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。     

Enzyme-linked immunosorbent assay for A and B water soluble blood group substances

The conditions affecting an enzyme-linked immunosorbent assay for salivary blood group substances were investigated. It was found that A, B, and O secretor saliva samples would each bind both anti-A and anti-B typing reagents. The conditions that affected the assay response were optimized for maximum sensitivity and to give the highest resolution possible between the result for an antiserum binding to homologous antigen and the response for heterologous antigen-antibody combinations. Monoclonal antibodies eliminated the heterologous binding indicating that this binding was due to a lack of specificity of the routine typing reagents. A sensitive assay using the monoclonal antibodies to distinguish between samples of A and B secretor saliva is described.     

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.     

成人肾脏ABO组织血型1-4型糖链H物质的分布

目的观察1型H、2型H及3/4型H糖链在成人肾组织中的分布及其与分泌状态的关系。方法 应用抗ABO抗体及3种糖链特异的单克隆抗H抗体的免疫组织化学方法,检查分泌型与非分泌型个体肾组织中相应抗原物质的分布。结果 在分泌型和非分泌型人的肾远曲小管均表达2型H和3/4型H物质,1型H和3/4型H物质只在分泌型人肾集合管表达,在非分泌型人中不表达。另外集合管的2型H物质的表达与分泌状态无关。结论 人肾组织有ABH物质的表达,不同肾组织细胞表达的H物质结构差异与AB0型分泌状态有关。     

非分泌型的基因型与血型物质的分泌研究

阐明非分泌型的基因型与血型物质分泌量和Lewis表型的关系。应用时间决定性荧光免疫测定法（TR－FIA），检测传统的A型Lewis阳性非分泌型个体唾液中H、A及Lewis抗原含量，并以序列特异性PCR，确定其基因型。非分泌型个体唾液中检出了高含量的Lea抗原，其中8例不同程度的检出了少量H、A和Leb抗原，其FUTZ位点为se2／se2纯合型，属Le（a＋b＋）型；1例基因型为se3／se5杂合型，未能检出H、A及Leb抗原，属Le（a＋b－）型。se2是弱分泌基因，se3及se5是非分泌基因。     

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.     

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.     

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.     

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, B and H group specific substances in human vitreous humor

The presence of A, B and H group specific substances in vitreous humor taken from 105 human corpses was determined. Good agreement was obtained between these group substances and the ABO blood group. The relationship with the secretor type is discussed.     

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.     

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.     

唾液中Lewis血型物质的定量研究

应用时间决定性荧光免疫测定法（TR－FIA法），对129例健康成人唾液中Lweis及H1血型物质进行定量检测。Le阳性个体均不同程度地检出了Lea和Leb物质。Lea物质含量：Le（a＋b-）型＞Le（a－b＋）型；Leb物质含量：Le（a－b＋）型＞Le（a＋b－）型。Le（a＋b－）型的Lea物质＞Leb，Le（a－b＋）型的Leb物质＞Lea物质。Le阴性的部分个体未能检出Lewis物质，其余个体也仅检出微量。根据Lea和Leb物质在唾液中的相对含量，可以推测红细胞Lewis型，并提示Leb物质可能由Lea物质转化而形成。     

Immunoenzyme determination of ABO and secretor status in paraffin-embedded autopsy material

Using the indirect immunoperoxidase technique (PAP method), A, B, and H antigens were identified on formaldehyde-fixed, paraffin-embedded kidney tissue from 100 autopsies. Comparison with the serologic findings showed all our blood group determinations to be correct. The labeling of the collecting tubules was evaluated as characteristic of the secretor. The secretor status determined according to this parameter was unequivocally confirmed by the Lewis constellation in 78 of 82 cases; group Le(a-b-) could be differentiated with immunohistochemical methods in secretors and nonsecretors. Determination of ABO blood groups and secretion behavior with immunohistochemical methods was correct even in those cases where classic serology failed due to hemolysis and decomposition. Immunohistologic results obtained with monoclonal antibodies were better than those obtained with human sera.     

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 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.     

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。     

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。     

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.     

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.