Dot—ELISA在用酶标单克隆抗体检验体液斑和血痕ABH血型物质中的应用研究

本文应用Dot-ELISA法用酶标单克隆抗体,直接检验出稀释5万倍的精斑、10万倍的唾液斑及阴道分泌物等体液斑和血痕中ABH物质,还可检验出1～17年的体液斑中ABH物质。全部操作在室温中进行,以肉眼观察颜色变化判断结果,试验周期30～60分钟,准确率100％,实验结果可长期保存。     

人体组织中ABH物质分布的研究

本研究采用特异性红细胞粘附试验(SRCA 试验)方法,系统地研究了11例已知 ABO 血型及分泌状态尸体的38种组织器官 ABH 物质的定位与分布,发现粘膜、粘液腺及前列腺中均含有丰富的 ABH 物质,并受分泌状态控制。血管内皮细胞、复层上皮细胞、胰腺腺泡上皮及汗腺中也含有较多的 ABH 物质,但不受分泌状态的影响。新发现肺泡上皮、肝小胆管粘膜上皮亦含有 ABH 物质。其它组织器官除自身的血管内皮及红细胞含有 ABH 物质外,均未测出 ABH 物质。采用 SRCA 试验,室温放置13天的皮肤组织亦能正确地测出其 ABO 血型。     

Detection of ABH blood group antigens in the saliva of Koreans and their stability according to storage of saliva samples

The purpose of the present study was to identify salivary molecules carrying the ABH blood group antigens in Koreans and to investigate the changes in these antigens according to processing and storage of saliva samples. Secretor or non-secretor phenotypes and salivary components carrying the ABH antigens were identified in 90 subjects, 30 subjects in each ABO blood group, by SDS-PAGE and immunoblotting. Saliva samples were then obtained from 12 secretors-two males and two females in each ABO blood group and aliquots of both fresh saliva samples and their supernatants after centrifugation were stored at room temperature, 4, -20 and -70 degrees C. The same experiments were performed after 1, 3 and 6 months to investigate changes in the blood group antigens. In all 68 secretors, high-molecular-weight salivary mucin (MG1) was found to be the primary carrier of the ABH antigens. A salivary component of approximately 80 kDa also carried H antigen in seven saliva samples of 22 blood type O secretors. The blood group antigens were better detected in centrifuged samples. In saliva samples preserved at room temperature and 4 degrees C, the blood group antigens were either not detected or detected as degraded molecules. No change was found in the blood group antigens in saliva samples preserved at -20 and -70 degrees C for 6 months.     

An ELISA using avidin-biotin complex for the determination of ABH group from saliva

The ABH group in a trace amount of saliva could be determined by an enzyme-linked immunosorbent assay (ELISA) using an avidin-biotin-peroxidase complex (ABC) technique. In this method ABH blood group substances as a solid phase are adsorbed to wells of a microtiter plate made of polystyrene. The primary antibody corresponding to the blood antigen adheres onto the wells, and reacts with the biotinylated secondary antibody. The previously formed ABC reagent is then added to the above wells, and finally the absorbance produced by the interaction of the peroxidase activity with a chromogenic substance is measured at 492 nm. This method proved to be clearly more sensitive for the detection of ABH blood groups in secretor-saliva than the conventional hemagglutination inhibition test. Also the ABH group of non-secretor-saliva could be easily determined by this method.     

The rapid determination of the ABO group from body fluids (or stains) by dot enzyme-linked immunosorbent assay (dot-ELISA) using enzyme-labeled monoclonal antibodies

Using ABH enzyme-labeled monoclonal antibodies, the authors could rapidly detect the ABO group from body fluids and body fluid stains by the dot enzyme-linked immunosorbent assay (dot-ELISA). In this test, the antigen was immobilized on nitrocellulose paper; the entire piece of paper was coated with an appropriate dilution of enzyme-labeled McAb directly against the antigen of interest; and, finally, 3,3'-diaminobenzidine (DAB) substrate solution was added. The site of a positive reaction is clearly visible as a brown spot. We analyzed 521 samples and got satisfactory results. We also analyzed 99 practical case samples by this method and achieved the same results as those obtained by other researchers using other methods. This method is accurate, simple, direct, rapid, and sensitive; it also produces easily observed results, requires no equipment, and can be completed in 30 min. The test proved to be clearly more sensitive for the detection of the ABO blood group in secretor saliva than the conventional hemagglutination inhibition test. Also saliva diluted 10(-4) to 10(-5) and the ABO group of nonsecretor saliva and urine could be easily detected by this method.     

A promising microbiological test for the diagnosis of drowning

A number of biological and chemical tests have been developed over the years to determine whether a person was drowned. This study focuses on the potential of a microbiological test for detecting common bacterial markers of water faecal pollution such as faecal coliforms (FC) and faecal streptococci (FS) as possible indicators of drowning. A promising previous study was carried out on central and peripheral blood samples of 42 drowned victims (20 cases in saltwater and 22 cases in freshwater) and 30 not-drowned bodies. To improve the accuracy of our previous results and also in order to investigate a possible cause of a false positive due to pulmonary passive diffusion and subsequently endogenous or exogenous bacterial invasion of the blood in the post-mortem interval (PMI), the FC and FS test was applied to bodies submerged in water but died from causes other than drowning. In the present study, blood samples collected from the left ventricle (LV), right ventricle (RV), femoral artery (FA) and, femoral vein (FV) of 10 drowned victims (5 cases in freshwater and 5 cases in seawater) and 3 not-drowned individuals with bodies submerged in water for a while after death have been analysed. Preliminary results are in agreement with other reports dealing with diatoms and marine bacteria that suggest to exclude the hypothesis of a passive penetration of sufficient quantities of drowning medium into circulation after death or during the agonal period. Based on our results there is also no evidence of a relevant dissemination of endogenous micro-flora from the gastrointestinal tract affecting the FS and FC test. There are still several other factors that could influence the applicability of post-mortem FS and FC cultures for the diagnosis of drowning and they need further investigations. The present article provides only a glimpse of the potential of the FS and FC test as bacteriological method for the diagnosis of drowning.     

An extremely sensitive species-specific ARMs PCR test for the presence of tiger bone DNA

The survival of the tiger (Panthera tigris) is seriously threatened by poaching to provide raw materials for Traditional Chinese Medicines (TCMs). Most highly prized are the tiger's bones, which are used in combination with other animal and plant derivatives in pills and plasters for the treatment of rheumatism and other ailments. Hundreds of patent remedies have been produced which claim to contain tiger bone, but proof of its presence is needed, if legislation prohibiting the trade in endangered species is to be enforced. A highly sensitive tiger-specific real-time PCR assay has been developed to address this problem. Using primers specific to the tiger mitochondrial cytochrome b gene, successful amplification has been reliably achieved from blood, hair and bone as well as from a range of TCMs spiked with 0.5% tiger bone. Although capable of detecting fewer than 10 substrate molecules, the seven varieties of TCM pills and plasters tested showed no detectable trace of tiger DNA before spiking. Furthermore, sequencing several "tiger bone" fragments seized from TCM shops has shown that they actually originated from cattle and pigs. The potential effects of traditional bone preparation methods, evidence that much lower concentrations are used than alleged on TCM packaging, and substitution of bones from other species all suggest a low likelihood of detecting tiger DNA in patent medicines. Despite this, the basic methods have been thoroughly proven and can be readily applied to derivatives from other CITES protected species providing a rapid and highly sensitive forensic test for species of origin. Potential applications to the monitoring of wild populations are demonstrated by the successful identification of shed hairs and faecal samples.     

New possibilities of detecting blood and seminal fluid in traces on material evidence using Fluorat-02-3M analyzer

Fluorat-02-3M liquid analyzer essentially extends the range of hemoglobin measurements, increasing its concentration in the studied extract from 0.1 to 100 micrograms in a 100 microliters sample (the lower threshold concentration remains unchanged: 0.005 in a 100-microliters sample) in detection of the blood in stains on material evidence pieces by fluorescent hemotest. This allows detection of blood in both washed and clearly seen blood stains on pieces of material evidence. Examples of using this method for measuring mass concentrations of zinc in water extracts on a Fluorat-02-3M analyzer are presented. The analyzer was developed at the Bureau of Forensic Medical Expert Evaluations of the Leningrad region for tentative detection of seminal fluid in stains on material evidence pieces. The results of this method are objectively recorded.     

因输血感染所致损害的法律救济探析

在因输血使患者感染疾病的场合,如果血液提供者存在过错,由于过错责任原则的存在,患者可获得的法律救济较为简单。如果血液提供方没有过错,患者会求助于货物销售、默示担保、产品责任法律制度,诉讼前景不容乐观。货物与服务的二分法排除了在医疗输血领域货物销售法律制度的适用。血液提供的必要性、不可替代性和风险的不可避免性否定了默示担保和严格责任的适用余地。要获得产品责任制度的益处,患者要跨过"产品"、"缺陷"、"发展抗辩"三道关口,结果可想而知。     

The examination of vaginally inserted plastic tampon applicators for genetic markers and evidence of prior sexual intercourse

Vaginally inserted plastic tampon applicators were obtained from 42 female volunteers. The applicators were examined for the presence of ABH blood group substances, phosphoglucomutase (PGM), amylase, acid phosphatase, P30, and intact spermatozoa. Each applicator was accompanied by a control blood sample, a saliva specimen, a brief sexual and menstrual history, and method of birth control of the donor. Eight of the male sexual partners of the donors submitted blood and saliva samples. One male sexual partner submitted only a saliva sample. ABH blood group substances corresponding to the donor were recovered from 36 of the 42 applicators. The remaining 6 applicators revealed a combination of the donor's and sexual partner's ABH substances. The female's PGM type was recovered from 34 of the applicators. The remaining 8 applicators failed to show PGM activity. Of the applicators, 15 indicated evidence of prior sexual intercourse by the detection of ABH substances not consistent with the applicator donor (6 samples), high levels of acid phosphatase (11 samples), or recovery of spermatozoa (8 samples) or some combination of these. All applicator samples failed to show the presence of either P30 activity or PGM factors foreign to the female.     

Lewis blood group determination in bloodstains by planimetric measurement of eluted monoclonal antibodies

Planimetric measurements were employed for reading the results of an elution test to determine Lewis blood groups in dry human bloodstains. In the absorption-elution test, two varieties of indicators were used to detect eluted Lewis antibodies. First, 64 blood-stains aged between 2 to 8 months were tested with glutaraldehyde (GLA)-treated erythrocytes (planimetric hemagglutination assay, PMHA). This method demonstrated that dry stains weighing approximately 0.4 mg (equivalent to 3 microliters of whole blood) were sufficient for detection of Lea or Leb antigen. Results were obtained within 1 h. Then, 37 of these stains were tested with Lewis substance-coated latex particles (planimetric latex agglutination assay, PMLA). The presence of Lea and Leb antigen were detected from dry stains weighing 0.1 mg (equivalent to 1 microL of whole blood) within 3 h. Both these assays are faster and simpler with accuracy than the enzyme-linked immunosorbent assay (ELISA). Latex particles coated with Lewis substance are, in particular, strongly agglutinated and show agglutination patterns more clearly than erythrocytes. The blind tests using these two methods properly classified 7 Le(a + b-) and 23 Le(a-b + ) bloodstains; whereas, 5 Le(a-b-) stains were undetermined by the criteria for these tests. These results indicate the usefulness of the PMHA and PMLA for typing Lewis blood groups from small bloodstains.     

An extremely sensitive species-specific ARMS PCR test for the presence of tiger bone DNA

The survival of the tiger (Panthera tigris) is seriously threatened by poaching to provide raw materials for traditional Chinese medicines (TCMs). Most highly prized are the tiger's bones, which are used in combination with other animal and plant derivatives in pills and plasters for the treatment of rheumatism and other ailments. Hundreds of patent remedies have been produced which claim to contain tiger bone, but proof of its presence is needed if legislation prohibiting the trade in endangered species is to be enforced.A highly sensitive tiger-specific real-time PCR assay has been developed to address this problem. Using primers specific to the tiger mitochondrial cytochrome b gene, successful amplification has been reliably achieved from blood, hair and bone as well as from a range of TCMs spiked with 0.5% tiger bone. Although capable of detecting fewer than 10 substrate molecules, the seven varieties of TCM pills and plasters tested showed no detectable trace of tiger DNA before spiking. Furthermore, sequencing several "tiger bone" fragments seized from TCM shops has shown that they actually originated from cattle and pigs. The potential effects of traditional bone preparation methods, evidence that much lower concentrations are used than alleged on TCM packaging, and substitution of bones from other species all suggest a low likelihood of detecting tiger DNA in patent medicines. Despite this, the basic methods have been thoroughly proven and can be readily applied to derivatives from other Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) protected species, providing a rapid and highly sensitive forensic test for species of origin. Potential applications to the monitoring of wild populations are demonstrated by the successful identification of shed hairs and faecal samples.     

一次检测微量血痕的种属来源、ABO血型及红细胞酶EsD、PGM_1的表型

本文介绍一种一次检测微量血痕的种属来源、ABO 型、红细胞酶 EsD、PGM_1表型的方法。用盲法共测151份已知血痕种属来源 ABO 血型、EsD 与 PGM_1表型的血痕纤维,均能正确判型、吻合率为100％。     

用酶标抗体免疫组化法测定性交后阴道内容物中精子的ABO血型

应用间接酶标抗体免疫组化法测出了53例新鲜精液、5例陈旧精斑及40例阴道分泌液中的精子与阴道脱落上皮细胞的ABO血型,30例精子与不同血型分泌型阴道分泌液孵育,未发现精子吸附阴道液中血型物质的现象,同时发现人类睾丸曲精细管中部份生精细胞、精子细胞,精子;直细精管部份上皮细胞、精液、精子;睾丸网大部份上皮细胞及副睾管中的精液与精子均含ABH抗原,故认为精子上的ABH抗原主要是精子固有抗原,13例性交后阴道内容物中精子的ABO型测定结果:7例与供者血型吻合,6例不吻合。6例中5例从O型精子中测出了女方分泌型阴道分泌液中的A或AB物质,1例B型精子未测出B及H抗原,文中对这种现象进行了讨论。     

Detection of glycosphingolipids of ABH and Le antigens on thin-layer plates using the PAP method

Stroma from hemolyzed erythrocytes of blood groups 0, A1, B and A1B were obtained and subjected to butanol/phosphate buffer extraction. This extract was separated using HPTLC, and the ABH and Le substances were detected on the chromatogram using the PAP technique. The staining of the bands allowed specific demonstration of the serologically active glycosphingolipids present in the ABH and Le blood group substances. The antigens of the AB0 system showed a 3- to 12-band pattern. Each of the antigens Lea and Leb presented 3 bands. The slight differences in the levels of glycosphingolipids of equal chain lengths are probably due to differences in their chemical structures.     

对我国民事执行中实体争议救济的考量

我国现行法律在民事执行当事人和大量实体问题争议事项上存在救济缺失的同时,也为处理民事执行中实体问题争议提供了僭妄的条件。在对关乎缺失和僭妄的民事执行中实体问题争议救济主体、事项、处理方式和处理机构四个方面的考察论证后认为,针对民事执行中各种实体问题争议,应设置不同层次和充分空间的救济;处理民事执行中实体问题争议既应通过诉讼的方式,也不能排斥非诉(异议)的方式;其处理机构应在多元模式中,根据我国的具体情况确立。     

宪法审查的穷尽法律救济原则

"穷尽法律救济原则"是各国宪法审查制度普遍采行的启动要件,它在集中式审查模式中主要存在于宪法诉愿程序中,而在分散式审查模式中主要体现为"穷尽行政救济"。然两类规范形态具有相通的双层结构规范内涵:在判断是否已穷尽相关法律救济之后,对于未穷尽者,进一步判断其是否属于具有普遍性意义或者会产生重大且无法避免之损害的例外情形,以谨慎决定宪法审查是否可提前。这既体现出宪法审查的备位性,也说明备位性须受到人权保障之价值目标的限制。     

Sensitivity of various study technics in blood stains

Quadratic pieces of fleece measuring 16 mm2 were soaked with 10 different blood-samples in the dilution steps of 1:1, 1:10, 1:100, 1:1000, respectively, and were tested in blood group typing and identification tests of forensic serology. The above spezified dilutions correspond with 5 microliters, 0.5 microliter, 0.05 microliter and 0.005 microliter of blood, respectively. The detection limit of the microspectrometric test for blood was the dilution 1:10, of the porphyrine test a dilution above 1:100, whereas the preliminary test for blood (peroxidase) succeeded always up to a dilution of 1:1000 and the species determination by the radial immunodiffusion test in agar gels succeeded in most cases op to a dilution of 1:1000. The detection limit of the anti-human globulin inhibition test was between the dilution steps 1:10 and 1:100 when non-titrated and undiluted anti-human globulin serum was used. Gc- and ABO-grouping were possible up to a dilution of 1:100 and were thus the most sensitive grouping systems. Phenotyping of the enzyme-systems and the Gm/Km-system usually required stains with considerably higher blood concentrations i.e. stains of undiluted blood.     

Study of the diagnostic value of iron in fresh water drowning

The aim of our study was to test the diagnostic value of iron (Fe) in fresh water drowning by investigating the postmortem levels of hemodilution in drowning cases compared to control cases. Twenty-six typical fresh water drowning cases were selected from 128 immersion cases autopsied in our Department of Forensic Pathology between 1998 and 2004. The exclusion criteria were a long postmortem interval and other causes of death than drowning. For all selected cases, the diagnosis of drowning was based on the presence of autopsy findings and positive diatom test. A control population of 12 cases was also selected. For each drowning and control case, iron blood levels were measured in the left ventricle (LV) and right ventricle (RV) of the heart. The mean difference of iron concentration (RVFe-LVFe) between the drowning group and the control group was statistically compared. Furthermore, iron measurements were performed in 19 drowning cases showing advanced putrefaction. The mean difference of iron concentration was significantly higher in the drowning cases compared with controls (P<0.001). All drowning cases showed hemodilution. No overlap was found in the RVFe-LVFe levels between the two groups. Resuscitation attempts seemed to have no effect on the results. In cases of drowning showing advanced putrefaction, the iron test was not reliable because biochemical iron measurement was often prevented by no sufficient blood in the heart or postmortem clots. In conclusion, according to our results, iron seems to be a good biochemical marker in fresh water drowning with a short postmortem interval.     

H-抗原缺乏分泌型个体FUT1及FUT2等位基因的分子基础

一例H-缺乏分泌型个体被发现。其血清学表现为：红细胞上无A、B、H抗原；唾液中有B、H抗原分泌；血清中检出了抗A抗体。推测其为类孟买OHmB型。在该个体FUT1等位基因编码区上发现两处单碱基突变（T460C、G1042A）。这两个点变异，将导致两个氨基酸的置换（Y154H、E348K）。同时也破坏了限制性内切酶RsaⅠ和AvaⅠ的作用部位。用PCR－RFLP法可检出此两种变异。用PCR－RFLP法证实，该个体为T460C、G1042A变异的纯合子。在136例随机个体中未能查出上述变异。将该FUT1基因转染COS－7细胞未能检出α2－FUT活性及证实H抗原的表达。该个体的FUT2基因与野生型一致。