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.     

Determination of ABH secretor status by an electronic quantitation method

Blood Group A and B substances in secretor (Se) and nonsecretor (se) salivas were tested by means of an electronic data processing-hemagglutinin-inhibition test (EDP-HAIT) with immunoglobulin M (IgM) isohemagglutinins. Besides a difference in quantity, the blood group substances in Se saliva showed high binding efficiencies compared with those in se saliva. EDP-HAIT with IgG isohemagglutinins proved no difference in the binding efficiencies of Se and se salivas. The determination of secretor status by EDP-HAIT with IgM isohemagglutinin was accurate because the conclusion was obtained based on two different quantitative results. Secretor status of some salivas in gargled water could be determined by comparing the binding efficiencies.     

Immunohistological studies on ABH-activities in secretory cells of human major salivary glands--correlation between ABH-activities in the secretory cells and secretor-nonsecretor

The activities of A, B and H in serous cells (S-cells), mucous cells (M-cells) and excretory duct cells were examined in a large number of paraffin sections of three major salivary glands obtained from 91 corpses, using the immunofluorescence technique. The results are: By taking H activity in S-cells of the submandibular gland or A, B and H activity in M-cells of the sublingual gland as an indicator, the salivary glands were classified as Type I showing activity and Type II showing no activity. No glands corresponding to the intermediate type, as seen in the case of saliva, were noted at all. Among 91 corpses, 70 cases were classified as Type I and 21 as Type II. The results matched well with those of Lewis type tested on blood. The frequencies of the typing (Type I; 76.9%, Type II; 23.1%) were approximately in concordance with those of secretor and nonsecretor in Japanese saliva. From these results, it was assessed that the former corresponded to the secretor type in the case of saliva, and the latter to the nonsecretor type. Even in the same individual, both S-cells and M-cells exhibited different productivities of substances, depending on the glands to which they belonged. Namely, only S-cells in the submandibular gland belonging to Type I showed only H activity independent of the blood group of the individual, but the other S-cells in the other major glands did not show any activity for A, B and H. M-cells exhibited strong activity for H and/or A and/or B in the sublingual and submandibular gland and belonged to Type I, but little activity in the sublingual gland belonged to Type II. In the submandibular gland of Type II, some M-cells showed activity and others did not. On the basis of the above results, we discuss the applicability of the present genetic theory concerning the secretor and nonsecretor type in saliva to salivary glands and cells, and further refer to the reasons for appearance of the weak secretor type or intermediate type in saliva.     

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.     

抗人N单克隆抗体的制备及其特性的研究

用ON型人红细胞免疫BALB/c小鼠脾细胞与SP2/O骨髓瘤细胞融合,获得分泌IgM类抗人N单克隆抗体杂交瘤细胞株(ON_1D_3)。其培养上清液抗体效价为128～256,诱生小鼠腹水的抗体效价为8192;与Panel A红细胞反应呈抗N特异性;检测273名成人红细胞,只特异性凝集N(?)MN型人红细胞,不凝集M型红细胞。与猴等9种动物红细胞无交叉反应;酶修饰试验结果表明,ON_1D_3识别的抗原决定簇是唾液酸依赖性的。     

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。     

The detection of soluble ABH blood group substances in semen and saliva using monoclonal blood grouping reagents

Using an enzyme-linked immunosorbent assay (ELISA), this study investigated the use of monoclonal antibodies for detecting secreted ABH blood group substances in semen and saliva. The results demonstrated that the behavior of some monoclonals were unpredictable and often failed to detect the corresponding antigen in a number of the specimens tested. The suitability of the monoclonal reagents for detecting soluble blood group antigens could not be predicted by their behavior with red cell antigens. Consequently, care must be taken in the selection of monoclonal reagents for use in the detection of secreted blood group antigens.     

Concentration of urine samples by three different procedures: ABO typing from concentrated urine samples

Urine samples from 28 donors with known blood group and secretor status were concentrated by three different procedures, and ABO typing on the concentrated samples was successfully performed after 12 weeks of storage. The effects of storage with or without sodium azide on ABO typing and on the pH values at several different temperatures were also studied.     

Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.     

The detection of gamma globulin and Inv-factors in semen and saliva and of haptoglobins in semen (author's transl)]

Gm- 1-, 2-, and Inv 1-factors can be demonstrated in semen and saliva. For the examination of traces it is necessary to verify the suitable dilutions for the different charges of antisera and to test the eluates of the samples if they have a sufficient concentration. We observed some incorrect negative results in seminal and saliva stains which were apparently caused by insufficient material. The demonstration was independent of the secretor type. Haptoglobin could not be determined in semen and saliva.     

唾液血型抗原分泌时限变化及温度对抗原活性的影响

探讨人唾液中ABH血型抗原不同时限的分泌量,以及保存温度对血型抗原活性的影响。应用时间决定性荧光免疫测定法（TR．FIA）对O型分泌型10例和非分泌型5例人在不同条件下唾液中H抗原量进行检测。唾液血型抗原的分泌量随时间而波动,进餐后降低明显,但不干扰分泌型的判定。37℃保存48h抗原活性完全丧失,6℃保存1周抗原活性几乎没有变化。结果表明,唾液分泌时段不影响分泌型判定,将唾液制成斑痕可长期保存样品。     

Detection of hereditary enzyme characteristics in vaginal secretion

Upon investigation of semen- and blood-free vaginal swabs using starch gel electrophoresis the Phosphoglucomutase type was clearly identified in about 40%. Using cellulose acetate membrane electrophoresis PGM could not be demonstrated. In all cases the results correspond with those obtained in blood. No relation was found between secretor type (determined in saliva) and PGM typing. In vaginal material the following could not be determined: Adenylatkinase (AK) using starch gel electrophoresis, Esterase D (EsD) using cellulose acetate membrane electrophoresis, and Glyoxalase I (GLO) using agarose gel thin-layer electrophoresis.     

人类A、B抗原单克隆抗体的研制和初步应用

作者用人类A、B型红细胞及A、B型分泌型唾液免疫8周龄的BALB/C小鼠,用经免疫的脾细胞与SP2/O骨髓瘤细胞融合,用血凝法筛选出分泌抗A抗体和抗B抗体的细胞株各3株。经一年多的传代,仍然能稳定地分泌抗A、抗B抗体,抗体特异性专一,培养上清液效价能达到2048倍,并初步应用于血液及体液(斑)的检验。     

Alpha-amylase kinetic test in bodily single and mixed stains

Recently, in Italy, a murder and a putative sexual violence was accomplished on a child. A bodily fluids mixture on the child's underwear between the victim (female) and the suspect (male) was ascertained by short tandem repeat (STR) DNA typing and, due to the absence of seminal fluid, saliva from the suspect and urine from the child was hypothesized. In order to investigate the possibility of specifically and rapidly detecting saliva stains both alone and mixed with other bodily fluids, we used a quantitative spectrophotometric technique, named Amylase test, for the detection of alpha-amylases. We determined alpha-amylase activity and reaction kinetic curves in several samples collected from the child's underwear. In order to confirm our intuition, we first tested saliva, perspiration, and urine, singularly and in mixtures; second, several forensic stains including saliva, perspiration, urine stains, saliva/perspiration, and saliva/urine mixture stains were tested. Evaluating alpha-amylase activity values and time-course curves' behavior of alpha-amylase reactions we were able to recognize successfully, in all cases, the presence of saliva and to distinguish it specifically from other bodily fluids containing alpha-amylase. A further confirmation of our result was provided by STR DNA typing on several areas of the underwear: a clear correlation between alpha-amylases activity and male DNA was detected on all the samples evaluated.     

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.     

唾液及含唾液检材的DNA分析

提取160份唾液及含唾液的检材中的DNA，并根据DNA的质和量进行了DNA指纹图检验或应用聚合酶链反应（PCR）进行了DNA分型。结果表明，唾液和含唾液检材是很好的DNA来源．对其进行DNA分析是可行的。     

A sensitive and simple assay of saliva on stamps

Identification of saliva on stamps or envelope flaps remains yet a not widely studied problem. In most forensic laboratories it is seldom carried out, but this fact does not reduce the importance of the assay. Most authors consider amylase a sufficiently specific marker of the presence of saliva; really, the only other human body fluid that contains high amounts of this enzyme is the pancreatic juice (and therefore feces). Here we present a simple and sensitive assay for the determination of alpha-amylase that uses a commercially available and well-known substrate. It is hydrolyzed by amylase with the production of soluble blue fragments, that can be measured by photometry, obtaining objective results. The presented assay identifies 1 X 10(-6) diluted saliva or that present on 0.5 mg of a stamp; 16-year-old samples can also be identified. Intra-assay and day-to-day CV resulted in 10.8% and 13.7%, respectively. Owing to the high sensitivity of the test, handling samples or reagents can introduce contamination with saliva traces, giving false-positive results. Addition of EDTA 0.1 mol/l to the incubation mixture, lowering the sensitivity to 1 X 10(-3) diluted saliva, overcomes this problem.     

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.     

Evaluation of Direct PCR Amplification Using Various Swabs and Washing Reagents

DNA profiles were generated via direct amplification from blood and saliva samples deposited on various types of swab substrates. Each of the six non-FTA substrates used in this research was punched with a Harris 1.2 mm puncher. After 0.1 μL of blood or 0.5 μL saliva, samples were deposited on each of these punches, samples were pretreated with one of four buffers and washing reagents. Amplification was performed using direct and nondirect autosomal and Y-STR kits. Autosomal and Y-STR profiles were successfully generated from most of these substrates when pretreated with buffer or washing reagents. Concordant profiles were obtained within and between the six substrates, the six amplification kits, and all four reagents. The direct amplification of substrates which do not contain lysing agent would be beneficial to the forensic community as the procedure can be used on evidence samples commonly found at crime scenes.     

抗Le~a和抗Le~b血清的研制

利用进口抗Le~a、Le~b血清筛选OLe(a+b-)和OLe(a-b+)型人,测定其唾液中Le~a和Le~b型物质含量,择其型物质含量高者唾液,用家兔和山羊免疫,合格后采全血,分离血清,用O、A、B型红细胞吸收、除去种属及α和β等凝集素。再用O型Le非相应型红细胞吸收,精制出含不完全抗体的抗Le~a和抗Le~b血清。该血清对Le相应型红细胞均产生明显凝集反应,而不发生非特异的交叉凝集反应。经过盲测鉴定,17份红细胞的Lewis型测定完全准确。制备的抗Le~a和抗Le~b血清在效价和特异性方面达到了引进的同种血清水平,填补了我国抗Le~a、抗Le~b血清制造的空白。