分泌抗人类血型-H抗原单克隆抗体杂交瘤细胞株建立及初步应用

应用杂交瘤技术,建立了两株分泌抗 H 的单克隆抗体细胞株 H_(2-6)H_8和 B_(2-5)D_9。经体外培养半年以上,生物学性状稳定。两株细胞分泌的抗体属 IgM 类,特异性识别 H 型物质,与几种常见动物的红细胞无交叉反应。培养上清液和腹水的抗体效价最高分别达1024倍和1.28×10~5倍,可用在中和试验、免疫斑点法中,在实际办案中应用理想。     

The investigation of blood and secretion stains by the absorption-elution reaction using anti-H monoclonal antibodies

Possible use of monoclonal antibodies anti-H in absorption-elution reaction was studied. Blood and secretion stains on gauze were analysed. Practical usefulness of monoclonal antibodies anti-H for investigation of human blood and secretions was stated. Differences in interaction of monoclonal antibodies with traces of different origin were found.     

Use of an anti-H extract of elder berries in the absorption-elution reaction during the examination of blood stains, saliva and sperm

Parameters of using anti-H extract from elder berries in absorbtion-elution reaction in order to detect H antigen in blood and excretion stains were established.     

抗丁丙诺啡单克隆抗体的制备

目的建立抗丁丙诺啡单克隆抗体的杂交瘤细胞株，制备高特异性的丁丙诺啡单克隆抗体，并对其免疫学特性进行鉴定。方法在丁丙诺啡的分子上连接活性羧基基团，通过缩合反应将丁丙诺啡半抗原连接于血蓝蛋白（KLH）和小牛血清白蛋白（BSA），形成完全抗原。以完全抗原免疫Balb／c小鼠，通过细胞融合，筛选等杂交瘤技术，建立稳定的分泌抗丁丙诺啡单克隆抗体的杂交瘤细胞株。通过腹腔注射杂交瘤细胞，诱导小鼠产生含有单抗的腹水。用辛酸-硫酸铵加亲和层析法纯化抗丁丙诺啡单克隆抗体。采用酶联免疫反应和胶体金膜层析实验测定丁丙诺啡单抗的特异性以及免疫反应动力学参数。结果共获得3株分泌抗丁丙诺啡单克隆抗体的杂交瘤细胞株，分别命名为7E6，6G4和3C2。7E6，6G4抗体灵敏度为10．0ng／ml，3C2抗体灵敏度为20．0ng／ml。7E6，6CA和3C2抗体的亲和常数分别为3．6×10^-9 mol／L，4．3×10^-9 mol／L和6．3×10^-9 mol／L。特异性测试结果表明7E6和6G4抗体与40种药物、毒品无任何交叉反应，而3C2抗体与吗啡有交叉反应。结论杂交瘤细胞株7E6和6G4产生的抗丁丙诺啡单克隆抗体具有很高的特异性和灵敏度。     

The use of monoclonal anti-M and anti-N antibodies in forensic medicine

Anti-M and anti-N monoclonal antibodies (MA) may be useful for bloodstain analysis by absorption-elution reaction. In order to detect N antigen in bloodstains aged up to 4 weeks the material tested must be treated by methanol. The material fixation is not recommended for analysis of "aged" bloodstains as well as for M antigen detection. Anti-M MA may be used for analysis of liquid blood using agglutination reaction.     

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.     

A novel fluorescence-based method in forensic science for the detection of blood in situ

Full DNA profiles can be generated from just a few cells; however these profiles can be contaminated from other cell types present at the crime scene. We report here on the development of an immunofluorescent technique to spatially locate human-specific blood in situ and also on the ability of this technique to detect individual leukocytes and the DNA contained within them. Four monoclonal mouse anti-human antibodies were evaluated; anti-glycophorin A to detect erythrocytes and anti-CD45, anti-myeloperoxidase (MPO) and anti-histone H1 to detect the nucleated leukocytes. Each antibody was labeled with either Alexa Fluor 488 or 568 for direct application to blood smears which allowed the simultaneous detection of erythrocytes and leukocytes. Furthermore, because histones are DNA binding proteins, the application of anti-histone H1 allowed the detection of DNA within a blood smear. Importantly it was found that full DNA profiles could be achieved after using this method with similar peak area ratios compared to untreated cells. The fluorescent antibodies were found to be human-specific with the exception of anti-histone H1 due to its conserved sequence. However, used in combination with anti-CD45 or anti-MPO the location of DNA from human-specific leukocytes could be detected. The technique was also tested on older blood stains and was still found to be sensitive and cell-specific after 4 months. Following the optimization of the methodology, the fluorescent antibodies were applied to short lengths of black cotton fibres covered with human blood spots. Although the background fluorescence from the cotton was found to be high, erythrocytes and even individual leukocytes could easily be detected, indicating that this technique could be used to detect extremely minute amounts of blood. Used in combination with laser capture microdissection (LCM), this method could be used to pick off individual leukocytes for LCN DNA techniques.     

高特异性三唑仑代谢物单克隆抗体的制备

目的建立分泌抗三唑仑代谢物α-羟基三唑仑单克隆抗体的杂交瘤细胞株,制备高特异性的三唑仑代谢物单克隆抗体,为三唑仑及其代谢物免疫分析方法的开发奠定基础。方法在三唑仑分子的6位苯环对位上引入活性氨基基团,然后通过缩合反应分别与匙孔血蓝蛋白(KLH)和牛血清白蛋白(BSA)相偶联形成完全抗原。以三唑仑-KLH免疫Balb/c小鼠,通过细胞融合,筛选等杂交瘤技术建立稳定的分泌特异性单克隆抗体的杂交瘤细胞株。纯化后的单克隆抗体,分别用SDS-PAGE电泳法、间接ELISA法和胶体金免疫层析法对其纯度、效价及灵敏度和特异性进行测定。结果获得3株能稳定分泌三唑仑代谢物单克隆抗体的杂交瘤细胞株,分别命名为2G4,4B2和5H6。2G4和4B2抗体只与三唑仑代谢物α-羟基三唑仑有反应,灵敏度分别为500ng/mL和750ng/mL。与其他参试物无交叉反应。因5H6抗体为IgM,考虑到纯化难度和实际应用的限制,暂未做深入研究。结论本研究制备的2G4和4B2单克隆抗体仅识别三唑仑代谢物α-羟基三唑仑,具有高度特异性和灵敏度。     

Monoclonal antibodies with anti-N specificity

In this article serologic characteristics of monoclonal antibodies with anti-N specificity is given. Antibodies are directed to homologous sequence of N-form of A glycophorine (group-specific N-antigen) and B glycophorine, expressed both on N- and on M-erythrocytes. Higher titre and avidity of monoclonal antibodies with anti-N specificity in relation to erythrocytes with N-phenotype make it possible to detect N-antigen in material subjected to expert evaluation.     

Immunoenzyme AB0 blood group determination using a single hair. I

The immunoenzyme technique was used to determine the ABO blood group of strands of human scalp hair. The hair was obtained from 168 individuals of known blood groups (A1: n = 58; A2: n = 11; B: n = 28; O: n = 46; A1B: n = 16; A2B: n = 9). Immunostaining was carried out by using monoclonal anti-A, anti-B and anti-H as primary antibodies. Group-specific staining was clearly observed within the medulla of the hair. The ABO blood group of all hair samples was determined correctly by the Sternberger (PAP) or APAAP (immunoalkaline phosphatase) technique. The present study indicates that immunoenzyme techniques can be regarded as practical methods for determining ABO blood group of hair.     

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.     

氯胺酮、甲基苯丙胺和吗啡金标单抗试剂盒的研制

目的建立同步检测氯胺酮、甲基苯丙胺和吗啡的方法。方法将胶体金标记的抗氯胺酮、抗甲基苯丙胺和抗吗啡单克隆抗体浸涂在玻璃纤维膜上，将氯胺酮、甲基苯丙胺和吗啡的完全抗原以及羊抗鼠多克隆抗体喷涂在硝酸纤维素膜上，分别标定为检测区（T）和质控区（C）。样本中游离的氯胺酮、甲基苯丙胺和吗啡分别与包被的完全抗原免疫竞争结合胶体金标记抗氯胺酮、抗甲基苯丙胺和抗吗啡单克隆抗体。以质控区和检测区是否出现紫红色条带判读结果。结果对66种药品和毒品的特异性测试表明，该试剂盒仅识别氯胺酮及其代谢物、甲基苯丙胺及其衍生物和吗啡类；对人体尿样中的氯胺酮、甲基苯丙胺和吗啡检测阈值分别为1000ng／ml、1000ng／ml和300ng／ml；与GC／MS对照试验结果一致；试剂盒稳定性较好，在常温下可较长时间保存。结论本文研制的试剂盒可用于样本中氯胺酮、甲基苯丙胺和吗啡成分定性的同步检测。     

分泌抗人红细胞膜单克隆抗体细胞株的建立

以人的红细胞膜为抗原，采用选择性免疫抑制的程序，免疫BALB／C小鼠。经免疫的小鼠脾细胞与SP2／0骨髓瘤细胞融合，用血凝法筛选出3株分泌抗人红细胞膜种属特异性单克隆抗体的杂交瘤，即M1A7C4．M3A5B7和M3D9F1。连续传代2个月及复苏冻存半年的杂交瘤，仍能稳定地分泌抗人红细胞膜单克隆抗体。初步鉴定证明：该抗体具有稳定的种属特异性，可鉴别人与其它动物，特别是猴的红细胞，不与己知的血型特异性成分交叉，可凝集成人及脐带血的红细胞。     

Application of an immunological method to detect trace amounts of dried human semen

An immunological assay based on a monoclonal antibody was used for identification of trace amounts of dried human semen in forensic science evidence. The monoclonal antibody (Mab 4E6) produced recognizes a human sperm-coating antigen which is specific to human seminal plasma. This antigen seems to be a protein secreted by the epithelial cells of the ejaculatory duct, which is stable indefinitely at room temperature. Mab 4E6 reacts positively with semen samples from individuals independently to their ABO group or secretory status, but does not react with semen from bull, ram, boar, horse, rabbit and dog. In the assay system developed, Mab 4E6 can detect human seminal plasma at concentrations of 0.5 micrograms/ml total protein. A similar sensitivity is found when human semen stains are eluted from forensic science samples and tested by the same assay. This method shows a good correlation with the microscopic methods routinely used. The method described is very sensitive and reproducible, it is time saving and special laboratory equipment is not needed.     

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。     

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.     

Establishment of a group classification of isolated cells using anti-A and anti-B monoclonal antibodies

Anti-A and anti-B monoclonal antibodies may be used for determination of ABO system A and B antigens in mixed agglutination reaction. The authors suggested a variant of this reaction which ensures reliability of antigen detection in cells of people of secretor state. Investigation of mixed traces (liquid salivary part and buccal epithelium cells) showed that three-fold washing of cells couldn't provide for reliable identification of their blood group.     

抗人同种异型G1m(3)单克隆抗体研制及特异性鉴定

用快速液相色谱仪从G1m（3）阳性人血浆中纯化IgG1蛋白，用其免疫BALB／C／小鼠．建立了一株分泌抗人G1m（3）单克隆抗体的细胞株（D7E8）。经抑制ELISA，直接ELISA及斑点ELISA分析，证明D7E8抗体具有G1m（3）单一特异性。其培养上清液效价为512倍，并初步应用于法医办案。     

检验人精浆特异蛋白P30免疫胶体金试剂条的研制

目的制备用于检验人精浆特异蛋白P30-主要是来自法医学案件的免疫胶体金层析试剂条.方法选取针对不同抗原决定簇的抗P30单克隆抗体细胞株,并制备其小鼠腹水,分离纯化单克隆抗体.制备胶体金并以纯化抗体包被,制成免疫胶体金,以免疫胶体金浸泡玻璃纤维.选取适宜的硝酸纤维素膜并于其上不同位置以未金标的另一株P30单克隆抗体和羊抗鼠IgG包被.搭建试剂条并检测其灵敏度和特异性.结果所制成的试剂条灵敏度至少可达4ng/ml;对6人份混合的人精液物质在稀释20万倍后仍出阳性结果,且无非特异性反应.结论检验人精浆特异蛋白P30的免疫胶体金试剂条可对嫌疑人精物质做出排查,有利于法医物证检验.     

Light-microscopic examination of ABH and Lewis antigens in human tracheal and epiglottic glands using the avidin-biotin-peroxidase complex technique

The localization of ABH and Lewis antigens was examined in formalin-fixed, paraffin-embedded human tracheal and epiglottic glands using monoclonal anti A, B, H, Lea and Leb antibodies. The mucous cells of the glands showed reactivity with antibodies corresponding to the respective ABO blood groups of the tissue donors. The mucous cells from one blood group A, Le(a-b-) individual showed no reactivity with any antibodies and those from another blood group A, Le(a-b-) individual showed reactivity only with anti A antibody. In individuals from blood group Le(a + b-) of all ABO groups, the mucous cells reacted exclusively with anti Lea. In blood group O, Le(a-b+) individuals, the mucous cells showed intense reaction with anti H and Leb antibodies and weak to moderate reactivity with anti Lea. In Le(a-b+) individuals of A1, B and A1B blood groups, the mucous cells showed strong reactivity with anti A and/or B antibodies, moderate with anti Leb, weak or no activity with anti Lea and absent with anti H. In blood group A2 Le(a-b+) individuals, the mucous cells stained with anti A were weakly stained or completely unstained with anti H antibody, but cells negative with anti A gave strong positive reactions with anti H antibody.