J Korean Soc Microbiol.  1997 Dec;32(6):633-644.

Production and Characterization of Monoclonal Antibodies to a 75-kDa Major Outer Membrane Protein of Porphyromonas gingivalis

• Lee JY

Abstract

Porphyromonas gingivalis has been implicated in adult periodontitis. A 75-kDa major outer membrane protein of P. gingivalis is an immunodominant antigen and is often associated with fimbrial proteins during its purification. The present study was performed to generate monoclonal antibodies (MAbs) to the 75-kDa protein and characterize the protein and the MAbs. Kight-week-old BALB/c mice were immunized with the partially purified 75-kDa protein from P. gingivalis 2561. Spleen cells of the mice were hybridized with SP2/0 Ag-14 mouse myeloma cells and then fused with polyethylene glyeol 3400. Hybrid cells surviving hypoxanthine/aminopterin/thymidine medium were tested for antibody secretion by ELISA. Positive clones were subcloned by limiting dilution and then three subclones were obtained. The subclones were injected peritoneally into Pristane-primed mice to produce MAbs. Immunoreactivity of the MAbs was confirmed by an immunoblot analysis. In the immunoblot using a partially denatured crude 75-kDa protein preparation as an antigen, the 75-kDa protein reacting with the MAbs revealed a ladder- like pattern which has been known as a characteristic of a native polymeric form of fimbriae. The intervals of the ladders, however, were different from those of fimbrial protein. The MAbs also recognized the completely denatured 75-kDa protein. Immunogold localization using IgG fraction of the MAbs demonstrated that the gold particles were deposited on globules on the surface of P. gingivalis 2561, but not on fimbriae. These results suggest that the 75-kDa protein is antigenically different from fimbrial protein and in nature, is not associated with, but may be physicochemically associated with the fimbrial protein during extraction and purification procedures for these proteins. Immunoreactivity of these 3 MAbs was tested by immunoblot using sonic extracts from various P. gingivalis strains. All the MAbs reacted only with the strains having proteins in their sonic extracts with a molecular mass of 75 kDa, but failed to recognize the strains having proteins with molecular masses of 61, 61.5, 76, and 78 kDa. These results indicate that strains of P. gingivalis have antigenically different major outer membrane proteins and the MAbs in the present study might have been raised against the same common, exposed immunodominant epitope in the 75-kDa proteins.

MeSH Terms

Animals
Antibodies, Monoclonal*
Chronic Periodontitis
Clone Cells
Enzyme-Linked Immunosorbent Assay
Hybrid Cells
Immunoglobulin G
Membrane Proteins*
Membranes*
Mice
Polyethylene
Polymers
Porphyromonas gingivalis*
Porphyromonas*
Spleen
Antibodies, Monoclonal
Immunoglobulin G
Membrane Proteins
Polyethylene
Polymers