摘要
目的制备人类白蛋白来源一段小分子多肽的多克隆抗体,建立毛细管电泳检测该小肽方法,为临床早期诊断GVHD提供新的手段.方法人工合成序列为LVRYTKKVPQVSTPTL的16个氨基酸残基的人来源多肽,按兔100μg/kg体重肽抗原标准制成免疫原.按常规方法,每次取2.5 ml抗原悬液,在脊柱两侧皮下和腹股沟处多点注射,分3次免疫动物,制备多克隆抗体.采用异硫氰酸酯荧光素（FTTC）标记小肽抗原,用获得的抗体通过LIF-CE-IA建立该肽的检测方案.结果获得该肽的多克隆抗体的效价为1∶1000,且它与正常人血清、尿液以及牛血清间没有交叉反应;标记抗原出峰时间为5.93 min,抗原-抗体复合物分离时间为6.47 min,所建立的检测方法的线性范围为16～512 ng/ml,最低检出限为10 ng/ml.结论实验所获得的多克隆抗体具有较高的滴度和特异性,所建立多肽的检测方法简便、快速,可为今后临床早期诊断GVHD提供一种新的手段.
Objective To develop an assay with polyclonal antibodies against a fragment derived from human albumin for determination of a peptide in urine, and to provide an early diagnostic tool for GVHD. Methods A small peptide composed of 16 amino acids （LVRYTKKVPQVSTPTL） was synthesized artificially. The immunogen was then diluted into 100 μg/kg body weight of rabbit. Subcutaneous injection in the immune animals was performed on both sides of spine and groin with 2.5 ml antigen suspension for three times, in order to prepare the polyclonal antibodies. The peptide antigen was then labeled with fluorescein isothiocyanate （FITC）, and detected by LIF-CE-IA with the pre-prepared antibodies. Results The titer of serum. The migration time of the labeled antigen was 5.93 min, while the migration time of antigen-antibody complex was 6.47 min. The linear range of the method was 16 to 512 ng/ml, and the minimum detection limit was 10 ng/ml. Conclusions The polyclonal antibodies against the peptide antigen was isolated successfully, which possessed high titer and specificity. These results indicated that the assay was simple and rapid and could be applied for the early diagnosis of patient with GVHD.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2010年第10期963-966,共4页
Chinese Journal of Laboratory Medicine
关键词
干细胞移植
尿分析
肽类
电泳
毛细管
Stem cell transplantation
Urinalysis
Peptides
Electrophoresis,capillary
