Immunologic characterization of human seminal leucine aminopeptidase (LAP) and its medicolegal use

A new method for identification of seminal stains is described, based on the immunologic demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen and specific for the prostate as well as semen. An antiserum against human seminal plasma was obtained by repeated immunization of rabbits with seminal plasma and Freund's adjuvant. Ouchterlony's double immunodiffusion test and Culliford's precipitin electrophoresis were performed to demonstrate specific proteins of seminal plasma. LAP activity was visualized with L-leucyl-beta-naphthylamide as substrate and with Fast Garnet GBC as coupler. The immunologic analysis of LAP produced two precipitin lines with enzyme activity. One was observed in kidney, jejunum, pancreas, prostate, as well as in semen, and was completely absorbed with kidney homogenates. The other was found only in semen and the prostate and was not absorbed with kidney homogenates. When the anti-seminal plasma serum absorbed with the kidney was used, the semen-specific LAP could be demonstrated by precipitin electrophoresis only in seminal stains stored for up to 2 months, whereas it was not demonstrated in stains from other human body fluids. By means of precipitin electrophoresis the detection of the semen-specific LAP was possible at semen dilutions of up to 1:32. The method described here greatly enhances the value of semen identification and is quite recommendable for the examination of stains in medico-legal practice.     

ELISA检测精浆特异蛋白P_(30)鉴定人精斑

<正> 1978年美国Sensabaugh分离出精浆特异蛋白P30,并成功的制备出相应的抗P30血清。1987年我国也研制出抗人精浆特异蛋白P30血清,并应用该种抗血清建立了几种琼脂扩散和电泳方法鉴定人精斑。由于人精液中P30含量不高,平均为1.52±0.676mg/ml,鉴定微量精斑存在困难。为了更有效的提高方法的灵敏度。我们建立了鉴定微量人精斑的ELISA固相P30抗体法,现报告如下。     

用酶免疫斑点法快速检测精浆特异蛋白P_(30)鉴定人精斑

作者用自制的辣根过氧化物酶标记的抗精浆特异蛋白P_(30) 单克隆抗体,建立了简便快速检测精斑中P_(30) 鉴定人精斑的斑点法。通过颜色变化判定结果,阳性为紫蓝色斑点,阴性为无色。结果表明,仅人类精斑和前列腺浸液出现阳性斑点,人体其他体液及组织器官浸液均为阴性。对动物血、精斑也无交叉反应。标定精斑稀释到1600倍亦可得到阳性结果。     

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.     

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.     

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.     

Comparative study of the sensitivity and specificity of the zinc and acid phosphatase spot tests for the detection of seminal stains

The sensitivity and specificity of a zinc spot test for the detection of semen were compared with those of an acid phosphatase detection method. As screening techniques both tests were found to be very sensitive, but the zinc test was more specific and was more reliable in older and especially in deteriorated specimens. It is concluded that the zinc spot test deserves at least the same place as the acid phosphatase test in the primary investigation of suspected semen stains and might well be the test of choice in older and poorly preserved stains.     

Developmental validation of RSID™-Semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen

Abstract: Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate‐specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID?‐Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID?‐Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross‐react with other human or nonhuman tissues tested. RSID?‐Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA‐based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID?‐Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.     

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.     

Evaluation of the 190H analogues of prostaglandins E1, E2, F1 alpha and F2 alpha as specific markers for the identification of human semen in body fluid mixtures

The specificity of antisera raised against each of the prostaglandin series 190H E1/E2 and 190H F1 alpha/F2 alpha, produced in males, was evaluated by radioimmunoassay. Further, the ability of these antisera to detect semen specific prostaglandins in mixtures of body fluids was examined. Antisera directed against the 190H E1/E2 series cross-reacted with prostaglandin E1 and marginally with E2. Antisera raised to the 190H F1 alpha/F2 alpha series were, however, highly specific to the semen specific prostaglandins 190H F1 alpha/F2 alpha and 190H E1/E2. It was possible to detect picogramme quantities of contaminating 190H F1 alpha/F2 alpha on vaginal swabs taken up to 72 h after intercourse and on vaginal swabs stored at room temperature for up to 2 years. These prostaglandins were not detected on semen free vaginal swabs, in faecal material, saliva, urine or in a sample of human milk (stain). A limited study of casework material is also described. Detection of the 190H F series, as a group, has considerable potential in the identification of human semen at picogramme levels, eliminating the need for alternative chemical tests and extensive microscopic examination.     

The zinc test as an alternative for acid phosphatase spot tests in the primary identification of seminal traces

The value of the acid phosphatase spot test in the primary visualization and identification of seminal traces is hampered by the sensitiveness of the enzyme to biodegradation. An alternative spot test is proposed, based on the high concentration of the more stable zinc metal in seminal plasma. The proposed zinc spot test is simple and suitable for on site investigation. Although the sensitivity in fresh stains is lower than that of the acid phosphatase spot test, this is largely compensated by the lower sensitiveness to biodegradation. The specificity for semen is higher than that of the acid phosphatase spot test. In vaginal swabs it was nevertheless seen, that samples should be taken within 24 h after alleged sexual assault to give reliable results.     

An improved method for semen alpha-L-fucosidase typing--distribution in the Wuhan population of China

An improved method for the separation of the genetic variants of alpha-L-fucosidase in human semen is described. The method involves, isoelectric focusing in ultrathin-layer PAG containing a mixture of ampholines, pH 5-7 and pH 3.5-9.5, and separator HEPES. The Fu pattern obtained is simple, easy to interpret, and reproducible. The occurrence of fucosidase phenotypes in 189 semen samples from the population of Wuhan was investigated, and the frequencies of the Fu alleles were calculated.     

高效价鸡抗人精液血清的制备和应用

本文介绍一种制备高效价鸡抗人精血清的方法。给10只2kg 以上的公鸡同时静脉和肌肉注射5～10％混合人精液7～8次,获得特异性很强,效价达50,000倍左右的抗血清。通过实验和检案证明,对由于腐败污染、水浸或水洗而含微量精斑的检材,均能检出。     

Use of a Y chromosome probe as an aid in the forensic proof of sexual assault

Currently, the most common procedures for the forensic identification of semen that may be present due to a sexual assault include the microscopic identification of spermatozoa, acid phosphatase activity, or the detection of PSA. However, not all cases of sexual assault result in the deposit of semen. Fluorescent In Situ Hybridization (FISH) has been found to be a very sensitive and specific method for detection of the Y chromosome from male cells. This study was undertaken to demonstrate the presence of epithelial cells of male origin in the postcoital vaginal tract using a commercially available probe. Results identified Y chromosome in intact epithelial cells on postcoital Days 1 through 4, and on Day 7. Additionally, Y chromosome positive epithelial cells were identified in vaginal swabs obtained following intercourse with no ejaculation. The method developed in this study demonstrates that FISH is a sensitive method for the identification of the presence of male epithelial cells in the postcoital vagina.     

The evaluation of an ELISA for semen-specific 19-OH prostaglandin F1 alpha/F2 alpha using ex-casework swabs and stains

The application of an enzyme-linked immunosorbent assay (ELISA) for 19-OH Prostaglandin F1 alpha/F2 alpha (19-OH PG F) to casework analysis of seminal contamination of swabs and stains is reported. The results are compared to those where the identification of semen was based on the presence of acid phosphatase and spermatozoa. Five hundred and one samples were analysed and there was good agreement between the results of ELISA and conventional methods. The determination of 19-OH PG F confirmed both the presence of semen where spermatozoa were absent and indicated semen was present when acid phosphatase and spermatozoa were both absent. The results indicate that 19-OH PG F represents a useful marker for the casework identification of semen and is particularly valuable where spermatozoa are absent.     

microRNA Detection in Blood,Urine, Semen,and Saliva Stains After Compromising Treatments

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.     

DNA指纹图在法医学鉴定中应用的研究(Ⅲ)——应用“Myo”DNA探针进行混合斑中精液的个体认定

本文利用蛋白酶K、SDS对精液和阴道液、精液和血液的混合斑进行前处理,除去女性阴道脱落上皮细胞和血液细胞成份获得精子。提取精子DNA,用“Myo”小卫星DNA探针杂交进行DNA指纹检验,获得了高度多态性的精子DNA指纹图谱,与同一个体血液DNA指纹图谱比较完全一致,实现了混合斑中精液来源的个体认定。在对20多起强奸案例混合斑的实际应用中,成功地认定了强奸罪犯。     

The evidential value of peptidase A as a semen typing system

Human erythrocyte peptidase A (Pep A) displays a genetic polymorphism in blacks. Its occurrence in human semen was examined for its possible use as a semen typing system. Studies by starch gel electrophoresis, in which the Pep A was located by an improved method, were carried out on semen, semen stains, and vaginal swabs taken at known times after intercourse. In addition, a large number of vaginal swabs, negative for semen, were taken from females throughout their menstrual cycles and examined for Pep A activity. The results indicated that Pep A typing could be carried out on semen and semen stains. However, it was possible to determine the Pep A type on vaginal swabs only when they had been taken within about 3 h after intercourse.     

Human semen stain analysis in casework sample by HRM-qPCR

Determining the biological origin of body fluid evidence from crime scenes is extremely important for criminal investigation, especially in sexual assault cases. In Brazil, sexual crimes still present low-resolution rates, where approximately 8% of cases are judged. The determination of the presence of semen in samples from crime scenes as a test prior to DNA analysis is a mandatory requirement in forensic analysis and can help to better understand the dynamics of the event. This report aims to present the methodological strategy used in a criminal case of a home invasion where a t-shirt containing visible stains similar to human semen was found at the site. Convencional tests to detect the presence of PSA and sperm cells were performed on the fabric cutouts which showed negative results. We then processed the fabric samples for genetic analysis after two-years-storage, where were performed automated method for the genetic material isolation (QIAcube, Qiagen). The Real-time PCR analysis were carried out using the SOLIScript 1-step SolisGreen (SolisBiodyne) kit, with specific primers to the TGM4 (Transglutaminase 4) gene, in the Rotor Gene Q-5Plex HRM equipment (Qiagen). The results obtained for the melt curve indicated the presence of human semen in the analyzed samples. After the HRM-qPCR assay we also analyzed the a-STR and Y-STR markers. All the results were useful for the criminal process, which led to the identification of the author of the crime.