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  • Author or Editor: Jerry K. McVicker x
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Abstract

Objective—To use antibodies produced by calves in response to infection with Mannheimia haemolytica in immunoaffinity chromatography for the identification and subsequent isolation of the dominant immunogenic antigens from bacteria grown in irondeficient media.

Sample Population—Serum from 10 calves actively infected with M haemolytica.

Procedure—An outer membrane protein fraction was obtained from sonicated salt-extracted M haemolytica cells by extraction with N-lauroyl sarcosinate. The immunoglobulin fraction of serum from calves actively infected with M haemolytica was used to prepare an immunoaffinity column. The immunoaffinity column was used to isolate the dominant immunogenic proteins from the outer membrane protein fraction. The resultant immunogenic protein fraction was subjected to ELISA and immunoblot methods as well as carbohydrate quantification. Sequencing of the N-terminal was performed on the most prominent protein.

Results—5 immunogenic proteins with molecular weights of 42, 30, 24, 20, and 15 kd were isolated. The immunogenic protein fraction was found to contain 51% carbohydrate. The immunoaffinity column capacity was 1 µg of immunogenic protein/mL of gel. The N-terminal sequence of the 42-kd protein was Tyr-Gln-Thr-Tyr-Gln-Ser-X-Leu-Gln, where X could not be identified.

Conclusions and Clinical Relevance—Immunogenic proteins were isolated by use of immunoaffinity chromatography. A substantial amount of carbohydrates was co-purified in the process. Additional experiments are needed to determine whether the carbohydrates would hinder or enhance development of vaccine preparations. This method could potentially allow a more rapid production of antigens for use in vaccines. (Am J Vet Res 2002;63:1634–1640)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the predictive ability of a commercially available lateral-flow immunoassay used for determining passive transfer of immunoglobulins in calves.

Animals—204 male Holstein calves ranging from 4 to 8 days old.

Procedure—Serum samples were obtained from each calf. Results of refractometry, zinc sulfate turbidity technique, and the lateral-flow immunoassay were determined. Sensitivity, specificity, accuracy, and predictive ability were calculated on the basis of IgG concentrations determined by turbidimetric immunoassay (TIA).

Results—Mean IgG concentration in the study was 10.9 mg/ml as determined by TIA. Rate of failure of passive transfer in this study population was 56%. Associations between the values for the refractometry and zinc sulfate turbidity techniques were established by regression analysis. Accuracy for the lateral-flow immunoassay, refractometry, and zinc sulfate turbidity methods was 95, 80, and 73%, respectively.

Conclusions and Clinical Relevance—The lateral-flow immunoassay was better at determining the status of passive transfer of immunoglobulins, compared with the refractometry or zinc sulfate turbidity methods. The ability of the lateral-flow immunoassay to provide accurate results should enable clinicians to make immediate management or intervention decisions. (Am J Vet Res 2002;63:247–250)

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in American Journal of Veterinary Research