高迁移组染色体蛋白-17的分离提取及其抗体ELISA测定方法的建立

ISOLATION AND EXTRACTION OF HMG 17 CHROMOSOMAL PROTEINS AND ESTABLISHMENT OF ELISA FOR DETECTION ANTIBODIES TO HMG 17

纯化高迁移组染色体蛋白－１７（ＨＭＧ－１７），建立检测抗ＨＭＧ－１７自身抗体的ＥＬＩＳＡ方法，探讨抗ＨＭＧ－１７抗体与临床疾病的关系。用水煮沸法提取，甲酸－吡啶缓冲液酸化和ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤技术，自小牛胸腺组织中分离ＨＭＧ－１７，用此纯化物包被，方阵滴定，建立测定抗ＨＭＧ－１７抗体的间接ＥＬＩＳＡ方法，并进行方法学考核和临床标本检测。纯化的ＨＭＧ－１７抗原经十二烷基硫酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）分析，呈现一条分子量约９２００的蛋白带；建立的ＥＬＩＳＡ方法批内平均变异系数（ＣＶ）为５．８％，批间ＣＶ值为９．６％，ＥＬＩＳＡ抑制试验最高抑制率达８７．２％；临床检测结果表明，在系统性红斑狼疮（ＳＬＥ）、混合性结缔组织病（ＭＣＴＤ）和干燥综合征（ＳＳ）患者中分别有１７．４％～３７．３％的抗ＨＭＧ－１７抗体的阳性检出率（Ｐ＜０．０１或Ｐ＜０．０５），而在其他自身免疫病和内科疾病者中，抗ＨＭＧ－１７抗体均为低水平。抗ＨＭＧ－１７抗体ＥＬＩＳＡ测定法具有较好的特异性和重复性，便于常规检测。

To purificate high mobility group 17(HMG 17) chromosomal proteins, develope enzyme linked immunosorbent assay(ELISA) for HMG 17 autoantibody, and investigate the relationship between this antibody and clinical diseases. Using boiling water extraction, formic acid pyridine buffer solution acidation and Sephadex G 150 gel filtration technique,the HMG 17 was isolated from calf thymus. With the purification as antigen coated the microtitre plates, square titration, an indirect ELISA for detection anti HMG 17 antibody was developed, and was used to evaluate its methodology as well as detect clinical samples simultaneously. The purified HMG 17 antigen was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS PAGE),one band of 9 200 peptide present on SDS polyacrylamide gel. The intra and inter assay coefficient of variation of ELISA for HMG 17 antibody were 5.8% and 9 .6%, respectively. Homologous inhibition using HMG 17 produced up to 87.2% inhibition. The results of clinical detection indicated that the prevalene of anti HMG 17 antibodies were 17.4%～ 37.3 % in patients with systemic lupus erythematosus(SLE), compared with patients with primary sjogrens syndrome(SS) and patients with mixed connective tissue disease (MCTD), respectively( P <0.01 or P <0.05). While this antibody was low levels in patients with other autoimmunne and medical diseases. An ELISA method for detection anti HMG 17 antibody was specific, stability, and was convenient for routine applications. The presence of anti HMG 17 antibody appeared to be associated with some autoimmune diseases(especially SLE).