Enzyme-linked immunosorbent assay, immunoblot analysis and RAST fluoroimmunoassay analysis of serum responses against crude larval antigens of Anisakis simplex in a Spanish random population

1996 ◽  
Vol 70 (4) ◽  
pp. 281-289 ◽  
Author(s):  
L. García-Palacios ◽  
M.L. González ◽  
M.I. Esteban ◽  
E. Mirabent ◽  
M.J. Perteguer ◽  
...  
Keyword(s):  
Optical Density ◽  
Immunoblot Analysis ◽  
Specific Ige ◽  
Negative Control ◽  
Anisakis Simplex ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Sera ◽  
Specific Ige Antibodies ◽  
Reference Serum ◽  
Diagnostic Criterion

AbstractThe results obtained in a study of seroprevalence by means of ELISA and immunoblot with crude larval extracts of Anisakis simplex using 1008 human sera from Spanish people showing no clinical suspicion of anisakidosis are given. For the evaluation of the results obtained by ELISA the Diagnostic Index (DI) was used, as the ratio between the optical density resulting from the test serum and the optical density of the negative control. Forty-seven sera showed DIs between 1.5 and 2, and 14 sera were greater than 2. After comparison of the immunoblot analysis with the immunorecognition pattern of a human anisakidosis reference serum, a diagnostic criterion could be established for those sera that, at a 1/100 dilution, showed a DI by ELISA greater than 1.5. Seven of 14 selected sera with DIs in ELISA higher than 1.3 showed anti-Anisakis specific IgE antibodies by RAST fluoroimmunoassay.

scholarly journals Effect of Antigen Coating Conditions on Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibody to Neisseria meningitidis Serogroup Y and W135 Capsular Polysaccharide Antigens in Serum

2003 ◽  
Vol 10 (6) ◽  
pp. 1136-1140 ◽  
Author(s):  
Peter C. Giardina ◽  
Renee E. Evans ◽  
Daniel J. Sikkema ◽  
Dace Madore ◽  
Stephen W. Hildreth
Keyword(s):  
Immunoglobulin G ◽  
Capsular Polysaccharide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Meningococcal Vaccine ◽  
Igg Antibody ◽  
Human Sera ◽  
Polysaccharide Antigens ◽  
Reference Serum ◽  
Adult Volunteers ◽  
Assay Conditions

ABSTRACT Human sera collected from 28 consenting adult volunteers were used to define assay conditions for meningococcal vaccine clinical trial serology. Immunoassay parameters were optimized with these test sera and the standard reference serum, CDC1992. Coating conditions for serogroup Y and W135 polysaccharide antigens were found to influence the predicted serum immunoglobulin G (IgG) antibody concentrations. Sera that displayed IgG antibody binding profiles most unlike that of CDC1992 were influenced the most by coating conditions. Our results suggest that presentation of specific epitopes is influenced by antigen-coating concentrations for serogroup Y and W135 polysaccharides.

scholarly journals Colonic Bacteria Express an Ulcerative Colitis pANCA-Related Protein Epitope

2000 ◽  
Vol 68 (3) ◽  
pp. 1542-1548 ◽  
Author(s):  
O. Cohavy ◽  
D. Bruckner ◽  
L. K. Gordon ◽  
R. Misra ◽  
B. Wei ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Monoclonal Antibody ◽  
Immune Response ◽  
Disease Process ◽  
Immunoblot Analysis ◽  
Culture Conditions ◽  
Enzyme Linked Immunosorbent Assay ◽  
E Coli ◽  
Colonic Bacteria ◽  
Human Sera

ABSTRACT Bacteria are a suspected pathogenic factor in inflammatory bowel disease, but the identity of the relevant microbial species remains unresolved. The pANCA autoantibody is associated with most cases of ulcerative colitis (UC) and hence reflects an immune response associated with the disease process. This study addresses the hypothesis that pANCA identifies an antigen(s) expressed by bacteria resident in the human colonic mucosa. Libraries of colonic bacteria were generated using aerobic and anaerobic microbiologic culture conditions, and bacterial pools and clonal isolates were evaluated for cross-reactive antigens by immunoblot analysis using the pANCA monoclonal antibody Fab 5-3. Two major species of proteins immunoreactive to pANCA monoclonal antibodies were detected in bacteria from the anaerobic libraries. Colony isolates of the expressing bacteria were identified as Bacteroides caccae andEscherichia coli. Isolation and partial sequencing of theB. caccae antigen identified a 100-kDa protein without database homologous sequences. The E. coli protein was biochemically and genetically identified as the outer membrane porin OmpC. Enzyme-linked immunosorbent assay with human sera demonstrated elevated immunoglobulin G anti-OmpC in UC patients compared to healthy controls. These findings demonstrate that a pANCA monoclonal antibody detects a recurrent protein epitope expressed by colonic bacteria and implicates colonic bacterial proteins as a target of the disease-associated immune response.

scholarly journals Efficacy of Recombinant S1 Protein Expressed in Pichia pastoris in Chicken & Using for Detection Titer Antibodies against Avian Infectious Bronchitis by ELISA

2019 ◽  
pp. 1-9
Author(s):  
Mahdi Ahmadi ◽  
Khosrow Aghayipour ◽  
Arash Ghalyanchi Langroudi ◽  
Hossein Godarzi ◽  
Masood Hashemzadeh
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Protein ◽  
Optical Density ◽  
Infectious Bronchitis Virus ◽  
Sodium Dodecyl ◽  
Optical Gain ◽  
Positive Control ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infectious Bronchitis

Infectious bronchitis (IB) is an acute, extremely contagious upper respiratory disease in chickens. The objective of this study was expressed and using of recombinant S1 Protein serotype 793/B expressed in pichia pastoris in order to serodiagnosis against avian infectious bronchitis antibody. The complete S1 gene (1623bp) was cloned in PTZ57 plasmid and transferred to E. coli (Escherichia coli)-XL1blue bacterium. Next cloned to pPICZB vectors and transferred to p. pastoris(Km71H). Produced protein were visualized by SDS (sodium dodecyl sulfate) PAGE gel that was about with 62KDa. Finally, the S1 recombinant protein was used to coat 96 well plate to recognize antibodies in sera against live Infectious bronchitis virus (IBV 4/91). As a result, this recombinant S1 protein Antigen can be used in an enzyme-linked immunosorbent assay (ELISA) for recognizing antibody titer against IBV. This recombinant protein was evaluated by Elisa, using a panel of field sera for known IBV titer That in the commercial kits, the optical gain of positive control is 0/741 ± 0/007 and the negative control is 0/167 ± 0/002, next the average Optical Density (OD) in the three farms were 2.585, 2.001, 1.657, resulting to the S/P ratio of was 4.212, 2.886, 2.484, respectively. The results of our effort for positive control and the negative control are 0/9 ± 0/010, 0/067 ± 0/008 respectively. The mean Optical Density of sera in 3 Flocks were showed 3.390, 2.737, 2.921, and the ratio of S/P 4. 01, 3.242 and 3.46. These results showed that our outcomes are neared to Biochek kit. The results also showed that recombinant protein S1 was able to identify different kinds of infectious bronchitis virus serotypes in poultry. It can be used for commercial ELISA kit.

Positive associations between infections ofToxoplasma gondiiand seropositivity withAnisakis simplexin human patients suffering from chronic urticaria

2014 ◽  
Vol 89 (6) ◽  
pp. 707-713 ◽  
Author(s):  
V. Fernández-Fígares ◽  
M. Rodero ◽  
A. Valls ◽  
C. De Frutos ◽  
A. Daschner ◽  
...  
Keyword(s):  
Chronic Urticaria ◽  
Odds Ratio ◽  
Specific Ige ◽  
Anisakis Simplex ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Atopic Sensitization ◽  
Prick Test ◽  
Food Borne ◽  
The Relationship

AbstractToxoplasma gondiiis a food-borne and orofecal microorganism which produces chronic infection, and attempts have been made to prove its negative association with atopy in the context of the hygiene hypothesis.Anisakis simplexis a fish parasite associated with chronic urticaria (CU) in endemic regions. We analysed the relationship between both infectious agents in CU. We included 42 patients with chronic urticaria (18 patients with CU associated withA. simplexsensitization and 24 not sensitized CU patients). Patients were assessed for atopy by a skin prick test (SPT) against common aeroallergens and for respiratory symptoms.Anisakis simplexsensitization was assessed by SPT and specific IgE by CAP fluoro-enzyme immunoassay (CAP-FEIA). Anti-T. gondiiIgG levels were measured by enzyme-linked immunosorbent assay (ELISA). CU patients were analysed with respect toT. gondiiseropositivity,A. simplexsensitization, atopy and immigrant status. The seroprevalence ofT. gondiiwas 40.5% in CU patients and 42.1% in the control group. Immigrants were more frequently infected byT. gondii(41.2% versus 12%;P =0.036). Anti-T. gondiiIgG antibodies were associated with pastA. simplexparasitism (odds ratio 6.73;P =0.03) and independently with atopic sensitization (odds ratio 5.85;P =0.04). In CU patients,T. gondiihas no protective effect on atopic sensitization orA. simplexsensitization.

scholarly journals Optimization of a Human Papillomavirus-Specific Enzyme-Linked Immunosorbent Assay

2002 ◽  
Vol 9 (3) ◽  
pp. 577-582 ◽  
Author(s):  
Kevin L. Karem ◽  
Alysia C. Poon ◽  
Cynthia Bierl ◽  
Rosane Nisenbaum ◽  
Elizabeth Unger
Keyword(s):  
Human Papillomavirus ◽  
Immunosorbent Assay ◽  
Critical Evaluation ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Optimal Cutoff ◽  
Optimal Method ◽  
Specific Immunoglobulin ◽  
Human Sera ◽  
Evaluation Techniques

ABSTRACT A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA.

scholarly journals Diagnosing Human Anisakiasis: Recombinant Ani s 1 and Ani s 7 Allergens versus the UniCAP 100 Fluorescence Enzyme Immunoassay

2010 ◽  
Vol 17 (4) ◽  
pp. 496-502 ◽  
Author(s):  
A. M. Anadón ◽  
E. Rodríguez ◽  
M. T. Gárate ◽  
C. Cuéllar ◽  
F. Romarís ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Immunosorbent Assay ◽  
Specific Ige ◽  
Enzyme Linked Immunosorbent Assay ◽  
Recombinant Allergens ◽  
Ige Antibodies ◽  
Specificity And Sensitivity ◽  
Specific Ige Antibodies

ABSTRACT Commercially available serological methods for serodiagnosis of human anisakiasis either are poorly specific or do not include some of the most relevant Anisakis allergens. The use of selected recombinant allergens may improve serodiagnosis. To compare the diagnostic and clinical values of enzyme-linked immunosorbent assay (ELISA) methods based on Ani s 1 and Ani s 7 recombinant allergens and of the UniCAP 100 fluorescence enzyme immunoassay (CAP FEIA) system, we tested sera from 495 allergic and 25 non-food-related allergic patients. The decay in specific IgE antibodies in serum was also investigated in 15 positive patients over a period of 6 to 38 months. Considering sera that tested positive by either Ani s 1 or Ani s 7 ELISA, the CAP FEIA classified 25% of sera as falsely positive, mainly in the group of patients with the lowest levels of anti-Anisakis IgE antibodies, and 1.28% of positive sera as falsely negative. Considering allergens individually, the overall sensitivities of Ani s 7 ELISA and Ani s 1 ELISA were 94% and 61%, respectively. The results also showed that anti-Anisakis IgE antibodies can be detected in serum for longer with Ani s 1 ELISA than with Ani s 7 ELISA and CAP FEIA (P < 0.01). Our findings suggest that ELISA methods with Ani s 7 and Ani s 1 allergens as targets of IgE antibodies are currently the best option for serodiagnosis of human anisakiasis, combining specificity and sensitivity. The different persistence of anti-Ani s 1 and anti-Ani s 7 antibodies in serum may help clinicians to distinguish between recent and old Anisakis infections.

scholarly journals Blocking ELISA to Distinguish Pseudorabies Virus-Infected Pigs from Those Vaccinated with a Glycoprotein gIII Deletion Mutant

1990 ◽  
Vol 2 (1) ◽  
pp. 14-23 ◽  
Author(s):  
Saul Kit ◽  
Yukikazu Awaya ◽  
Haruki Otsuka ◽  
Malon Kit
Keyword(s):  
Optical Density ◽  
Deletion Mutant ◽  
Pseudorabies Virus ◽  
Standard Dose ◽  
False Negative ◽  
Elisa Test ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blocking Elisa ◽  
Test Kit

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV)-infected pigs from those immunized with a glycoprotein g92(gIII) deletion mutant, PRV(dlg92dltk). The blocking ELISA utilizes 96-well microtiter test plates coated with a cloned PRV g92(gIII) antigen, a mouse monoclonal antibody against gIII antigen (moMCAgIII): horseradish peroxidase (HRPO) conjugate, and undiluted test sera. Analyses can be completed in less than 3 hours with results printed out by an automated plate reader. Analyses on over 300 pig sera from PRV-free farms, on sera from other species, and on control sera containing antibodies to microorganisms other than PRV showed that the ratio of the optical density at 405 nm for the test sample to the optical density at 405 nm for the negative control (S/N value) was >0.7 for all sera. No false positives were identified. Likewise, the S/N values were >0.7 for over 400 sera obtained from pigs vaccinated twice with more than 1,000 times the standard PRV (dlg92dltk) dose or 1–4 times with the standard dose (2 × 105 TCID50/pig). Following challenge exposure to virulent PRV, the S/N values of the vaccinates were 0.1, showing that g92(gIII) antibodies in the sera of experimentally challenged pigs strongly blocked the binding of the moMCAgIII: HRPO conjugate to the antigen-coated wells. Sera of 233 pigs from PRV-infected herds with virus neutralization (VN) titers of 1:4 or greater were tested. All except 2 of these sera had S/N values <0.7 and more than 175 had S/N values <0.1. Sixteen sera from feral pigs with VN titers of 1:4 or greater had S/N values of 0.24 or less, but 2 sera with VN titers of 1:4 when tested 5 years prior to the PRV g92(gIII) blocking ELISA test gave false negative S/N values. Twenty-four of 29 pig sera from PRV-infected herds with VN titers < 1:4 were positive for g92(gIII) antibodies, illustrating the sensitivity of the PRV g92(gIII) blocking ELISA test. Analyses on 7 sera with VN titers of 1:4–1:64 showed that titers obtained with the PRV g92(gIII) blocking ELISA test were from 2- to 16-fold greater than the VN titers. The accuracy and sensitivity of the PRV g92(gIII) blocking ELISA test was further demonstrated by analyses of 40 unknown sera supplied in the National Veterinary Services Laboratories 1988 PRV check test kit.

scholarly journals Combined Cell Culture Enzyme-Linked Immunosorbent Assay for Quantification of Poliovirus Neutralization- Relevant Antibodies

2000 ◽  
Vol 7 (6) ◽  
pp. 915-919 ◽  
Author(s):  
A. F. Wahby
Keyword(s):  
Cell Culture ◽  
Sensitivity And Specificity ◽  
Immunosorbent Assay ◽  
Humoral Response ◽  
Enzyme Linked Immunosorbent Assay ◽  
Titration Curves ◽  
Microneutralization Assay ◽  
Human Sera ◽  
Reference Serum ◽  
Presence And Absence

ABSTRACT A combined cell culture enzyme-linked immunosorbent assay (CCC-ELISA) was developed for measuring the neutralizing antipoliovirus antibodies in human sera. The binding of different concentrations of each of the three poliovirus types to BGM cells in the presence and absence of a constant dilution from each test and reference serum was measured in the CCC-ELISA. The titers of the viruses neutralized by each serum were measured with the titration curves and used for interpretation of neutralizing titers to the three poliovirus types. Analysis of human sera revealed that the sensitivity and specificity of the CCC-ELISA and the microneutralization assay were comparable. The CCC-ELISA is nonsubjective, rapid, and highly reproducible. Furthermore, the CCC-ELISA could potentially be used as a seroepidemiologic tool for assessment of the humoral response to the cell culture infectious viruses.

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