Flow cytometric analysis of 68 monoclonal anti-D reagents

68 monoclonal IgG anti-D were assessed for quantitative and qualitative binding to D variant red cells using a flow cytometric indirect immunofluorescence test. One antibody failed to bind to normal RhD pos. cells, but reacted weakly with D cat VI and D Cat VII cells. Quantitatively, binding varied approximately twenty-fold between different MAbs and different D variants. D cat III cells gave the lowest level of binding when compared with the D pos. controls. Qualitatively, R₁^VIr, R₂^VIr, R₁^VIIr and DFR samples failed to react with various MAbs regardless of the levels of binding of these MAbs to the D pos. controls, thus supporting the idea of missing epitopes in such variants.     

Coordinator's report on the flow cytometric analysis of the Rh antibodies

One hundred and four IgG monoclonal antibodies with specificity within the Rh Blood Group System were evaluated by flow cytometry as part of the Third International Workshop. Standardisation of data to permit interlaboratory comparison of antibody binding was achieved by adherence to a standard red blood cell staining protocol, defined control cells and a standard FITC-labelled antibody. In addition, calibration standards were provided to permit the calculation of Molecules of Equivalent Soluble Fluorochrome (MESF) values from the mean channel fluorescence. For the majority of anti-D antibodies mean MESF values obtained with D positive cells were far higher than with the negative controls (D negative cells), with D variants having intermediate although very varied MESFs. In general MESF values obtained with non anti-D antibodies were less than for the anti-D antibodies although some of the anti-E antibodies were very strong. The highest MESF values were obtained with an anti-CD antibody.     

Serological analysis of monoclonal anti-E and -e with Japanese E/e variant red cells

The agglutination patterns have been analyzed for the reaction between 24 monoclonal antibodies (MAB) with specificity for the Rh antigen E or e and red cells of E/e variant from Japanese blood donors. The MABs were tested for direct agglutination of papain treated cells at 20°C. The reactions of a full titration series of each MAB with a E/e variant cell were compared to the reactions of that MAB with normal E+e+ cells. Sixteen anti-E were divided into a minimum of 6 different agglutination patterns with 8 examples of E variant cells. Eight anti-e gave a minimum of 5 different agglutination patterns with 6 examples of e variant cells.     

Rh血型抗-D阳性个体中未见DEL型

目的分析Rh血型系统抗-D抗体阳性个体的RhCcEe表型及检测DEL等位基因,观察产生抗-D同种免疫反应个体中是否存在DEL型。方法对因输血或妊娠产生抗-D同种抗体的个体,采用间接抗人球蛋白试验(IAT)排除部分D型或弱D型,然后进行RhCcEe表型检测,同时采用PCR方法检测RHD1227A等位基因鉴别DEL型和真实Rh(D)阴性表型。同时检测99名IAT确认的、抗-D抗体阴性的Rh(D)阴性健康人作对照。结果 99名抗-D抗体阴性组共检出C抗原阳性(包括CC或Cc)个体44名(44.4%);DEL型24名(24.2%)。抗-D抗体阳性组经IAT确认共118名Rh(D)阴性,C抗原阳性22名(18.6%),未发现DEL型个体,均与抗体阴性组存在显著性差异(P<0.01)。结论抗-D抗体阳性组未见DEL型,提示DEL型个体或不会因输血或妊娠针对Rh(D)抗原发生抗-D同种免疫反应;由于DEL型多携带C抗原,因此造成抗-D阳性组C抗原频率显著降低。     

用血影细胞从低效价血浆中分离抗-D抗体

目的 研究从低效价血浆中分离抗-D抗体的方法.方法 用遗传表现型CCDee的RhD阳性O型红细胞制备血影细胞,经亲和层析法从抗-D含量为0.814μg/mL的健康人血浆中分离抗-D抗体,检测相关指标.结果 经此法成功获得抗-D抗体,抗-D抗体回收率达到84.65%,所得制剂经检测激肽释放酶原激活剂含量、IgG单体加二聚体含量、抗补体活性及y-球蛋白纯度等指标符合中国生物制品规程的要求.结论 血影细胞亲和层析法简化了抗-D抗体提取和纯化步骤,提高了从低效价血浆中提纯抗-D抗体的回收率.     

Reactivity of 62 Rh MAbs with weak and variant antigens

We characterized serologically 5 anti-C (4 IgM and 1 IgG), 3 anti-c (2 IgM and 1 IgG), 4 anti-E (1 IgM and 3 IgG), 4 anti-e (3 IgM and 1 IgG) and 46 anti-D (16 IgM and 30 IgG) monoclonal antibodies, provided by the Rh Section of the Third International Workshop and Symposium on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens (1996) for their ability to detect weak and variant antigens. The agglutination patterns were established using untreated and papain-treated red blood cells in a column agglutination technology system (BioVue, Ortho). Significant differences were found between the IgM and IgG antibodies. The papain treatment seemed to be important for IgM but not for IgG antibodies. Almost all of the IgM anti-D antibodies detected untreated D^IV samples and almost all of the IgG anti-D antibodies detected untreated weak D samples. Both IgM and IgG anti-D antibodies showed the highest number of negative reactions with D^VI and Rh 33 red blood cells. The C^WC^W sample was detected by only one of the 4 anti-C IgM MAbs using enzyme-treated red blood cells. All anti-c MAbs were able to detect treated C^x samples. Because of the small number of weakly expressed E and e samples, definitive conclusions cannot be drawn on the ability of these antibodies to detect these antigens.     

Flow cytometric evaluation of non anti-RH1(D) monoclonal Rh antibodies

IgG non anti-RH1(D) monoclonal Rh antibodies were evaluated by flow cytometry. The values obtained with these antibodies were less strong than those obtained with anti-RH1(D) antibodies. For a significant number of antibodies, the signal was not high enough to give reliable results for the antibody specificity. Despite these drawbacks, flow cytometry was an efficient tool to appreciate the variation of reactivity by different antibodies with normal or variant cells. These variations were not always obvious by serological means.     

Specificity C and e: tests using Rh variant cells

Monoclonal antibodies, specificity C, e, and Rh subgroup ?e in the Section 1-Rh were tested with selected blood samples of various Rh variant types in KwaZulu-Natal, South Africa. The standard red cell serological techniques supplied were used.     

Detection and quantitation of rotavirus using monoclonal antibody coupled red blood cells: comparison with ELISA

A total of 125 faecal extracts from infants were tested by reverse passive haemagglutination (RPH) using red cells coated with a monoclonal antibody against the major group-specific rotavirus antigen (VP 6). Results were compared with those obtained using a rabbit anti-rotavirus capture, guinea pig anti-rotavirus detector-based ELISA. The specificity of the assay was confirmed by use of 'normal' immunoglobulin coupled red cells and by inhibition with rabbit antiserum. The antibody-coated red cells could be stabilised by treatment with glutaraldehyde and subsequent freeze-drying with no detectable loss of activity even after storage at 45 degrees C for 4 wk. Good correlation was obtained between RPH and ELISA. Purified bovine rotavirus could be detected by RPH down to approximately 10(5) particles in a 25 microliters vol. Similar results were obtained with polyclonal antibody coupled cells and an ELISA using monoclonal antibody. Experiments using subgroup-specific monoclonal antibodies indicated the feasibility of rapid subgroup determination.     

孕妇及胎儿Rh(D/E)免疫性溶血病的诊断与血液治疗

目的：探讨孕妇及胎儿Rh（D/E）血型不合的免疫性溶血病的治疗,预防孕妇早孕自然流产或不足月死胎.方法：用大量去除血浆和换血疗法对孕妇及胎儿或新生儿Rh（D/E）血型不合者进行治疗,以降低IgG抗D/E的含量,提高胎儿或新生儿的成活率.结果：2例孕妇及胎儿Rh（D）血型不合者去除血浆前IgG抗-D分别为512和1 024,去除血浆3周后,IgG抗-D效价降至16和32,以后间断性的治疗,减少治疗频率,保持IgG抗-D≤64,置足月行剖宫术,产下正常男、女婴儿各1名;1例Rh（E）血型不合者未经治疗而胎死宫内;另1例Rh（D）新生儿溶血病（HDN）经换血治疗而得救.1年后随访,3例婴儿发育均良好,与正常婴儿无差异.结论：大量去除血浆,同时补充高蛋白饮食是治疗孕妇及胎儿RhD/E血型不合的免疫性溶血病的好方法,换血疗法是HDN治疗的最佳选择.     

Epitope mapping of monoclonal antibodies raised to recombinant Mengo 3D polymerase

The cDNA coding sequence of the RNA-dependent RNA polymerase (3D^pol) of Mengovirus was cloned and expressed in a bacterial system. Eleven monoclonal antibodies were raised against the recombinant Mengo 3D^pol (rM3D). All of them recognized the recombinant and the viral-induced form of the protein. The panel of monoclonals belonged to the IgG1 and IgG2a isotypes and were mapped to four different epitopes in the 3D molecule by competition assays. All monoclonals recognized Mengo 3D^pol in western blots and cross-reacted with the homologous polymerases of seven other cardioviruses but failed to react with 3D^pol from poliovirus type 1 and 3 or rhinovirus type 14 and 16.     

Detection of both hepatitis B e antigen and antibody in a single assay using monoclonal reagents

Monoclonal antibodies raised against HBeAg were used to develop a HBeAg and anti-HBe detection assay. Monoclones containing anti-HBe were used both for the coating of the solid phase and for the fluid phase label in a sandwich type assay. The percentage binding of 125I-labelled anti-HBe to serum HBeAg was much greater than that seen in a similar assay using only polyclonal reagents. Therefore it was possible to add a small quantity of HBeAg for neutralising any anti-HBe present in a test serum without affecting HBeAg detection. This small amount of serum HBeAg was incorporated into each test sample thus allowing the determination of the e status of a patient using only one aliquot of test serum. This single test assay could be performed either as a radioimmunoassay or as an ELISA. The sensitivity of these assays was found to be greater than the conventional polyclonal assay particularly with regard to sera containing anti-HBe.     

Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody

A monoclonal antibody to HLA-G has been generated by immunizing HLA-A2.1/human β²-microglobulin (β²m) double transgenic mice with murine L cells transfected with both human β²m and HLA-G. This monoclonal antibody, designated as G233, has been found not to cross-react with other HLA class I antigens when tested on numerous cell lines by flow cytometry. With immunohistology, all populations of extravillous trophoblast (cell columns, interstitial trophoblast, endovascular trophoblast, placental bed giant cells) were stained. An extensive range of adult and fetal tissues was also tested but none reacted with monoclonal antibody G233, including those previously reported to express HLA-G mRNA, indicating that the protein has a highly restricted distribution. Failure to detect HLA-G in the fetal thymus raises the question as to how T-cell tolerance to this antigen is induced. Immunoprecipitation of trophoblast surface proteins with monoclonal antibody G233 revealed a heavy chain of 39 kDa and a light chain of 12 kDa, indicating that HLA-G expressed on the surface of trophoblast is complexed with p2m. However, sequential immunoprecipitation with monoclonal antibody W6/32 followed by monoclonal antibody G233 continued to detect a residual band of 39 kDa, suggesting that trophoblast surface HLA-G may also occur as free heavy chains not associated with p2m. Immunoprecipitation followed by two dimensional gel electrophoresis showed that monoclonal antibody G233 recognizes several iso-forms of HLA-G from trophoblast similar to the characteristic spot array previously described for HLA-G. This monoclonal antibody G233 will be highly useful in future experiments to elucidate the function of HLA-G.     

Deficiency of monoclonal antibody (Leu 7) defined NK cells in newly diagnosed insulin-dependent diabetes mellitus

Peripheral blood from 11 newly diagnosed patients with insulin-dependent diabetes mellitus (IDDM) was studied for the proportion of monoclonal antibody (HNK 1, Leu 7) defined natural killer (NK) cells using a fluorescence-activated cell sorter analyzer. The proportion of Leu 7+ cells in patients with IDDM (7.0 +/- 4.0) was significantly (P less than 0.001) lower than in simultaneously studied healthy controls (16.8 +/- 7.0). A 2-yr-old boy with recent onset IDDM had a deficiency of Leu 7+ NK cells (6.1%), while his healthy identical twin had normal proportions of Leu 7+ cells (22.2%), when compared to a simultaneously studied healthy control. Two patients reexamined in remission and one other studied in remission alone, showed deficiency of Leu 7+ NK cells. This study demonstrates a quantitative deficiency of monoclonal antibody (Leu 7+) defined NK cells in newly diagnosed patients with IDDM that persists during remission of the disease and therefore appears to be independent of metabolic abnormality. The deficiency of NK cells may predispose genetically susceptible individuals to viral-induced islet cell injury, contributing to the pathogenesis of IDDM.     

Discrepancies in Rh(D) typing of sensitized red blood cells using monoclonal/polyclonal anti-D reagents: case report and review

Instructions included with monoclonal Rh(D) typing reagents do not require routine use of an Rh control as immunoglobulin-coated red blood cells (RBCs) rarely yield falsely positive results with low protein reagents. However, the American Association of Blood Banks (AABB) Technical Manual recommends a concurrent control be performed on patients' RBCs that type as group AB, D(+). Proficiency testing surveys presented sensitized AB, D- RBCs, which resulted in a positive direct antiglobulin test and, in some samples, spontaneous agglutination in saline. One intent of the surveys was to monitor the accuracy of the reported Rh(D) type. On an initial survey, 19 of 115 (16.5%) participants reported the RBCs as D(+). Of these laboratories, 63.2 percent (12/19) had used a monoclonal/ polyclonal blend anti-D reagent. On a subsequent survey, after educational material had been distributed, only five of 113 (4.4%) participants reported the Rh type as D(+). Two of these five laboratories had used a monoclonal/polyclonal blend anti-D reagent. As RBCs coated with immunoglobulin may give unreliable results with Rh typing reagents, laboratories should follow the guidelines of the AABB Technical Manual. An appropriate control should be performed whenever RBCs from patients type as AB, D(+).     

Development of an ELISA for nitrazepam based on a monoclonal antibody

We report the production of a specific monoclonal antibody against Nitrazepam (NZP). First, the hapten 7-aminonitrazepam (7-ANZP) was synthesized, then the hapten was coupled with bovine serum albumin and ovalbumin by the diazotization method, and used as immunogen and coating antigen. Factors affecting binding, ionic strength, pH, and methanol concentration in phosphate-buffered saline, were optimized. The monoclonal antibody had a satisfactory IC₅₀ of 0.2 ng/mL for NZP. The cross-reactivities with other analogs were all lower than 0.1% except for 23% with 7-NZP, which suggested that the enzyme-linked immunosorbent assay method possessed high specificity. The recoveries were in the range of 84–95%, indicating that the method could be applied for the detection of NZP in urine.     

Interest of immunomodulation as a mean to improve the preparation of polyclonal and monoclonal antibody reagents

Immunomodulation by monoclonal antibodies (mAbs) was investigated in mice in order to improve the preparation of antibody reagents. Three different types of representative immunogens were chosen: a human soluble protein (secretory immunoglobulin A, SIgA), a bacterial polysaccharide from E. coli K1 and an envelope protein from the hepatitis B virus. These Ag are all of importance for diagnosis and exhibit different levels of immunogenicity. Antibody-mediated enhancement was observed against restricted and defined regions of each immunogen i.e.: the Fab epitopes of SIgA, the preS1 domain of the HBV envelope and associated cell wall components of the capsular PS. The epitopes which were enhanced appeared to be different from those recognized by the modulating mAb. Negative modulations were also observed. Moreover, new epitopes seemed to be generated. In both cases the level and direction of the modulation were irrespective of isotypy and affinity of the mAbs. Interestingly the positive modulatory effect was found to be correlated with an in vitro assay based on the binding of immune complex to antigen-presenting cells.     

鼠抗人Syndecan-1分子功能性单克隆抗体的制备及其生物学特性

制备鼠抗人Syndecan-1(CD138)分子的功能性单克隆抗体,研究其对高表达CD138分子细胞的生物学效应。以人多发性骨髓瘤(MM)细胞株8226作为免疫原,8226和B淋巴瘤细胞株Daudi作为检测细胞株,利用B淋巴细胞杂交瘤技术制备单克隆抗体。以快速定性试纸分析法鉴定单抗所属的IgG亚类,采用间接免疫荧光标记法及竞争抑制实验分析单抗的亲和力和特异性,以高表达Syndecan-1的人多发性骨髓瘤细胞株XG-1为靶细胞,分析单抗对其生长的影响。结果:成功地获得1株鼠抗人Syndecan-1分子的功能性单克隆抗体杂交瘤细胞株(克隆4B3),经体外长期传代培养,液氮冻存半年以上反复复苏良好,分泌特异性抗体性能稳定。4B3分泌的单克隆抗体识别位点与单抗BB4识别位点相同或相近,重链为IgG1亚类,轻链为κ型,纯化后腹水中单克隆抗体蛋白的得率为2.9 mg/ml,加入4B3单克隆抗体后的XG-1细胞体外生长受到抑制,并可用于神经胶质瘤细胞的免疫组织化学检测。由此表明,4B3为功能性抗人Syndecan-1单克隆抗体,具有一定的基础研究和临床应用前景,这对进一步研究Syndecan-1分子的生物学特性也具有重要的价值。