New, practical approach to detecting antibody to pertussis toxin for public health and clinical laboratories

Abstract

A new, practical method for determining antibody to pertussis toxin (PT) by enzyme-linked immunosorbent assay for public health and clinical laboratories is possible because of recent advances in understanding the pathobiology of Bordetella pertussis and the physicochemical properties of PT. The new approach does not require the use of highly purified PT antigen, whiçh is difficult and expensive for most laboratories to obtain. Moreover, it employs only reagents that are commercial readily available. The method combines the purification of PT antigen and an assay for PT antibody into one process. It depends on (i) growth ofB. pertussis in a simple, defined medium to obtain a PT-rich supernatant with little contamination of cellular antigens; (ià) a simple, one-step concentration of PT in the culture supernatant with Affi-Gel Blue (Bio-Rad Laboratories, Richmond, Calif.); and (iii) specific adsorption of PT as the test antigen to microtiter wells coated with fetuin for the enzyme-linked immunosorbent assay. The procedure is sensitive and specific for PT antibody. It is technically simple, reproducible, and can be performed in a modestly equipped laboratory.

Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction

“…The yields of FHA and PT in culture filtrate and the purification efficiency of the system derived from a representative experiment are summarized in Table 1. B. pertussis NIH 165 produced about 40 to 80 mg of FHA and 8 to 16 mg of PT in 1 liter of the modified CL medium as estimated by ELISA with specific antiserum to FHA or PT (18). The yields were consistent with those observed in our previous studies (18,19).…”

“…Purified FHA and PT have been under intensive investigation for use as immunogens in experimental acellular pertussis vaccines (1, 14) and as antigens for the serodiagnosis of pertussis (2,4,(18)(19)(20). Because of meager antigen yields in the commonly used growth media (3,16) and the relatively complicated purification techniques, these antigens have been quite expensive and hard to obtain.…”

“…B. pertussis NIH 165 was grown in the CL medium (6) as modified in our previous studies (18,19). After incubation in a shaker water bath at 37°C for 48 h, the bacteria were separated from the supernatant by centrifugation (12,000 x g, 30 min, 4°C), and the supernatant was sterilized by * Corresponding author.…”

“…passing it through a filter (pore size, 0.2 nm; Nalge/Sybron Corp., Rochester, N.Y.). Antigen yields in the culture filtrate were estimated by enzyme-linked immunosorbent assay (ELISA) (18,19) to be approximately 40 to 80 mg of FHA per liter and 8 to 16 mg of PT per liter in reference to purified FHA (FHA-R) and PT (PT-R). FHA-R was obtained from Merieux Institute, Paris, France, and PT-R was obtained from the Michigan Department of Health, Lansing, Mich.…”

Laboratory diagnosis of pertussis: state of the art in 1997

“…For ELISA and other purposes, whole-cell sonicates can be used (37), and these have been shown to have sensitivities comparable to those of purified virulence factors of the bacteria, but they tend to lack specificity. Another method with semipurified antigens used copurified extracellular products (120). For most ELISA techniques, however, purified antigens are used.…”