Development of urtrasensitive noncompetitive enzyme immunoassay for hypothalamic hormone and it's clinical application

采用夹心法建立了灵敏的下丘脑激素(生长激素释放激素，GHRH；生长抑素，SRIF)非竞争性酶免疫分析(EIA)方法，用抗原载体蛋白复合体免疫兔，获得特异性抗体。我们首先尝试获得两种抗体，分别识别多肽的C-末端和N-末端，以建立夹心EIA，但只获得了GHRH(1-44)的C-末端部分的抗体，而没有获得SRIF(1-14)的中间部分的抗体。用亲和层析法纯化这些抗血清，并将其免疫球蛋白组份用于EIA。EIA采用夹心法，利用亲和素与生物素的高亲和力和抗原抗体反应。用生物胺已酸N-羟基闪锌酰亚胺酯对多肽进行生物素化，并与固定在平板上的亲和素反应，纯化得到上述特异性抗体。TH…进一步与羊抗兔Ig G-过氧化物酶结合物和邻苯二胺(OPD)反应，在420 nm波长处测定显色。用这种方法，多肽的定量范围为5.0-1000pg/管(GHRH)和0.61-100pg/管(SRIF)。该方法的灵敏度与常规竞争性放射免疫分析法基本相同。正常人血浆GHRH和SRIF水平分别为27(SY+-)9.7 pg/ml和15(SY+-)6.4 pg/ml(平均SY+-SD)。然而，通过在检测系统中加入正常山羊血清(10%)，以及使用非特异性反应较低的抗血清，OPD由于非特异性反应而产生的显色作用被降低到可以忽略的程度。今后可以考虑用320 nm、405 nm的荧光法代替OPD比色法来提高检测灵敏度。为了减少非特异性反应，目前正在研究将生物素化多肽固定在包被抗体的聚苯乙烯小球上的方法。较少

Sensitive noncompetitive enzyme immunoassay (EIA) for hypothalamic hormone (growth hormone-releasing hormone, GHRH ; somatostation, SRIF) was developed using sandwich method.Specific antibody was raised in rabbits by immunizing them with an antigen-carrier protein complex. We first tried to obtain two kinds od antibody, which recognize the C-terminal or the N-terminal portion of the peptides, to establish sandwich EIA.However, only antibodies that C-terminal portion but not the N-terminal portion of GHRH(1-44), and the middle portion of SRIF(1-14) were obtained. Thus these antisera were purified by affinity chromatography, and their IgG fractions were used to EIA.EIA was conducted by sandwich method utilizing high affinity of avidin (streptavidin) to biotin and antigen-antibody reaction. The peptides were biotinylated using biotinamidocaproate N-hydroxysccinimide ester and reacted with avidin, which had been immobilized to a plate, and the purified specific antibody described above. Th … More is complex formed was further reacted with goat anti-rabbit IgG-peroxidase conjugate and o-phenylenediamine (OPD), and the color development at 420 nm was measured. By means of this assay method, the peptides could be quantified in a range of 5.0-1,000 pg/tube (GHRH) and 0.61-100 pg/tube (SRIF). The assay sensitivity was almost the same as that by the conventional competitive radioimmunoassay. In normal subjects, the Plasma GHRH and SRIF levels were 27 (〕SY.+-.〔) 9.7 pg/ml and 15 (〕SY.+-.〔) 6.4 pg/ml(mean (〕SY.+-.〔) SD), respectively.The problem to be solved was a relatively strong color development due to nonspecific reaction irrespective of the good dose-response curves. However, the color development of OPD due to non-specigic reaction was decreased to a negligible level by adding normal goat serum (10%) to the assay system, and by the use of antiserum with low non-specific reaction. In the future, Fluorometry at 320 nm, 405 nm, instead of colorimetry using OPD will be considered to increase the assay sensitivity. To reduce non-specific reaction, the method to trap biotinylated peptide to specific anti-peptide IgG-coated polystirene balls is now under investigation. Less