Complement-fixing Monoclonal Antibody Research Articles

Depletion of CD8+ T cells from vaccinated goats does not affect protection from challenge with wild-type peste des petits ruminants virus.

Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East and Asia. The disease is caused by peste des petits ruminants virus (PPRV); cell culture‐attenuated strains of PPRV have been shown, both experimentally and by extensive use in the field, to be effective vaccines and are widely used. We have previously demonstrated that these vaccines elicit both serological (PPRV‐specific antibody) and cell‐based (PPRV‐specific CD4+ and CD8+ T cells) immune responses. However, it is not known which of these responses are required for protection from PPRV, information that would be useful in the evaluation of new vaccines that are being developed to provide the capability to differentiate infected and vaccinated animals (DIVA capability). To begin to address this issue, we have used a complement‐fixing monoclonal antibody recognizing caprine CD8 to deplete >99.9% of circulating CD8+ T cells from vaccinated goats. Animals were then infected with wild‐type PPRV. Despite the absence of the CD8+ T‐cell component of the vaccine‐induced immune response, the vaccinated animals were almost fully protected, showing no pyrexia or viraemia, and almost no clinical signs. These data suggest that a virus‐specific CD8+ T‐cell response is not critical for protection against PPRV and that virus‐specific antibody and/or CD4+ T cells are the main mediators of protection. We have also shown that the leucopenia caused by infection with wild‐type PPRV affects all major classes of circulating leucocytes.

Neutralizing and complement-fixing monoclonal antibodies as an aid to the diagnosis of equine herpesvirus-1 infection.

One complement-fixing (C-MAb) and three complement-dependent neutralizing monoclonal antibodies (N-MAbs) were raised against Hisar-90-7 equine herpesvirus-1 (EHV-1) strain. The target antigen of the C-MAb (2A5) and two of the N-MAbs (1H6, 9C4) was identified as a 140 kDa polypeptide in Western blotting. The target antigen of N-MAb (9C6) could not be identified. Purified polypeptides of five EHV-1 strains isolated from different regions and at different times gave intense bands at 140 kDa when reacted with N-MAb (1H6) in Western blots. In sandwich ELISA, all four MAbs captured the viral antigen from clinical materials, giving a reliable and rapid diagnosis of EHV-1 infection in equines.

High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization.

Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.

Hot pressing of boron carbide at low temperatures: J. Guoxin, W. Shenghong (Central Iron and Steel Research Inst., Beijing, China). PM Technol., vol 13, no 2, 1995, 108–111. (In Chinese.)

Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement- and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.

Variable-Region Gene Family Usage for Type II Collagen Autoantibodies in Arthritis-Susceptible DBA/1 Mice

Collagen-induced arthritis is mediated by autoantibodies to type II collagen (CII). This experimental model has proven useful in determining the molecular and cellular mechanisms responsible for autoimmune arthritis. We have shown that polyarthritis can be transferred to normal mice by administrating combinations of three or four complement-fixing monoclonal antibodies (mAbs) which recognize cross-reactive epitopes on the α1(II)-CB11 region of chick and mouse CII. Currently, the light- and heavy-chain variable-region structures of a panel of α1(II)-CB11-specific mAbs that cross-react with chick and mouse CII, or react solely with chick CII, have been analyzed. The results indicate biased usage of VK19 and VK21 families of light-chain variable-region genes but random VHgene usage. Interestingly, two mAbs derived from different mice recognized identical epitopes on mouse CII and had nearly identical light- and heavy-chain variable-region structure including junctionally derived sequence.

Removal of Fibroblasts from Human Epithelial Cell Cultures with Use of a Complement Fixing Monoclonal Antibody Reactive with Human Fibroblasts and Monocytes/Macrophages

A complement fixing IgM monoclonal antibody (1B10) that reacts with surface membrane molecules of human fibroblasts, tissue macrophages, and peripheral monocytes was produced. In Western blot analysis of detergent extracts of cultured human foreskin fibroblasts, antibody 1B10 detected protein bands of Mr 43,000 and 72-80,000. We used the 1B10 antibody with complement to eliminate most 1B10 positive nonepithelial cells from thymic epithelial (TE) cell cultures, thereby allowing us to grow highly enriched populations of human TE cells.

Heterogeneity among the non-Hodgkin's lymphomas. Implications for autologous bone marrow transplantation with in vitro purging using monoclonal antibodies.

To investigate the possible implications of heterogeneity among the non-Hodgkin's lymphomas for bone marrow purging using complement-fixing monoclonal antibodies to lymphoma-associated antigens, a panel of large cell lymphoma cell lines of diverse phenotypes was treated with monoclonal antibodies DLC-48 and LN-1. An association was demonstrated between the percentage of suppression of colony formation by the cell line and both the percentage of cells staining with the antibody and the intensity of its binding. Flow cytometric analysis of cells surviving treatment with antibody and complement demonstrated that the population that escaped lysis showed weak immunofluorescent staining. Similarly, 40% of the clones derived from cells surviving treatment with antibody and complement stained weakly compared with the parent cell line. For a given fluorescence intensity, cells differed in their susceptibility to treatment. Some cells with moderate to strong staining survived incubation with antibody and complement. In five cases, treatment of bone marrow contaminated with malignant lymphoma cells resulted in complete eradication of even cells with weak staining. In two cases, a population of cells that stained dimly survived treatment with either antibody. DNA-content analysis showed that the cell cycle distribution of cells surviving treatment with DLC-48 or LN-1 and complement was similar to that of cells treated with control antibody 46-1G7 and complement. Phenotypic heterogeneity may hinder efforts to purge malignant lymphoma cells from human bone marrow with complement-fixing monoclonal antibody reagents. Relative resistance to complement-mediated lysis may underlie differences in the susceptibility of cells to treatment and also limit the effectiveness of this technique.

4654210 Methods and compositions using complement fixing monoclonal antibody to human T cells: Patrick C Kung, Gideo Goldstein assigned to Ortho Pharmaceutical Corporation

4654210 Methods and compositions using complement fixing monoclonal antibody to human T cells: Patrick C Kung, Gideo Goldstein assigned to Ortho Pharmaceutical Corporation

4658019 Complement-fixing monoclonal antibody to human T cells: Patrick C Kung, Gideo Goldstein assigned to Ortho Pharmaceutical Corporation

4658019 Complement-fixing monoclonal antibody to human T cells: Patrick C Kung, Gideo Goldstein assigned to Ortho Pharmaceutical Corporation

A monoclonal antibody detecting an HLA‐DQwl‐related determinant

A complement fixing monoclonal antibody (moab) was prepared which reacts with a polymorphic determinant on HLA class II molecules. The moab IIB3 recognises all DQwl (DC1, MB1, LB-E12) positive cells as well as some DR4, DR7, DRw8 and DRw9 positive cells. The moab reacts mainly with B-cells and not or with only a minority of the monocytes. Segregation of the determinant with HLA-DR could be shown. The determinant is strongly expressed on DR2, DR4 and DRw6 positive cell lines but only weakly on DR1 lines. In contrast to a monoclonal antibody against a monomorphic determinant on class II molecules IIB3 did not give a distinct inhibition of the MLC nor did it inhibit the generation of CTLs in MLC as has been described for the DQwl like moab BT 3/4 (Corte et al. 1982). Immunoprecipitation indicates that IIB3 reacts with DQ-like molecules.

Refinement of HLA gene mapping with induced B-cell line mutants.

The lymphoma cell line BJAB.B95.8.6 was gamma-irradiated to induce mutations of major histocompatibility complex (MHC) encoded genes. Cloned "wild-type" cells were phenotyped HLA-A1, A2, B13, B35, Bw4, Bw6, Cw4, DR5, DRw52, DQw1, DQw3, DPw2, DPw4, GLO1 1, PGM3 2-1, and ME1 0 and possessed two apparently normal chromosome 6s prior to mutagenesis. Loss mutants were selected 5 days after 3 Gy gamma-irradiation employing three complement-fixing monoclonal antibodies specific for HLA-A2 (TU101) and Bw4 (TU48, TU109). Fifteen independently arising mutants were isolated and cloned. Typing with monospecific alloantisera and cell-mediated lympholysis revealed the presence of HLA-A1, B35, Bw6, Cw4, DR5, DRw52, DQw3, and DPw4 specificities on all mutant clones. HLA-A2, B13, and Bw4 were absent. Mutants differed in their expression of class II antigens. One group retained DQw1 and DPw2, another was DQw1-, DPw2+, and a third was DQw1-, DPw2-. Karyotyping of the "wild-type" line and selected mutant clones showed that the loss of HLA specificities correlated with deletions which map the HLA-A and -B loci directly to the distal part of the 6p21.33 region and the class II genes to the region 6p21.33 (proximal) to 6p21.31 (distal) on the short arm of chromosome 6.

Biology of Langerhans Cells: Analysis by Experiments to Deplete Langerhans Cells from Human Skin

In vivo studies have demonstrated that various treatments of skin, e.g., UV irradiation, topical corticosteroids, and tape-stripping, will temporarily deplete the epidermis of Langerhans cells (LC). Whether this loss represents simply a loss of cell surface markers unique to LC, or actual depletion of cells, is unknown. By design, normal human skin transplanted to the congenitally athymic (nude) mouse is a system devoid of circulating precursors for human LC. Because LC have been shown to be of bone marrow origin, any depletion of these cells in this system should be permanent. Treatments to deplete LC from human skin grafts on nude mice after grafting included: (a) large doses of UV radiation (400 mJ/cm2 every 48 h, X 3), (b) potent high-dose topical corticosteroids (2.5 mg betamethasone valerate/cm2 every day, X 5), (c) tape-stripping (X 20). Treatments before grafting included: (a) treating donor skin with 900 R of gamma irradiation, (b) complement fixing monoclonal antibody to Ia-like antigens of LC, followed by fresh complement, (c) monoclonal antibody conjugated to toxins. Quantitation of the number of LC was analyzed on control and treated epidermal sheets using immunodiagnostic reagents, anti-HLA-DR, and surface ectoenzymes , ATPase. Results show that both UV irradiation and topical corticosteroids reduce the number of LC by these analyses. However, within 3 weeks, recovery to pretreatment levels has occurred. X-irradiation and tape-stripping were without effect. Despite evidence that the monoclonal antibody, complement, and toxic systems were delivered to the LC within the epidermis, there is no evidence that these treatments resulted in a decrease in LC. It appears that LC are currently either long-lived or replaced locally from a proliferative pool and that certain cell membrane determinants of human LC are somewhat differentially sensitive to UV radiation and corticosteroids.

Phenotypic characterization of acute leukemia with monoclonal antibodies using the microlymphocytotoxicity assay

Surface antigens of lymphoblasts from 56 pediatric ALL patients were studied with a set of complement fixing monoclonal antibodies. This group of lymphoblasts was comprised of 22 T-cell ALL, 22 CALLA + Ia + ALL and 12 non-T-non-B, CALLA − Ia + ALL. For comparison, two adult T-cell CLL and six B-cell CLL were also studied. It was found that by using the microlymphocytotoxic technique, the lymphoblasts can be assigned their immunophenotype and thus be classified into their respective lineage and stage of differentiation. In the samples tested, concordant reactivity was observed when FACS fluorescence profile was compared with that of microlymphocytotoxic suggesting that the letter can be used especially when qualitative estimates are required.

Antigenic expression and proliferative status of multilineage myeloid progenitor cells (CFU-GEMM) in normal individuals and patients with chronic granulocytic leukaemia.

We assayed the number of multilineage myeloid progenitor cells (CFU-GEMM) in the blood and marrow of patients with newly diagnosed chronic granulocytic leukaemia (CGL). The mean number of CFU-GEMM in the blood was increased 600-fold and CFU-GEMM in the marrow was doubled in the CGL patients compared with normal. A complement-fixing monoclonal antibody with HLA-DR specificity inhibited the proliferation of CFU-GEMM from CGL blood to a greater extent than that of comparable cells in normal marrow. Using a hydroxyurea 'suicide' method we found that the proportion of CFU-GEMM in proliferative cycle was higher in CGL blood than in normal marrow. We conclude that (1) CFU-GEMM numbers are greatly increased in the blood of patients with CGL, (2) CFU-GEMM express HLA-DR antigens on their surface, and (3) the apparently increased expression of the antigen on CFU-GEMM from CGL blood in comparison with CFU-GEMM from normal marrow may parallel the relatively higher proportion of CGL CFU-GEMM in cell cycle.

4361549 Complement-fixing monoclonal antibody to human T cells, and methods of preparing same: Patrick C. Kung, Gideon Goldstein, assigned to Ortho Pharmaceutical Corporation

4361549 Complement-fixing monoclonal antibody to human T cells, and methods of preparing same: Patrick C. Kung, Gideon Goldstein, assigned to Ortho Pharmaceutical Corporation

4361550 Complement-fixing monoclonal antibody to human suppressor T cells and methods of preparing same: Patrick C. Kung, Gideon Goldstein, assigned to Ortho Pharmaceutical Corporation

4361550 Complement-fixing monoclonal antibody to human suppressor T cells and methods of preparing same: Patrick C. Kung, Gideon Goldstein, assigned to Ortho Pharmaceutical Corporation

HLA-DR monoclonal antibodies inhibit the proliferation of normal and chronic granulocytic leukaemia myeloid progenitor cells.

We studied the capacity of murine monoclonal antibodies with HLA-DR specificity to inhibit the proliferation in vitro of erythroid (BFU-E and CFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells in normal bone marrow and the blood of patients with chronic granulocytic leukaemia (CGL). Two IgG2 antibodies (CA 2.06 and L243) inhibited the proliferation of normal BFU-E and CFU-GM at relatively high dilution; a third antibody, DA2, had no effect on either progenitor cell. A complement-fixing monoclonal antibody with T-cell activity (OKT3) produced only minor reduction in progenitor cel proliferation. Further studies with L243 showed that BFU-E and CFU-GM from the blood of patients with CGL were inhibited to the same degree as normal marrow progenitor cells. The inhibition of progenitor cell proliferation by a given antibody was always complement dependent and was therefore presumed to be due to a direct cytotoxic effect. The inhibitory effect of monoclonal antibodies is a valuable approach to the characterization of antigenic determinants on myeloid progenitor cells and the differential cytotoxicity of selected monoclonal antibodies might be exploitable for therapy.

Murine monoclonal antibodies specific for virulent Treponema pallidum (Nichols).

Murine anti-Treponema pallidum (Nichols) lymphocyte hybridoma cell lines secreting monoclonal antibodies against a variety of treponemal antigens have been generated. Hybridomas isolated were of three major types: those that were directed specifically against T. pallidum antigens, those that were directed against treponemal group antigens (as evidenced by their cross-reactivity with T. phagedenis biotype Reiter antigens), and those that cross-reacted with both treponemal as well as rabbit host testicular tissue antigens. The majority (31 of 39 clones) of these anti-T. pallidum hybridomas, which produced monoclonal antibodies of mouse isotypes immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3 or IgM, were directed specifically against T. pallidum and not other treponemal or rabbit antigens tested by radioimmunoassay. Four of these T. pallidum-specific hybridomas secreted monoclonal antibodies with greater binding affinity for "aged" rather than freshly isolated intact T. pallidum cells, suggesting a possible specificity for "unmasked" surface antigens of T. pallidum. Six anti-T. pallidum hybridomas produced complement-fixing monoclonal antibodies (IgG2a, IgG2b, or IgM) that were capable of immobilizing virulent treponemes in the T. pallidum immobilization (TPI) test; these may represent biologically active monoclonal antibodies against treponemal surface antigens. Three other hybridomas secreted monoclonal antibodies which bound to both T. pallidum and T. phagedenis biotype Reiter antigens, thus demonstrating a possible specificity for treponemal group antigens. Five hybridoma cell lines were also isolated which produced IgM monoclonal antibodies that cross-reacted with all treponemal and rabbit host testicular tissue antigens employed in the radioimmunoassays. This report describes the construction and characteristics of these hybridoma cell lines. The potential applications of the anti-T. pallidum monoclonal antibodies are discussed.

Functional analysis of human T cell subsets defined by monoclonal antibodies. II. Collaborative T-T interactions in the generation of TNP-altered-self-reactive cytotoxic T lymphocytes.

In previous reports we have demonstrated that human T cells, responding to soluble and alloantigens, release helper factor(s) that amplify primary in vitro hapten-altered-self-reactive CTL responses. In the present studies, we have employed complement-fixing monoclonal antibodies (OKT4 and OKT8) that recognize functionally distinct human T cell subsets to investigate the role of T-T interaction in the generation of these killer cells. In all experiments, purified OKT4+ responder T cells were deficient in cytotoxic activity, whereas responder populations containing OKT8+ T cells generated substantial cytotoxicity; demonstrating that TNP-altered-self-reactive CTL precursors are contained within the OKT8+ T cell subset. Further, optimal cytotoxic responses were obtained from responder populations containing both OKT4+ and OKT8+ T cells, suggesting that cooperative interaction between these subsets may result in an amplification of killer cell activity. This interpretation was supported by the following observations: (1) the amplifying effect of soluble antigen required the presence of both OKT4+ and OKT8+ responders; (2) during MLC, OKT4+ but not OKT8+ responder T cells generate helper factor(s) that amplify TNP-altered-self-reactive CTL responses; (3) helper factor(s) bypass the requirement for direct OKT4-OKT8 T cell interaction, triggering a CTL response that is proportional to the percentage of OKT8+ T cells present within the responder population. In additional studies, we determined that the TNP-altered-self-reactive effector CTL maintain the OKT3+, OKT4-, OKT8+ surface phenotype displayed by the CTL precursor.