Immunochemistry最新文献

Repeated injections in rabbits of pneumococcal vacxine strains R 36 A induce antibodies of restricted heterogeneity as evidences by electrophoresis and isoelectric focusing. An immunoadsorbent containing purified C-polysaccharide was used to isolate and characterize the induced antibodies. Specificity for phosphorylcholine was demonstrated by hapten elution and by inhibition of precipitation. Addition of the ligand caused an enhancement of the intrinsic fluorescence of the anti-phosphorylcholine antibodies. The association constants determined by fluorometric titration were of the order of 10³M⁻¹ and were only slightly influenced by temperature.

The complement activating capacity and complement dependent complex release activity (CRA) of immune complexes (IC) formed at different antigen:antibody ratios were investigated. The correlation between antibody avidity, precipitating capacity and the effect of these parameters on Fcdependent biological functions was studied in BSA-anti-BSA and OA-anti-OA systems. The results suggest that antibody excess favours the complement activating effect of ICs. No correlations between antibody precipitating capacity and avidity was found. However, antibody avidity and complement activating capacity were related: the higher the avidity index of antibodies, the higher the complement activation. The kinetics of CRA are not directly influenced by these characteristics of antibodies. The intercalation of C3b into the lattice interferes not only with the primary antigen—antibody bonds, it may result in the rearrangement of the lattice through the disruption of the non-specific intermolecular interactions.

Intensive research in the author's laboratory over a 10-year period has now culminated in the precise determination of the entire antigenic structure of native hen egg-white lysozyme. This is the second antigenic structure of a protein to be precisely defined, the first one being that of sperm-whale myoglobin which was reported by this author in 1975. The antigenic structures of the two proteins are compared and contrasted. The novel and unorthodox concept of ‘surface-simulation’ synthesis which we introduced and developed during the definition of the antigenic sites of lysozyme is described and its powerful implications and potential in protein chemistry and immunochemistry are outlined.

The Fd fragment of a homogeneous rabbit antibody to type III pneumococcal polysaceharide has been prepared directly from the heavy chain by cleavage with cyanogen bromide. Cyanogen bromide cleavage of heavy chain produces Fd in 50–60% yield, free of the usual light chain contaminant. Fd combined with its homologous light chain to form an Fab fragment that had antigen binding activity indistinguishable from the recombinant of native light and heavy chains.

Tuftsin, the phagocytosis-stimulating peptide, was labeled with [¹⁴C] or [¹²⁵I] at the C-terminal or N-terminal portions of the molecule and the specific interactions of the corresponding radiolabeled tuftsin with human neutrophils, lymphocytes, or monocytes were studiedin vitro. Neutrophils bound 72.2 ± 10.3%, of the¹⁴C- or¹²⁵I-labeled tuftsin in the incubation medium. When the label was incorporated at the N-terminal portion, the binding was reduced to 10%, indicating that the N-terminus is essential for the activity. Lymphocytes and monocytes also showed specific binding sites for labeled tuftsin, but the percentage binding was lower. The differences were not significant. Addition of varying amounts of unlabeled tuftsin to the labeled tuftsin-neutrophil complex, indicated quantitative competition for the binding sites on the cells. Preincubation of the neutrophils with chicken antituftsin abolished the binding.

These studies indicate that neutrophils, lymphocytes and monocytes possess receptor sites for the phagocytosis-stimulating peptide. The enzymatic cleavage and generation of tuftsin from leukokinin by the action of leukokininase on neutrophils has been suggested to be a major event in phagocytosis. The receptor sites on neutrophils for the tetrapeptide tuftsin, evidenced by our present work, provide a valuable link towards the elucidation of the mechanism of phagocytosis-stimulation. The presence of similar binding sites for tuftsin on lymphocytes and monocytes indicates a probable role for these cell populations as well, in the sequence of events during phagocytosis. Splenectomized or asplenic patients who have frequent infections may be having defective F_c receptors on these cells or these patients may be deficient in the particular subclass of IgG and/or the specific enzyme which cleaves tuftsin from it.

Previous studies (Onicaˇ et al., 1978) have shown that glutaraldehyde polymerized homologous albumin is immunogenic in rabbits. The reaction mixture consisted of high proportion of polymeric and oligomeric molecules, but a small quantity of monomeric ones. The present investigation shows that the monomeric fraction isolated from the reaction mixture is altered in comparison with untreated monomeric albumin. The modification produced in the homologous monomeric albumin by glutaraldehyde conferred to the molecule immunogenic properties, due to the presence of new antigenic sites (haptenic determinants). Glutaraldehyde treatment induced similar haptenic determinants on albumins of different origins as shown by the cross-reactivity between glutaraldehyde treated human and rabbit albumin, which precipitate both with human and rabbit antibodies. Rabbit antibodies were shown to be specific both for the glutaraldehyde modified monomeric albumin and the “aged” fraction which exist in albumin stored either in dry state (commercial albumin) or in solution at 37°C. The same antibodies failed to react with freshly prepared albumin. Our data strongly suggest that the glutaraldehyde treatment alter the monomeric albumin in a similar way asin vitro “ageing” does.

C57BL thymocytes synthesize preferentially μ-heavy chains, in constrast to the BALB/c thymocytes which produce mainly α chains. In thymocytes obtained from F₁ hybrids and their backcrosses to BALB/c and C57BL mice, both α and μ chains were synthesized. However, distinct differences were found in the relative amount of nascent α and μ chains among the various crosses and the observed amount of μ-chain synthesis in the hybrids thymocytes was significantly lower than that expected according to simple mendelian modes of inheritance. We therefore assume that the expression of either of the genes which control α- or μ-chain synthesis is regulated by an additional gene, whose presence or absence will determine the relative quantities of α and μ chains produced in any of the strains.

Cadmium binding protein (CdBP)^* has been isolated from rat liver and separated by DEAE ionexchange chromatography into its two forms: CdBP-1 and CdBP-2. An analysis of the purity of these two proteins has been made by disc gel clectrophoresis and slab gel electrophoresis. It was found that the first cadmium binding protein peak to elute from the DEAE separation, referred to as CdBP-1, contained significant amounts of impurities, whereas the second peak, referred to as CdBP-2, was electrophoretically pure. Rabbits were immunized with either monomeric CdBP or glutaraldehyde-trcated CdBP. Antisera from these rabbits have been tested for their ability to bind CdBP and ZnMt. Antigen binding capacity (ABC), double diffusion in gels and competitive binding assays were used to characterize the strength and specificity of the antisera. The anti-CdBP produced cross reacts completely with CdBP-1 and CdBP-2 as well as with ZnMt-A and ZnMt-B.

Splitting of native polymeric IgM to tree non-covalently linked half sub-units and free J-chains was obtained by reduction with 0.2M mercaptoethanol. Removal of the reducing agent by dialysis for 24 hr or several days in the presence of 10 mm of cheluting agents gave no polymers, while dialysis in the presence of 20 μM zinc ions gave up to 97% of polymers. The polymers showed a sedimentation rate and protein profile in the ultracentrifugc and J-chain content similar to that of native IgM.

The polymers made by dialysis for 24 hr were non-covalently linked since they were split by iodoacetamide orN-ethyl maleimide to free non-covalently linked half sub-units and free J-chains and by sodium dodecyl sulphate mostly to free chains. Of the polymeric molecules prepared by dialysis for several days, some of the molecules were found to be stabilized by disulphide bridges.

It was concluded that in order to obtain IgM polymersin vitro from reduced IgM, the sub-units and J-chains must have their sulfhydryl groups intact and zinc ions must be present. It seems likely that the formation of covalently linked IgM polymers alsoin vivo is preceded by the formation of non-covalently linked polymers.

Fluorescein isothiocyanate was reacted with purified rabbit anti-fluorescyl IgG antibodies under defined conditions. Relative to the normal IgG control, significantly more ligand was conjugated to the purified antibody which was then inhibited from further binding with radioactively labeled ligand (³H-N-acetylfluorescein amine). Fluorescyl ligand conjugated to purified antibody preparations exhibited a red spectral shift characteristic of specifically bound fluorophore. Fluorescence of ligand conjugated to antibody was quenched about 95% relative to ~ 90% for ligand reversibly bound. The quantum yield (Φ) of the former is indicative of a thiocarbamyl linkage within the antibody-active site. Fluorescence lifetime (t) measurements by phase and modulation showed a close correlation between liganded and affinity-labeled antibody relative to free ligand and fluorescyl conjugated non-antibody proteins. The fluorescyl-affinity label was not dissociated from the antibody-active site upon denaturation of the protein in 6M guanidine-HCl. Reduction, alkylation and resolution of H and L chains in two different dispersing agents showed that the ligand remained attached to either chain. Fluorescyl ligand bound to high affinity IgG (i.e. non-affinity labeled) was quantitatively released under conditions required for H and L chain production. The fluorescence of affinity labeled ligand was enhanced in high concentrations of deuterium oxide. The latter is characteristic of ligand bound specifically to the antibody active site and is not observed with fluorescein covalently conjugated to non-specific Ig.