Abe S, Kunii S, Fujita T, Hiraiwa K
Department of Legal Medicine, Fukushima Medical College, Japan.
Forensic Sci Int. 1998 Jan 9;91(1):19-28. doi: 10.1016/s0379-0738(97)00169-2.
A sensitive and specific sandwich ELISA for human seminal gamma-glutamyl transpeptidase (gamma-GTP) was developed using a combination of monoclonal antibodies. SG1 and SG3, which we produced. For semen identification in forensic samples, we modified the assay so as to be more sensitive and to establish efficient extracting conditions. After testing the extracting abilities of several detergents, CHAPS and deoxy-BIGCHAP were chosen as the solubilizer. Polystyrene beads coated with SG1 were incubated with samples extracted by the detergents, and further with biotinylated SG3, followed by peroxidase-labeled streptavidin. gamma-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal gamma-GTP equivalent to 10(7) times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal gamma-GTP was detected in other biological stains such as blood, saliva and vaginal smear. The extract of a 500 fold diluted seminal stain, 8 months old, showed the detection limit. Seminal gamma-GTP was detectable even in 14-year-old stains.
利用我们制备的单克隆抗体 SG1 和 SG3 的组合，开发了一种用于检测人精液γ-谷氨酰转肽酶（γ-GTP）的灵敏且特异的夹心酶联免疫吸附测定法（ELISA）。为在法医样本中鉴定精液，我们对该测定法进行了改进，使其更灵敏，并确定了高效的提取条件。在测试了几种去污剂的提取能力后，选择 CHAPS 和脱氧-BIGCHAP 作为增溶剂。将包被有 SG1 的聚苯乙烯珠与用去污剂提取的样本孵育，再与生物素化的 SG3 孵育，随后与过氧化物酶标记的链霉亲和素孵育。仅在精液样本中检测到γ-GTP。该测定法的灵敏度为 0.01 ng/ml 的精液γ-GTP，相当于稀释 10⁷ 倍的精液，比之前的平板测定法灵敏度高十倍。在其他生物污渍如血液、唾液和阴道涂片样本中未检测到显著的精液γ-GTP。一份稀释 500 倍、保存 8 个月的精液污渍提取物显示出检测限。即使是 14 年之久的污渍中也可检测到精液γ-GTP。
