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Identification of Afipia felis antigens in culture medium: reaction with human sera
K Engbaek1, L O Uttenthal, C Koch
1Department of Immunological Research and Development, Statens Seruminstitut, Copenhagen, Denmark.
Abstract:
Fourteen protein antigens were identified on SDS-PAGE of Afipia felis culture supernatant. Immunoblotting against 10 monoclonal antibodies obtained from mice infected with live A. felis showed that 4 antibodies reacted with a 56 kDa band and 3 with both 56 kDa and 62 kDa bands. Compared with A. felis sonicate, the reacting proteins in culture supernatant showed an increase in molecular mass of 2-3 kDa, suggesting that they were more glycosylated. Purified antigen obtained by affinity chromatography of culture supernatant on the seven immobilized antibodies was tested against antibodies reacting with the 56 kDa and 62 kDa bands. All eluates contained both components, suggesting that the antibodies were directed against different epitopes of a double antigen held together during the affinity chromatography but cleaved by reduction and SDS-PAGE. The molecular size of the uncleaved protein in culture supernatant was determined by size-exclusion chromatography as > 1000 kDa. Testing of pre- and post-infection rabbit sera in immunoblotting against culture supernatant demonstrated that the 56 kDa and 62 kDa components gave the most prominent specific reactions with post-infection sera. One of fifty human sera submitted for testing for cat-scratch disease and 1 of 50 sera from healthy blood donors reacted with several bands in A. felis culture supernatant, including the 56 kDa and 62 kDa bands.
Insights
Researchers identified key protein antigens in Afipia felis culture supernatant, crucial for diagnosing cat-scratch disease. These antigens, particularly 56 kDa and 62 kDa proteins, show promise for serological tests.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Afipia felis is the causative agent of cat-scratch disease.
- Understanding A. felis antigens is vital for developing diagnostic tools.
Purpose of the Study:
- To identify and characterize protein antigens in A. felis culture supernatant.
- To evaluate their potential as diagnostic markers for cat-scratch disease.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins.
- Immunoblotting with monoclonal antibodies to detect antigen-antibody reactions.
- Affinity chromatography for antigen purification.
- Size-exclusion chromatography to determine molecular size.
Main Results:
- Fourteen protein antigens were identified in A. felis culture supernatant.
- Specific antibodies reacted with 56 kDa and 62 kDa protein bands, suggesting increased glycosylation.
- Purified antigens indicated antibodies targeted different epitopes on a larger complex.
- Post-infection rabbit sera showed strong reactions with 56 kDa and 62 kDa components.
- One human serum from a cat-scratch disease patient reacted with these antigens.
Conclusions:
- The 56 kDa and 62 kDa proteins are significant antigens of A. felis.
- These antigens show potential for use in serological diagnosis of cat-scratch disease.