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首页> 外文期刊>Journal of Clinical Microbiology >Preparation of filamentous hemagglutinin from Bordetella pertussis and assay for serum antibodies to filamentous hemagglutinin and pertussis toxin for clinical and public health laboratories.
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Preparation of filamentous hemagglutinin from Bordetella pertussis and assay for serum antibodies to filamentous hemagglutinin and pertussis toxin for clinical and public health laboratories.

机译：从百日咳博德特氏菌制备丝状血凝素，并测定用于临床和公共卫生实验室的血清丝状血凝素和百日咳毒素抗体。

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A procedure that is sufficiently simple and economical for use in clinical and public health laboratories for producing and purifying filamentous hemagglutinin (FHA) and determining antibodies to this major antigen of Bordetella pertussis in serum is described. High yields of FHA (40 to 80 mg/liter) were obtained in the supernatant by cultivating B. pertussis in modified CL medium. The FHA antigen was separated from pertussis toxin (PT) and other antigens by chromatography on hydroxylapatite. Removal of residual PT activity in the FHA fraction was effected by affinity absorption of PT with Fetuin immobilized to Sepharose 4B. The FHA was used as the antigen for determining titers of immunoglobulin G (IgG), IgA, and IgM to FHA in sera of patients with pertussis by an improved enzyme-linked immunosorbent assay. Development of the interfering background color commonly observed in conventional FHA enzyme-linked immunosorbent assay procedures was eliminated by washing the reaction wells with a buffer of high ionic strength before adding the peroxidase conjugates. In the absence of nonspecific background color, the reaction endpoints were easy to read. The FHA prepared by the procedure described was identical to a reference preparation of purified FHA in sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and serological specificity assays. High yields of FHA were obtained from all four strains of B. pertussis tested in this study, indicating that the procedure for enhanced production of FHA may be generally applicable to other strains of B. pertussis. Results from tests of 50 serum specimens with clinical information on pertussis for FHA and PT antibodies by the assay procedures described exemplified the usefulness and caveats of serodiagnosis for pertussis.
机译：描述了一种在临床和公共卫生实验室中用于生产和纯化丝状血凝素（FHA）并确定血清中百日咳博德特氏菌主要抗原抗体的足够简单和经济的方法。通过在改良的CL培养基中培养百日咳博德特氏菌，可在上清液中获得高产量的FHA（40至80 mg / L）。通过在羟基磷灰石上色谱分离FHA抗原与百日咳毒素（PT）和其他抗原。通过用固定在琼脂糖凝胶4B上的胎球蛋白亲和力吸收PT来除去FHA级分中残留的PT活性。 FHA用作抗原，通过改进的酶联免疫吸附测定法确定百日咳患者血清中免疫球蛋白G（IgG），IgA和IgM对FHA的滴度。通过在加入过氧化物酶结合物之前，用高离子强度的缓冲液洗涤反应孔，可以消除常规FHA酶联免疫吸附法中通常观察到的干扰背景色的产生。在没有非特异性背景色的情况下，反应终点易于阅读。通过所述方法制备的FHA与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱和血清学特异性测定中的纯化FHA的参考制剂相同。从本研究中测试的所有四种百日咳博德特氏菌菌株均获得了高产的FHA，这表明增强FHA产生的程序通常可适用于其他百日咳博德特氏菌菌株。通过描述的测定方法从50个血清样本中获得有关百日咳的FHA和PT抗体临床信息的测试结果，证明了百日咳血清学诊断的有效性和注意事项。

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