抗人Hb试剂盒的应用

抗人Hb试剂盒是采用金标单克隆抗体技术,检验血痕种属的一种新的试剂盒。通过对不同稀释度的人血及动物血的检测及不同温度、pH值等环境因素下的检验,证实使用抗人Hb试剂盒检验血痕的方法是一种简单、快速、灵敏、稳定、特异性好的实验方法。     

用酶标抗人Hb单克隆抗体检测微量血痕种属的实验

法医学血痕种属检验的方法很多，如环状沉淀试验，琼脂糖单扩散试验以及对流免疫电泳试验等。这些方法操作简便，但灵敏度低，易受血痕所附着的客体以及化学合成物等因素的干扰。笔者参照文献［1］介绍的方法，用酶标抗人Hb单克隆抗体检测微量血痕种属，取得良好的效果，现报道如下。材料与方法一、材料酶标扰人Hb单克隆抗体和8管反应板（华能公司博塞试剂研究所生产）；抗人Hb单克隆抗体（白求恩国际和平医院提供）；抗人Hb多克隆抗体（公安部第二研究所血清室提供）；三羟甲基氨基甲烷等试剂（北京化学试剂厂）；牛血清白蛋白（BSA）（…     

反向间接血凝(RPHA)法应用在血痕检验中的研究

血痕检验目前采用的方法有肉眼观察、预试验、确证试验、种属鉴别、血型检查及出血部位检验等.用免疫法制备抗人血红蛋白(Hb)的抗血清进行人血液的法医学种属鉴别已沿用很久,但国内还未见到RPHA法应用在血痕检验中的报导.本实验室建立了该法检测人粪便隐血,并对模拟血痕的检测也作了研究.认为本法对微量血痕     

5种免提取试剂盒检验滤纸血痕结果的比较

目的探讨5种免提取试剂盒对滤纸血痕样本检验的效果。方法陈旧滤纸血痕(存放时间12～14个月)及新鲜滤纸血痕(存放时间小于1个月)各920份,分别随机分为5组。应用AGCU 17+1、Goldeneye 20A、Powerplex16HS、Identifiler Plus、Identifiler Direct 5种免提取试剂盒进行检验,对比各组检验结果。结果陈旧滤纸血痕5种试剂盒的检验成功率为98.91%～100%,各组间无差异(P>0.05);新鲜滤纸血痕,Identifiler Plus和Identifiler Direct试剂盒检验成功率高于AGCU17+1、Goldeneye 20A及Powerplex 16HS试剂盒(P<0.01);将样本做陈旧化处理后再用成功率较低的3种试剂盒进行检验,成功率分别升至100%、99.46%、99.46%;Identifiler Plus试剂盒扩增循环27次效果优于28次。结论本文5种试剂盒均可用于滤纸血痕的直接扩增检验,但使用AGCU17+1、Goldeneye 20A及Powerplex 16HS试剂盒需将新鲜血痕做陈旧化处理;Identifiler Plus试剂盒需将循环次数降为27次。     

抗人红细胞凝集素血清的应用研究

本文通过吸收抑制试验证明了抗人血红蛋白沉淀素血清中存在的那种能凝集人类红细胞的凝集素,实际上就是用人的红细胞免疫家兔而产生的抗人红细胞凝集素.为考察抗人红细胞凝集素血清对血痕种属的鉴识能力,本文用此抗血清作解离试验,对不同稀释度的新鲜人血痕、室内存放的久暂不一的人血痕和15种动物血痕进行了检测.得到的结果是,新鲜人血痕稀释至1/1000仍呈阳性;500例室内存放的人血痕中,存放时间在11年以下的448例全部能够准确测出;存放12-16年的52例人血痕中,有33例能够测出,余19例呈阴性;15种动物血痕全部呈阴性.     

应用聚乙二醇—抗人Hb液检验血痕种属

<正> 聚乙二醇(Polyethylene glycol,PEG)是一种棒状分子结构的多糖,可以促进抗原抗体反应,形成免疫复合物。在临床检测中,引入 PEG 可明显提高临床免疫检验的灵敏度,最少可测到0.1mg/L 的蛋白抗原。作者根据这一原理,在常规的抗人 Hb 血清沉淀试验作血痕种属鉴定中,     

新鲜血痕样本直接扩增试验条件比较

目的探讨新鲜血痕在不同直接扩增试剂盒的试验条件。方法存放2d内的新鲜血痕FTA卡540份和存放1~3m的陈旧性血痕FTA卡270份,各分别随机均分为3组,每份血痕打3片1.0mm纸片,分别用DNATyperTM15Plus试剂盒、GoldeyeTM20A试剂盒和华夏TM试剂盒在标准条件(说明书条件)、优化条件1(标准条件+1μL DMSO)和优化条件2(标准条件+室温浸泡1h)扩增检验,比较在3种条件下各组STR检验成功率。结果陈旧血痕样本用3种直扩试剂盒,在3种条件下,检验成功率均97.00%,且均高于新鲜血痕。新鲜血痕在标准条件和优化条件1下,成功率为27.22%~31.67%,在优化条件2下,检验成功率相似(97.00%),与标准条件和优化条件1比较,有统计学差异(P0.01)。结论新鲜血痕在加入直接扩增试剂后于室温浸泡1h,可有效提高STR检验成功率。     

酶标2G8单克隆抗体斑点ELISA快速检验人血痕G2m(23)因子

利用自制的抗人 G2m(23)酶标2G8酶标单克隆抗体,首次应用直接斑点 ELISA 方法检测血痕中 G2m(23)因子。该法最小检出量为0.000048μl 血清;整个试验可在15分钟内完成。对常见的13种动物血和8种鱼血无交叉反应;盲测450例干血痕结果全部正确。该方法具有快速、准确、灵敏、简便等特点,有较大的实用价值。     

用酶标抗人IgG单克隆抗体直接ELISA法检测微量血痕种属

<正> 酶联免疫吸附试验(ELISA)在法医物证检验中已有许多研究和报道。国外不仅用于血痕种属鉴别,而且还应用于血型及器官特异性的检测。笔者采用酶标抗人 IgGMcAb 作直接 ELISA 法鉴别血痕种属.获得满意效果。     

ABO基因分型与多重STR联合检测在法医学中的应用

目的建立ABO基因型和Goldeneye16A试剂盒联合检测的方法,并评价其在法医学实践中的应用价值。方法将6种ABO基因型(A/A,A/O,B/B,B/O,A/B,O/O)的序列特异性引物(PCR-SSP)检测方法与Goldeneye16A试剂盒相整合进行同步分型。通过对460份男性个体血痕样本、9947A DNA及90份案件样本进行检测,考察方法的一致性、灵敏度及对法庭科学检材的适用性。结果应用本文方法可同时检出6种ABO基因型和15个常染色体STR基因座及性别决定基因座,检测灵敏度为125pg,其中ABO基因检测灵敏度达63pg。460份男性血痕和90份案件检材证实该联合分型方法用于各类检材结果准确、稳定。结论本文ABO基因分型与多重STR联合检测方法,适用于各类含有核细胞的生物检材,在法庭科学DNA鉴定中有较好的应用前景。     

Validation studies of rapid stain identification-blood (RSID-blood) kit in forensic caseworks

When bloodstains are detected at crime scene using presumptive tests (e.g. luminol, phenolphthalein, leuchomalachite green), it is important to establish the real human nature of each stain. This is possible using confirmatory tests. One of these is rapid stain identification-blood (RISD-blood) a lateral flow immuno-chromatographic strip test format which allows the identification of human blood by detection of glycophorin A, a red blood cell membrane antigen, using two anti-human glycophorin A (GPA) monoclonal antibodies.The aim of this study is to assess the sensitivity of RSID-blood test in old, degraded bloodstains and in some bloodstains previously treated with BlueStar Forensic, a presumptive test which is often used in crime scene investigations to detect latent bloodstains. The genetic analysis of all bloodstains of confirmed human nature was subsequently performed using the AmpF1STR Identifiler PCR Amplification Kit (Applied Biosystems), to validate the possibility of obtain a consistent and reliable DNA typing results.     

检测丁丙诺啡的胶体金标记单克隆抗体免疫试剂盒研制

目的采用免疫竞争法原理,建立一种简便快速检测丁丙诺啡的方法。方法将20nm胶体金颗粒标记的抗丁丙诺啡单克隆抗体,均匀浸在吸水玻璃纤维上,将丁丙诺啡-BSA和纯化后的羊抗鼠IgG多克隆抗体在硝酸纤维薄膜分别划1mm宽的检测线(T线)和质控线(C线),制成丁丙诺啡胶体金标记单克隆抗体免疫层析检测条,并组装成检测板。结果以GC/MS为标准对照,100例样品检测结果显示,本检测板的准确率为99.0%。丁丙诺啡胶体金标记单克隆抗体免疫层析检测板在对45种药(毒)物的测试中,仅识别丁丙诺啡及其主要代谢产物。结论丁丙诺啡胶体金标记单克隆抗体免疫层析检测板在5min内可检出样品中的丁丙诺啡及其代谢物,检测丁丙诺啡的阈值为100ng/mL。     

PuriTyper~(TM)法医学纯化试剂盒的性能验证

目的验证PuriTyperTM纯化试剂盒各项性能指标和法医学应用价值。方法收集及制备抗凝血液、常见案件检材(唾液、烟头、精液、毛发、指甲、骨骼及组织块)、斑痕样本(血斑、唾液斑、精斑)以及模拟添加抑制剂和模仿自然环境中放置的血斑。采用PuriTyperTM纯化试剂盒提取纯化并进行DNA定量,IdentifilerTM复合扩增试剂盒扩增,产物经ABI 3130遗传分析仪进行检测,Genemapper软件分析结果,对该试剂盒灵敏度、稳定性、重复性、检材适应性进行测试。结果采用该试剂盒提取0.1～40μL血液分别获得0.042～26.45ng/μL的DNA。3种斑痕样本DNA产量平行试验结果稳定。不同类型检材重复检验所获IPC的CT平均值在27.60至28.03之间。常见案件检材所得分型与已知结果均一致。结论 PuriTyperTM纯化试剂盒能够满足法医DNA检验的要求,对法医学实践具有重要的应用价值。     

A highly specific and sensitive sandwich enzyme immunoassay for human hemoglobin A

A highly specific and sensitive sandwich enzyme immunoassay for human hemoglobin A (Hb A) is described. A rabbit anti-human Hb A IgG-coated polystyrene ball was incubated with human Hb A and subsequently with affinity-purified anti-human Hb A Fab'-horseradish peroxidase conjugates, which had been prepared before and after absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. Bound peroxidase activity was measured by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. The detection limits of human Hb A using the conjugate before and after the absorption were 0.65 pg/tube (3 X 10(10)-fold dilution of whole blood) and 2.0 pg/tube (1 X 10(10)-fold dilution of whole blood), respectively. Human Hb A could be discriminated from Hb of animals such as Japanese monkey, dog, cat, pig, horse, sheep, chicken and cow by measuring bound peroxidase activity in the presence and absence of the conjugates prepared before and after the absorption. Human Hb A in bloodstains on cotton gauze could be discriminated from Hb of animals described above even after seven washings. Human Hb A in 220,000-fold diluted bloodstains on cotton gauze could also be discriminated from Hb of animals described above.     

Transferrin subtyping of human bloodstains

A method was described for subtyping transferrin derived from human bloodstains. Bloodstain cuttings were extracted in 0.5% ferrous ammonium sulfate. The extracts were subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. After isoelectric focusing, transferrin was detected by silver staining. This method permitted the successful typing of Tf in 6-month-old blood stains maintained at -20 degrees C and room temperature and 3-month-old bloodstains maintained at 37 degrees C.     

琼脂糖凝胶电泳法同步检测血液和血斑型特异性成分及转铁蛋白型

作者采用琼脂糖凝胶电泳法和免疫固定技术,同时分析血液和血斑的型特异性成分(Gc)及转铁蛋白(Tf)两种血清型,并调查了北京地区229名汉族健康献血员两种血清型的表型分布及基因频率。     

微量生物物证提取套装在现场微量血痕DNA检验中的应用

目的探讨微量生物物证提取套装应用于提取现场微量血痕DNA的检验效果。方法将静脉血制成地面血痕。分别应用微量生物物证提取套装法和普通法提取血痕。分别于恒温摇床放置2、24、48、72、96h(每组50份)进行血痕DNA检验,对比各组检验结果。结果恒温摇床上分别放置24、48、72、96h后,微量生物物证提取套装法的DNA检出率(分别为100.00%、100.00%、100.00%、96.00%)均高于普通法(分别为62.00%、26.00%、10.00%、0),差异均具有统计学意义(P<0.05)。恒温摇床上放置2h,两种方法的DNA检出率差异无统计学意义(P>0.05)。结论微量生物物证提取套装可有效提升放置时间较久的现场微量血痕的检出率和检出时限。     

国产Goldeneye~(TM) 20A试剂盒性能指标验证

目的测试国产GoldeneyeTM20A试剂盒技术性能指标,评估其法医学应用能力。方法从方法学验证、准确性、峰值均衡性、灵敏度、批次间试剂及稳定性测试、耐受性、不同检材的适应性与一致性、种属特异性、混合样本等9个方面对GoldeneyeTM20A试剂盒进行测试。结果阳性DNA样本分型正确,内标和等位基因分型标准物符合要求;等位基因间的均衡性≥83%,同一荧光标记基因座间的均衡性≥55%,不同标记物间的均衡性≥52%;0.125ng DNA阳性样本可检出全部STR基因座分型,不同批次间和反复冻融后试剂盒测试可以获得正确分型,对降解检材和混有抑制剂的样本等具有一定的耐受性,能对案件中多种检材进行分型且分型结果一致,具有一定的种属特异性和混合DNA样本检测能力。结论国产GoldeneyeTM 20A试剂盒可用于法庭科学实际检案与建库。     

Human chorionic gonadotropin detection by means of enzyme immunoassay: a useful method in forensic pregnancy diagnosis in bloodstains

This paper reports on human chorionic gonadotropin (HCG) detection in bloodstains using a commercial kit based on enzyme immunoassay. The specificity and sensitivity of the method is tested, as well as HCG stability over time in the samples. Detection of this hormone in bloodstains is of special interest for pregnancy diagnosis in forensic science applications. The experimental series comprises 60 bloodstains prepared in our laboratory and obtained from blood samples taken from 30 pregnant women and 30 healthy, nonpregnant individuals. The bloodstains were studied at different stages of aging over a 6-month period, each sample being stored at 2 different temperatures throughout the process. The experimental evidence proves HCG to be a useful and stable diagnostic indicator of pregnancy in bloodstains. In addition, the technique used is fast, as well as highly sensitive and specific.