Heterogeneity in the ability of IgG1 monoclonal anti-D to promote lymphocyte-mediated red cell lysis

Thirty-four IgG anti-D human monoclonal antibodies (mAb) derived from 18 donor were assessed for their ability to mediate lysis of D+ red cells by lymphocytes in antibody-dependent cell-mediated cytotoxicity assays. Cell-bound antibody was quantified and the mAb were compared at similar levels of sensitization. The majority (23/31) of IgG1 and all (3/3) IgG3 mAb were ineffective; two donors produced both lytic and non-lytic anti-D mAb. Greater sensitivity was achieved using fluid-phase antibody (as culture supernatants) in the assay than was obtained with pre-sensitized red cells. Minimum levels of 2000 anti-D molecules per cell were required for lysis using pre-sensitized cells. Partial D red cells (DIVa, DVa and DVI) were lysed by three mAb that were lytic with normal D+ cells. There was no relationship between lytic ability and Gm allotype or D epitope specificity of the antibodies. Four mAb to other blood group specificities were tested: two (anti-E and anti-G) were lytic and two (anti-c and anti-Kell) were not lytic. Possible reasons for the heterogeneity of the lytic activity by the mAb are discussed.     

Comparison of tube and gel techniques for antibody identification

There are several methods for antibody detection and each technique has advantages and limitations. We compared the performance of the tube (polyethylene glycol-indirect antiglobulin test [PEG-IAT]) and gel test technique for antibody identification. From January to May 1999, we performed antibody screening tests by gel and tube techniques on 10,123 random blood samples submitted to our reference laboratory. Six hundred and twenty-eight (6.2%) reactive samples were tested for antibody specificity by both methods. One hundred and ninety-six were reactive only by gel: 25 anti-D, 33 anti-C, 76 anti-E, 13 anti-c, 5 anti-e, 18 anti-K, 7 anti-Jka, 2 anti-Dia, 3 anti-S, 8 combination Rh antibodies (1 with anti- K), and 6 other antibody specificities. Two samples were reactive only by PEG-IAT: 1 anti-K and 1 anti-Dia. Four hundred and thirty were positive by the two methods: 156 anti-D, 9 anti-C, 68 anti-E, 15 anti-c, 6 anti-e, 61 anti-K, 12 anti-Jka, 17 anti-Dia, 5 anti-S, 73 combination Rh antibodies (2 with anti-K), and 8 other antibody specificities. Based on this study, the gel test is more sensitive (p <.01) than the tube test for identifying potentially clinically significant antibodies.     

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.     

A critical study of the IgG subclasses of Rh anti-D antibodies formed in pregnancy and in immunized volunteers

The IgG subclasses of Rh anti-D antibodies were investigated in a hundred anti-Rh sera using IgG subclass-specific antisera. Antibody activity was found to be entirely restricted to the IgG1 and IgG3 subclasses. Immunized volunteers tended to produce both IgG1 and IgG3 antibodies whereas antibodies from incompatible pregnancies were more often composed of only one subclass.It is suggested that some supposedly subclass `specific'' antiglobulin sera that have been standardized using weakly coated test red blood cells may show cross-reaction with more strongly coated cells and this may lead to the erroneous assumption that certain high titre anti-Rh sera also contain IgG2 and IgG4 antibodies.     

The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.     

Rh血型抗体的检测及结果分析

目的:调查Rh血型抗体的检出率及其特异性分布特点.分析Rh血型抗体的临床意义及产生规律.方法:采用微柱凝胶抗球蛋白技术筛查和鉴定红细胞血型不规则抗体,对鉴定为Rh血型抗体者,采用单克隆抗-D、抗-C、抗-c、抗-E、抗-e鉴定红细胞Rh血型抗原,以确认抗体的准确性;检测抗体的效价、Ig类型及37℃反应性,以明确其临床意义;询问孕产史、输血史,如果为新生儿检测其母亲血浆中是否有相同特异性的抗体,以分析抗体产生的原因.结果:就诊者54000例,共检出Rh血型抗体47例,检出率为0.087%,其中有妊娠史者27例,有输血史者13例,既有妊娠史又有输血史者1例,抗体来自母体的新生儿6例;抗体的特异性为:抗-E 29例(61.70%)、抗-D 8例(17.02%)、抗-cE5例(10.64%)、抗-c 4例(8.51%)、抗-C 1例(2.13%);47例Rh血型抗体均为IgG或IgG IgM类,37℃均可与具有相应抗原的红细胞反应,抗体效价介于1～4096.结论:被检就诊者Rh血型抗体的检出率低于白种人;在检出的Rh血型抗体中,抗-E占绝对多数,而抗-D的检出率呈逐步减少的趋势;妊娠和输血引起的同种免疫是Rh血型抗体产生的原因,新生儿自母体被动获得的Rh血型抗体是Non-ABO-HDN最主要的致病抗体.     

母婴ABO合并Rh-ccdee/ccDEe血型不合HDN实验诊断与鉴别诊断的研究

目的：探讨母婴ABO合并Rh血型不合和Rh两座位3位点表型不合HDN实验诊断与鉴别诊断,为临床早期防治提供实验依据。方法：用吸收放散和直抗作为实验诊断,用A-c、B-c和O-c阴性红细胞鉴别诊断,O型Rh-ccdEe、O型Rh-CCDee及A型Rh-CCdee组合谱细胞鉴别两座位多位点表型不合Rh-HDN,盐水法鉴定抗体性质。结果：①母婴ABO/Rh血型：母婴1血型鉴定为 O-A及Rh-ccdee/ccDEe交叉不合、母婴2鉴定为A-A及Rh-ccdee/CcDEe两座位3位点表型不合;②抗体筛选：母婴1不规则抗体均为强阳性（4+）;母2为不规则抗体强阳性（4+）、婴儿2为阳性（+）。③抗体鉴定与鉴别诊断：母1为 IgG抗A合并IgG抗D,IgG抗E缺失,IgG抗A效价 256,IgG抗D效价128;母2为IgM抗E合并IgG抗D,IgM抗C缺失,IgG为抗D效价128,IgG抗E及抗C缺失。④鉴别诊断：婴儿1证实为IgG抗A合并IgG抗D-HDN;婴儿2 证实为Rh抗D-HDN,IgG抗E及抗C缺失。结论：用A-c、B-c、O-c阴性红细胞和Rh单特异性组合谱细胞,可以鉴别诊断ABO合并Rh HDN以及Rh单一性或混合性HDN。     

Gel-test: interpretation and value of a new technique for the detection of irregular antibodies.

Here we report on our experience with the use of a 'Gel-Test' (DiaMed-ID Micro Typing System) technique for the detection and identification of irregular antibodies in a general hospital. This easy-to-use, standardized technique poses the question of the impact of its sensitivity on the specificity of the results. Of the 10% of observed positive reactions, 3.7% were irregular antibodies, 3.8% papain auto-antibodies, 1% cold antibodies and 2% not elucidated. Two hundred and eighteen irregular antibodies identified and titred with the 'gel-test' system were tested in parallel by 'tube' method. Sixty-three of these antibodies (29%) were not detected by the 'tube' method. While anti-Kell was always detected by both methods, we found the following false natives with the tube method: 15% anti-D, 32% anti-E, 42% anti-Cw and 58% anti-Lea. 68% of these false negatives had a low titre. The immunoglobulin class of the anti-E was studied; the sensitivity of the 'gel-test' system was associated with IgM in the anti-E. The sensitivity and standardization of the 'gel-test' technique guarantee greater safety in blood transfusion and increase efficiency in the prevention of foeto-maternal stimulation of anti-D.     

Charge differences between human IgG isoantibodies associated with specificity

Human sera containing various blood group antibodies were fractionated on carboxymethylcellulose, using stepwise elution, and fractions were assayed for total IgG concentration and for antibody titre. Whereas the highest concentration of total IgG was regularly found in the 0.06 M phosphate fraction, the highest titres of anti-D, anti-E and anti-Fy^a were found in the 0.1 and 0.15 M fractions indicating that these antibodies are less negatively charged than the bulk of the IgG. In two cases in which the concentration of anti-D was estimated with radioiodine-labelled antiglobulin, the amount in the 0.15 M fraction, as a percentage of total IgG present, was respectively eight and seventeen times higher than in whole serum. With the Rh antibodies anti-c and anti-e, the difference from total IgG in elution pattern was less striking, the antibodies being found mainly in the 0.06–0.1 M fractions. Finally, the distribution of anti-K, anti-A and anti-B in the different fractions did not differ from that of total IgG.     

Evaluation of Rh monoclonal antibodies for flow cytometry tests

As participants of the “III International Workshop on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens”, we tested 43 RH monoclonal antibodies by the flow cytometry technique. Besides the anti-D antibodies (not included in this paper), we tested the following antibodies: three (3) anti-C; one (1) anti-C^w; six (6) anti-c; eight (8) anti-E; three (3) anti-e; two (2) anti-G; one (1) anti-CcEe (total = 24 antibodies). These antibodies (from different lab sources) were tested against antigen positive cells (homozygous or heterozygous) and antigen negative cells. When available, some of them were tested against “rare” phenotypes like r_yr, r″^Gr, r^Gr, R₂R_z. All the three anti-C tested, showed poor discrimination between positive and negative cells; from the six anti-c tested, only three had good results (Workshop n° 18, 20, 21) with a superior performance of one of them (Workshop n° 18). From the eight anti-E tested, we found two (Workshop n° 139 and 153) with good performance; all the three anti-e were non reactive; the two anti-G failed to react with r′r red cells; the anti-C^w reacted better with R₁^wr cells than R₁^wR₁; and the anti-CcEe antibody showed good results with all the phenotypes tested. From the 24 antibodies tested, we found six (25%) antibodies with a good performance.     

Functional activity of human Rh monoclonal antibodies (MABS)

Anti-D and anti-c monoclonal antibodies (MABS) were found to be heterogeneous with respect to their activity in five cell-mediated functional assays. The two IgG3 MABS promoted greater rosette formation of red cells with monocytes than the IgG1 MABS. Discrepant results were obtained by different laboratories for the relative effectiveness of the MABS in promoting monocyte-mediated red cell phagocytosis and lysis, which may have been due to variations in the assay methods used. The IgG3 anti-D MAB promoted greater monocyte chemiluminescence than the IgG3 anti-c MAB, but of these two MABs only the anti-c mediated lysis of red cells in lymphocyte ADCC assays. The majority of the IgG1 anti-D MABS were ineffective at promoting red cell lysis by lymphocytes (K cells), but two were active even when diluted 1 in 300. There was no correlation of functional activity with red cell sensitization levels, IgG concentration or D epitope specificity of the MABS.     

A quantitative determination of IgG anti-D subclasses by Elisa in hemolytic disease of the newborn]

The quantification of IgG anti-D subclasses is one of the most important parameters considered in the assessment of the severity of hemolytic disease of the newborn. Traditionally IgG subclassing is performed using qualitative haemagglutination methods, difficult to interpret. A quantitative enzyme-linked immunosorbent assay (Elisa) was implemented for measuring IgG anti-D subclasses in 20 sera collected from 14 RhD-immunized pregnant women. All 4 IgG subclasses were detected in the 20 sera tested. The mean proportion of IgG1 was 52.8%. The mean proportion of IgG3 was 30.7%. The mean proportions of IgG2 and IgG4 were 14.5 and 1.9% respectively. A good correlation between the sum of IgG subclasses and the severity of HDN was found. Severe HDN occurred when both IgG1 and IgG3 were present. IgG1 anti-D was the predominant subclass in 4 of the 8 severe cases.     

Restriction of Human Immune Antibodies to Heavy-Chain Variable Subgroups

Human immune antibodies such as anti-Rh and anti-Kell antibodies were tested in hemagglutination and hemagglutination inhibition experiments for VH subgroup composition. A striking VH subgroup restriction was found in several of these groups of antibodies. In the majority of cases there was a restriction to one particular VH subgroup for a single antibody specificity in a given individual. In some cases there was also an overall restriction to one particular subgroup for antibodies with the same antibody specificity. This was particularly pronounced for anti-D antibodies, which was primarily restricted to VHII, and for the anti-Kell, which was particularly related to VHIII. Subgroup-specific antigens for all the main VH subgroups were blocked on combination of the antibody molecule with antigen. No relation was found between VH restriction and restriction to IgG subclass, or genetic markers or chi and lambda light-chain type.     

Constant isotype pattern of anti-dsDNA antibodies in patients with systemic lupus erythematosus

The isotype profile, particularly emphasizing IgG subclass distribution, of dsDNA antibodies in patients with systemic lupus erythematosus was evaluated using an especially adapted ELISA technique. Anti-dsDNA antibodies were quantified with class-specific antisera and subclass-specific monoclonal antibodies. IgG subclass specificity was proven with 20 myeloma proteins differing in light chains and allotypes. The standardization with myeloma proteins proved to be useful and reliable. Results from more than 100 anti-dsDNA positive sera from SLE patients showed specific antibodies within the three subclasses (IgG1: 52-100%, IgG2: 0-39%, IgG3: 0-48%). IgG4 was not detected in significant amounts. No correlation to the subclass distribution of total IgG was found. Each patients' serum displayed an individual isotype pattern that remained constant in longitudinal studies, independent of anti-dsDNA titre fluctuations. These results suggest a stable population of autoreactive clones in the progress of the disease.     

Antibody-Induced Hemolytic Activity of Human Blood Monocytes

The hemolytic effect of purified human blood monocytes was quantitated as the release of isotope from human erythrocytes labeled with ⁵¹Cr-chromate. We here demonstrate that IgM antibody (anti-A or cold agglutinin) was unable to confer monocyte-mediated hemolysis, and that lysis was induced by IgG anti-A antibody. In addition, IgG anti-D sera containing antibodies belonging to subclass IgG1 or IgG3 had approximately equal hemolytic efficiency, as calculated from the antigen-binding capacity of the sera at the concentrations inducing 50% monocyte-mediated hemolysis. The lytic reaction was suppressed by pooled IgG or by individual myeloma proteins of subclass IgGl or IgG3. The inhibitory effect of heat-aggregated IgG was not different from that of native IgG. IgG2 or IgG I had no or weak effects. Cross-inhibition of lysis induced by IgG1 and IgG3 anti-D antibody showed strong and equal suppression by IgG1 and IgG3 myeloma proteins. Hundredfold higher concentrations of IgG2 or IgG4 was required for inhibition of lysis, which may be an effect of contaminating IgGl or IgG3. Our data strongly suggest that the receptors on human blood monocytes for IgGl and IgG3 that participate in monocyte-mediated hemolysis are identical.     

Non-receptor muscle antibodies in myasthenia gravis are of IgG1 and IgG4 subclasses.

The IgG subclass distribution of non-receptor muscle antibodies was examined in 15 myasthenia gravis (MG) sera, employing an indirect haemagglutination-immunofluorescence technique. Four sera contained only IgG1, 4 contained only IgG4 and 7 contained both IgG1 and IgG4 muscle antibodies. IgG2 and IgG3 antibodies were not found. Among 11 patients with a defined thymus pathology 8 had thymoma and 3 had thymic atrophy, but there was no correlation between antibody subclass pattern and thymic pathology. Patients with both IgG1 and IgG4 antibodies tended to have the longest disease duration. We conclude that IgG non-receptor muscle antibodies in MG are of the IgG1 and/or IgG4 subclasses, irrespective of thymic pathology.     

Determination of IgG subclass antibodies against wheat flour antigens by an ELISA technique

We describe the assay conditions for an enzyme-linked immunoassay for the determination of IgG and IgG subclass antibodies in serum to water-soluble wheat flour antigens. The optimal antigen coating concentration was 5 micrograms/ml for total IgG, IgG1, IgG4 and 100 micrograms/ml for IgG2. Serial dilutions of test sera were used and commercially available monoclonal mouse anti-human IgG isotype antibodies (as ascites fluid) were diluted 1:500-1:1000. Specific wheat flour antibodies belonging to the IgG1, IgG2 and IgG4 subclasses were detected. Despite the lack of standardized isotype-specific second mouse monoclonal antibodies, the subclass antibody levels between flour-exposed bakers and controls could be compared. We observed significantly higher IgG1, IgG2, and IgG4 subclass antibodies among 23 bakers than among 12 non-exposed controls, but no IgG3 antibodies were detected. The differences in biological activities of the IgG subclass antibodies may explain the clinical and pathophysiological features for fluor-induced occupational allergic diseases among bakers.     

Synergistic effect of blending IgG1 and IgG3 monoclonal anti-D in promoting the metabolic response of monocytes to sensitized red cells.

Monoclonal antibodies specific for the Rh antigen D were used to sensitize red cells for use in a series of cellular assays. IgG3 anti-D was more efficient than IgG1 anti-D in promoting the attachment and lysis of red cells by human monocytes. In contrast, IgG1 anti-D was more efficient at mediating phagocytosis. The metabolic response of monocytes, measured by chemiluminescence (CL), was greater towards IgG3-sensitized red cells than IgG1-sensitized cells; however, the CL response was further increased when red cells were sensitized in antibody mixtures comprising both subclasses. Using monoclonal antibodies from five IgG1-secreting cell lines and from three IgG3-secreting cell lines, this synergistic increase was seen with 0/4 IgG1/IgG1 combinations, 0/2 IgG3/IgG3 combinations and 8/8 IgG1/IgG3 combinations. Synergism was observed only when both subclasses were present on the same red cells; mixing of IgG1-sensitized red cells with IgG3-sensitized red cells before addition to monocytes did not increase CL generation. The binding and phagocytosis of red cells by monocytes and the lysis of red cells by monocytes or lymphocytes were not greater when red cells were sensitized with IgG1 and IgG3 antibodies together compared to red cells coated with single subclasses.     

Human monoclonal antibodies specific for blood group antigens demonstrate multispecific properties characteristic of natural autoantibodies.

A panel of 72 human monoclonal antibodies with specificities for blood group antigens, A, Rh D, Rh C, Rh c, Rh E, Rh e, Rh G, Jka and Jkb, has been established from the peripheral blood of deliberately immunized donors. Previous work has established that the antibodies are highly specific for their respective blood group antigens, and a number of them are in routine clinical use as blood grouping reagents. This panel was screened for reactivity against six unrelated foreign and autoantigens by ELISA, for rheumatoid factor activity by ELISA and agglutination techniques, and for reactivity with a number of different tissues by immunofluorescence. Binding of the monoclonal antibodies to unrelated exo- and autoantigens was commonly seen amongst the antibodies of the IgM class, and to a lesser degree amongst the IgG class, with reaction patterns similar to those given by natural autoantibodies. Only five of the IgM antibodies failed to demonstrate any unexpected cross-reactivities and these included 1/13 anti-D, 2/7 anti-E, 1/13 anti-c and 1/2 anti-A. We propose that rather than natural autoantibodies representing a distinct population of immunoglobulins, multispecificity (polyspecificity, or polyreactivity) may be a feature of antibodies produced in response to exogenous antigens. The implications of this for the study of autoantibodies are discussed.     

Serum volumes should not be reduced when testing neonates for unexpected red cell antibodies

Serum-to-red cell ratio (volume:volume) significantly affects the sensitivity of antibody detection. Obtaining sufficient serum quantity can be a problem when testing blood from transported neonates not accompanied by maternal samples. Reducing serum volumes used for testing might result in missed antibodies. The authors studied 1,177 sera for unexpected red cell antibodies by comparing one versus two drops of patient serum using a technique with LISS at 37 degrees C through the antiglobulin phase. The serum-to-red cell ratio using two drops was approximately 50: 1. Results of 60% (58/96) of samples containing antibody benefited by the use of increased serum. Eighteen percent; (17/96) had antibodies detected only with two drops that were missed entirely by one, and 43% (41/96) showed stronger positive reactions using two drops versus one. Importantly, antibodies detected only with or enhanced by two drops were clinically significant (anti-D, anti-K, anti-M, anti-c, anti-E). Thus, reducing serum-to-cell ratio is potentially dangerous, and blood banks should insist on adequate sample size to ensure patient safety.