A computer program for the evaluation of ELISA data obtained using an automated microtiter plate absorbance reader

Caulfield, Michael J.; Shaffer, Diana

doi:10.1016/0022-1759(84)90288-6

Cited by 43 publications

(16 citation statements)

References 7 publications

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“…The plates were developed using p-nitrophenyl phosphate substrate (Sigma), and absorbance was measured at 405 nm. Titers were calculated as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Joyce

Cook

Chabot

et al. 2006

Journal of Biological Chemistry

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The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by ␥-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free ␣-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to >95% homogeneity and have demonstrated that the polymer contains >99% poly-␥-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the ␥-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5)triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of ␣-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.

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“…The plates were developed using p-nitrophenyl phosphate substrate (Sigma), and absorbance was measured at 405 nm. Titers were calculated as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Joyce

Cook

Chabot

et al. 2006

Journal of Biological Chemistry

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“…The background was determined as the mean OD 405 of wells not containing primary antibodies. The endpoint titer of antibodies was defined as the reciprocal of the highest serum dilution that gave a reading above an OD 405 of 0.1 as previously described (9). Each sample was run in duplicate on at least three different days.…”

Section: Methodsmentioning

confidence: 99%

Immunization with theHaemophilus ducreyiHemoglobin Receptor HgbA Protects against Infection in the Swine Model of Chancroid

Afonina¹,

Leduc²,

Nepluev³

et al. 2006

Infect Immun

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The etiologic agent of chancroid is Haemophilus ducreyi. To fulfill its obligate requirement for heme, H. ducreyi uses two TonB-dependent receptors: the hemoglobin receptor (HgbA) and a receptor for free heme (TdhA). Expression of HgbA is necessary for H. ducreyi to survive and initiate disease in a human model of chancroid. In this study, we used a swine model of H. ducreyi infection to demonstrate that an experimental HgbA vaccine efficiently prevents chancroid, as determined by several parameters. Histological sections of immunized animals lacked typical microscopic features of chancroid. All inoculated sites from mock-immunized pigs yielded viable H. ducreyi cells, whereas no viable H. ducreyi cells were recovered from inoculated sites of HgbA-immunized pigs. Antibodies from sera of HgbA-immunized animals bound to and initiated antibodydependent bactericidal activity against homologous H. ducreyi strain 35000HP and heterologous strain CIP542 ATCC; however, an isogenic hgbA mutant of 35000HP was not killed, proving specificity. Anti-HgbA immunoglobulin G blocked hemoglobin binding to the HgbA receptor, suggesting a novel mechanism of protection through the limitation of heme/iron acquisition by H. ducreyi. Such a vaccine strategy might be applied to other bacterial pathogens with strict heme/iron requirements. Taken together, these data suggest continuing the development of an HgbA subunit vaccine to prevent chancroid.

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“…The reaction was stopped by adding 50 p1 of 3 N NaOH to each well. The absorbance at 405 nm in each well was determined using an MR580 MicroELISA Auto Reader (Dynatech Laboratories), and antibody concentrations were determined relative to the standard using a computer program as previously described [20]. It should be noted that in the presence of a high IgM response to PnC, there may be a prozone effect on the IgG3 response.…”

Section: Assay For Serum Antibody and Idmentioning

confidence: 99%

Specific antigedantibody complexes induce the in vivo production of a parallel set of nonantigen‐binding idiotype‐positive antibodies

Caulfield

Černý

1988

Eur J Immunol

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Immune complexes prepared with the polysaccharide antigen (PnC) extracted from Streptococcus pneumoniae R36a and two different PnC-specific antibodies were found to differ in their regulatory properties depending on the isotype of the antibody. Thus, complexes formed in antibody excess with TEPC15 (IgA) were suppressive whereas complexes formed with 96-G (IgG3) antibodies enhanced the IgM response to PnC. During the course of these studies, we found that little or no PnC-specific IgG antibody was induced during the response to PnC coupled to sheep red blood cells (PnC-SRBC). Interestingly, however, immunization with 96-G/PnC complexes either alone or with PnC-SRBC resulted in the induction of IgG3 antibodies that express the T15 idiotype (Id) but which do not bind PnC. This unique IgG3 response occurred after injection of 96-G/PnC complexes formed in antibody excess but not when complexes were formed in antigen excess. The Id+ nonspecific IgG3 response peaked on day 5 and could be activated with 96-G/PnC complexes but not with free PnC antigen. The Id+ nonspecific response was not due to polyclonal activation of IgG3 production since there was no difference in IgG3 levels in mice injected with 96-G/PnC complexes with those injected with PnC-SRBC. Finally, mice that had been suppressed for expression of the T15 Id by neonatal injection of anti-Id antibody were able to produce Id+-unspecific IgG3 antibody after immunization with 96-G/PnC complexes, further suggesting that Id+ IgG3 was produced by different clones than those that usually comprise the antibody response to PnC. The results suggest that the formation of IgG immune complexes during an immune response may result in stimulation of idiotypically related clones thus resulting in degeneracy of the immune response.

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A computer program for the evaluation of ELISA data obtained using an automated microtiter plate absorbance reader

Cited by 43 publications

References 7 publications

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Immunization with theHaemophilus ducreyiHemoglobin Receptor HgbA Protects against Infection in the Swine Model of Chancroid

Specific antigedantibody complexes induce the in vivo production of a parallel set of nonantigen‐binding idiotype‐positive antibodies

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