Use of monoclonal antibodies with neutralizing effects on toxic antigens from human bacterial plaque to detect specific bacteria by colony blotting.
Inflammatory periodontal diseases are provoked by bacteria which adhere to teeth at the gingival margin and form plaques containing toxins detectable by their effect on mammalian cells in culture. The aim of this study was to make toxin-neutralizing monoclonal antibodies and determine whether they detect antigen in specific oral bacteria. Bacterial plaque was collected from teeth and homogenized, and the fluid phase (plaque extract) was boiled or first fractionated over Sephacryl S-300. Hybridomas from immunized mice secreted immunoglobulin M (IgM) antibodies which reacted to plaque antigens. Neutralization was detected by an increase in the growth of HL60 cells which were exposed to plaque toxins in the presence of IgM from hybridoma culture or ascitic fluids. However, the neutralization was obvious only when the plaque toxins reduced growth by 50% or less. Plaque toxin preparations were found to contain proteases which hydrolyzed all of the IgM in ascitic fluids within 24 h. Replenishing the IgM daily preserved protection compared with protection from IgM from other hybridomas or saline only. The decrease in the specific activity of plaque proteins caused by replenishing one such antibody (3hE5) was 2.5-fold compared with activity with unreplenished 3hE5, 3.8-fold compared with activity with saline only, and 10.7-fold compared with activity with replenished, unrelated antibody. The neutralizing IgM detected an array of 14,000- to 22,000-molecular-weight antigens. The native toxins may be aggregates of these antigens, or the array may indicate fragments of an undetected, larger antigen or a common, nonpeptide adduct. Only 0.5 to 0.8% of the bacteria from sites with periodontitis and grown on blood agar contained antigen. One group of reactive bacteria was identified as Actinomyces odontolyticus serotype I. Other isolates were identified as Staphylococcus epidermidis, but antigen disappeared from the these isolates within 6 weeks of subculture. Epitope-containing antigens were also found in streptococcal and Eikenella isolates, and it is likely that the antigens from only some of these bacteria are toxic.
Levine
M
M
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Miller
F C
FC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000633:toxicity*; D001419:Bacteria / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification*; D003101:Collodion; D003773:Dental Plaque / Q000276:immunology / Q000382:microbiology*; D010450:Endopeptidases; D006801:Humans; D006825:Hybridomas / Q000382:microbiology; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D010209:Paper
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D007074:Immunoglobulin G; D003101:Collodion; D010450:Endopeptidases