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The Effects of Proteolytic Enzymes on Agglutination by Bacterial Antibodies of Lipopolysaccharide Modified Erythrocytes¹

From the Clinical Laboratories of the Roswell Park Memorial Institute, the Laboratory of Bacteriology of the Children's Hospital, the Department of Bacteriology and Pediatrics of the University of Buffalo School of Medicine, Buffalo, New York, and the Wander-Forschungsinstitut, Freiburg, Germany

Abstract

The effects of various proteolytic enzymes on agglutination of antigen modified erythrocytes from both warm and cold blooded animal species were investigated, and the following results were obtained.

1. Red blood cells from alligator, axolotl and caiman after treatment with enterobacterial antigens (crude extracts and highly purified lipopolysaccharides) are not agglutinated by homologous bacterial antiserum of rabbit and human origin, in contrast to sheep and human erythrocytes.

2. Specific hemagglutination of lipopolysaccharide modified erythrocytes from these cold blooded animal species is made possible by treatment of the red blood cells with trypsin, chymotrypsin, protease (of pancreatic origin), ficin and papain. Identical results are obtained when enzyme treatment precedes or follows antigen modification.

3. The agglutination enhancing effect of trypsin is abolished by soy bean trypsin inhibitor.

4. Proteinase (of fungal origin), peptidase (from hog intestinal mucosa) and human plasmin are ineffective in this system.

5. Trypsin, chymotrypsin, protease, ficin and papain also enhance agglutination of alligator erythrocytes by alligator red blood cell antiserum, although to a substantially lesser degree than agglutination of lipopolysaccharide modified erythrocytes by homologous bacterial antibodies.

6. Alligator, axolotl and caiman erythrocytes after treatment with the proteolytic enzymes are not agglutinated by either homologous or heterologous bacterial antisera or normal rabbit serum in the dilutions used, indicating that a Thomsen-Friedenreich type of reaction is not encountered.

7. Lipopolysaccharide modified human erythrocytes of blood group A or B after treatment with the proteolytic enzymes are agglutinated to a slightly greater degree by both homologous bacterial and blood group antisera than are red blood cells not treated with enzymes.

8. Lipopolysaccharide modified sheep erythrocytes after treatment with trypsin, chymotrypsin, protease, ficin and papain are agglutinated to the same extent by homologous bacterial antiserum as are erythrocytes not treated with enzymes. Enzyme treatment, however, enhances agglutination by sheep cell antiserum (amboceptor).

9. It is postulated that erythrocytes from certain cold blooded animal species, in contrast to those from sheep and man, contain on their surface in the area of the receptors of the lipopolysaccharide a protein or protein-like material which interferes with the spatial orientation of bacterial antibodies and subsequent hemagglutination, and that certain proteolytic enzymes remove or alter this inhibitor making hemagglutination possible.

10. The bearing of the results obtained in this study on the nature of incomplete antibodies and of the agglutination by bacterial antibodies of erythrocytes modified by homologous bacterial antigen is discussed.

Footnotes

This investigation was supported by a research grant (E-658) from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. Presented in part at the Annual Meeting of the American Association of Immunologists, Philadelphia, Pennsylvania, April 16, 1958.