Schultz W W, Phipps T J, Pollack M
J Clin Microbiol. 1979 Jun;9(6):705-8. doi: 10.1128/jcm.9.6.705-708.1979.
An enzyme-linked immunosorbent assay (ELISA) is described for Pseudomonas aeruginosa exotoxin A. A double antibody sandwich method was used, employing polyvinyl microtiter plates as the solid phase, a primary coat of monospecific rabbit antitoxin serum, an outer layer composed of a horseradish peroxidase-sheep antitoxin immunoglobulin G conjugate, and an ortho-phenylene-diamine substrate. Absorbance (optical density) of hydrolyzed end product was read spectrophotometrically at 492 nm. ELISA detected as little as 30 pg (0.3 ng/ml) of purified toxin, and absorbance was linear over a 20-fold or greater concentration range. Toxin was demonstrated in culture filtrates from 42 of 48 (88%) consecutive clinical P. aeruginosa isolates compared with 37 of 48 (77%) positive by hemagglutination inhibition. Results of the two assays correlated closely (r = 0.82, P less than 0.001). Specificity was confirmed by neutralizability of ELISA activity with monospecific antitoxin. ELISA was thus a sensitive, specific, and quantifiable technique for the assay of P. aeruginosa exotoxin A in both purified and crude culture materials.
本文描述了一种用于检测铜绿假单胞菌外毒素A的酶联免疫吸附测定（ELISA）方法。采用双抗体夹心法，以聚乙烯微量滴定板为固相，用单特异性兔抗毒素血清进行初次包被，外层由辣根过氧化物酶-羊抗毒素免疫球蛋白G缀合物组成，并使用邻苯二胺作为底物。水解终产物的吸光度（光密度）在492nm处用分光光度计读取。ELISA可检测低至30pg（0.3ng/ml）的纯化毒素，且在20倍或更大的浓度范围内吸光度呈线性关系。在连续的48株临床分离铜绿假单胞菌中，42株（88%）的培养滤液中检测到毒素，相比之下，血凝抑制法检测阳性率为48株中的37株（77%）。两种检测方法的结果密切相关（r = 0.82，P < 0.001）。通过用单特异性抗毒素中和ELISA活性证实了其特异性。因此，ELISA是一种用于检测纯化和粗制培养物中铜绿假单胞菌外毒素A的灵敏、特异且可定量的技术。
