R. von Baehr

A rapid and sensitive enzyme immunoassay for Cu/Zn superoxide dismutase with polyclonal and monoclonal antibodies.

An enzyme immunoassay for the quantification of human Cu/Zn SOD in serum, urine and erythrocytes was developed applying monoclonal and polyclonal antibodies. The one-step assay is completed within 30 min and enables the detection of 0.3 microgram Cu/Zn SOD per litre. A Cu/Zn SOD concentration of 46 +/- 21.5 micrograms/l and of 1 +/- 0.6 micrograms/mmol creatinine was determined in the serum and the urine, respectively, of healthy individuals. A content of 15 +/- 1.7 ng Cu/Zn SOD was found in 10(6) erythrocytes. Patients with Downs syndrome exhibited a 3.8-fold, a 2-fold and a 1.6-fold higher concentration of Cu/Zn SOD in their serum, urine and erythrocytes.

The role of dipeptidylpeptidase IV positive T cells in wound healing and angiogenesis

In our former investigations we showed that dipeptides like Lys-Pro and others with the structure X-Pro are able to promote wound healing and angiogenesis in several experimental models (implantation of polyvinyl rings, open skin wounds, wound breaking strength). These dipeptides can originate from free amino termini of various polypeptides (collagenopeptides, cytokines, growth factors etc.) by degradation by dipeptidylpeptidase IV (DPP IV, EC 3.4.14.5). This enzyme occurs, among other sources, in exocrine epithelia, hepatocytes, renal tubuli, endothelia, and myofibroblasts. T-lymphocytes also reveal this enzyme activity (CD26 antigen) [1]. T-lymphocytes, monocytes, and macrophages generate numerous polypeptides and other factors capable of stimulating and modulating the proliferation and function of fibroblasts, endothelial cells and other cell types [2]. It is known that in cases of immunosuppressive therapy, disturbances of would healing may occur and that patients suffering from immunodeficiencies, among others, have impaired wound healing. In contrast immunostimulation may improve wound healing. We have shown that Wistar rats, systemically treated by complete Freunds adjuvant produce more granulation tissue than untreated animals and that T cells are involved in this process [3]. Other investigators showed that nude mice have a lower capa-

Establishment of human Ig producing heterohybridomas by fusion of mouse myeloma cells with human lymphocytes derived from peripheral blood, bone marrow, spleen, lymph node, and synovial fluid: Effect of polyclonal prestimulation and cryopreservation

50 fusion experiments were carried out to analyse heterohybridization efficiencies on mouse myeloma cells of the P3 X63 Ag8/653 line with human lymphocytes derived from peripheral blood, bone marrow, lymph node, spleen or synovial fluid. We found higher yields of growing and human Ig-producing hybridoma lines when lymphocytes from spleen or lymph node were fused. Although primary hybridomas could be established from fusions with bone marrow-derived cells, only in nine out of 1616 initially seeded wells was Ig production registered. Four fusions using immune cells from synovial fluid were made without success. Independently of the source of lymphocytes pokeweed mitogen (PWM) prestimulation had no enhancing effect on the percentage of wells with cell growth and this did not alter the IgM:IgG ratio in primary hybridomas (9:1), although cells from all compartments used here (with the exception of bone marrow cells) could be stimulated with PWM to produce both IgG and IgM in cultures. Cryopreserved lymphocytes from different sources could be used for fusions with comparable results registered for the fresh material.

Stabilization of the Substrate Reaction of Horseradish Peroxidase with o-Phenylenediamine in the Enzyme Immunoassay

When the horseradish peroxidase reaction is stopped with acid, the decay of unconverted hydrogen peroxide is responsible for the further oxidation of o-phenylenediamine. This leads to a time-dependent flattening of the standard curve in the enzyme immunoassay, after the reaction has been stopped. Addition of reducing agents, such as sulphite ions, to the stopping solution, prevents the further oxidation of o-phenylenediamine by completely reducing the remaining hydrogen peroxide. The developed colour is then stabilized.

Human hybridomas derived from CDS+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies

Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B‐CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT‐sensitive heteromye‐loma line (CB‐F7). A fusion frequency of up to 10‐5 allowed us to analyse hundreds of initial hybridoma lines per fusion. In all culture supernatants in three out of five fusions IgM lambda antibodies were detected, in two experiments only IgM kappa was measured, suggesting monoclonality of the primary hybridoma cell lines. The later fusions resulted in hybridomas producing multi‐specific antibodies against both an autoantigen and an infectious agent: (i) dsDNA/influenza virus haemagglutinin; (ii) dsDNA/class V outer membrane protein type C from Neisseria meningitidis. However, no antibodies of the described specificity were detected in blood sera of patients, indicating a ‘switch‐on’ of the immunoglobulin secretion capacity of malignant B cells during fusion to a myeloma partner. We discuss the results as further evidence for the natural multi‐reactive antibody repertoire of CD5+ B cells.

Two-colour combination enzyme-linked immunosorbent assay for the simultaneous detection of HBV and HIV infection

To reduce the cost, time and waste in screening for HIV and HBV infections a combined assay for HIV-1 and -2 antibodies and HBsAg has been developed. Monoclonal anti-HBs antibodies were co-immobilized with synthetic peptides representing immunodominant regions of HIV-1 and -2. The presence of anti-HIV antibodies in the samples was detected with alkaline phosphatase-labelled anti-human IgG and of HBsAg with horseradish peroxidase-labelled monoclonal anti-HBs antibodies by a sequential substrate reaction. In this assay, HBsAg was detectable in a concentration range between 0.25 and 0.30 U/ml and the results were available within 3 h. The specificity, tested on 5000 serum samples from blood donors after confirmation, was 99.8% for HBsAg and 99.5% for anti-HIV antibody detection. All serum samples taken from 600 HIV-1- and 115 HIV-2-infected individuals were correctly classified as reactive. The two-colour HBsAg-anti-HIV-1/-2 combination ELISA meets all the requirements of single parameter assays with regard to precision, stability and robustness.

Expansion of a B-lymphocyte clone producing IgM auto-antibodies encoded by a somatically mutated VHI gene in the spleen of an autoimmune patient

SummarySeventy-six human B-cell hybridomas were obtained by fusing B lymphocytes from the spleen of a patient with chronic idiopathic thrombocytopenia (ITP). Two independent hybridoma clones producing IgM autoantibodies reacting with platelets and other antigens from both the internal and the external environments were established from this fusion experiment. The IgM autoantibodies produced by the two hybridoma clones were found to be encoded by identical VHDJH and VLJL genes. The comparison of the VHI gene expressed in both hybridomas with the germline equivalent cloned from the patients DNA showed two somatic mutations in the complementarity-determining regions CDR1 and CDR2 resulting in amino acid replacements. These data suggest the selection and expansion of an autoantibody-producing B-cell clone in the spleen of an ITP patient, probably as a result of (auto)antigen-driven stimulation.

Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells

In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Colo205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human JH probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumour-bearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.

Polyclonal stimulation of human B lymphocytes derived from fetal liver and spleen cells at different stages of ontogeny

The functional capacity of human lymphocytes derived from fetal liver and spleen at different stages of ontogeny (16-34 weeks of gestation) was studied using in vitro models (increase in cell volume, [3H]thymidine incorporation, Ig secretion) reflecting various stages of activation induced by mitogens (LPS, PWM) in vitro. Lymphocytes differed in their reactivity to LPS depending on the period of intrauterine development: cells from the early liver could respond with enhanced IgM production whereas lymphocytes derived from this organ after more than 25 weeks failed. The opposite was found to apply to spleen cells: only lymphocytes derived from the organ after more than 25 weeks showed significant LPS-induced in vitro differentiation. These data were in correlation with the proliferative response to LPS. It was clear that the number of CD20-positive mature B cells in the lymphocyte preparations was not responsible for these results, since comparable yields were found throughout the period of fetal development of the liver studied, whereas in the spleen increasing numbers of B cells were seen.

An anti-lipid a antibody obtained from the human fetal repertoire is encoded by VH6-Vλ1 genes

Abstract The human hybridoma cell line CB-201 has been obtained from a fusion of fetal spleen lymphocytes (31st gestational week) with heteromyeloma cells. The IgM(λ) secreted was found to bind to lipid A, whereas other endogenous and exogenous antigens were not recognized. The CB-201 antibody is encoded by the unmutated V H 6 gene recombined with DN4 and J H 3 elements and a V λ subgroup 1 gene. Therefore, a V H gene, which was previously described to be over-represented in the fetal repertoire and to be expressed in autoantibody-producing B cells, may encode an anti-bacterial specificity.