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Isolation of immunogenic outer membrane proteins from Mannheimia haemolytica serotype 1 by use of selective extraction and immunoaffinity chromatography

Jerry K. McVickerDepartment of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Ames, IA 50011.

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Louisa B. TabatabaiUSDA-Agricultural Research Service, National Animal Disease Center, 2300 Dayton Ave, Ames, IA 50010.

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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)

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)