Use of monoclonal Jk(a) and Jk(b) reagents in phenotyping red cells with a positive direct antiglobulin test

Twenty-five red cell samples with a positive anti-IgG direct antiglobulin test QAT) were tested with human monoclonal IgM Jka and jkb antibodies. Red cell samples were first tested by a 5- minute incubation tube test with the monoclonal antibodies (MAbs). The same red cells were then chloroquine diphosphate (CDP)-treated, and retested. Eleven of the CDP-treated samples were also tested with conventional polyclonal antibodies (PAhs) that required a 37 degrees C incubation for 30 minutes, followed by an indirect antiglobulin test. The Jka and Jkb MAbs consistently gave the same phenotype results both on untreated DAT-positive red cells and on the same cells after CDP treatment. Two of the CDP- treated samples had diminished antigen expression with the MAbs, a finding that may have been caused by the CDP treatment. One untreated sample, which spontaneously agglutinated in a low- protein medium, was incorrectly phenotyped with the anti-Jka MAb, but both MAbs and PAbs gave the same correct results with the CDP-treated cells. These findings illustrate that the use of Jka and Jkb MAbs in phenotyping DAT-positive specimens is practical and beneficial.     

Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography

The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked.     

Serological studies of monoclonal RH antibodies with RH1 (D), RH2 (C), RH3 (E) and RH5 (e) variant RBCs.

One hundred and forty five Mabs against RH antigens were tested. In this paper, we chose to detail reactivity of MoAbs directed against variant RBCs of the CNRGS collection for which we studied the molecular background. Because we developed procedures to identify variants of the RhD, RhC, RhE and Rhe antigens, we were especially interested in finding new monoclonal antibodies that could help us to characterize more accurately these variants. Therefore, we drew parallels between our procedures and results obtained with the 2001 workshop antibodies.     

Analysis of human class II antigens by cloned cytolytic T cell reagents: a study using HLA loss mutant lymphoblastoid cell lines and monoclonal antibodies detecting the HLA-DP product(s)

We have utilized cloned T cell reagents and ionizing radiation-induced mutants of an HLA heterozygous lymphoblastoid cell line (LCL) to investigate the determinants detected by the cell-mediated lympholysis (CML) assay. Cells of an LCL clone, 721.501, an HLA haplotype loss mutant expressing the HLA-A2-Cw1-Bw51-DR1-Dw1-DQw1-DPw2-GLO haplotype were used as sensitizing cells for responder cells in vitro. "Cloned" reagents were generated by single-cell deposition of cells of a bulk reagent primed against 721.501 cells. Those clones were screened for cytolytic activity against HLA loss mutant targets (derived from LCL 721) of four different categories; HLA-A2 loss only, A2-Cw1-Bw51 loss, A2-Cw1-Bw51-DR1-DQw1 loss, and the entire HLA haplotype loss. Of 196 clones tested, 36 were cytolytic, including three anti-A2, five anti-Bw51/Cw1, 12 anti-DR1/DQw1, 13 anti-DP region associated with DPw2, and three of undetermined specificity, based on cytolytic patterns against the HLA loss mutant targets. Of 25 anti-HLA class II lytic clones, 23 (92%) fitted the characteristics of helper cell-independent cytolytic T cells (HITc), whereas only two of eight (25%) anti-class I clones were HITc. The 13 anti-DP region clones were divided into three subgroups defined by blocking by anti-FA and not Tü39 monoclonal antibodies (MoAb), by Tü39 and not anti-FA, and by both MoAbs.     

Generation and characterization of a monoclonal antibody (1C5) to human migration inhibitory factor (MIF)

A monoclonal antibody was raised in mice against human MIF of the Mr 14,000 kd, produced by Concanavalin A-stimulated peripheral blood mononuclear cells. A hybridoma (1C5) secreting an IgG 1 antibody was selected which binds, yet does not neutralize MIF in the macrophage migration assay. MIF activity may be released from immobilized antibodies by acidic buffer elution. The eluate consists of three major bands at Mr 8,000, 14,000 and 28,000 as revealed by SDS-polyacrylamide gel electrophoresis and molecular sieve chromatography (HPLC). By radioimmunoassay and enzyme-linked immunoassay, it could be shown that the antibody binds material with isoelectric points of 4.5 to 5.0 and of 3.0, which coincides precisely with the biological activities. Similar congruencies between the distribution of biologically reactive and binding material were found in molecular sieve and ion exchange chromatography. It is concluded that the antibody 1C5 reacts with most molecular weight entities of MIF which seem to be structurally related and which display similar characteristics as described for guinea pig and mouse MIF.     

Human complement (C3b) receptors defined by a mouse monoclonal antibody.

The aim of the present study was to prepare a monoclonal antibody against human C3 receptors. The monoclonal antibody termed C3RTo5, which is characterized in this study, inhibited the ligand binding of C3b receptors of human erythrocytes, neutrophils and lymphocytes, but did not block the ligand binding of C3bi and C3d receptors. C3RTo5 reacted only with cells that express C3b receptors. It did not react with cells that do not express C3 receptors or with cells that express C3 receptors other than C3b receptors. The reactivity of C3RT05 against C3b receptors of human erythrocytes, neutrophils, glomerular cells, tonsil cells, or spleen cells could be removed by absorption with human erythrocytes or tonsil cells, whereas absorption with human peripheral T cells or sheep erythrocytes had no effect. In immunoprecipitation studies, a glycoprotein with a mol. wt of 205,000 could be isolated with C3RTo5 from non-ionic detergent lysate of tritiated tonsil cells. A rabbit antiserum prepared against this glycoprotein was able to stain C3b receptor-positive cells, inhibit C3b receptor ligand-binding activity and, furthermore, to precipitate a 205,000 mol. wt component. The results of this study indicate that C3RTo5 is a monoclonal antibody with selective reactivity to C3b receptors and presumably to the binding sites within the receptor molecule. Using C3RTo5 further strong evidence was obtained that membrane-bound C3b receptors have a mol. wt of 205,000.     

A monoclonal antibody (HO-No-1) with specificity for a human nucleolar protein

A murine monoclonal antibody (named HO-No-1) which specifically reacts with a target antigen of 72,000 mol. wt (p72) in the nucleoli of human cells, has been isolated. Using an avidin-biotin peroxidase immunoperoxidase method, it was found that this antibody stains exclusively the nucleolus and not other portions of the nucleus and cytoplasm. This reaction pattern was observed consistently, although to different degrees in 14 human normal tissues and human malignant cell lines. However, after pretreatment with actinomycin D (250 ng/ml) for 2 h, the antibody stains the nucleoplasm uniformly. Thus this study demonstrates evidence for an antibody which reacts specifically with human nucleoli to a protein which could play an important role in rRNA synthesis.     

Immunohistochemical study of colorectal adenomas with monoclonal antibodies against blood group antigens (sialosyl-Le(a), Le(a), Le(b), Le(x), Le(y), A, B, and H)

We studied 40 colorectal adenomas with monoclonal antibodies against blood group antigens (sialosyl-Le(a), Le(a), Le(b), Le(x), Le(y), A, B, and H). Sialosyl-Le(a), Le(a), Le(x), and Le(y) are usually present in different compartments of the crypts of normal colorectum and can be considered markers of normal differentiation antigens (NDA). The former two antigens represent markers for differentiated colonic epithelium and the latter two, markers for "undifferentiated" crypt base epithelium. Le(b), A, B, and H are normally absent from the distal colon and rectum, but are expressed by fetal colon and carcinomas and are considered oncofetal tumor-associated antigens (OF-TAA). Individual adenomas could be characterized as to whether or not they expressed OF-TAA and NDA. Of the adenomas, 35% were OF-TAA+/NDA+, 40% were OF-TAA+/NDA-, 17.5% were OF-TAA-/NDA+, and 7.5% were OF-TAA-/NDA-. When NDA were present, they were expressed in the same compartment of the crypt as in the normal colon and rectum. In adenomas there was proliferation of the undifferentiated enterocyte marker Lex throughout the entire length of the crypt when compared with controls (p less than 0.01). Of the adenomas, 75% expressed OF-TAA, however, 35% of adenomas concomitantly expressed NDA in the same distribution as normal colon and rectum indicating that adenomas have features of both carcinoma and normal colorectum epithelium.     

A monoclonal antibody (HO-No-1) with specificity for a human nucleolar protein.

A murine monoclonal antibody (named HO-No-1) which specifically reacts with a target antigen of 72,000 mol. wt (p72) in the nucleoli of human cells, has been isolated. Using an avidin-biotin peroxidase immunoperoxidase method, it was found that this antibody stains exclusively the nucleolus and not other portions of the nucleus and cytoplasm. This reaction pattern was observed consistently, although to different degrees in 14 human normal tissues and human malignant cell lines. However, after pretreatment with actinomycin D (250 ng/ml) for 2 h, the antibody stains the nucleoplasm uniformly. Thus this study demonstrates evidence for an antibody which reacts specifically with human nucleoli to a protein which could play an important role in rRNA synthesis.     

Phagocytosis by receptors for C3b (CR1), iC3b (CR3), and IgG (Fc) on human peritoneal macrophages

Human peritoneal macrophages (HPM) obtained via laparoscopy were examined for the presence and functional capacity of complement and Fc receptors. Between 5 and 20 ml of peritoneal fluid containing 1-2 X 10(6) macrophages/ml was available for each study. Macrophages made up 80-95% of the cells in the fluid. Fc and C3 receptors on HPM were characterized by rosette formation with, and phagocytosis of, IgG- and C3-coated sheep erythrocytes (E). ElgG were bound by 82% and ingested by 63% of HPM, with 4-15 E ingested/HPM. The HPM formed rosettes with EC3b (56%) and EC3bi (71%) but not EC3d,g or EC3d. Antibodies to complement receptors type 1 (CR1) and type 3 (CR3) inhibited rosette formation with EC3b and EC3bi, respectively, indicating that HPM possessed separate and distinct receptors for the C3b and iC3b ligands. In 60% of the samples studied, HPM demonstrated the ability to ingest both EC3b and EC3bi, as well as ElgG. Because of the heterogeneous nature of the cells obtained in peritoneal fluid, due to their progressive change from monocytelike cells into mature macrophages, HPM were separated by 1 g velocity sedimentation into fractions of increasing maturity. They were then examined for phagocytosis via Fc and complement receptors. Fc receptor mediated phagocytosis occurred throughout the monocyte-to-macrophage maturation sequence, while the ability of HPM to ingest via CR1 and CR3 was maturation dependent, with ingestion via CR3 occurring before CR1, in a manner analogous to in vitro differentiation of monocyte-derived macrophages.     

A monoclonal antibody (BIP 45) detects Ag(c,g) polymorphism of human apolipoprotein B

A monoclonal antibody (BIP 45) against human apolipoprotein B (apo B) was used to study the polymorphism of apo B in families and in unrelated subjects. BIP 45 bound to apo B-containing lipoprotein particles in one of three distinct patterns of immunoreactivity (strong, weak and intermediate). Family studies showed that these binding patterns result from co-dominant transmission of apo B allelic pairs which are temporarily designated allele BIP- and allele BIP+; allele BIP+ would code for the apo B BIP 45 epitope. Analysis of plasma samples from 244 unrelated men randomly chosen from the North French population indicated that 46.7% of them bound BIP 45 with low affinity (weak reactors), 44.7% with intermediate affinity (intermediate reactors) and 8.6% with high affinity (strong reactors). According to the Hardy-Weinberg equilibrium, this corresponds to gene frequencies of 0.690/0.310 for the type BIP-/BIP+ alleles. This corresponds to the gene frequencies of 0.695/0.305 at the Ag(g)/Ag(c) locus previously found in a Caucasian population. Furthermore, the investigation of Ag(c,g) and of monoclonal BIP 45 antibody immunoaffinity for 30 individual plasma samples showed that BIP 45 bound strongly to Ag(c) factor, whereas it bound weakly to the allelic Ag(g) factor. This monoclonal antibody will be useful for the detection of the two corresponding apo B species designated apo B (Ag(c) factor, BIP+) and apo B (Ag(g) factor, BIP-).     

Serological, functional, and immunochemical characterization of a monoclonal antibody (MoAb Q2/70) to human Ia-like antigens

Serological and immunochemical studies showed that monoclonal antibody Q2/70 (MoAb Q2/70), produced by the hybridoma technique, is specific for human Ia-like antigens. This antibody recognizes an antigenic determinant which is different from those defining the serologic polymorphism of Ia-like antigens, and is expressed on subsets of human Ia-like molecules and on lymphoid cells from other species. MoAb Q2/70 inhibits unidirectional MLRs^* between allogenic human lymphocytes, but not between murine and human lymphocytes. In ADCC^* assays, MoAb Q2/70 mediates lysis of cultured human B lymphoid cells RPMI 4098, effected by murine splenocytes. The antibody is suitable to isolate immunologically functional B lymphocytes from human peripheral blood.     

Quantitative assay of a human monoclonal IgM antibody (HA-1A) in human serum

A rapid, specific and sensitive radiometric assay was developed capable of quantitating serum levels of HA-1A, a human IgM monoclonal antibody to endotoxin. 'Private' anti-idiotypic murine monoclonal antibodies were produced and utilized in the assay to avoid cross-reactivity with normal human IgG, IgM, IgA, IgE or IgD. The presence of E. coli or gram-negative lipopolysaccharide in the sera did not affect the ability of the assay to detect HA-1A. The sensitivity of the assay was calculated to be 25 ng/ml with an interassay coefficient of variation of less than 10%. In one patient given 100 mg of HA-1A, peak serum concentration was 101.5% of the predicted value with a mean plasma half life of 24.5 h. This assay will be useful in establishing the pharmacokinetics of HA-1A and in monitoring serum levels during phase II and phase III clinical trials.     

The production of a human monoclonal antibody defining a split of HLA-DRw13 (DRw13b)

By use of the heterohybridoma technique we have produced a human monoclonal antibody (NDS40) which detects a split of HLA-DRw13 (DRw13b) which is in linkage with HLA-DQw1. In addition, the antibody reacts with cells positive for HLA-DRw8 and DRw11, but does not react with the commonly found split of DRw13 (DRw13a) which is associated with DQw1. NDS40 is cytotoxic and is of the lambda IgM class.     

Influence of monoclonal antibodies to human neutrophil antigens, HNA-1a/b and HNA-2a on phagocytosis

Neutrophil antibodies frequently cause severe conditions such as transfusion-related acute lung injury, alloimmune/autoimmune neutropenia. As it was thought that surviving neutrophils would also be damaged from antibodies binding with the neutrophil membrane, we studied the functional influence of neutrophil antibodies on natural phagocytosis and immune phagocytosis by using neutrophil-specific monoclonal antibodies (MoAbs), TAG1: HNA-la on FgammaRIIIb, TAG2: HNA-1b on FgammaRIIIa/b, TAG3: FgammaRIIIa/b and TAG4: HNA-2a. In an inhibition assay of carbon particle phagocytosis as a representation of natural phagocytosis, neutrophils binding with TAG3 or TAG4 were inhibited from carbon particle-phagocytosis by 42.5% and 53.2% (% inhibition), respectively, but in an inhibition assay using TAG3 MoAb, HNA-2a strongly positive neutrophils were more weakly inhibited than HNA-2a weak-positive neutrophils, 39.2% and 54.0% (% inhibition), respectively. These results suggested that HNA-2a salvaged the inhibition process of natural phagocytosis by anti-FcgammaRIII antibodies. These results also suggested that natural phagocytosis would be inhibited when antibodies combined with every antigen on the cell membrane. In an inhibition assay of EA-rosette formation as a representation of immune phagocytosis, FcgammaRIII-specific MoAbs, TAG1, TAG2 and TAG3 inhibited EA-rosette formation, but HNA-2a-specific MoAb, TAG4, did not markedly inhibit EA-rosette formation. It was thought that neutrophil immune-phagocytosis would be inhibited by antibodies binding with FcgammaRIII, and that HNA-2a was not related to immune phagocytosis. Further investigation of the relationship between clinical symptoms and antibody specificity or antibody quantity is needed.     

14C1, an antigen associated with human ovarian cancer, defined using a human IgG monoclonal antibody.

We have selected a human EBV-transformed cell line from the involved lymph nodes of an ovarian cancer patient which secretes an IgG1 kappa antibody, able to recognize an antigen present on the surface of ovarian cancer cells. The antigen, termed '14Cl,' has previously been shown by immunohistological techniques to be present on the surface of the malignant cells within tumour specimens. Western blotting analysis has shown that the majority of primary ovarian cancer specimens and three continuous cell lines derived therefrom express 14Cl; other tissue types were negative. Preliminary biochemical characterization has been carried out, which shows that the 14Cl antigen has a molecular weight range of 25-32 kD and an isoelectric point from pI 6.3 to 6.8. We believe that the 14Cl antigen is immunologically relevant to ovarian cancer patients and may therefore represent a novel target for both active and passive immunotherapy.     

Characterization of the D, c, E and G antigens of the Rh blood group system with human monoclonal antibodies

The human MAbs, anti-D, -c, -E and -G of the Rh blood group system, produced by Epstein-Barr virus transformed B-cell lines, were purified by protein A-Sepharose chromatography and used to characterize the Rh antigens of human red cells. Scatchard plot analyses performed with the radiolabelled MABs indicated that each R2R2 red cell carries 0.43, 0.32 and 0.38 x 10(5) D, c and E binding sites, respectively. About half this number of antigen sites are present on erythrocytes from heterozygote individuals using the appropriate antibody. We found, however, that only 0.18 x 10(5)G antigenic sites were present on each R1R1 red cell. The affinity constants of the anti-D, -E and -G were similar varying from 0.6 to 1.5 x 10(8) M-1 whereas that of the anti-c was much lower (0.035 x 10(8) M-1). The blood group specificity and binding properties indicate that the MAbs behave like the polyclonal anti-Rh reagents. Immunoprecipitation experiments carried out with membranes from R2R2 red cells show that a 30-32 kDa component can be identified whatever the antibody used. The immune complexes involving anti-c, -E or -G antibodies could be formed with the detergent lysates from red cell membranes. In contrast, membrane integrity was a prerequisite for the binding of the anti-D antibodies. Finally, from extraction studies of immunocomplexes with non-ionic detergents it was concluded that all the Rh-active components are bound to the membrane skeleton, suggesting that these molecules may have important function for maintaining red cell shape and viability.     

CD226(PTA1)单抗诱导NK细胞克隆杀伤靶细胞的研究

目的 :研究CD2 2 6分子在NK细胞克隆上的表达及功能。方法 :用有限稀释法建立NK细胞克隆并鉴定。采用重导向杀伤实验 (redirectedcytotoxicityassay ,RCA)观察CD2 2 6单克隆抗体对NK细胞克隆杀伤作用的影响 ,并用ELISA方法检测杀伤相培养上清中细胞因子水平的动态变化。结果 :获得 1株NK细胞克隆。在重导向杀伤实验中 ,CD2 2 6单克隆抗体能明显提高NK细胞克隆的杀伤水平 ;促进NK细胞克隆IFN γ和GM CSF的分泌。结论 :CD2 2 6是一种新的NK细胞活化性受体 ,IFN γ和GM CSF水平升高可能与NK细胞克隆功能有关。     

Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells,and its association with the infectious salmon anaemia virus cell receptor

Endothelial cells (ECs) line the luminal surfaces of the cardiovascular system and play an important role in cardiovascular functions such as regulation of haemostasis and vasomotor tone. A number of fish and mammalian viruses target these cells in the course of their infection. Infectious salmon anaemia virus (ISAV) attacks ECs and red blood cells (RBCs) of farmed Atlantic salmon (Salmo salar L.), producing the severe disease of infectious salmon anaemia (ISA). The investigation of ISA has up to now been hampered by the lack of a functional marker for ECs in Atlantic salmon in situ. In this study, we report the characterisation and use of a novel monoclonal antibody (MAb) detecting Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells and scavenger ECs) and RBCs. The antibody can be used with immunohistochemistry, IFAT and on Western blots. It appears that the epitope recognised by the antibody is associated with the ISAV cellular receptor. Besides being a tool to identify ECs in situ, it could be useful in further studies of the pathogenicity of ISA. Finally, the detection of an epitope shared by ECs and RBCs agrees with recent findings that these cells share a common origin, thus the MAb can potentially be used to study the ontogeny of these cells in Atlantic salmon.