Chen Y F, Ramirez V D
J Histochem Cytochem. 1982 Sep;30(9):926-31. doi: 10.1177/30.9.6813375.
To generate anti-thyrotropin-releasing hormone (TRH) antibodies TRH was rendered antigenic presumably by reaction of its histidine residue with bis-diazotized benzidine (BDB) coupled to bovine serum albumin (BSA). Six California white rabbits were each injected with 330 micrograms protein/ml of emulsified immunogen by the multisite intradermal immunization technique. Seven repeated injections were given at 30-day intervals using half of the original quantity of antigen. Antibodies binding 125I-TRH appeared in the serum of four of the six rabbits three months after the first injection. Five months later the sera of two rabbits bound 50% of the labeled TRH at 1:6000 final dilution. Using this antiserum a radioimmunoassay for TRH was developed in which as little as 10 pg/300 microliter unlabeled TRH can be detected. The linear range of detectable TRH was 10 to 10000 pg. No cross-reaction with various hypothalamic and pituitary hormones, neurotransmitters, neuropeptides, and BSA were detected in this immunoassay. Extracts from rat and frog hypothalami produced 125I-TRH-binding inhibition curves parallel to synthetic TRH. Samples from elutes of rat medio-basal hypothalami superfused in vitro were examined by using this antiserum. Serial dilution of superfusate showed a similar inhibitions curve. Stimulatory effect of K+ depolarization on TRH release from superfused hypothalami was inhibited in Ca2+-free ethylenediaminetetraacetic acid medium. Ouchterlony double diffusion test of the TRH antisera revealed that low levels of antibodies against albumin but not immunoglobulin G or ovalbumin were also produced. However, the immunoprecipitates could only be detected with undiluted serum. In conclusion, this antiserum generated one of the most sensitive TRH radioimmunoassay currently available in the literature. The anti-TRH serum produced by this method can be used to examine both content of TRH from several tissues as well as release from hypothalamic tissue in vitro and might be useful to trace brain TRH pathways by immunocytochemistry.
为了产生抗促甲状腺激素释放激素（TRH）抗体，TRH可能通过其组氨酸残基与偶联至牛血清白蛋白（BSA）的双偶氮联苯胺（BDB）反应而被抗原化。通过多点皮内免疫技术，给6只加利福尼亚白兔每只注射330微克蛋白质/毫升的乳化免疫原。每隔30天进行7次重复注射，每次使用原始抗原量的一半。首次注射3个月后，6只兔子中有4只的血清中出现了结合125I-TRH的抗体。5个月后，2只兔子的血清在最终稀释度为1:6000时结合了50%的标记TRH。使用这种抗血清开发了一种TRH放射免疫分析方法，该方法可检测低至10 pg/300微升的未标记TRH。可检测的TRH线性范围为10至10000 pg。在该免疫分析中未检测到与各种下丘脑和垂体激素、神经递质、神经肽以及BSA的交叉反应。大鼠和青蛙下丘脑提取物产生了与合成TRH平行的125I-TRH结合抑制曲线。使用这种抗血清检查了体外灌流的大鼠中基底下丘脑洗脱液的样品。灌流液的系列稀释显示出类似的抑制曲线。在无钙的乙二胺四乙酸培养基中，K+去极化对灌流下丘脑释放TRH的刺激作用受到抑制。TRH抗血清的双向琼脂扩散试验表明，还产生了低水平的抗白蛋白抗体，但未产生抗免疫球蛋白G或卵白蛋白的抗体。然而，免疫沉淀物只能用未稀释的血清检测到。总之，这种抗血清产生了目前文献中最灵敏的TRH放射免疫分析方法之一。通过这种方法产生的抗TRH血清可用于检测几种组织中TRH的含量以及体外下丘脑组织的释放，并且可能有助于通过免疫细胞化学追踪脑内TRH通路。
