Immunochemistry最新文献

Chicken antibodies raised to the (Fab')₂ fragment of mouse IgG recognize determinants expressed on both normal mouse serum and T-cell-associated immunoglobulin, as assessed by immune diffusion and competition radioimmunoassay. Analyses of reactions with both normal and myeloma immunoglobulins indicate that chicken anti-(Fab')₂ recognizes determinants expressed on light and heavy (Fd fragment) chains of mouse IgG and IgM. The relatively low efficiency of reaction of purified γ heavy and light chains (compared to intact IgG) with the anti-(Fab')₂ suggests that these antibodies recognize determinants expressed only when heavy and light chains are combined in the native molecule.

Serological studies with T-cell-associated immunoglobulin show that this molecule expresses determinants cross-reactive with both light and heavy immunoglobulin polypeptide chains. The antigenic determinants seen on the T-cell immunoglobulin light and heavy chains are common to IgM and IgG and are not isotype specific.

Both lipopolysaccharides and lipid A moiety prepared fromVibrio el-tor exhibited almost equal endotoxic activities in eliciting the local Shwartzman reaction, in Limulus-lysate gelation, in chick-embryo lethality and pyrogenicity. The structural part responsible for these activities of lipopolysaccharides is the lipid A moiety. The high anti-complementary activities present in lipopolysaccharides and lipid A moiety was destroyed by alkaline digestion of the samples. Lipid A antiserum can protect against some of the toxic activities of the endotoxin in animals.

Three out of five rabbits subjected to successive irradiations and immunized againstSalmonella abortus-equi produced IgG which, transiently, were not precipitated by anti-a3 anti-allotypic sera, although they carried all the determinants of the a3 allotypic pattern. As a3 IgG from normal rabbits are precipitated by anti-a3 sera, it appeared that the molecular distribution of the a3 allotypic determinants was different on the IgG produced by these irradiated rabbits compared to IgG produced by normal rabbits.

After a second irradiation, one of these rabbits produced a high level (50 mg per ml) of anti-Salmonella antibodies of restricted heterogeneity.

An anti-idioytpic serum prepared against anti-Salmonella antibodies produced by this rabbit after the first irradiation precipitated the idiotypes it recognized in the serum collected after the first irradiation, while it did not precipitate the idiotypes it recognized in the serum collected after the second irradiation, although they carried all the determinants of the idiotypes of the serum collected after the first irradiation. That probably means that there is a different molecular distribution of the idiotypic determinants between antibodies produced after the first irradiation and antibodies produced after the second irradiation. An antiidiotypic serum prepared against anti-Salmonella antibodies produced after the second irradiation did not distinguish by precipitation in gel medium or by radioimmunoassay between the idiotypes it recognized in antibodies produced after the first irradiation and those in antibodies produced after the second irradiation. The idiotypic similarity thus detected, and the fact that we did not detect unexpected allotypes, is in better agreement with the expression of the potentiality of radiation-resistant cells than with the expression of new antibody producing cell clones arising from virgin stem cells.

Anti-sDNA antibodies were purified from various human SLE sera by immunoadsorption and analyzed for their nucleotide binding properties. Equilibrium dialysis studies with radioactivity labeled nucleotides show that the average intrinsic association constants were relatively low (10⁴–10⁵M⁻¹). Heterogeneity index values in general indicated that all antibody populations were quite heterogeneous. Comparative r values, calculated for antibody samples displaying multi-base specificity, indicated both mono-specific and cross reactive sub-populations. Antibody samples were also studied for binding properties with³²P-sDNA, and nucleotide inhibition of the polymer interaction. Certain purified antibody samples were studied using mononucleotide and dinucleotide inhibitors of the apparent homologous nucleotide reaction. Results of binding specificities are discussed in terms of pathological implications.

Antibodies toEscherichia coli β-d-galactosidase form distinct sub-populations which segregate independently during the response: thus precipitating and activating molecules are distinguished. The latter molecules react with a defective mutant enzyme. AMEF, and activate its catalytic site, which then interacts with the subsrate. As a step toward elucidating the mechanism of AMEF activation, some properties of the relevant anti-enzyme antibodies were determined. Antibody class and valence do not affect activation: equal activation was obtained with IgM, IgG and Fab molecules over a 100-fold range of AMEF concentrations. Precipitating antibodies do not influence activation or compete with activating molecules. Most or all antienzyme antibodies produced by 15 rabbits were IgG. Three rabbits also produced small amounts of IgM antibodies, but only within 2 weeks after immunization. Non-activating IgM molecules from immune sera did not compete with activating IgG molecules, whether or not these IgM molecules were able to bind the enzyme, as demonstrated by precipitation or by coprecipitation with anti-μ-chain antisera.

Soluble anti-DNP antibodies competed or collaborated in the binding of dinitrophenylated human serum albumin to anti-DNP or anti-HSA antibodies attached to Sepharose depending, directly, on the ratio of the concentrations of soluble to insoluble antibodies and, inversely, on that of the antigen. Soluble antibodies competed or collaborated with the insoluble ones by polymerizing the antigen and either incorporating or not incorporating the insoluble antibodies into the polymer. Furthermore, a clear advantage of insoluble antibodies over soluble ones in the binding of antigen was found, and is perhaps indicative of the importance of the reactions' micro-environment and of the secondary non-specific forces that may be participating. Thus, by analogy, the role of circulating anti-hapten and anti-carrier antibodies on the regulation of the immune response may be either positive or negative depending on their concentration, on the number of cellular receptors available, on the micro-environment of the cell surface and on the magnitude of the antigenic challenge. A simple rule that relates some of these variables with the immune response is discussed.

Previous investigations which have referred to red cell Fc receptor in rabbit, guinea pig. sheep and man, have been extended to other vertebrates, such as reptiles, amphibia, birds and mammalians.

In these investigations, red cells from different animal species have been analyzed. As ligand DNP-BSA anti-DNP non-precipitating antibody, aggregated IgG by the bis-diazotized benzidine method. IgM 7S and non-modified IgG, IgA and IgM were used. The ligand binding to red cells was detected by Coombs test with specific anti-immunoglobulin serum.

The analyzed red cells showed exposed Fc receptor. In the case of human red cells, to render such receptor evident it is necessary to submit the cells to trypsinization.

All the analyzed erythrocytes bind IgM 7S, Ab-Ag complex and aggregated IgG, and some of these cells bind non-modified immunoglobulins, although none of them bind IgM 19S. These bindings were inhibited by Fcγ, Fcμ and Ag-Ab complex, but they were not inhibited by Fabγ. When specific anti-immunoglobulin serum was added to red cells with bound IgM 7S or Ag-Ab, the clusters which formed by nucleated erythrocytes were bigger than those obtained with non-nucleated red-cells.

In the case of chicken (nucleated) and sheep (non-nucleated) erythrocytes. the interaction between¹³¹I-IgM_s-red cells at equilibrium showed a similar K₀ for both red cells (chicken K₀ = 1.8 × 10⁸ L/M; sheep K₀ = 1.1 × 10⁸ L/M). The number of Fc receptors by red cell were 1.23 × 10⁶ and 1.21 × 10⁵ for chicken and sheep erythrocytes, respectively. Taking into account these results, the different behaviour of nucleated and non-nucleated red cells with bound IgM 7S, when they react with anti-IgM antibody, would be the consequence of different avidity for both equilibrated systems and not a particular characteristic of their Fc receptors.

A novel method for labeling immunoglobulin G by substitution of the sugar moiety with amino compounds is described. The method consists of mild treatment of IgG with sodium metaperiodate and subsequent Schiffs base formation of the product with amino compounds. Favourable conditions for the reaction were established. The method was sucessfully applied for the preparation of enzyme-labeled IgG antibody. Rabbit anti-(human IgG) labeled with peroxidase was used for the detection of human IgG and anti-Toxoplasma IgG by enzyme-linked immunoassay. Furthermore, substitution of oxidized IgG with acidic amino acid resulted in modification of the electric charge of IgG without affecting the antibody titre. Rabbit anti-(human IgG) labeled with aspartic acid was used for detecting antigenicity of human IgG by counter immunoelectrophoresis.

Substituent constants and regression analysis have been used to formulate structure-activity relationships for the interaction of two groups of haptens (phenylsuccinamates and pyridines) with anli-3-azopyridine antibodies. The strength of the interactions of both types of haptens is primarily governed by dispersion forces of the substituents as modeled by molar refractivity. The most important conclusion is that the binding sites of the antibody do not appear to be typically hydrophobic in so far as this can he assessed by the parameter π which suggests that the binding sites are composed of hydrophilic ami no acid residues rather than hydrophobic. This result confirms a similar earlier finding for benzoates acting as haptens.