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

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.

Download Full-text

A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

Frontiers in Immunology ◽

10.3389/fimmu.2021.817604 ◽

2022 ◽

Vol 12 ◽

Author(s):

Katharina Radakovics ◽

Claire Battin ◽

Judith Leitner ◽

Sabine Geiselhart ◽

Wolfgang Paster ◽

...

Keyword(s):

Limit Of Detection ◽

Ectopic Expression ◽

High Sensitivity ◽

High Specificity ◽

Reporter System ◽

Specificity And Sensitivity ◽

Highly Sensitive ◽

Definition Of ◽

The Individual ◽

Allergen Extracts

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

Download Full-text

Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

Molecules ◽

10.3390/molecules23092337 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2337 ◽

Cited By ~ 4

Author(s):

Xixia Liu ◽

Qi Lu ◽

Sirui Chen ◽

Fang Wang ◽

Jianjun Hou ◽

...

Keyword(s):

Gold Nanoparticles ◽

High Throughput ◽

High Throughput Sequencing ◽

Limit Of Detection ◽

Retention Rate ◽

Pcr Amplification ◽

Cross Reactivity ◽

Enriched Library ◽

Amplification Curve ◽

Specificity And Sensitivity

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

Download Full-text

Development of Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies

10.21203/rs.3.rs-24925/v1 ◽

2020 ◽

Author(s):

chihai ji ◽

Jingyu Wang ◽

Yuchen Zeng ◽

Haoming Pan ◽

Yingfang Wei ◽

...

Keyword(s):

Pseudorabies Virus ◽

High Specificity ◽

Antibody Detection ◽

Economic Losses ◽

Igg Antibodies ◽

Wild Type ◽

Swine Industry ◽

Igg Antibody ◽

Fluorescent Microsphere ◽

Specificity And Sensitivity

Abstract Background Pseudorabies, also known as Aujezsky’s disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies, therefore, the disease brings huge economic losses to the swine industry. Establishment of a differential diagnosis technique that can distinguish between wild-type infected and vaccinated pigs, and monitor vaccine-induced IgG is crucial for eventual eradication of pseudorabies.Results The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52–238 and gB codons at aa 539–741 were expressed to obtain recombinant PRV gE and gB proteins by pMAL-c5x vector. After purification with Qiagen Ni–NTA agarose affinity, the two proteins were analyzed by SDS-PAGE and immunoblotting assay. Two single fluorescent-microsphere immunoassays (FMIA) were established by coupling two recombinant proteins (gE and gB) with two magnetic microbeads and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection and 95.74% and 96.3% for gB IgG antibody detection by dual-FMIA.Conclusion We provide a new method for monitoring PRV protective antibody in vaccinated pigs and differentiating wild-type-PRV-infected from vaccinated pigs

Download Full-text

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

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822012000100008 ◽

2012 ◽

Vol 45 (1) ◽

pp. 35-44 ◽

Cited By ~ 11

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.

Download Full-text

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

mSphere ◽

10.1128/msphere.00484-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 3

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.

Download Full-text

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

Scientific Reports ◽

10.1038/s41598-020-73491-5 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 6

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.

Download Full-text

Benign Prostate-specific Antigen (BPSA) in Serum Is Increased in Benign Prostate Disease

Clinical Chemistry ◽

10.1373/49.2.253 ◽

2003 ◽

Vol 49 (2) ◽

pp. 253-259 ◽

Cited By ~ 81

Author(s):

Harry J Linton ◽

Leonard S Marks ◽

Lisa S Millar ◽

Christine L Knott ◽

Harry G Rittenhouse ◽

...

Keyword(s):

Prostate Specific Antigen ◽

Limit Of Detection ◽

Specific Antigen ◽

Clinical Information ◽

High Specificity ◽

Wide Distribution ◽

Cross Reactivity ◽

Free Psa ◽

Control Serum ◽

Benign Prostate

Abstract Background: BPSA is a “benign” form of free prostate-specific antigen (PSA) that is increased in prostate transition zone tissues of men with pathologic benign prostatic hyperplasia (BPH). We developed an immunoassay to determine the concentration of BPSA in the serum of men with BPH. Methods: The BPSA antigen was purified by HPLC, and murine monoclonal antibodies were prepared by standard methods. A fluorogenic ELISA was developed with high specificity for BPSA and no cross-reactivity with other forms of PSA. Results: The BPSA immunoassay had a lower limit of detection of 6 ng/L and a cross-reactivity of <1% with all other clipped and nonclipped forms of PSA. The BPSA antibody was specific for the internal Lys182 cleavage site that characterizes BPSA. Biopsy-negative men with a median total PSA of 4.8 μg/L had a median of 0.22 μg/L BPSA, representing 25% of the free PSA in serum. BPSA ranged from 0% to 60% of the free PSA in serum. BPSA in a cohort of cancer serum also comprised 25% of the free PSA. Control serum from women or men without increased PSA had nondetectable BPSA. Conclusions: BPSA is a significant percentage of the free PSA in BPH serum but not in control serum. The presence of prostate cancer does not alter the relative proportions of BPSA in sera with <10 μg/L PSA. BPSA has a wide distribution of concentrations in the serum and may provide clinical information for the study of men with BPH.

Download Full-text

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

10.20944/preprints202005.0188.v2 ◽

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.

Download Full-text

Development of Vasoinhibin-Specific Monoclonal Antibodies

Frontiers in Endocrinology ◽

10.3389/fendo.2021.645085 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nils Müller ◽

Juan Pablo Robles ◽

Magdalena Zamora ◽

Johannes Ebnet ◽

Hülya Markl-Hahn ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Serum Proteins ◽

Limit Of Detection ◽

Sandwich Elisa ◽

Vascular Diseases ◽

High Specificity ◽

Cross Reactivity ◽

Dimensional Structure ◽

Serum Samples ◽

Limit Of Quantitation

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

Download Full-text

THE IMPORTANCE OF BRUCELLIN ALGERIC SKIN TEST FOR DIAGNOSIS OF BOVINE BRUCELLOSIS

ВЕТЕРИНАРСКИ ЖУРНАЛ РЕПУБЛИКЕ СРПСКЕ ◽

10.7251/vetjen1802392v ◽

2019 ◽

Vol 18 (2) ◽

Author(s):

Lejla Velić ◽

Toni Eterović ◽

Silvio Špičić ◽

Željko Cvetnić ◽

Amina Hrković Porobija ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Skin Test ◽

Yersinia Enterocolitica ◽

High Specificity ◽

Cross Reactivity ◽

Bovine Brucellosis ◽

Specific Antibodies ◽

Humoral Immune ◽

Allergic Skin

Infection with Brucella results in the induction of both humoral andcellular immune responses. Humoral immune resposne is based on monitoringthe occurrence of specific antibodies against smooth lipopolysaccharide (S-LPS)of Brucella. However, in cattle, classical serological methods can detect antigenicdeterminants for other types of microorganisms (cross reactivity) such as Escherichiacoli 0:157, Yersinia enterocolitica 0:9, Salmonella urban, Pseudomonas malthopilia andPasteurella. The aim of our work was to determine the immunological responsebased on the use of standardized and purified allergen in which lypopolysaharid hasbeen removed and doesn’t induce humoral immune response. A total of 16 dairycattle previously tested positive using RBT (Rose Bengal test) and CFT (complementfixation test) were tested for confirmation with BST (brucelline skin test) accordingto the instructions of the producer. B. melitensis B115 (Synbiotics BrucellergeneOCB) was used in the test. 14 of 16 cattle reacted with skin thickening >1 mm after72 hours from the application of brucellin. 2 animals with no skin thickening orthickening <1mm also reacted negative in CFT. This outcome can be attributed tocross reactions with other antigens than Brucella that commonly occurs in RoseBengal test.Brucellin allergic skin test is not recommended as a standalone diagnostic toolbecause all infected animals do not react therefore this test cannot be recommendedas a self-sufficient diagnostic test or for the purpose of international trade.However, due to high specificity and adequate sensitivity at the herd level, it can berecommended for the control of herds in areas free of brucellosis.

Download Full-text