scholarly journals Development and Evaluation of a Multiplex Microsphere Assay for Quantitation of IgG and IgA Antibodies against Neisseria meningitidis Serogroup A, C, W, and Y Polysaccharides

2015 ◽  
Vol 22 (7) ◽  
pp. 697-705 ◽  
Author(s):  
Guro K. Bårnes ◽  
Paul A. Kristiansen ◽  
Dominique A. Caugant ◽  
Lisbeth M. Næss
Keyword(s):  
Coefficient Of Variation ◽  
High Specificity ◽  
Sample Volume ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Standard Curve ◽  
Iga Antibodies ◽  
Repeated Analysis ◽  
Reference Serum ◽  
Specific Igg

ABSTRACTWe developed and evaluated a rapid and simple multiplex microsphere assay for the quantification of specific IgG and IgA antibodies against meningococcal serogroup A, C, W, and Y capsular polysaccharides in serum and saliva. Meningococcal polysaccharides were conjugated to distinct magnetic carboxylated microspheres, and the performance of the assay was assessed using the CDC1992 standard meningococcal reference serum and a panel of serum and saliva samples. The standard curve was linear over an eight 3-fold dilution range in the IgG assay and a seven 3-fold dilution range in the IgA assay. No cross-reactivity was discovered, and the assay showed high specificity with ≥91% homologous inhibition and ≤11% heterologous inhibition for all serogroups and immunoglobulin classes. Lower limits of detections were ≤280 pg/ml for IgG and ≤920 pg/ml for IgA antibodies. The assay was reproducible, with a mean coefficient of variation of ≤5% for intra-assay duplicates, a mean coefficient of variation of ≤20% for interassay repeated analysis with different conjugations of microspheres, and a mean coefficient of variation within 25.8% for interoperator variation. The assay showed good correlation to the standard meningococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies. This multiplex assay is robust and reliable and requires less sample volume, and less time and workload are needed than for ELISA, making this method highly relevant for serological and salivary investigations on the effect of meningococcal vaccines and for immunosurveillance studies.

Development and Characterization of Anti-Estradiol Polyclonal Antibody

2012 ◽  
Vol 461 ◽  
pp. 67-70 ◽  
Author(s):  
Chao Ying Li ◽  
Jin Qing Jiang
Keyword(s):  
Polyclonal Antibody ◽  
Dynamic Range ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Uv Absorbance ◽  
Standard Curve ◽  
Ic50 Value ◽  
New Zealand White Rabbits

This paper reports an indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody (pAb) for estradiol (E2) residues. After derivation, E2 haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method, and New Zealand white rabbits were immunized to produce anti-E2 pAb. The conjugation ratio of E2-BSA was proved to be 18.6:1 by an UV absorbance method. Based on the square matrix titration, an icELISA standard curve was developed. The dynamic range was from 0.16 to 128 ng/mL, with LOD and IC50 value of 0.08 ng/mL and 3.76 ng/mL, respectively. Except for a little cross-reactivity (16.2%) to estrone, this assay showed negligible cross-reactivity to other analogues tested. The results suggest that the produced anti-E2 pAb could be used to develop an icELISA method for the determination of E2 residues in animal-originally products.

scholarly journals Assignment of Weight-Based Antibody Units for Seven Additional Serotypes to a Human Pneumococcal Standard Reference Serum, 007sp

2015 ◽  
Vol 22 (11) ◽  
pp. 1154-1159 ◽  
Author(s):  
D. Goldblatt ◽  
C. Y. Tan ◽  
P. Burbidge ◽  
S. McElhiney ◽  
L. McLaughlin ◽  
...  
Keyword(s):  
Polysaccharide Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reference Standard ◽  
Concordance Correlation ◽  
Clinical Protocol ◽  
Serum Samples ◽  
Reference Serum ◽  
Specific Igg ◽  
Standard Serum ◽  
Adult Volunteers

ABSTRACTThe pneumococcal enzyme-linked immunosorbent assay (ELISA) reference standard serum, lot 89SF, has been in use since 1990 and was replaced in 2013 with a new reference standard, 007sp, that is projected to be available for the next 25 years. 007sp was generated under an FDA-approved clinical protocol; 278 adult volunteers were immunized with the 23-valent unconjugated polysaccharide vaccine Pneumovax II, and a unit of blood was obtained twice from each immunized subject within 120 days following immunization. Pooled serum was prepared from the plasma of 262 subjects, filled at 6 ml per vial, and lyophilized. Five independent laboratories participated in bridging the serotype-specific IgG assignments for 89SF to the new reference standard, 007sp, to establish equivalent reference values for 13 pneumococcal capsular serotypes (1,3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) by using the WHO reference ELISA. In a second study involving three laboratories, a similar protocol was used to assign weight-based IgG concentrations in micrograms per ml to 007sp of seven serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) also present in the 23-valent pneumococcal unconjugated polysaccharide vaccine. In addition, the IgG assignments for a 12-member WHO quality control (QC) serum panel were also extended to cover these seven serotypes. Agreement was excellent, with a concordance correlation coefficient (rc) of >0.996 when each laboratory was compared to the assigned values for the 12 WHO QC serum samples. There are four remaining pneumococcal serotypes (2, 9N, 17F, and 20) found in Pneumovax II for which IgG assignments exist for 89SF and remain to be bridged.

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 Immunoglobulins G, M, and A against Sporothrix schenckii Exoantigens in Patients with Sporotrichosis before and during Treatment with Itraconazole

2007 ◽  
Vol 14 (9) ◽  
pp. 1149-1157 ◽  
Author(s):  
Rodrigo Almeida-Paes ◽  
Monique Amorim Pimenta ◽  
Paulo Cezar F. Monteiro ◽  
Joshua D. Nosanchuk ◽  
Rosely Maria Zancopé-Oliveira
Keyword(s):  
Sporothrix Schenckii ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Number Of Patients ◽  
Highly Sensitive ◽  
Iga Antibodies ◽  
Itraconazole Treatment ◽  
Few Data ◽  
Humoral Responses

ABSTRACT Sporotrichosis is an important subcutaneous mycosis, with an increasing worldwide incidence. However, few data are available regarding the immunological aspects of Sporothrix schenckii infection, particularly the humoral responses to the fungus. In this study we measured immunoglobulin G (IgG), IgM, and IgA in sera from 41 patients with sporotrichosis before antifungal treatment and from another 35 patients with sporotrichosis during itraconazole treatment by using a recently described S. schenckii exoantigen enzyme-linked immunosorbent assay (ELISA). More than 95% of patients had detectable IgA antibodies, and more than 85% had IgM and IgG antibodies before treatment. The number of patients with IgG antibodies increased to 91% during treatment. Conversely, significantly fewer samples from treated patients were positive for IgM (71%) and IgA (89%). Overall, 78% of patients had detectable levels of all isotypes tested at diagnosis, and this percentage dropped to 62.9% in patients receiving itraconazole. Testing of all three isotypes improved the sensitivity; at least two isotypes were detected in 93% of patients before and 89% after treatment. The reactivity of 94 sera from patients with other diseases and healthy individuals was also tested. Cross-reactivity occurred in 33% of the heterologous sera. Most of them were positive only in one isotype, 8.5% were positive for at least two isotypes, and only one serum (1.1%) was positive for the three isotypes. Antibodies produced during S. schenckii infection are diverse, and we demonstrate that an exoantigen ELISA for the detection of combinations of IgA, IgG, and IgM antibodies is a highly sensitive and specific diagnostic assay for sporotrichosis.

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.

scholarly journals SARS-CoV-2 antigens expressed in plants detect antibody responses in COVID-19 patients

2020 ◽  
Author(s):  
Mohau S. Makatsa ◽  
Marius B. Tincho ◽  
Jerome M. Wendoh ◽  
Sherazaan D. Ismail ◽  
Rofhiwa Nesamari ◽  
...  
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Viral Proteins ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Robust Detection ◽  
Recombinant Plant ◽  
Specific Igg ◽  
African Patients ◽  
Global Threat

AbstractBackgroundThe SARS-CoV-2 pandemic has swept the world and poses a significant global threat to lives and livelihoods, with over 16 million confirmed cases and at least 650 000 deaths from COVID-19 in the first 7 months of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings.MethodsWe established an indirect enzyme-linked immunosorbent assay (ELISA) using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n=77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of six weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n=58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva.ResultsWe demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and OD values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers.ConclusionsWe demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

scholarly journals The optimization of an ultra-sensitive single molecule assay for the multiplexed detection of SARS-CoV-2 antigens

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S145-S145
Author(s):  
Y Senussi ◽  
Z N Swank ◽  
D R Walt
Keyword(s):  
Single Molecule ◽  
Respiratory Infections ◽  
Sample Volume ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
N Protein ◽  
Children And Young Adults ◽  
Inflammatory Syndrome

Abstract Introduction/Objective SARS-CoV-2 antigens, including the nucleocapsid (N) protein, spike protein, and its S1 subunit have served as key biomarkers for research and diagnostic purposes. We previously developed quantitative single molecule array (Simoa) assays to measure the concentration of spike, S1 subunit and N protein in plasma samples with femtomolar limits of detection. We aimed to test antibodies that were not available early in the pandemic, reduce assay cross-reactivity, develop a multiplexed assay for spike, S1, and N protein in order to minimize the sample volume needed. Methods/Case Report Using the Simoa platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 17 S1 subunit and spike antibodies for a total of 130 antibody-pair combinations, we performed dilution linearity experiments to determine the ideal dilution factor, spike and recovery experiments, tested the assay using S1 subunit from other human coronavirus HKV1, NL63, and 229E, pre-pandemic plasma samples from patients that were sick with viral or bacterial respiratory infections. We then used the best antibody pairs to measure S1 and spike in plasma samples collected from patients with severe SARS-CoV-2. Lastly, we conjugated the best-performing capture antibodies for spike, S1 and N to beads labeled with different fluorophores to test if the assay for all three antigens could be multiplexed. Results (if a Case Study enter NA) We observed no cross-reactivity with S1 from other coronavirus strains, no detection of S1 or spike in a cohort of 30 pre-pandemic samples and successfully developed a multiplexed assay for the detection of spike, S1, and N protein, enabling us to use 50% less sample volume. Conclusion Reduction of necessary sample volume is important for studies involving multisystem inflammatory syndrome in children (MIS-C), and possible adverse effects of SARS-CoV-2 vaccinations on children and young adults. An improved assay with minimal cross-reactivity will also be useful to study individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

scholarly journals Seroepidemiology ofHelicobacter pyloriinfection in vegans and meat-eaters

1992 ◽  
Vol 108 (3) ◽  
pp. 457-462 ◽  
Author(s):  
M. J. Webberley ◽  
J. M. Webberley ◽  
D. G. Newell ◽  
P. Lowe ◽  
V. Melikian
Keyword(s):  
Helicobacter Pylori ◽  
Risk Factor ◽  
Campylobacter Jejuni ◽  
Meat Consumption ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Specific Igg ◽  
H Pylori ◽  
Age And Sex

SUMMARYAn enzyme-linked immunosorbent assay has been used to diagnose serologically the prevalence ofHelicobacter pyloriinfection in Asian life-long vegans. There was no difference in the seropositivity between these individuals and a group of age-and sex-matched Asian meat-eaters, indicating the meat consumption is not a risk factor forH. pyloriinfection. However, both Asian groups had a higher prevalence of infection than age- and sex-matched Caucasian meat-eaters. Additionally, the Asian individuals had a wider range of specific IgG antibody concentrations than the Caucasians. This did not appear to be due to antigenic cross-reactivity betweenH. pyloriandCampylobacter jejuni. The significance of these observations to the establishment of cut-off levels for the serodiagnosis of certain ethnic groups is discussed.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay Based on the Multiepitope Peptide for the Synchronous Detection of Staphylococcal Enterotoxin A and G Proteins in Milk

2015 ◽  
Vol 78 (2) ◽  
pp. 362-369 ◽  
Author(s):  
MINGYAN LIANG ◽  
TINGTING ZHANG ◽  
XUELAN LIU ◽  
YANAN FAN ◽  
SHENGLIN XIA ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Signal To Noise Ratio ◽  
Food Poisoning ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Synchronous Detection ◽  
Standard Curve ◽  
Stability And Accuracy

Staphylococcal food poisoning (SFP), one of the most common foodborne diseases, results from ingestion of staphylococcal enterotoxins (SEs) in foods. In our previous studies, we found that SEA and SEG were two predominant SE proteins produced by milk-acquired S. aureus isolates. Here, a tandemly arranged multiepitope peptide (named SEAGepis) was designed with six linear B-cell epitopes derived from SEA or SEG and was heterologously expressed. The SEAGepis-specific antibody was prepared by immunizing rabbit with rSEAGepis. Then, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on rSEAGepis and the corresponding antibody was developed to simultaneously detect SEA and SEG. Under the optimized conditions, the ic-ELISA standard curve for rSEAGepis was constructed in the concentration range of 0.5 to 512 ng/ml, and the average coefficients of variation of intra-and interassay were 4.28 and 5.61% during six standard concentrations. The average half-maximal inhibitory concentration was 5.07 ng/ml, and the limit of detection at a signal-to-noise ratio of 3 was 0.52 ng/ml. The anti-rSEAGepis antibody displayed over 90% cross-reactivity with SEA and SEG but less than 0.5% cross-reactivity with other enterotoxins. Artificially contaminated milk with different concentrations of rSEAGepis, SEA, and SEG was detected by the established ic-ELISA; the recoveries of rSEAGepis, SEA, and SEG were 91.1 to 157.5%, 90.3 to 134.5%, and 89.1 to 117.5%, respectively, with a coefficient of variation below 12%. These results demonstrated that the newly established ic-ELISA possessed high sensitivity, specificity, stability, and accuracy and could potentially be a useful analytical method for synchronous detection of SEA and SEG in milk.

Sign in / Sign up

Export Citation Format

Share Document