A bovine monoclonal antibody detecting a class I BoLA antigen.

The bovine IgG1 monoclonal antibody (mAb) ILA70 was made by immunizing a calf with peripheral blood mononuclear cells (PBM) from a BoLA-w10 homozygous heifer and subsequently fusing lymphocytes from the local lymph-node with the heterohybridoma 53B3. Family and population studies, antibody binding inhibition and immunoprecipitation of the target antigen all indicate that ILA70 detects a polymorphic epitope on a bovine class I major histocompatibility complex (MHC) molecule. The antibody is complement fixing and so may be used in a standard cytotoxicity assay. Ascitic fluid with antibody activity many times greater than that of the tissue-culture supernatant has been prepared in nude mice. The antibody-producing heterohybridoma has been subcloned three times and appears to be stable. Such heterohybridomas may prove to be a valuable source of particularly discriminating and informative mAbs for the serological analysis of the products of the bovine MHC.     

Immunoregulation by antigen/antibody complexes. I. Specific immunosuppression induced in vivo with immune complexes formed in antibody excess

Specific immune complexes, prepared at different ratios of antibody to antigen, were examined for their effects on the antibody response of BALB/c mice to the cell wall polysaccharide antigen (PnC) extracted from Streptococcus pneumonia R36a. Mice immunized with complexes formed in antigen excess developed a PnC-specific antibody response that was equivalent to that in mice injected with free antigen. On the other hand, mice injected with complexes formed in antibody excess developed very little PnC-specific antibody. Furthermore, administration of immune complexes (formed in antibody excess) resulted in suppression of the response to an immunogenic dose of PnC given concurrently or 1 day after injection of immune complexes but not when the antigen was given 1 day before injection of the immune complexes. Injections of free antibody (TEPC-15) also resulted in suppression of the response to antigenic challenge; however, suppression was greatest when the antibody was injected concurrently with the antigen, suggesting that the suppression was mediated through the formation of immune complexes in vivo. The suppression appears to be specific for the antigen (PnC), since in mice injected with TEPC-15/PnC complexes (formed in antibody excess) and challenged with PnC coupled to sheep RBC, only the response to PnC was suppressed.     

Desensitization of delayed-type hypersensitivity by antigen and specific antibody.

The role of antibody in the desensitization of delayed-type hypersnsitivity (DTH) to dinitrophenylated bovine gammaglobulin (DNP-BGG) was studied in rats. Rats sensitized by a subcutaneous injection of DNP₃₂-BGG in Freund's complete adjuvant (FCA) were desensitized 14 days later with various doses of DNP₃₂-BGG injected intravenously. It was found that only certain doses (100–500 μg) of DNP-BGG effectively desensitized, antigen doses outside this optimum range being ineffective in suppressing DTH. In adoptive cell transfer experiments, it was shown that sensitized peritoneal cells incubated with optimum doses of the antigen in the presence of specific antiserum in vitro failed to transfer the delayed response to normal recipients, whereas the treatment of the sensitized cells with the antigen or with the antiserum separately did not impair the ability of these cells to transfer DTH. The effect of desensitization is specific and is not permanent. The DTH reappears 3–4 wk after desensitizing injection.     

Characterization of Epstein-Barr virus antigens. I. Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen.

The Epstein-Barr virus-soluble (S) antigen extracted from RAJI cells was characterized by sucrose gradient centrifugation, gel filtration, and ion-exchange chromatography. The sedimentation coefficient was estimated to be 8.5S corresponding to a molecular weight of 180,000. The S antigen binds to DEAE-A25 ion exchanger from which it can be eluted with 0.3 M NaCl in Tris buffer (pH 7.2). All fractions which contained complement-fixing S antigen also inhibited the anticomplement immunofluorescence reaction as used to detect the Epstein-Barr virus-associated nuclear antigen. These results are consistent with the hypothesis that the S and Epstein-Barr virus-associated nuclear antigens are either a single antigen or that both activities are present on the same molecule.     

Two-step mobilization of arachidonic acid in platelet activation induced by low concentrations of TP 82, a monoclonal antibody against CD9 antigen.

Arachidonic acid mobilization in platelets activated by low concentrations (less than or equal to 1.6 micrograms/ml) of TP 82, a monoclonal antibody against CD9, appears to consist of two distinct phases. In the first phase, limited arachidonic acid release occurs concomitantly with a shape change induced by TP 82. This appears to be dependent upon phospholipase A2 activation, since it is well preserved in the presence of aspirin, which completely blocked both intracellular Ca2+ elevation and phosphatidic acid formation which would indicate phospholipase C activation. The Na+ Exchange was also found to participate in the first phase of arachidonic acid mobilization, since extracellular Na+ depletion and ethylisopropylamiloride, a specific inhibitor of the Na+/H+ exchanger, effectively blocked this limited mobilization of arachidonic acid. The second, much larger, phase of arachidonic acid mobilization occurs with the beginning of platelet aggregation. A limited amount of thromboxane A2 formed during the first phase of arachidonic acid release plays an important role in induction of the massive arachidonic mobilization in the second phase. Factors, as yet unidentified, also appear to work synergistically with thromboxane A2 to induce the full picture of arachidonic acid mobilization.     

Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry

BACKGROUND: Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous. METHODS: Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. RESULTS: Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. CONCLUSIONS: The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.     

Hepatitis B core antigen. Detection of antibody by radioimmunoprecipitation.

Radioactive cores were prepared from concentrated Dane particles by DNA polymerase reaction followed by CsCl density gradient centrifugation. Two radioactive peaks were obtained: one peak with an average density of 1.36 g/cm-3 in CsCl contained cores that possessed full serologic reactivity; the other peak, with a density of 1.28 to 1.32 g/cm-3, contained cores associated with globulin. A double antibody immunoprecipitation test was developed, using the radioactive heavy cores as a source of antigen. The test was at least 300 times as sensitive as complement fixation for detecting antibody to core.     

Immunosuppression by procarbazine. I. Sites of action of the drug and effect on adjuvant arthritis and circulating antibody responses

The effect of procarbazine hydrochloride on adjuvant arthritis and on circulating antibody responses in the rat was studied. Procarbazine had a profoundly depressing effect on both adjuvant arthritis and the circulating antibody response to ovalbumin (OA). In contrast, it had no effect on the immune response to sheep erythrocytes (SRBC) alone; the presence of adjuvant, however, enhanced the response to SRBC, and this enhancement was suppressed by procarbazine. Preinjection with adjuvant enhanced the response to OA given at a different site: procarbazine treatment at the time of adjuvant injection suppressed this enhancement. These results taken in conjunction with histological observations, reinforce the idea that the initial effect of the drug is predominantly on the thymus and thymus-dependent cell populations, though prolonged treatment may also affect the bone marrow. They also give a clue to the mode of action of adjuvant in enhancing immune responsiveness. It is suggested that judicious use of procarbazine may assist fine dissection of various components of the immune response.