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 MixedChlamydia trachomatisPeptide Antigens Provide a Specific and Sensitive Single-Well Colorimetric Enzyme-Linked Immunosorbent Assay for Detection of Human Anti-C. trachomatisAntibodies

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
K. Shamsur Rahman ◽  
Toni Darville ◽  
Harold C. Wiesenfeld ◽  
Sharon L. Hillier ◽  
Bernhard Kaltenboeck
Keyword(s):  
B Cell ◽  
High Specificity ◽  
Cross Reactivity ◽  
Specific Detection ◽  
B Cell Epitopes ◽  
Content Type ◽  
Peptide Antigens ◽  
Specificity And Sensitivity ◽  
Single Well ◽  
Individual Peptide

ABSTRACTSensitive and specific detection of anti-Chlamydia trachomatisantibodies in standard enzyme-linked immunosorbent assays (ELISAs) is compromised by cross-reactivity and poor sensitivity of classicalC.trachomatisantigens. Previously, we discovered 48 strongly reactive peptide antigens ofC. trachomatis-specific B-cell epitopes from 21 immunodominant proteins. By comprehensive individual testing of 11 top-ranked peptide antigens, we found very high sensitivity and specificity for detection of anti-C. trachomatisantibodies in chemiluminescent ELISAs. The current study established a labor-saving colorimetric ELISA by using a mixture of 12 strongly reactiveC. trachomatispeptide antigens (Ctr Mix1) in a single well/serum rather than assaying reactivity to each individual peptide. For performance evaluation, we used a simulated population of 212 anti-C. trachomatisantibody-positive and -negative sera from 125 women with NAAT-confirmed activeC. trachomatisinfection and from 87 healthy women at low risk forC. trachomatisinfection. In comparison to a composite reference standard (CRS) for anti-C. trachomatisantibody status, the Ctr Mix1 IgG ELISA achieved 93.9% sensitivity, significantly superior to the 49% to 79% sensitivities of four commercial anti-C. trachomatisIgG ELISAs, and 98% specificity of all tested assays. Compared to the labor-intensive individual peptide testing, this mixed peptide ELISA retained high specificity with only marginal, ∼5% sensitivity loss. By ROC-AUC, likelihood ratio, and predictive value analyses, the Ctr Mix1 ELISA performed satisfactorily at 10% to 75% prevalence range of anti-C. trachomatisantibodies but significantly better than commercial ELISAs. Thus, the labor-saving mixed peptide colorimetric ELISA format provides simultaneously high specificity and sensitivity for detection of anti-C. trachomatisantibodies.IMPORTANCEFor detection of anti-C. trachomatisantibodies by serological assays, use of classical chlamydial antigens results in high cross-reactivity and poor sensitivity. Previously, we discovered 48 strongly reactive peptide antigens ofC. trachomatis-specific B-cell epitopes from 21 immunodominant proteins, and individual testing and combined scoring of 5 to 11 peptide antigens provided highly sensitive and specific detection of anti-C. trachomatisantibodies in chemiluminescent ELISAs. To simplify this method, this study established a single-well labor-saving colorimetric ELISA using a mixture of 12 strongly reactiveC. trachomatispeptide antigens (Ctr Mix1) for detection of anti-C. trachomatisantibodies. This Ctr Mix1 ELISA (94% sensitivity and 98% specificity) outperformed 4 commercial ELISAs (49% to 79% sensitivity and 98% specificity). This ELISA can be easily implemented and commercialized, with convenient setup for use in nonspecialized laboratories. Thus, this mixed peptide assay with superior specificity and sensitivity will improve serodiagnosis ofC. trachomatisinfections.

scholarly journals In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

2012 ◽  
Vol 45 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Lilian da Silva Santos ◽  
Rosália Morais Torres ◽  
Girley Francisco Machado-de-Assis ◽  
Maria Terezinha Bahia ◽  
Helen Rodrigues Martins ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
Chagas Disease ◽  
Clinical Status ◽  
High Specificity ◽  
Disease Diagnosis ◽  
Cross Reactivity ◽  
Serological Method ◽  
Serum Dilution ◽  
Specificity And Sensitivity ◽  
American Tegumentary Leishmaniasis

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

scholarly journals SARS-CoV-2 S1 and N-based serological assays reveal rapid seroconversion and induction of specific antibody response in COVID-19 patients

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sharif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamri ◽  
...  
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Human Coronaviruses

Abstract As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

scholarly journals SARS-CoV-2 S1 and N-Based Serological Assays Reveal Rapid Seroconversion and Induction of Specific Antibody Response in COVID-19 Patients

2020 ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sherif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamri ◽  
...  
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Human Coronaviruses

As the coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses. The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

Production, Characterization of a Monoclonal Antibody against Aristolochic Acid-II and Development of its Assay System

2008 ◽  
Vol 36 (02) ◽  
pp. 425-436 ◽  
Author(s):  
Min Tian ◽  
Hiroyuki Tanaka ◽  
Ming-Ying Shang ◽  
Seiko Karashima ◽  
Zhi Chao ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Aristolochic Acid ◽  
High Specificity ◽  
Myeloma Cell ◽  
Cross Reactivity ◽  
Hybridoma Cells ◽  
Dilution Method ◽  
Elisa Method ◽  
Chemical Structures ◽  
Specificity And Sensitivity

Aristolochic acid-II (AA-II) conjugated with bovine serum albumin (BSA) was used as an antigen for immunizing BALB/c mice. Isolated splenocytes from the immunized mice were fused with an aminopterin-sensitive mouse myeloma cell line, SP2/0-Ag14, to produce hybridoma cells that secreted a monoclonal antibody (MAb) against AA-II. The selected hybridoma was subsequently cloned by limited dilution method. For MAb, the isotype and an estimated dissociation constant ( K D ) of the MAb were determined. The MAb was used to establish an ELISA method. Accuracy and variation assays, as well as determinations of the specificity and sensitivity, were also carried out and the linear range was 0.19–13 μg/ml. The anti-AA-II MAb showed a very high specificity for AA-II and had low cross-reactivities against the other aristolochic acid (AAs) (CR: AA-I, 3.4%; AA-VIIa, 0.86%) or aristololactam-I (AL-I) (CR < 0.07%) except AA-IIIa which has 17% of cross activity. Anti-AA-II MAb also showed negligible cross-reactivity (< 0.5%) toward other natural compounds with different chemical structures including barbaloin, sennoside A, rutin, glycyrrhizin, caffeic acid etc. This is the first time that an ELISA method was successfully established for the application of anti-AA-II MAb.

scholarly journals Development and validation of an enzyme immunoassay for detection and quantification of SARS-CoV-2 salivary IgA and IgG

2021 ◽  
Author(s):  
Veronica Costantini ◽  
Kenny Nguyen ◽  
Zoe Lyski ◽  
Shannon Novosad ◽  
Ana C Bardossy ◽  
...  
Keyword(s):  
Immune Response ◽  
Enzyme Immunoassay ◽  
Limit Of Detection ◽  
Natural Infection ◽  
High Specificity ◽  
Cross Reactivity ◽  
Igg Antibodies ◽  
Antibody Isotype ◽  
Salivary Iga ◽  
Specificity And Sensitivity

Oral fluids offer a non-invasive sampling method for the detection of antibodies. Quantification of IgA and IgG antibodies in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG antibodies against the prefusion-stabilized form of the SARS-CoV-2 spike protein. Normalization against total antibody isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 pre-pandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA: 95.5%; IgG: 89.7%) without compromising specificity (IgA: 99%; IgG: 97%). No cross reactivity with seasonal coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/mL and 0.30 ng/mL, respectively. Salivary IgA and IgG antibodies were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary antibody titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 weeks in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

scholarly journals Multiple Indirect ELISAs for Serological Detection of SARS-CoV-2 Antibodies

2020 ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sherif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamari ◽  
...  
Keyword(s):  
Antibody Response ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
Specificity And Sensitivity ◽  
Novel Coronavirus

As the coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus SARS-CoV-2, continues to spread rapidly around the world, there is an urgent need for validated serological assays to evaluate viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological tests to study the antibody response in COVID-19 patients. In order to validate the assays, we determined the cut-off values, sensitivity and specificity of the developed assays using sera collected from COVID-19 patients in Saudi Arabia at different time points after disease onset, as well as sera that are seropositive to other human CoVs; namely MERS-CoV, hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1. The SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N) ELISAs that we developed here not only showed high specificity and sensitivity, but also did not show any cross-reactivity with other CoVs. We also showed that all RT-PCR confirmed COVID-19 patients included in our study developed both virus specific IgM and IgG as early as one week after the onset of disease. The availability of these validated assays will enable us to determine the nature and duration of the antibody response mounted in response to SARS-CoV-2 infection. It will also allow conducting large-scale epidemiological studies to determine evidence of previous exposure to the virus and assess the true extent of virus spread within communities.

scholarly journals Production and Characterization of a Polyclonal Antibody of Anti-rLipL21-IgG againstLeptospirafor Early Detection of Acute Leptospirosis

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Arivudainambi Seenichamy ◽  
Abdul Rani Bahaman ◽  
Abdul Rahim Mutalib ◽  
Siti Khairani-Bejo
Keyword(s):  
Polyclonal Antibody ◽  
Diagnostic Marker ◽  
Bacterial Species ◽  
High Specificity ◽  
Cross Reactivity ◽  
Zoonotic Diseases ◽  
Igg Antibody ◽  
E Coli ◽  
Specificity And Sensitivity

Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world. LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen. It is widely considered as a diagnostic marker for leptospirosis. In this study, we evaluated the serodiagnostic potential of LipL21 protein ofLeptospira interrogansserovar Pomona. We have successfully amplified, cloned, and expressed LipL21 inE. coliand evaluated its specificity by immunoblotting. Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibody. Characterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay. Only sera from leptospirosis patients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction in immunoblotting. We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species. Results observed showed that anti-rLipL21-IgG antibody has high specificity and sensitivity to leptospires. The findings indicated that the antibody could be used in a diagnostic assay for detection of leptospires or their proteins in the early phase of infection.

scholarly journals SELEX-Based Direct Enzyme-Linked Aptamer Assay (DELAA) for Diagnosis of Toxoplasmosis by Detection of SAG1 Protein in Mice and Humans

2021 ◽  
Author(s):  
Jilong Shen ◽  
Wei Wang ◽  
Yuanhong Xu ◽  
Xuhang Shen ◽  
Wen Cui ◽  
...  
Keyword(s):  
Acute Infection ◽  
Pcr Amplification ◽  
High Specificity ◽  
Laboratory Diagnosis ◽  
Specificity And Sensitivity ◽  
Recognition Probe ◽  
Human Sera ◽  
Aptamer Assay ◽  
Oligonucleotide Library ◽  
Major Surface

Abstract Background: Toxoplasma gondii is a single-celled parasite commonly found in mammals. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma-derived DNAs due to the presence of tissue dwelling duplicating tachyzoites, or quiescent cysts in latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key marker for laboratory diagnosis. However, there are no methods available yet for SAG1 detection using aptamer-based technology.Methods: Recombinant truncated SAG1（r-SAG1）of Toxoplasma WH3 strain (type Chinese 1) was prokaryotically expressed and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamers which target the r-SAG1, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The specific aptamer-2 was screened out and used in direct enzyme-linked aptamer assay (DELAA) for detection of native SAG1 obtained from tachyzoite lysates (n-SAG1), mouse sera of acute infection, and human sera that had been verified to be positive for Toxoplasma DNAs by PCR amplification. Results: The soluble r-SAG1 protein was obtained from E.coli lysates by purification and identification with immunoblotting, and then labelled with biotin. The selected aptamers were amplified by PCR, followed by DNA sequencing. The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals in the four aptamer candidates, has a high specificity and sensitivity when used in detection of n-SAG1 in the sera of humans when compared with the commercial kit of ELISA for Toxoplasma circulating antigen test.Conclusions: A new direct enzyme-linked aptamer assay (DELAA), with aptamer-2 as the recognition probe, was developed for detection of native SAG1 protein of Toxoplasma. With increased sensitivity and specificity, stability, easy and cheap preparation, the aptamer-based technology is considered as a efficient method for the diagnosis of active and reactivated toxoplasmosis.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay Method for the Detection of Rhein in Rheum officinale

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Min Chen ◽  
Tie-Gui Nan ◽  
Jie Xin ◽  
Li Cui ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Quality Control ◽  
Immunosorbent Assay ◽  
High Specificity ◽  
Cross Reactivity ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Simple Method ◽  
Specificity And Sensitivity ◽  
Important Quality ◽  
Aloe Emodin

Rhein is an important quality-control marker of Rheum officinale. The aim of this study was to develop an indirect competitive enzyme-linked immunosorbent assay (icELISA) for rhein detection, which acts as a powerful tool for quality control and proper usage of Rheum officinale. First, a specific and sensitive monoclonal antibody (mAb) against rhein was produced from a stable hybridoma cell line, 1F8, generated by the fusion of mouse myeloma sp2/0 with spleen cells obtained from a Bal b/c mouse immunized with rhein-BSA. Then, an icELISA method was developed with an IC50 value and working range of 0.05 μg L−1 and 0.02–0.11 μg L−1, respectively. The icELISA revealed high assay specificity, since it only had a relatively high cross reactivity with aloe-emodin (27%) and almost no cross reactivity with any other anthraquinones (<1%). When spiked with 0.2–2 mg kg−1 of rhein, the recoveries ranged from 84.19% to 102.90%. Finally, icELISA was used to detect rhein contents of Rheum officinale collected from different regions, and the results corresponded well with those of HPLC. Overall, the developed icELISA with high specificity and sensitivity provided a rapid and simple method for rhein detection, and it may be a powerful tool for quality control and proper usage of Rheum officinale.

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