Comparison between radioimmunoassay and direct and indirect enzyme-linked immunosorbent assays for determination of antibodies against Haemophilus influenzae type b capsular polysaccharide

Lagergård, Teresa; Trollfors, Birger; Claesson, Bo A.; Schneerson, Rachel; Robbins, John B.

doi:10.1128/jcm.26.12.2554-2557.1988

Cited by 13 publications

(3 citation statements)

References 13 publications

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“…However, unlike the radiolabeled-antigen binding assay, which is subject to inconsistencies in the method and intensity of antigen radiolabeling (1), the ELISA uses simple reagents accessible to most laboratories and is therefore easily standardized (29). Results with ELISA also have been shown to correlate well with those of the radiolabeled-antigen binding assay in studies of H. influenzae type b (5,20,25). In addition, the ELISA is easily modified with appropriate reagents to quantitate class-specific antibodies and immunoglobulin G subclasses, as demonstrated in assays with H. infiuenzae type b (5,19,31).…”

Section: Discussionmentioning

confidence: 79%

Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay

et al. 1994

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A standardized enzyme-linked immunosorbent assay (ELISA) was used by 11 laboratories to measure levels of total serum antibody to Neisseria meningitidis serogroup C capsular polysaccharide in 16 unpaired pre-and postvaccination serum samples. Twelve serum samples were from adults, and four were from children aged 2, 3, 5, and 9. The between-laboratory coefficient of variation for pre-and postvaccination sera ranged from 16 to 59% and 11 to 21%, respectively. The average percent difference (absolute value) from the betweenlaboratory means for all prevaccination sera measured by each laboratory was 24%, whereas the average percent difference was 13% for all postvaccination sera. A postvaccination quality control serum was diluted three times to give optical densities on the high, middle, and low portions of the standard reference curve. The three dilutions were assayed by the 11 laboratories a total of 241 times and yielded an overall coefficient of variation of 20%. Antibody-binding inhibition curves showed that the standardized ELISA was specific for N. meningitidis serogroup C capsular polysaccharide antibody. Fifty percent inhibition of seven serum samples was obtained after reaction with an average concentration of 0.9 ,ug of meningococcal serogroup C polysaccharide per ml; an average of 93% inhibition was obtained with 50 ,ug of polysaccharide per ml. The acceptance and use of this standardized ELISA will reduce between-laboratory assay variability and ensure a more accurate and reproducible assessment of immunogenicity for vaccines under development.

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Section: Discussionmentioning

confidence: 79%

Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay

et al. 1994

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“…Serum and lung extracts were analyzed for specific antibody content using ELISA. For analysis of anti-HibCPS antibodies, ELISA plates were coated with avidin and biotinylated HibCPS as previously described (15,24). As a reference and positive control for Hib-ELISA a serum pool from mice immunized subcutaneously with HibCRM conjugate was used.…”

Section: Elisamentioning

confidence: 99%

Antibody responses in serum and lung to intranasal immunization with Haemophilus influenzae type b polysaccharide conjugated to cholera toxin B subunit and tetanus toxoid

Bergquist

Lagergård

Holmgren

1998

APMIS

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responses in serum and lung to intranasal immunization with Haemophilus infuenzae type b polysaccharide conjugated to cholera toxin B subunit and tetanus toxoid. APMIS 106: 800-806, 1998. Aim: Cholera toxin B subunit (CTB) has previously been used as a mucosal carrier for various vaccine candidate antigens. The objective of this study was to see if coupling a bacterial polysaccharide, Haemophilus infuenzae type b capsular polysaccharide (HibCPS), to CTB, either directly or through prior coupling to tetanus toxoid (TT), would improve the immunogenicity of HibCPS after nasal immunization. Methods: HibCPS was conjugated to CTB, TT or via TT to CTB, using glutaraldehyde or I-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The conjugates were characterized and used for intranasal (IN) and subcutaneous (SC) immunizations of mice. The anti-Hib, -TT and -CTB antibody titers in serum and lungs after the immunizations were measured with ELISA. Results: The HibCTB was poorly immunogenic both given IN and SC compared with HibTT and HibTTCTB, probably because of inefficient coupling. In contrast, the conjugation of CTB to the HibTT conjugate resulted in a preparation which was superior both to the HibTT and the HibCTB conjugates in inducing local IgA and IgG anti-HibCPS antibodies in the lungs. The anti-HibCPS serum IgG titers after IN immunization with the HibTTCTB conjugate were similar to the titers after IN immunization with HibTT, or SC immunization with a commercial HibCRM conjugate vaccine. In contrast to the other conjugates, the HibTTCTB conjugate also gave rise to anti-Hib serum IgA titers. Conclusion: We conclude that appropriate conjugation to CTB increases the mucosal immunogenicity of HibCPS, and that intranasal immunization with such a conjugate can give rise to both local and systemic anti-HibCPS antibody responses.

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“…The high sensitivity of the radioimmunoassay results in the detection of antibodies to crossreacting antigens, unless serum samples are highly diluted (12). The ELISA seems to offer better performance than the radioimmunoassay in the detection of antibodies to Haemophilus influenzae type b capsular polysaccharide (15). In the serodiagnosis of A. pleuropneumoniae serotype 5, the ELISA also offers a better sensitivity than other serological tests (10,20,37).…”

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Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5

1992

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An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal withinand between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H202) and hydrogen donor [2,2'-azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both withinand between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.

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Comparison between radioimmunoassay and direct and indirect enzyme-linked immunosorbent assays for determination of antibodies against Haemophilus influenzae type b capsular polysaccharide

Abstract: Levels of antibodies against Haemophilus influenzae type b capsular polysaccharide were determined in acute-phase and convalescent-phase serum samples obtained from 21 children with invasive H. in.fluenzae type * Corresponding author.

Cited by 13 publications

References 13 publications

Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay

Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay

Antibody responses in serum and lung to intranasal immunization with Haemophilus influenzae type b polysaccharide conjugated to cholera toxin B subunit and tetanus toxoid

Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5

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