Immunochemistry最新文献

MBP-SF, a prominent serum factor in suckling Lewis rats, that additively inhibits the binding of¹²⁵I-labeled and unlabeled MBP to syngeneic Lewis rat anti-MBP, has now been found in low but significant amounts in adult Lewis rats using a new and more sensitive radioimmunoassay procedure. The serum factor appears to delay the time of appearance of free circulating anti-MBP antibody in immunized rats by at least a day, but the disappearance curve of MBP-SF is immediately joined by the antibody time-response curve, both elements appearing to be mutually antagonistic. The generation of significant amounts of anti-MBP antibody appears to begin on the 8th day after immunization even though free antibody does not become measurable until the 9th day. From the nature of the timing of events leading to EAE, in which the 9th day is also critical, it is therefore conceivable that MBP-SF may function as the proposed endogenous autoneurotolerogen of Orgad and Cohen and that its efficient removal by anti-MBPat least a day before anti-MBP actually becomes detectable sets the stage for the subsequent cellular immune reactions that signal EAE.

Macromolecular insoluble cold globulin (MICG) is synthesized selectively by mouse T-cells. In support of this conclusion is evidence derived from mitogenic stimulation of thymic and splenic lymphocytes. PHA and Con A stimulated a disproportionately large increase in MICG compared to total protein synthesis in spleen and thymus cells, while IgM synthesis in spleen cells only rose parallel with the increase in total protein synthesis. In LPS-treated spleen cells, MICG synthesis rose only in proportion to total protein. Therefore, selective enhancement of MICG synthesis, i.e. as a proportion of total protein synthesis, only occurred under conditions where T-cells were activated. In the presence of complement, antibody to MICG was cytotoxic to virtually all thymocytes and half of the spleen cells. Furthermore, antibody to MICG eliminated the mitogenic effect of PHA and Con A on spleen cells, while the response of these lymphocytes to LPS was normal. Cytotoxicity with antibody and complement also caused a significant diminution of MICG synthesis in spleen cells. Finally, isolated T-cells demonstrated a significant amount of MICG synthesis, while only a trace of MICG synthesis was atrributable to B-cells.

We have studied the effect of several lectins that have different sugar specifications on the functional activity of purified guinea-pig and human complement components 1 to 9. We tested castor bean type II (specific for galactose and lactose), wheat germ agglutinin (specific forN-aceytl-glucosamine), lotus bean (specific for fucose), soybean and phytohemagglutinin (PHA) (specific forN-aceytl-d-galactosamine). Leucoagglutinin, a purified constituent of PHA with no known suger specificity, was also included. Soy bean, wheat germ agglutinin and PHA, when immobilized, interacted with GP or Hu C1. Only PHA affected the activity of human or guinea-pig fluid phase Cl. Castor bean type II was found to inhibit the hemolytic action of fluid phase or cell-bound Cl and C4 but inhibited C2 only in the fluid phase. Castor bean type II binds to EACH cells with only minimal impairment of the binding and hemolytic functioning of C2; however, the presence of the lectin inhibited the decay of the hemolytically effective EAC142 sites generated. Lotus bean and leucoagglutinin did not affect the activity of any of the complement components under the conditions tested. Our results suggest that specific sugar residues (e.g. glucose, mannose, lactose or galactose) may play a significant role in the functional activity of human and guinea-pig C1, C4 and C2.

Tetrameric Ig anti-fluorescyl antibody was purified from sera of immunized coho salmon by immunoadsorption and the ligand binding properties studied from a series of bleedings over a 120-day period. Purified antibody was assayed by fluorescence quenching and equilibrium dialysis for determination of the average intrinsic association constant (K_a) and heterogeneity index (a). All antibody preparations showed an equilibrium constant of ~4–5 × 10⁵ M⁻¹ for the fluorescyl ligand, regardless of the time of bleeding after primary or secondary immunizations. Heterogeneity indices, derived from Sips plots, indicated relatively restricted heterogeneity in all purified antibody populations. The ligand binding results are discussed in terms of apparent restricted regulatory functions in salmon with respect to affinity maturation.

Murine thymus and spleen cells synthesize a 225,000 dalton macromolecule (MICG). This protein is insoluble in the cold in non-ionic detergents and migrates as a β-globulin on electrophoresis. MICG can be isolated by a two-step procedure consisting of cold precipitation of cellular lysates in NP-40, followed by gel chromatography in detergent solution. Thymocytes synthesized three times more MICG than splenocytes. Immunological analysis demonstrated that anti-MICG antiserum reacted with the cold insoluble protein. Trypsin digestion of radiolabeled, isolated MICG illustrated the incorporation of¹⁴C-leucine into a number of peptides. MICG is a glycoprotein which accounts for 3.5% of the protein synthesized in thymus cells and is not secreted from thymocytes or spleen cells.

In the present work, we have studied the antigenically reactive regions of S.C. with immune sera of well-defined specificity. Immunochemical analyses conducted with an I.S. containing only precipitating antibodies against a hidden S.C. group of determinants, unravels the existence of a new antigenic specificity that we chose to call provisionally “R” (from mercapto-resistant). R was shown to be completely independent from Brandtzaeg's I and A₁ by its resistance to strong reduction and to be distinct from A₂ by the fact that anti-R antibodies do not precipitate sIgA or tryptic S.C.

The experiments described show that R occurs unaltered on spontaneously degraded S.C. as well as on the 35.000 daltons fragment from peptic digestion.

By crossed passive hemagglutination inhibition two distinct parts of the R specificity were found to occur on sIgA and tryptic S.C. respectively.

The R specificity could be located on the S.C. near the site(s) where this molecule is fixed on IgA.

An improved procedure for the isolation of carcinoembryonic antigen (CEA) has been developed. Lengthy dialysis, ultrafiltration and lyophilization steps following perchloric acid extraction are replaced by a simple ethanol precipitation of the CEA prior to the Chromatographie purification steps. Chemical and immunochemical characterization of CEA prepared by this procedure showed no significant differences compared to CEA prepared by the more lengthy procedure not using ethanol precipitation.

Clq was purified from rat and human serum. Rat and human Clq were indistinguishable functionally as assessed by the capacity of isolated preparations to bind to aggregated IgG from either source. By polyacrylamide disc electrophoresis in the presence of sodium dodecyl sulphate, rat Clq exhibited two noncovalent subunits and, following reduction and alkylation, three smaller polypeptide chains, the apparent molecular weights of which were comparable to those of human Clq.

An analysis is presented of the limits of resolution of Scatchard and Sips plots as methods for detecting affinity heterogeneity in receptor populations. We estimate using statistical criteria for defining resolution that Scatchard plots can resolve two groups present at equal concentration, provided they differ by more than a factor of about six in affinity, and that resolution drops rapidly as the concentration difference between the groups increases. In addition, because of the non linear relation between affinity and free energy, and because the Sips analysis must necessarily be formulated in terms of free energies, the Scatchard plot is the more sensitive of the two methods for detecting heterogeneity.