The LAP test. A new method for identification of seminal stains by a qualitative color reaction of leucine aminopeptidase.

A new simple method for identification of seminal stains is described. It employs a qualitative color reaction based on histochemical technique for demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen. The method herein reported (the LAP test) is quite suitable for medicolegal examination of seminal stains as a preliminary test.     

Application of an immunological method to detect trace amounts of dried human semen

An immunological assay based on a monoclonal antibody was used for identification of trace amounts of dried human semen in forensic science evidence. The monoclonal antibody (Mab 4E6) produced recognizes a human sperm-coating antigen which is specific to human seminal plasma. This antigen seems to be a protein secreted by the epithelial cells of the ejaculatory duct, which is stable indefinitely at room temperature. Mab 4E6 reacts positively with semen samples from individuals independently to their ABO group or secretory status, but does not react with semen from bull, ram, boar, horse, rabbit and dog. In the assay system developed, Mab 4E6 can detect human seminal plasma at concentrations of 0.5 micrograms/ml total protein. A similar sensitivity is found when human semen stains are eluted from forensic science samples and tested by the same assay. This method shows a good correlation with the microscopic methods routinely used. The method described is very sensitive and reproducible, it is time saving and special laboratory equipment is not needed.     

gamma-Glutamyltransferase test as a new tool for identification of seminal stains

A simple qualitative method for identification of seminal stains based on a high activity of gamma-glutamyltransferase (gamma-GTP) in human semen is described. It employs the release of alpha-naphthylamine from N-gamma-glutamyl-alpha-naphthylamide by the gamma-GTP action: alpha-naphthylamine couples with Fast Garnet GBC salt to produce a strong brownish-red color. The data on its simplicity, specificity, and stability show that the present method is suitable for medicolegal examination of seminal stains as a preliminary test.     

Zinc test as a new tool for identification of human seminal stains

An extremely simple qualitative method for identification of seminal stains based on high levels of zinc in human semen is described. It uses reaction of 1-(2-pyridylazo)-2-naphthol with zinc to develop a deep red color. The data are presented on the sensitivity, stability and specificity of the present method. We can recommend it for identification of human semen especially in old or denatured samples.     

A dot-blot-immunoassay for semen identification using a polyclonal antibody against semenogelin, a powerful seminal marker

Among various seminal plasma proteins, semenogelin (Sg), produced in the seminal vesicle, has been considered a candidate for demonstrating the presence of semen. Sg consists of two proteins, one 52 kDa (Sg-I) in size, and the other a mixture of 71 and 76 kDa proteins (Sg-II). Recombinant Sg-I and Sg-II proteins were obtained using a baculovirus system and then injected into a rabbit to produce the respective antibodies [Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells, Int. J. Mol. Med. 2 (1998) 693]. When liquefied seminal plasma was immunoblotted with the anti-Sg-I and Sg-II antibodies, the anti-Sg-II antibody identified a wider range of the polypeptides originating from Sg than did the anti-Sg-I antibody. A dot-blot-immunoassay using anti-Sg-II antibody revealed a clear immunoreactive spot even when the semen was diluted 6400-fold. However, this assay showed that the Sg antigen was undetectable in saliva, urine, vaginal secretions, sweat, nasal secretions and serum. To determine the stability of Sg antigenic activity, filter paper with dried semen stains were kept at 37, 4 and 22 degrees C for 1, 6 and 18 months, respectively, and the Sg antigenic activity was examined. The activity was detectable in an area not less than 0.5 cm x 0.5 cm under all of the above environmental conditions during each period. Finally, semen was mixed with saliva or blood at various volumetric ratios, and used as a source of dried stains. The Sg antigenic activity was detectable in the stains until the ratio of semen to saliva or blood reached 1:8. These results suggest that Sg may be useful as a marker for semen identification.     

人精浆特异蛋白P_30的分离纯化与鉴定

本文介绍人精浆特异蛋白P_(30)分离纯化及鉴定的方法。人精浆经蒸馏水透析后经Sephadex G100、G150和G75三种不同的柱层析即可获得精浆特异蛋白。SDS-PAGE、免疫电泳、免疫双扩散证明其纯度和特异性均符合要求。它与已知抗P_(30)血清呈强阳性反应,用其免疫制备的抗血清和已知抗P_(30)血清,在免疫电泳中只与精浆或精浆特异蛋白形成一条完全相同的沉淀线,SDS-PAGE测得分子量约为30,000。故笔者将其亦定名为P_(30)。     

Identification of human semenogelin in membrane strip test as an alternative method for the detection of semen

Semenogelin (Sg), a protein originating in the seminal vesicles and a substrate for prostate specific antigen (PSA or p30), is a useful marker for the identification of semen. And detection of Sg has been available commercially in a membrane test recently. PSA is commonly used to detect semen in forensic significant samples taken from sexual assault cases. The strip PSA test has been available commercially from various manufacturers for many years. In this study, we evaluated two immunochromatographic membrane tests, one for Sg and the other for PSA by analyzing human semen, other human bodily fluids/materials including urine, blood, saliva, sweat, breast milk, vaginal secretion and fecal materials, semen from various animals and forensic casework samples. The data demonstrate that both Sg and PSA strip tests provide rapid and sensitive method for identification of seminal plasma. These results show that the immunochromatographic method for Sg detection is useful for the identification of seminal plasma in forensic samples, an alternative to the method for PSA detection.     

用酶标抗人精液单克隆抗体A_(10)C_6建立ELISA斑点法快速鉴定人精(液)斑

作者用过氧化物酶标记自己研制的抗人精液单克隆抗体A10C6建立了简便快速鉴定人精斑的ELISA斑点法，直接通过酶标抗体上的酶催化底物显示颜色变化，来判断结果，阳性呈灰色斑点，阴性为无色。其结果只对人精斑浸出液显示阳性斑点，对人的其他组织器官及体液均呈阴性，对动物精斑也无交叉反应，精斑浸出液稀释到1：3200倍仍呈阳性结果。     

Detection of human seminal γ-glutamyl transpeptidase in stains using sandwich ELISA

A sensitive and specific sandwich ELISA for human seminal γ-glutamyl transpeptidase (γ-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. γ-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal γ-GTP equivalent to 10⁷ times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal γ-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 γ-GTP was detectable even in 14-year-old stains.     

分泌抗人精液特异蛋白P_(30)单克隆抗体杂交瘤细胞系的建立和初步应用

作者通过杂交瘤技术建立了9株产生抗精浆特异蛋白 P_(30) 单克隆抗体的杂交瘤细胞系。它们是由 SP2/0骨髓瘤与经 P_(30) 免疫的 BALB/C 小鼠脾细胞按常规方法进行细胞融合、并经克隆化筛选得出.这些细胞株均经体外培养3个月以上能够稳定分泌抗 P_(30) 单克隆抗体。该抗体只能识别纯化的 P_(30) 和精液中的 P_(30) ;与人精液以外的其他体液和多种人体组织无交叉反应;与几种常见动物的精液和血液无交叉反应。这些 P_(30) 单克隆抗体均属 IgG 类和 IgG_1亚类。其培养上清液和腹水的抗体效价最高分别达到320和128,000。以 ELISA 法应用这些单克隆抗体能很好地鉴定精液和精斑。     

Semen finger print.

A correlation between testosterone (T) and dihydrotestosterone (DHT) exists in seminal plasma. These androgens play a role in sperm maturation. The T/DHT ratio is different from one person to the other, due to the heterogenicity of seminal plasma which stems for the most part from the male accessory sex glands, the prostate and seminal vesicles, and also from the 'epididymo-testicular unit'. This ratio is useful in identifying the person's semen. Consequently, the steroid values from assailant semen or accused persons and semen on the victim's clothes are of cardinal importance in matching. The results reported include data on the validation of this technique as a tool for semen identification. Coital and masturbated semen were correlated, and consecutive coital semen were also analysed. It may be concluded that the T/DHT value is specific for each person.     

人类岩藻糖基转移酶5特异性分布及其在精细胞的表达与定位

目的研究人类岩藻糖基转移酶5(fucosyltransferase 5,FUT5)的特异性分布及在精细胞的表达与定位。方法收集健康志愿者的精液(分离精细胞并提取精细胞膜蛋白)、阴道拭子、唾液及静脉血,应用免疫印迹方法检测FUT5在人类精细胞膜、精浆、阴道液、唾液及血清中的表达量,采用免疫荧光技术检测FUT5在精细胞中的表达与定位。结果免疫印迹方法结果显示FUT5在精细胞膜及血清中有较高表达,但在精浆、阴道液及唾液中未被检测到。免疫荧光实验结果显示FUT5主要存在于精细胞头部。表明人类精细胞膜存在一定表达量的特异性FUT5,可采用抗原-抗体反应分离精液和阴道液混合斑中的精细胞。结论人类FUT5表现出分布特异性,可应用到法医学性犯罪案件中混合斑的鉴定。     

人精浆蛋白双向谱型特征及其器官特异性的研究

本文应用双向聚丙烯酰胺凝胶电泳（2D—PAGE）技术分析了60例正常人精浆中的蛋白成分。阐明了人精浆蛋白双向电泳谱型的特征，并将人精浆蛋白双向电泳谱型与其他体液蛋白双向谱型进行了比较，证明人精浆蛋白双向电泳谱型具有器官特异性。本文还用2D－PAGE对不同条件下保存的精斑进行了认定。并将其应用于强奸案的鉴定。     

Detection of orosomucoid 1 phenotypes in semen and semen stains

Orosomucoid 1 phenotypes were detected in seminal plasma by isoelectric focusing and immunoprinting. The orosomucoid 1 phenotypes in seminal plasma correlated with the types found in the corresponding serum specimens. Semen stains stored for ten days could be typed for orosomucoid 1. The present work revealed that orosomucoid 1 is a useful genetic marker for the medicolegal grouping of semen stains.     

Glucose phosphate isomerase variants in human bloodstains and seminal stains

Glucose phosphate isomerase (GPI) variants occurring in human red cells were also demonstrated in human semen. Phenotyping was possible from bloodstains of 6 weeks storage and seminal stains of 12 weeks storage. The GPI system may be a supplemental tool for medicolegal individualization of seminal stains.     

用等电聚焦酶免疫分析法检测人类精斑GC表现型

本文用IEF结合使用过氧化物酶标记第二抗体的酶免疫分析法检测了17名键康成年男性的精液(斑)和唾液斑及10名健康成年女性的阴道分泌液中的GC表现型。结果发现17份人类精斑均可测出三种GC蛋白普通表现型。在10份阴道液中测出一份样本的GC表现型,3份样本有不甚清楚的GC带,不能定型,其他样本均无GC带。17份唾液斑未测出GC。本法的灵敏度(0.675ng)比文献报道的用过氧化物酶标记第二抗体的酶免疫分析(5.6ng)高。     

mRNA荧光复合扩增系统鉴别正常和异常精液初探

目的构建mRNA荧光复合扩增体系,实现对不同种类精液(尤其是无精症精液)的区分鉴别。方法收集正常、少精症及无精症的精液样本,制备精斑样本后提取细胞总RNA,利用逆转录PCR技术扩增2个精子特异mRNA标记(PRM1、PRM2)、2个精浆特异mRNA标记(TGM4、SEMG1)和2个管家基因mRNA标记(TEF、UCE)。结果正常精液样本可检测到全部精液mRNA标记表达;少精症精液样本虽然可检测到全部mRNA标记表达,但精子特异mRNA标记表达量较低;无精症精液样本不能检测到精子mRNA标记,只能检测到精浆特异mRNA标记。结论利用mRNA荧光复合扩增系统可以实现对正常和无精症精液的区分,而正常精液和少精症精液相比差异无统计学意义。     

The establishment of the presence of sperm in stains by an agar gel electrophoretic method]

Comparative investigation of seminal stains and other secretions from human body and blood as well as objects of plant origin by gel electrophoresis using alkaline values of pH buffer solutions and subsequent enzymography for acid phosphatase made it possible to develop optimal conditions for detection of seminal presence in stains. Good preservation of seminal acid phosphatase in stains during several years is shown.     

An evaluation of gamma-glutamyl transpeptidase (GGT) and p30 determinations for the identification of semen on postcoital vaginal swabs

The following study entails the investigation of gamma-glutamyl transpeptidase (GGT) and p30 for the identification of seminal stains in sexual assault cases. A commercial kit was used to test for GGT activity, while p30 was demonstrated with a crossed electrophoresis technique. Specificity, sensitivity, and stability of both markers were studied. Postcoital swabs from lab staff were tested for GGT and p30. In addition, 144 postcoital swabs from case material were tested for p30, spermatozoa, and acid phosphatase. Results show p30 to be a useful semen marker particularly in cases of azoospermia. However, GGT was found to be unsuitable for forensic science casework.     

Evaluation of prostate-specific antigen (PSA) membrane test assays for the forensic identification of seminal fluid.

Prostate specific antigen (PSA, also known as p30), a glycoprotein produced by the prostatic gland and secreted into seminal plasma, is a marker used for demonstrating the presence of seminal fluid. Methods for the detection of PSA include Ouchterlony double diffusion, crossover electrophoresis, rocket immuno-electrophoresis, radial immunodiffusion, and ELISA. The extremely sensitive ELISA technique can detect PSA in concentrations as low as approximately 4 ng/mL. However, all these techniques are cumbersome and time consuming to perform in forensic laboratories, especially when only a few samples per week are processed. Various membrane tests are currently used in clinical settings to screen a patient's serum for the presence of PSA at levels greater than 4 ng/mL. In this study we evaluated three immunochromatographic PSA membrane tests by analyzing semen stains stored at room temperature for up to 30 years, post-coital vaginal swabs taken at different time after intercourse, semen-free vaginal swabs, and various female and male body fluids, including urine. The data demonstrate that PSA membrane test assays offer the same sensitivity as ELISA-based tests and provide a rapid approach for the forensic identification of seminal fluid. Furthermore, when the supernatant from a DNA extraction is used for the assay, there is essentially no DNA consumption for determining the presence of PSA in a forensic sample.