The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells.
A microplate assay, based on the clustering effect induced by pertussis toxin (PT) in Chinese hamster ovary (CHO) cells, has been developed and standardized. Toxin titration is done directly in the culture microplate by twofold dilutions of 25 microliters of test material to which are added 10 000 freshly trypsinized cells in 200 microliters of culture medium per well. The dilution causing the clustering effect is determined by direct microscopic observation after 48 h of incubation. The method allows detection of 50-100 pg toxin per millilitre. For determination of neutralizing antibodies (antitoxin), twofold dilutions of 25 microliters of antiserum are first made directly in the culture microplate. Thereafter 25 microliters of toxin, containing four times the minimal clustering concentration, is added to each well. After three hours for neutralization at +37 degrees C, cells are added, incubated and examined as above. The assay has been found to be simple and reproducible for measuring the antibody response to PT in human and different animal sera. For titration of bacteria associated toxin, the CHO cells are seeded and incubated for 24 h before the addition of bacteria. Incubation and examination are done as described for toxin titration.
D000818:Animals; D001427:Bacterial Toxins / Q000592:standards*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006168:Guinea Pigs; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D010053:Ovary / Q000166:cytology; D037342:Pertussis Toxin; D010567:Pertussis Vaccine / Q000592:standards*; D011817:Rabbits; D013696:Temperature; D013997:Time Factors; D010566:Virulence Factors, Bordetella
D001427:Bacterial Toxins; D010567:Pertussis Vaccine; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin