Improved specificity and reduced subtype cross-reactivity for antibody detection by ELISA using globular head domain recombinant hemagglutinin

A stable hybridoma cell line (DD9) has been produced secreting a monoclonal antibody specific for porcine muscle proteins. The DD9 monoclonal antibody (mAb) failed to show a significant cross-reactivity when tested against beef, horse, chicken, and soya proteins, as well as bovine caseins, gelatin, and bovine serum albumin. The DD9 mAb was further used in an indirect ELISA format for detection of defined amounts of porcine meat (1–100%) in beef and chicken meat mixtures immobilized on 96-well plates. Immunorecognition of monoclonal antibodies adsorbed to porcine meat was made with rabbit anti-mouse immunoglobulins conjugated to the enzyme horseradish peroxidase.

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Robustness of Serologic Investigations for Chikungunya and Mayaro Viruses following Coemergence

mSphere ◽

10.1128/msphere.00915-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Carlo Fischer ◽

Fernando Bozza ◽

Xiomara Jeanleny Merino Merino ◽

Celia Pedroso ◽

Edmilson F. de Oliveira Filho ◽

...

Keyword(s):

Latin America ◽

False Positive ◽

Chikungunya Virus ◽

Cross Reactivity ◽

Positive Test ◽

Antibody Detection ◽

Test Results ◽

Specific Patient ◽

False Positive Test ◽

Mayaro Virus

ABSTRACT Since 2013, the arthropod-borne Chikungunya virus (CHIKV) has cocirculated with the autochthonous Mayaro virus (MAYV) in Latin America. Both belong to the same alphavirus serocomplex, termed the Semliki Forest serocomplex. The extent of antibody cross-reactivity due to the antigenic relatedness of CHIKV and MAYV in commonly used serologic tests remains unclear. By testing 64 CHIKV- and 37 MAYV-specific sera from cohort studies conducted in Peru and Brazil, we demonstrate about 50% false-positive test results using commercially available enzyme-linked immunosorbent assays (ELISAs) based on structural antigens. In contrast, combining ELISAs for CHIKV and MAYV significantly increased positive predictive values (PPV) among all cohorts from 35.3% to 88.2% for IgM and from 61.3% to 96.8% for IgG (P < 0.0001). Testing of longitudinally collected CHIKV-specific patient sera indicated that ELISA specificity is highest for IgM testing at 5 to 9 days post-onset of symptoms (dpo) and for IgG testing at 10 to 14 dpo. IgG cross-reactivity in ELISA was asymmetric, occurring in 57.9% of MAYV-specific sera compared to 29.5% of CHIKV-specific sera. Parallel plaque reduction neutralization testing (PRNT) for CHIKV and MAYV increased the PPV from 80.0% to 100% (P = 0.0053). However, labor-intense procedures and delayed seroconversion limit PRNT for patient diagnostics. In sum, individual testing for CHIKV or MAYV only is prone to misclassifications that dramatically impact patient diagnostics and sero-epidemiologic investigation. Parallel ELISAs for both CHIKV and MAYV provide an easy and efficient solution to differentiate CHIKV from MAYV infections. This approach may provide a template globally for settings in which alphavirus coemergence imposes similar problems. IMPORTANCE Geographically overlapping transmission of Chikungunya virus (CHIKV) and Mayaro virus (MAYV) in Latin America challenges serologic diagnostics and epidemiologic surveillance, as antibodies against the antigenically related viruses can be cross-reactive, potentially causing false-positive test results. We examined whether widely used ELISAs and plaque reduction neutralization testing allow specific antibody detection in the scenario of CHIKV and MAYV coemergence. For this purpose, we used 37 patient-derived MAYV-specific sera from Peru and 64 patient-derived CHIKV-specific sera from Brazil, including longitudinally collected samples. Extensive testing of those samples revealed strong antibody cross-reactivity in ELISAs, particularly for IgM, which is commonly used for patient diagnostics. Cross-neutralization was also observed, albeit at lower frequencies. Parallel testing for both viruses and comparison of ELISA reactivities and neutralizing antibody titers significantly increased diagnostic specificity. Our data provide a convenient and practicable solution to ensure robust differentiation of CHIKV- and MAYV-specific antibodies.

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Food Allergy

Nepal Medical College Journal ◽

10.3126/nmcj.v20i4.25124 ◽

2018 ◽

Vol 20 (4) ◽

pp. 109-115

Author(s):

Shiva Raj KC ◽

G. KC ◽

P. Gyawali

Keyword(s):

Antibody Titre ◽

Cross Reactivity ◽

Antibody Detection ◽

Allergic Reactions ◽

Food Allergens ◽

Colour Reaction ◽

Common Food ◽

Common Antigens ◽

The Many ◽

The Individual

Introduction: Any adverse effect on health resulting from repeated exposure to a substance against which the individual has been sensitized against is allergy. Food allergens are one of the many common causes of allergies. The aim of this study is to find out the common food allergens and also specify the type of food suspicious for allergy among the patients with allergic reactions. A total of 178 patients with allergic reactions were included. The test was carried out using kit with an enzyme-labelled antihuman IgE catalysing a colour reaction. The kit contains test stripes coated with 21 different common antigens. Concentration of antibody specific to certain allergen is calculated and graded into different classes. Among the patients sensitized to allergic food, mean age was 34.36 years with female to male ratio of 2.9:1. Ninty two patients (51.7%) patients were sensitized to 1 to 3 allergens, whereas, 3.9% w ere sensitized to more than 10 food allergens. Thirty three (27.9%) patients had definite antibody detection, 11(9.3%) patients had strong antibody titre and 4 (3.3%) patients had very high antibody titre. Most common food allergen was crab (20.1%) followed by potato (25.8%). Cross reactivity was seen in various food allergens. Cereals and rice showed significant reactivity (P <0.001). Other food like soybean with mustard, peanut with coconut and apple with grape also showed significant correlation (P<0.001). The most common food allergens were crab, potato, spinach, peanut, coconut, soybean, mustard, wheat, rice, shrimp/prawn, cucumber and onion. While determining the specific allergen, we should be aware of potential cross-reactivity against one or more food allergens.

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Protective Immunity Based on the Conserved Hemagglutinin Stalk Domain and Its Prospects for Universal Influenza Vaccine Development

BioMed Research International ◽

10.1155/2014/546274 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 24

Author(s):

Madhu Khanna ◽

Sachin Sharma ◽

Binod Kumar ◽

Roopali Rajput

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Natural Infection ◽

Influenza Viruses ◽

Cross Protection ◽

Influenza B ◽

Universal Vaccine ◽

Virus Surface ◽

Head Domain ◽

Globular Head

Influenza virus surface glycoprotein hemagglutinin (HA) is an excellent and chief target thatelicitsneutralizing antibodies during vaccination or natural infection. Its HA2 subunit (stem domain) is most conserved as compared to HA1 subunit (globular head domain). Current influenza vaccine relies on globular head domain that provides protection only against the homologous vaccine strains, rarely provides cross-protection against divergent strains, and needs to be updated annually. There is an urge for a truly universal vaccine that provides broad cross-protection against different subtype influenza A viruses along with influenza B viruses and need not be updated annually. Antibodies against the stem domain of hemagglutinin (HA) are able to neutralize a wide spectrum of influenza virus strains and subtypes. These stem-specific antibodies have great potential for the development of universal vaccine against influenza viruses. In this review, we have discussed the stem-specific cross-reactive antibodies and heterosubtypic protection provided by them. We have also discussed their epitope-based DNA vaccine and their future prospects in this scenario.

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Sensitivity and Kinetics of an NS1-Based Zika Virus Enzyme-Linked Immunosorbent Assay in Zika Virus-Infected Travelers from Israel, the Czech Republic, Italy, Belgium, Germany, and Chile

Journal of Clinical Microbiology ◽

10.1128/jcm.00346-17 ◽

2017 ◽

Vol 55 (6) ◽

pp. 1894-1901 ◽

Cited By ~ 48

Author(s):

Yaniv Lustig ◽

Hana Zelena ◽

Giulietta Venturi ◽

Marjan Van Esbroeck ◽

Camilla Rothe ◽

...

Keyword(s):

Czech Republic ◽

Immunosorbent Assay ◽

Zika Virus ◽

False Negative ◽

Cross Reactivity ◽

Diagnostic Methods ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibodies ◽

Kinetics Of

ABSTRACT Serological diagnosis of Zika virus is challenging due to high cross-reactivity of Zika virus with other flavivirus antibodies. Recently, a Zika NS1-based enzyme-linked immunosorbent assay (ELISA) was developed and shown to be highly specific for Zika antibody detection; however, sensitivity was evaluated for only a small number of confirmed Zika-infected patients. In this study, we measured the sensitivity and kinetics of Zika IgM and IgG antibodies using the Zika NS1-based ELISA in 105 samples from 63 returning travelers infected with Zika virus (proven by PCR or neutralization assay) from Israel, Czech Republic, Italy, Belgium, Germany, and Chile. Zika virus IgM was detected from 2 to 42 days post-symptom onset (PSO) with an overall sensitivity of 79% in the first month and 68% until 2 months PSO, while IgG antibodies were detected from 5 days to 3 years PSO with 79% sensitivity. Interestingly, significant differences in IgM sensitivity and IgM detection period were observed between Israeli and European/Chilean Zika-infected travelers, adding to the complexity of Zika infection diagnosis and suggesting that other diagnostic methods should be complemented to reduce false-negative results.

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Evaluation of the Truvian Easy Check COVID-19 IgM/IgG Lateral Flow Device for Rapid Anti-SARS-CoV-2 Antibody Detection

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqaa221 ◽

2020 ◽

Author(s):

Clarence W Chan ◽

Sajid Shahul ◽

Cheyenne Coleman ◽

Vera Tesic ◽

Kyle Parker ◽

...

Keyword(s):

Clinical Performance ◽

Reference Sample ◽

Rapid Test ◽

Antibody Test ◽

Cross Reactivity ◽

Antibody Detection ◽

Antibody Assay ◽

Widespread Application ◽

Global Pandemic ◽

Polymerase Chain

Abstract Objectives To evaluate the analytical and clinical performance of the Truvian Easy Check coronavirus disease 2019 (COVID-19) IgM/IgG anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody test. Serologic assays have become increasingly available for surveillance through the Food and Drug Administration emergency use authorization in the ongoing COVID-19 global pandemic. However, widespread application of serologic assays has been curbed by reports of faulty or inaccurate tests. Therefore, rapid COVID-19 antibody tests need to be thoroughly validated prior to their implementation. Methods The Easy Check device was analytically evaluated and its performance was compared with the Roche Elecsys anti-SARS-CoV-2 antibody assay. The test was further characterized for cross-reactivity using sera obtained from patients infected by other viruses. Clinical performance was analyzed with polymerase chain reaction-confirmed samples and a 2015 prepandemic reference sample set. Results The Easy Check device showed excellent analytical performance and compares well with the Roche Elecsys antibody assay, with an overall concordance of 98.6%. Clinical performance showed a sensitivity of 96.6%, a specificity of 98.2%, and an overall accuracy of 98.1%. Conclusions The Easy Check device is a simple, reliable, and rapid test for detection of SARS-CoV-2 seropositivity, and its performance compares favorably against the automated Roche Elecsys antibody assay.

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gAd-globular Head Domain of Adiponectin Increases Fatty Acid Oxidation in Newborn Rabbit Hearts

Journal of Biological Chemistry ◽

10.1074/jbc.m400347200 ◽

2004 ◽

Vol 279 (43) ◽

pp. 44320-44326 ◽

Cited By ~ 33

Author(s):

Arzu Onay-Besikci ◽

Judith Y. Altarejos ◽

Gary D. Lopaschuk

Keyword(s):

Fatty Acid ◽

Fatty Acid Oxidation ◽

Acid Oxidation ◽

Newborn Rabbit ◽

Head Domain ◽

Globular Head

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Evaluation of a Surrogate ELISA- Based Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) cPass Neutralization Antibody Detection Assay and Correlation with IgG Commercial Serology Assays

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2021-0213-sa ◽

2021 ◽

Author(s):

Vijayalakshmi Nandakumar ◽

Tracie Profaizer ◽

Bucky K. Lozier ◽

Marc G. Elgort ◽

Erin T. Larragoite ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Qualitative Agreement ◽

Respiratory Infections ◽

Cross Reactivity ◽

Antibody Detection ◽

Plaque Reduction Assay ◽

Healthy Donors ◽

Detection Assay ◽

Specific Igg ◽

Combined Data

ABSTRACT Context: Emerging evidence shows correlation between the presence of neutralization antibodies (nAbs) and protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently available commercial serology assays lack the ability to specifically identify nAbs. An ELISAbased nAb assay (GenScript cPass neutralization antibody assay) has recently received emergency use authorization from the Food and Drug Administration (FDA). Objective: To evaluate the performance characteristics of this assay and compare and correlate it with the commercial assays that detect SARS-CoV-2 specific IgG. Design: Specimens from SARS-COV-2 infected patients (n=124), healthy donors obtained pre-pandemic (n=100), and from patients with non-COVID (coronavirus disease 2019) respiratory infections (n=92) were analyzed using this assay. Samples with residual volume were also tested on three commercial serology platforms (Abbott, EUROIMMUN, Siemens). Twenty-eight randomly selected specimens from patients with COVID-19 and 10 healthy controls were subjected to a Plaque Reduction Neutralization Test (PRNT). Results: The cPass assay exhibited 96.1% (95% CI, 94.9%–97.3%) sensitivity (at >14 days post- positive PCR), 100% (95% CI, 98.0%–100.0%) specificity and zero cross-reactivity for the presence of non- COVID respiratory infections. When compared to the plaque reduction assay, 97.4% (95% CI, 96.2%–98.5%) qualitative agreement and a positive correlation (R2 =0.76) was observed. Comparison of IgG signals from each of the commercial assays with the nAb results from PRNT/cPass assays displayed >94.7% qualitative agreement and correlations with R2=0.43/0.68 (Abbott), R2=0.57/0.85 (EUROIMMUN) and R2=0.39/0.63 (Siemens), respectively. Conclusions: The combined data support the use of cPass assay for accurate detection of the nAb response. Positive IgG results from commercial assays associated reasonably with nAbs presence and can serve as a substitute.

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

PLoS ONE ◽

10.1371/journal.pone.0248444 ◽

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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Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

10.21203/rs.2.12443/v4 ◽

2020 ◽

Author(s):

Muhammad Ali-ul-Husnain Naqvi ◽

KaLiBiXiaTi Aimulajiang ◽

Muhammad Ali Memon ◽

Muhammad Waqqas Hasan ◽

Sana Zahra Naqvi ◽

...

Keyword(s):

Haemonchus Contortus ◽

Cold Shock ◽

Fasciola Hepatica ◽

Trichinella Spiralis ◽

Early Stage ◽

Cross Reactivity ◽

Antibody Detection ◽

Economic Losses ◽

Diagnostic Potential ◽

Specificity And Sensitivity

Abstract Background : Haemonchus contortus ( H. contortus ) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results : Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21-103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis , Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28μg/well at 37℃ 1h and overnight at 4°C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD 450 ). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion : These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

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