应用斑点ELISA快速进行体液(斑)的血型测定

<正> 在法医学上体液(斑)的血型测定一般常规应用中和试验、吸收试验、解离试验及混合凝集反应。这些试验既耗时,又需较高的技术,且要新鲜标准红细胞指示结果。本实验系应用我们自己提纯标酶的抗-A,-B与抗-H单克隆抗体A,利用斑点ELISA来进行体液(斑)的血型测定。通过颜色变化直接判断血型。     

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.     

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 ABO blood grouping from human oral squamous epithelium by the highly sensitive immunohistochemical staining method EnVision+

Using the highly sensitive immunohistochemical staining method EnVision+, which employs a dextran polymer reagent for the secondary antibody, the detection of the ABH antigens was attempted in the oral squamous epithelium. This new technique uses monoclonal antibody as a primary antibody and it takes about three hours for staining. The time is much shorter than conventional absorption-elution testing or absorption-inhibition testing for the determination of ABO blood grouping. Secretor saliva samples were stained at strong intensity by the antibody, which corresponded to its blood group and anti-H. On the one hand, nonsecretor saliva samples were stained at strong intensity only by the antibody that corresponded to its blood group, and at weak intensity only by anti-H. Since human oral squamous epithelium antigens were stained specifically by this method, we can examine the ABO blood group of saliva samples and perform cytodiagnosis at the same time. Our research suggested that the EnVision+ Method is a useful technique for ABO blood grouping of saliva in forensic cases.     

Lewis typing of human bloodstains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b)

The Lewis blood grouping of human dried bloodstains could be determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies with an avidin-biotin complex (ABC). The bloodstains aged 1 year were used as samples, and approximately 1 mg of the stains was enough to type each Lewis antigen reliably by this method. The Lewis substances of 106 individual stains were correctly typed regardless of their ABO blood group system.     

Detection of Lea substance in saliva stains by enzyme-linked immunosorbent assay (ELISA) using anti-gum arabic serum

It is known that rabbit anti-gum arabic (GA) serum has cross-reactivity with Lea antigen, and that, by using this cross-reactive anti-Lea antibody, the presence of Lea antigen in red blood cells and saliva can be demonstrated with accuracy. We have devised a rapid and highly sensitive method for detecting Lea substance in human saliva by the enzyme-linked immunosorbent assay (ELISA) method using an anti-Lea antibody isolated from anti-GA serum by affinity chromatography on Synsorb Lea. The ELISA plate, coated with the specific anti-Lea antibody, adsorbed the Lea substance in saliva which was subsequently identified by adding enzyme labeled anti-Lea IgG in that order. The method could detect the Lea substance in Le(a+) saliva stains as small as 0.1 by 0.1 cm in size that had been stored at room temperature for three weeks and in Le(a+) saliva stains 0.7 by 0.7 cm in size that had been stored for ten years. This method seems to be useful for quantitative analyses of the Lea substance in various body fluids.     

Development and application of hydrogel oligonucleotide microchips for forensic-medical personal identification as illustrated by ABO locus

The article describes the method defining 5 alleles of ABO blood group typing system by molecular hybridization in hydrogel oligonucleotide microchip. The testing points were SNP variants in positions 261 and 297of exon 6 and in positions 646 and 657 of exon 7. Therefore, 5 ABO blood groups can be easily revealed: A, B, 0(1), 0(1v), 0(2). The method was tested on 10 DNA samples isolated from blood and saliva of unrelated individuals. The results were confirmed by sequencing of the identified allelic fragments. Estimation sensitivity was 25 pg of total DNA input. This technique is cost-effective and easy for use and, therefore, promising for forensic-medical personal identification.     

An enzyme-linked immunosorbent assay (ELISA) for prostate-specific antigen.

A sandwich ELISA for human prostate-specific antigen (PSA) is described. Optimal assay conditions, resulting in a sensitive assay with a low background, are presented. The method uses a hyperimmune antiserum produced in the New Zealand white rabbit, against human semen PSA. The IgG fraction of the antiserum was conjugated with horseradish peroxidase and used in the sandwich ELISA method. The anti-PSA IgG showed no cross reactions with saliva, normal blood, female urine, vaginal fluid, or menstrual blood. On occasions, a blood sample showed a non-specific cross-reaction, which was detected by non-immune rabbit IgG. This reaction could be caused by rheumatoid factors, as indicated by experiments with a series of known IgG and IgM rheumatoid antibodies, although other heterophilic antibodies could not be eliminated. The recovery of PSA added to blood plasma, saliva and vaginal fluid was affected by three factors; (a) protein concentration (dilution) of body fluid; (b) nature of the protein; and (c) amount of PSA added.     

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.     

G3m(21) typing by ELISA and dot immunobinding with enzyme-labeled monoclonal anti-G3m(21) antibody

Simple, rapid methods are described for G3m(21) typing with peroxidase-labeled monoclonal anti-G3m(21) antibody. In G3m(21) typing by ELISA, microtiter wells were coated directly with the test antigen, which was detected with the enzyme-labeled monoclonal antibody. To further simplify the procedure, a dot immunobinding method was developed. The antigen in the test serum applied onto a nitrocellulose membrane was successfully detected with the enzyme-labeled monoclonal antibody. These methods, particularly the dot immunobinding, are suitable for forensic casework because they are rapid and simple and require no technical skill.     

型特异性沉淀素血清检验人唾液斑精斑ABO血型的研究与应用

本文介绍了利用型特异性沉淀素血清环状沉淀法检验人唾液斑、精液斑ABO血型的方法与实验结果,并与中和试验及解离试验进行了比较。实验结果表明,本法操作简便,对多种干扰条件下的唾液斑、精斑均具有高度的型特异性,并能从分泌液与血液的混合斑中准确地鉴别出分泌液的血型。本法仅需0.4cm的分泌斑纱线即可进行血型鉴定,其灵敏度高于中和试验而略低于热解离试验,并能有效地检出陈旧分泌液斑中的型物质,因此适于在实际检案中应用。     

One method of collecting fallen off epithelial cell

In this paper, the comparative analysis of ABO genotyping and serological typing was conducted in 360 unrelated blood samples from northern Chinese Han population using genotyping method and serological typing method, respectively. The results of ABO genotyping were obtained by Goldeneye 16BT STR plus ABO kit. The ABO serological types were determined by the antigen–antibody agglutination test. The ABO types were confirmed by the two methods and no contradiction types were found; two more types were obtained using the ABO genotyping method and the discrimination power was further improved; the information of ABO genotyping and 15 STRs could be obtained at the same time using the Goldeneye 16BT STR plus ABO kit.     

免疫抗A、抗B、抗H沉淀素血清的制备

本文介绍了用浓缩的分泌型人唾液免疫鸡,经吸收精制获得鸡抗A、抗B、抗H沉淀素血清的方法。用本法制备的抗血清,对相应血型人唾液的特异性沉淀价可达1:512倍以上,对非相应血型者的分泌液不出现非特异性沉淀反应。应用该血清进行环状沉淀试验、单向琼脂扩散试验等免疫学试验,可简便、准确地鉴定人唾液斑、精液斑等分泌液斑的ABO血型。     

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.     

Comparison of modern techniques for saliva screening

Saliva stains present a unique challenge in the forensic setting, often challenging the analyst to weigh the value of presumptive indication of the fluid versus the potential for DNA analysis to yield identification information. There are many situations in which determining the presence of a body fluid is probative and further corroborates DNA evidence. That said, even a minute portion of sample consumed by a screening test could mean the difference between a full, partial, or null profile obtained through DNA analysis. The basis of presumptive testing or screening of saliva has historically been based on the presence of amylase, a component found in relatively high concentrations in human saliva versus other body fluids and substances. Though the current available methods for the screening of saliva in a forensic application have grown in number, the popularity of these methods seemingly has not. This study attempts to identify a specific and sensitive saliva screening test by comparing three modern techniques--the recently released SALIgAE, Phadebas, and starch-iodine mini-centrifuge test--on the basis of sensitivity, specificity, mixtures, and simulated casework samples while also considering sample consumption. The Phadebas method for presumptive saliva testing detected dilutions of neat saliva down to 1:200 versus considerably less sensitive results with SALIgAE and the starch-iodine mini-centrifuge test. Utilizing a screening test with a high degree of sensitivity, such as Phadebas, allows an analyst to gain a maximum amount of information in the form of body fluid indication and DNA results because of the consumption of a small portion of sample.     

ABO typing studies on liquid urines

ABO typing was successfully performed on 46 urine samples whose ABO group and secretor status had been determined previously from blood and saliva. Twenty-four urine samples were collected on which blind studies, time studies, and storage studies were performed. Multiple urines from several individuals were collected to evaluate the duplicity of the test. Also, urines were collected from pregnant and menstruating females to determine if ABO typing was affected under these conditions. Results of these studies are discussed.     

Effect of blood group active micro-organisms on the ABO grouping of human whole saliva

Three saliva samples with false positive ABO grouping results were assayed for blood group active organisms, using a variety of selective media to isolate representative strains from the salivary microflora. Eight out of 40, 8 out of 40 and 4 out of 30 strains from the three samples, respectively, showed blood group activity, which correlated well with the false positive specificities of the saliva samples. In all cases the false reaction only lasted a few days. Investigation of one of these samples before and after the appearance of the false positive activity yielded only one out of 40 blood group active organisms, using the same methods. Similar investigation of two "normal" saliva samples found none out of 40 and one out of 40 blood group active organisms, respectively. It is concluded that occasional false positive ABO grouping reactions of saliva samples are probably caused by the presence of unusually high numbers of blood group active micro-organisms, due to disturbances in the ecological balance of the salivary microflora.     

Determination of ABO(H) blood group substances from finger and toe nails

Thirty-six finger and toe nails were analyzed for ABO(H) blood group substances by the modified absorption elution method. The blood groups from nails were successfully determined in all the samples.     

A novel method for ABO genotyping using a DNA chip

ABO genotyping is often performed to identify the blood type of decomposed samples, which is difficult to be determined by a serological test. In this study, we developed a simple method for ABO genotyping using a DNA chip. In this method, polymerase chain reaction-amplified and fluorescent-labeled fragments in the ABO gene and primate-specific D17Z1 were hybridized with DNA probes on a chip designed to detect single nucleotide polymorphisms (SNPs) in the ABO gene and part of the D17Z1 sequence. Using blood samples from 42 volunteers and 10 animal species, we investigated whether the chip could be used to detect SNPs in the ABO gene and the D17Z1 sequence. This method was then applied to various forensic samples, and it was confirmed that this method was suitable for the simultaneous analyses of ABO genotyping and species identification. This method fulfills the recent need for the development of rapid and convenient methods for criminal investigations.     

Lewis typing of human saliva stains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies

The Lewis blood grouping of human saliva stains could be detected by an enzyme-linked immunosorbent assay (ELISA) using anti-Le(a) and anti-Le(b) monoclonal antibodies with an avidin-biotin complex (ABC). The saliva stains (1.0 by 1.0 cm in size) were used as samples and not only could the Lewis substances of 57 individual stains be correctly typed by this method, but also it was clarified that there are several different secretion patterns of amounts of Le(a) and Le(b) substances in 3 individual Lewis types.