Hashida S, Ishikawa E, Mohri Z, Nakanishi T, Noguchi H, Murakami Y
Department of Biochemistry, Medical College of Miyazaki, Japan.
Endocrinol Jpn. 1988 Feb;35(1):171-80. doi: 10.1507/endocrj1954.35.171.
A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.
描述了一种使用单克隆抗体检测人生长激素(hGH)的灵敏夹心酶免疫测定法。将包被有抗hGH单克隆IgG的聚苯乙烯球与hGH一起孵育,随后与亲和纯化的兔抗hGH Fab'-辣根过氧化物酶偶联物孵育。使用3-(4-羟苯基)丙酸作为底物,通过荧光法测定结合到聚苯乙烯球上的过氧化物酶活性。血清和尿液中hGH的检测限分别为:使用20微升血清时为1.5纳克/升,使用0.15毫升尿液时为0.2纳克/升。特异性和测定精密度令人满意。使用包被有抗hGH单克隆IgG的聚苯乙烯球的本夹心酶免疫测定法测定的血清和尿液中的hGH水平与先前使用包被有兔抗hGH IgG的聚苯乙烯球的夹心酶免疫测定法测定的水平高度相关。19至28岁健康受试者晨尿中hGH的水平为6.4±3.2(标准差)纳克/克肌酐。然而,本测定法给出的hGH水平低于先前的测定法。这至少部分是由于尿液中的hGH与抗hGH单克隆IgG - 聚苯乙烯球的结合效率低于标准hGH,而尿液中的hGH和标准hGH与包被有兔抗hGH IgG的聚苯乙烯球的结合效率相同。此外,凝胶过滤显示尿液中的主要成分22K hGH与抗hGH单克隆IgG - 聚苯乙烯球的结合效率低于标准22K hGH。血清和尿液中hGH的性质仍有待研究。