded 18 previously discovered C. pneumoniae-specific B-cell epitopes to 48 peptide antigens from 12 C. pneumoniae immunodominant proteins. For specific detection of antibodies against C. pneumoniae, we developed novel ELISAs with strongly reactive individual peptide antigens and mixtures of these peptides. By comparison to a composite reference standard (CRS) for anti-C. pneumoniae antibody status of human sera, the top-performing CpnMixF12 peptide assay showed 91% sensitivity at 95% specificity, significantly higher than 4 commercial anti-C. pneumoniae IgG ELISAs (36-12% sensitivity at 95% specificity). Human C. pneumoniae (Cpn) and C. trachomatis (Ctr) seroreactivity was 54% biased towards co-positivity in commercial Cpn and Ctr ELISAs, but unbiased in Cpn and Ctr peptide antibody assays, suggesting severe cross-reactivity of commercial ELISAs. Using hyperimmune mouse sera against each of 11 Chlamydia spp., we confirm that commercial Cpn and Ctr ELISA antigens are cross-reactive among all Chlamydia spp., but Cpn and Ctr peptide antigens react only with antisera against the cognate chlamydial species. With simultaneously high specificity and sensitivity, and convenient use for non-specialized laboratories, these ELISAs have the potential to improve serodiagnosis of C. pneumoniae infection.

scholarly journals Identification of B Cell Epitopes of Blo t 13 Allergen and Cross-Reactivity with Human Adipocytes and Heart Fatty Acid Binding Proteins

2019 ◽  
Vol 20 (24) ◽  
pp. 6107 ◽  
Author(s):  
Marlon Múnera ◽  
Dalgys Martínez ◽  
Alexis Labrada ◽  
Luis Caraballo ◽  
Leonardo Puerta
Keyword(s):  
Fatty Acid ◽  
B Cell ◽  
Immunoglobulin E ◽  
Allergic Diseases ◽  
Cross Reactivity ◽  
Fatty Acid Binding Proteins ◽  
Serum Samples ◽  
Fatty Acid Binding ◽  
B Cell Epitopes ◽  
Human Sera

Cross-reactivity between allergens and human proteins could have a clinical impact in allergic diseases. Blo t 13 is an allergen from the mite Blomia tropicalis, which belongs to the fatty acid binding protein (FABP) family and has structural homology with human FABPs. This work aimed to map B cell epitopes on Blo t 13 and to identify epitopes involved in cross-reactivity with human heart FABP (FABP3) and adipocyte FABP (FABP4). Sera from 25 patients with house dust mite (HDM) allergy that were sensitized to Blo t 13 were used for testing the reactivity of immunoglobulin E (IgE) and IgG to FABP. The epitope mapping of Blo t 13 was performed using overlapping peptides, and cross-reactivity between Blo t 13 and human FABP was analyzed using human sera and anti-Blo t 13 monoclonal antibodies. IgE antibodies to all FABPs were detected in 14/25 serum samples, and IgG was detected in 25/25 serum samples. The cross-reactivity of Blo t 13 was 42% with FABP3 and 48% with FABP4. Two IgE-binding regions were identified in Blo t 13; one between residues 54 and 72 (the main cross-reacting region) and another between residues 111 to 129. Our results suggest that exposure to the Blo t 13 allergen could induce an auto-reactive response to endogenous FABP in allergic patients sensitized to Blo t 13.

scholarly journals Phosphorylcholine-Dependent Cross-Reactivity between Dental Plaque Bacteria and Oxidized Low-Density Lipoproteins

2001 ◽  
Vol 69 (11) ◽  
pp. 6612-6617 ◽  
Author(s):  
Harvey A. Schenkein ◽  
Collin R. Berry ◽  
Donald Purkall ◽  
John A. Burmeister ◽  
Carol N. Brooks ◽  
...  
Keyword(s):  
Dental Plaque ◽  
Serum Antibody ◽  
Significant Proportion ◽  
Cross Reactivity ◽  
Oral Bacteria ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Igg Antibody ◽  
Oxidized Low Density Lipoproteins ◽  
Human Sera

ABSTRACT Antibodies reactive with phosphorylcholine (PC) are ubiquitous in human sera, but the antigens stimulating their production and their function are not clear. Previous studies have shown that a significant proportion of dental plaque bacteria contain PC as determined by reactivity with PC-specific mouse myeloma proteins and monoclonal antibodies. Additionally, serum antibody concentrations of immunoglobulin (IgG) G anti-PC are higher in sera of individuals who have experienced periodontal attachment loss than those who are periodontally healthy. These data implicate the oral microflora as a source of antigen-stimulating anti-PC responses. Recent data also indicate that antibodies with specificity for PC are elevated in ApoE-deficient mice, a model for studies of athersclerosis, and that such antibodies bound oxidized low-density lipoproteins (LDL) (oxLDL) in atherosclerotic plaques. These data prompted the hypothesis that human anti-PC could bind to both oral bacteria and human oxLDL, and that these antigens are cross-reactive. We therefore examined the ability of human anti-PC to bind to PC-bearing strains of oral bacteria using enzyme-linked immunosorbent inhibition assays and by assessment of direct binding of affinity-purified human anti-PC to PC-bearingActinobacillus actinomycetemcomitans. Our results indicated that PC-bearing strains of Streptococcus oralis,Streptococcus sanguis, Haemophilus aphrophilus,Actinomyces naeslundii, Fusobacterium nucleatum, and A. actinomycetemcomitans, as well as a strain of Streptococcus pneumoniae, absorbed up to 80% of anti-PC IgG antibody from human sera. Furthermore, purified anti-PC bound to a PC-bearing strain of A. actinomycetemcomitansbut only poorly to a PC-negative strain. OxLDL also absorbed anti-PC from human sera, and oxLDL but not LDL reacted with up to 80% of the anti-PC in human sera. Furthermore, purified anti-PC bound directly to oxLDL but not to LDL. The data indicate that PC-containing antigens on a variety of common oral bacteria are cross-reactive with neoantigens expressed in oxLDL. We propose that PC-bearing dental plaque microorganisms may induce an antibody response to PC that could influence the inflammatory response associated with atherosclerosis.

scholarly journals Reactivity of Porcine Epidemic Diarrhea Virus Structural Proteins to Antibodies against Porcine Enteric Coronaviruses: Diagnostic Implications

2017 ◽  
Vol 55 (5) ◽  
pp. 1426-1436 ◽  
Author(s):  
Luis Gabriel Gimenez-Lirola ◽  
Jianqiang Zhang ◽  
Jose Antonio Carrillo-Avila ◽  
Qi Chen ◽  
Ronaldo Magtoto ◽  
...  
Keyword(s):  
Antibody Response ◽  
Porcine Epidemic Diarrhea Virus ◽  
Herd Immunity ◽  
Cross Reactivity ◽  
Structural Proteins ◽  
Transmissible Gastroenteritis Virus ◽  
Serum Samples ◽  
Igg Antibody ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACTThe development of porcine epidemic diarrhea virus (PEDV) antibody-based assays is important for detecting infected animals, confirming previous virus exposure, and monitoring sow herd immunity. However, the potential cross-reactivity among porcine coronaviruses is a major concern for the development of pathogen-specific assays. In this study, we used serum samples (n= 792) from pigs of precisely known infection status and a multiplex fluorescent microbead-based immunoassay and/or enzyme-linked immunoassay platform to characterize the antibody response to PEDV whole-virus (WV) particles and recombinant polypeptides derived from the four PEDV structural proteins, i.e., spike (S), nucleocapsid (N), membrane (M), and envelope (E). Antibody assay cutoff values were selected to provide 100% diagnostic specificity for each target. The earliest IgG antibody response, mainly directed against S1 polypeptides, was observed at days 7 to 10 postinfection. With the exception of nonreactive protein E, we observed similar antibody ontogenies and patterns of seroconversion for S1, N, M, and WV antigens. Recombinant S1 provided the best diagnostic sensitivity, regardless of the PEDV strain, with no cross-reactivity detected against transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), or porcine deltacoronavirus (PDCoV) pig antisera. The WV particles showed some cross-reactivity to TGEV Miller and TGEV Purdue antisera, while N protein presented some cross-reactivity to TGEV Miller. The M protein was highly cross-reactive to TGEV and PRCV antisera. Differences in the antibody responses to specific PEDV structural proteins have important implications in the development and performance of antibody assays for the diagnosis of PEDV enteric disease.

scholarly journals Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

2020 ◽  
Author(s):  
Saahir Khan ◽  
Rie Nakajima ◽  
Aarti Jain ◽  
Rafael Ramiro de Assis ◽  
Al Jasinskas ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Symptomatic Patient ◽  
Cross Reactivity ◽  
Human Sera ◽  
Antigenic Domains ◽  
Preliminary Study ◽  
Human Coronaviruses ◽  
Epidemiologic Risk ◽  
Limited Testing ◽  
Respiratory Specimens

AbstractThe current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.

scholarly journals A high-throughput microsphere-based immunoassay of anti-SARS-CoV-2 IgM testing for COVID-19 diagnostics

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0248444
Author(s):  
Dayu Zhang ◽  
Tianyang Xu ◽  
Eric Chu ◽  
Aiguo Zhang ◽  
Jinwei Du ◽  
...  
Keyword(s):  
High Throughput ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Serum Samples ◽  
Igg Antibody ◽  
Igm Antibody ◽  
Percent Agreement ◽  
Novel Coronavirus ◽  
The Individual

The pandemic of novel coronavirus disease COVID-19 is rapidly expanding across the world. A positive result of antibody tests suggests that the individual has potentially been exposed to SARS-CoV-2, thus allowing to identify asymptomatic infections and determine the seroprevalence in a given population. The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgM antibody detection on the Luminex MAGPIX platform. Clinical agreement studies were performed in 42 COVID-19 patient serum samples and 162 negative donor serum/plasma samples. Positive percent agreement (PPA) was 42.86% (95% CI: 9.90% to 81.59%), 71.43% (95% CI: 29.04% to 96.33%), and 28.57% (95% CI: 13.22% to 48.67%) for samples collected on 0–7 days, 8–14 days, and 2–8 weeks from symptom onset, respectively. Negative Percent Agreement (NPA) was 97.53% (95% CI: 93.80% to 99.32%). There was no cross-reactivity with the SARS-CoV-2 IgG antibody. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL), triglyceride (250 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. In conclusion, an anti-SARS-CoV-2 IgM antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up the anti-SARS-CoV-2 IgM testing.

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