用ELISA检测精液及精斑中精子抗原的研究

本实验应用单克隆抗人精子抗体和酶标记羊抗人精子抗体,采用ELISA方法确定精子抗原成份的存在。对10份新鲜精液,15份精斑进行了验测,其结果阳性率为100%。新鲜精液(精子数约10,000万个/ml)稀释100万倍,精斑浸出液稀释50万倍,均可出现阳性。对唾液斑、尿斑、乳汁斑、阴道斑、汗斑及输精管结扎的精液均为阴性。实验结果表明,本法检验精子抗原具有灵敏度高,特异性好的优点。     

MMP-11多克隆抗体的制备及其法医学意义

目的制备兔抗人基质金属蛋白酶-11(MMP-11)多克隆抗体,建立用MMP-11抗体检测月经血的可行方法,探讨其法医学意义。方法将用基因工程制备的人MMP-11融合蛋白免疫新西兰白兔,饱和硫酸铵法进行抗体纯化。运用蛋白印迹法检测月经血痕、外周血痕、阴道液斑、精液斑、唾液斑和尿液斑,盲测验证该方法的可靠性。结果高效价的兔抗人MMP-11多克隆抗体检测月经血MMP-11蛋白的阳性率为90.48%(93/105),而外周血痕、精液斑、唾液斑、尿液斑和阴道液斑均未检出MMP-11。结论用自制的抗MMP-11多克隆抗体所建立的蛋白印迹法检测月经血中的MMP-11特异性好,灵敏有效,可用于月经血及外周血的鉴别。     

人体不同体液内PSA含量及其法医学意义

目的检测人体不同体液内前列腺特异抗原(PSA)含量,探讨其法医学价值。方法收集成年人(19～63岁)晨尿40份(男28份、女12份)、血液58份(男45份、女13份)、唾液25份(男14份、女11份);青少年(10～15岁)男性晨尿205份;哺乳期(25～31岁)女性乳汁9份;使用Cobas e411型全自动电化学发光免疫分析系统及T-PSA定量测定试剂盒,检测各样本T-PSA含量;分析不同体液及不同年龄青少年男性尿液PSA含量差异。结果除男、女性唾液外,其它样本均可检测到PSA,其中成年男性尿液含量最高,与其它体液比较具有显著性差异(P<0.000 1);青少年男性各年龄组尿液PSA含量随年龄逐年增高,11岁及以下年龄组含量不足1ng/mL,14岁及以上年龄组可超过1 000ng/mL。结论前列腺发育成熟的男性尿液PSA含量较高,在进行精液斑的法医学检验时应给予充分注意。     

免疫磁珠法与差异裂解法检验混合样本的效果观察

目的分析应用免疫磁珠分离技术和差异裂解法对混合样本的DNA检验效果,评价其在法医学应用中的价值。方法制备含有不同数量精细胞与阴道上皮细胞混合悬液和植绒混合斑拭子,分别采用MOSPD3抗体、SPAG8抗体免疫磁珠法及差异裂解法对样本进行检验,观察分析不同比例的混合细胞悬液,以及不同保存时间植绒混合斑拭子DNA分型的正确率。结果当混合细胞悬液内精细胞与阴道上皮细胞比例约1∶10时,免疫磁珠法的正确率明显高于差异裂解法(P0.01);当比例等于或高于1∶1时,两种方法正确率的差异无显著性意义(P0.05)。植绒拭子混合斑的免疫磁珠法正确率明显低于差异裂解法(P0.01);随混合斑保存时间的延长,差异裂解法正确率无明显下降(P0.05)。结论免疫磁珠法适用于新鲜混合样本中精细胞的分离检验,且在精细胞含量较低时优势更明显,差异裂解法较适合陈旧混合斑的分离检验。     

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。     

阴道液与血液中PGM_1亚型、EsD、GLOI表型相关性的研究

本文用等电聚焦电泳法和琼脂糖淀粉混合凝胶电泳法,对阴道液、血液中的PGM_1亚型、EsD、GLOI表型进行了检测。在部分妇女的阴道液中未检出其本人血液中的这些酶型,测出的比例最高的是PGM_1亚型,EsD次之,GLOI最低。凡能显示清晰酶谱带的均与同一妇女血液中的表现型相同,未见不一致的现象。同时初步探讨了月经周期和性刺激对阴道液中PGM_1酶活性的影响。     

唾液中乙醇含量检测试剂条的研究

目的根据唾液和血液中乙醇含量相关性的实验结果,建立了一种简便快速、准确可靠的检测唾液中乙醇含量的方法。方法本方法利用酶学原理,将一定量乙醇氧化酶(ALO)和过氧化物酶以及底物四甲基联苯胺(TMB)固定于试剂条上,当样本中含有乙醇时,酶学反应使底物TMB显色,通过比对反应的不同颜色,对样本中乙醇质量浓度进行半定量。结果用本方法检测300个自愿者的唾液,和用GC/MS法对照检测志愿者唾液中的乙醇含量,定量结果基本一致。本产品检测过程仅需2min,其检测的阈值为0.1mg/mL,敏感度为96.5%,特异性为91%,准确性为94.7%。结论采用酶学方法制备的乙醇含量检测试剂条,通过显色反应对唾液中的乙醇含量进行半定量检测,其特点为快速简便、准确可靠,适合现场使用。     

用免疫酶组化法测定混合斑中精子ABO血型的案例分析

精液与阴道液混合斑的检测,一直是强奸案件中的棘手问题之一。传统的中和试验由于不能排除阴道分泌物中ABH物质对精液ABO血型测定结果的干扰,个人识别能力有限。国内外学者用多种方法研究证实了人类精子上含ABH物质。欧炯文等采用酶标抗体免疫组化法直接测出了混合斑中精子的ABO型,我们用该法对各市、县公安局送检的12例强奸案混合斑中精子的ABO型进行了测定,供个人识别之用,结果见表、图1、图2。     

基于microRNA表达谱初步构建PLS-DA体液识别模型

针对外周血、唾液、精液、月经血、阴道分泌物这五种法医学常见的体液样本，采用二代测序（NGS）平台检测获得microRNA(miRNA)表达谱，使用偏最小二乘判别分析（PLS-DA）建立基于这五种体液的识别模型，并探讨PLS-DA在法医体液溯源中的应用价值。经优化小分子RNA文库制备过程，采用Ion Torrent S5 XL测序系统对前述法医学五种体液样本（每种10例）进行小RNA测序，以PLS-DA构建体液识别模型，评估不同数量miRNA标记组合下预测的准确性。本研究获得法医学常见五种体液样本的miRNA表达谱，外周血与月经血中表达量前10名的miRNAs有6个重叠；唾液和阴道分泌物中表达量前10名的miRNAs有4个重叠。基于全数据集、107个和11个miRNAs构建的体液来源识别模型的准确率分别为0.95、0.94、0.89。本研究通过NGS测序分析获得了五种体液样本的miRNA组（miRNome），利用PLS-DA初步构建了体液识别模型，对于应用miRNome进行体液识别的相关研究具有参考价值。     

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。     

ABO genotyping by polymerase chain reaction.

ABO blood group system's genotyping by polymerase chain reaction in genomic DNA level is developed. The positions of nucleotide 258 and 700 of cDNA from A transferase were used to distinguish A, B, and O alleles by restriction enzyme digestion. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with Kpn I. For the 700th nucleotide, a 128-bp PCR amplified fragment was designed and digested with Alu I. By examining the DNA fragment digested patterns, ABO genotypes were easily determined. Results obtained using this method on 20 ABO-known peripheral blood samples showed that this new technique could provide accurate ABO genotype. Biologic forensic samples, such as, blood stains, saliva stains, semen stains, hair, bone tissue, and semen contaminated with vaginal secretion were also successfully typed. This rapid, sensitive and reliable method should be applicable not only in forensic identification but also in medical examination.     

Determination of the Lewis blood group substances in stains of forensically relevant body fluids

Forensic investigations often demand a clear definition of secretor status. Lewis-typing of secretion stains may help to verify non-secretor results and to identify mixtures of secretions from Le (a-b-) persons and secretors (or non-secretors). Furthermore it gives an additional check on secretor status, determined by ABO-grouping. Few problems may arise, when testing prepared saliva or semen stains. Therefore our interest was focussed on the possibility of Lewis-typing in stains appearing in forensic case work such as cigarette tips, stamps and envelope flaps, semen stains and vaginal swabs, nasal secretion, sweat and urine stains. All stains with the exception of sweat and urine were successfully Lewis-typed. In saliva stains Lewis substances could be determined even after 5 years and in semen stains for at least up to 40 days.     

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.     

A sandwich enzyme-linked immunosorbent assay for ABO blood typing of semen by using anti-p 84 monoclonal antibody as a marker of blood group substance in semen

A blood group substance (BGS), a protein with ABH antigenic activity, was isolated from human seminal plasma and designated as p 84 (Sato, 1995). We have developed a method for determining the ABO blood type of semen by performing a sandwich enzyme-linked immunosorbent assay (ELISA) in which p 84 is captured with an anti-p 84 monoclonal antibody, and evaluated the specificity and sensitivity of this method. Although BGS activity was detected in semen sensitively by this method, it was not detected in saliva, urine, breast milk, blood or vaginal secretions. Since the concentration of p 84 in semen was independent of the secretion status, the status can be determined as non-secretor when p 84 but not BGS activity was detected. To determine the stability of BGS activity on p 84, dried stains of semen on filter paper were kept at 4, 26, and 37 degrees C for 8 months, 2 years and 1 month, respectively, and their BGS activities were examined. After 8 months at 4 degrees C, over 60% of the original BGS activity was recovered from the stain. The activity could be detected even from a square as small as 0.25 by 0.25 cm. After 1 month at 37 degrees C and 2 years at 26 degrees C, 31 and 20% of the BGS activity, respectively, still remained. It could be detected from the pieces of 1.0 by 1.0 cm and 0.5 by 0.5 cm squares, kept for 1 month at 37 degrees C and 2 years at 26 degrees C, respectively. Finally, semen was mixed with saliva or blood at varying volumetric ratios and used for the sources of dried stains. The BGS activity of p 84 could be detected in the stains until the ratio between semen and saliva or blood reached 1:4. We conclude that this sandwich ELISA offers a more sensitive and specific method for determining the ABO blood type of semen samples obtained from sexual assault victims than existing methods, such as the conventional absorption-elution and classical hemagglutination-inhibition tests.     

Identification of saliva stains by determination of the specific activity of amylase.

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.     

Detection of saliva traces using test strips

The test strip Rapignost-Amylase (Behring) for the rapid determination of alpha-amylase in the urine is also suitable for the determination of salivary amylase in stains stored up to 6 weeks at room temperature. The stains are extracted with physiological saline (extraction time 30 min), then the application zone of the strip is wetted with the extract. Positive amylase-reaction is recognised as a reddish-violet colouration of the reaction zone. Biological stains with low amylase concentrations (urine semen, vaginal secretion, mucus) react amylase negative. The method is uncomplicated and can be completed within 30 min. The test strips are easily available and stable during storage. Therefore the determination of saliva with test strips should be preferred to the clinical methods if the storage times of the stain are not longer than 4-6 weeks. It is a suitable procedure to determine salivary stains for use in forensic biology.     

DIA3 phenotyping in human semen and seminal stains by isoelectric focusing

The polymorphism of DIA3 was investigated by isoelectric focusing in semen samples from 235 unrelated Japanese volunteers and patients. Besides the three common phenotypes seven samples of the type 3-1 were observed. However, readable isoenzyme patterns were not demonstrated in semen samples of oligospermia under about 10 X 10(6)/ml sperm cells. The allele frequencies were DIA3*1 = 0.821, DIA3*2 = 0.164, and DIA3*3 = 0.015. The DIA3*1 frequency in oligospermia (0.765) was lower than that in normospermia (0.836). The isoelectric focusing method was successfully applied to phenotyping DIA3 in seminal stains; each phenotype was demonstrated at 37 degrees C for up to 4 weeks, at room temperature for up to 8 weeks, and at 4 degrees C for over 12 weeks after stain formation. In vaginal swabs the isoenzyme bands were very faint and not identifiable.