Guesdon J L, Chevrier D, Mazié J C, David B, Avrameas S
J Immunol Methods. 1986 Feb 27;87(1):69-78. doi: 10.1016/0022-1759(86)90345-5.
An enzyme immunoassay to measure histamine has been developed. A histamine-bovine serum albumin conjugate was prepared using 1,4-benzoquinone as the coupling agent and was employed to immunize mice for the preparation of monoclonal antibodies against histamine. After an initial screening to identify antigen-binding monoclonal antibodies the clones were isolated by limiting dilution cloning, grown in ascites and antibodies which had been secreted into the ascitic fluid were precipitated by ammonium sulphate at 50% saturation. A systematic approach for the determination of epitope specificities of monoclonal antibodies was performed. It was found that for the most specific antibody the main epitope encompassed the 2-histaminyl-1,4-benzoquinone moiety and that the KD value determined by indirect ELISA was 1.5 X 10(-8) M for the hapten part of the immunogen and 4.6 X 10(-10) M for a histamine-Bq-ovalbumin conjugate. The selected monoclonal antibody could not recognize histidine or methyl-histamine. Using this antibody, we developed an enzyme immunoassay for histamine and pg amounts could be detected. The same assay was used to quantify the allergic release of histamine from guinea pig lung mast cells. Results obtained either by the present enzyme immunoassay or by a fluorometric assay were closely correlated (correlation coefficient r = 0.9702, n = 37).
已开发出一种用于测量组胺的酶免疫测定法。使用1,4-苯醌作为偶联剂制备了组胺-牛血清白蛋白结合物，并用于免疫小鼠以制备抗组胺单克隆抗体。在初步筛选以鉴定抗原结合单克隆抗体后，通过有限稀释克隆分离克隆，在腹水中培养，并通过50%饱和度的硫酸铵沉淀分泌到腹水中的抗体。对单克隆抗体的表位特异性进行了系统的测定。结果发现，对于最特异性的抗体，主要表位包含2-组胺基-1,4-苯醌部分，通过间接ELISA测定的免疫原半抗原部分的KD值为1.5×10^(-8) M，组胺-Bq-卵清蛋白结合物的KD值为4.6×10^(-10) M。所选单克隆抗体不能识别组氨酸或甲基组胺。使用该抗体，我们开发了一种组胺酶免疫测定法，可检测到皮克量。相同的测定法用于定量豚鼠肺肥大细胞组胺的过敏释放。通过本酶免疫测定法或荧光测定法获得的结果密切相关（相关系数r = 0.9702，n = 37）。
