Immunochemistry最新文献

Immunological properties of peptides from two different heavy chains of a3 allotype and restricted heterogeneity were studied using radioimmunoassays that involved inhibition by these peptides of a reaction between¹²⁵I labeled anti-a3 antibodies and Sepharose-bound a3 IgG. A purified fraction of a2-anti-a3 which cross reacts with at IgG was used for the assays of peptides from one of the heavy chains (AH80-5). The picomoles of various inhibitors required for 50% inhibition of the a3-anti-a3 reactions were determined and the corresponding ΔG⁰ values were calculated. Citraconylation of the lysine side chains (CH) markedly affected the inhibitory activity of one of the two heavy chains (AH80-5) and had a lesser effect [Δ(ΔG⁰) 1.4 kcal/mole] on the other. In both instances when the lysines of the tryptic peptides were deblocked by exposure to low pH, we observed gains in activity (1.3–1.7 kcal/mole). Although some of the primary structure which contributes to some a3-antigenic determinants may have been removed by tryptic digestion, the immunopeptides which we recovered inhibited at least 60% of the maximal binding of purified, labeled a 2 anti-a3 antibodies to a3 IgG. Thus major antigenic determmant(s) recognized by these particular anti-a3 antibodies, were retained on the portions of V_H region recovered from the tryptic digests. It is probable that losses in conformation contributed to the changes in ΔG⁰

This paper compares the combining site of the Dnp binding mouse IgA myeloma protein in the Fv and Fab fragments and in the intact IgA. This was done by monitoring the environment of the aromatic hapten protons of DnpO⁻ , Dnp-l-aspartate and Dnp glycine on binding to protein 315, using high resolution 270 MHz¹H NMR. The aromatic Dnp proton resonances undergo large upfield chemical shifts which can be measured directly. These shifts have been interpreted previously as arising from interactions of the hapten with aromatic residues in the combining site. These shifts are an intrinsic reporter of the environment of the hapten in the combining site for each hapten. The magnitudes of the shifts are similar for both the Fv and Fab fragments. The absolute magnitude of the shifts for the IgA could not be measured directly, but the shift ratios for each Dnp proton were found to be similar to those obtained for the Fv and Fab fragments, indicating that the combining site is the same in all three fragments. The line-widths of the bound Dnp proton resonances vary with fragment size and show that the constant regions impose rotational constraints on the combining site. This is in contrast to the rotational freedom of the CH₃ domain which appears to be independent of the rest of the molecule (Burtonet al., 1977).

Antibodies to poly d(A-T)‡ were elicited in two rabbits by immunization with complexes of heat-denatured poly d(A-T) and MBSA. Denaturation of poly d(A-T) was carried out in a salt solution and in distilled water. The antibodies were characterized by precipitation, hemagglutination and radioimmunoassay procedures. The antibody bound poly d(A-T) and cross reactions were observed with dAdo, dThd, poly(b) and ApU but not with UpA.V.faba satellite and calf thymus DNAs were bound at least as well as poly d(A-T); DNA fromB. subtilis andPseudomonas were poorly bound. After absorption of the serum with Ado-RSA, Thd-RSA and BSA, the antisera were directed primarily to heat-denatured poly d(A-T). Cross-reactions were observed with mouse myeloma DNA, poly(dT), poly(dA)-poly(dT) but not with poly(dA). The results indicated that anti-poly d(A-T) recognizes sequence and secondary structures. The immunochemical data are in accord with findings made by other workers on DNA and synthetic polynucleotides by physical studies, such as X-ray diffraction and scattering. Moreover, the immunochemical data support earlier contentions that local conformations in nucleic acids are responsive to primary sequences as is the case with proteins.

Also described are procedures for the synthesis of poly d(A-T)-[³H]A and poly ([³H]A-T). For the preparation of the latter, TTP was synthesized from TMP which was prepared enzymically from Thd.

Agar gel double-diffusion experiments demonstrated that the allodeterminants Lpm7 and Lpm8, very high density lipoproteins of mink, reside on different molecules. Molecules possessing Lpm7 and Lpm8 have no other known Lpm-allodeterminants. These results extend our earlier observations on the distribution of Lpm1, Lpm2, Lpm3, Lpm4 and Lpm5 on lipoprotein molecules. It is concluded that Lpm-lipoproteins from mink sera are represented by molecules of several types. identical in their species-specific antigenic properties and each having a unique allodeterminant. The distribution patterns of Lpm7 and Lpm8 among molecules support the suggestion that monogenically inherited allogroups are controlled by closely-linked structural genes.

The capsular polysaccharide fromCryptococcus neoformans serotype C has been studied using the usual methods for the elucidation of chemical structure. The results are consistent with the occurrence of a polysaccharide having an α 1,3 linked backbone ofd-mannopyranoside residues, every other one of which is doubly substituted with two terminald-xylopyranosyl residues linked β (1,2 and β 1,4 respectively, and with the remainder of the mannosyl residues substituted with a terminald-glucuronosidopyranosyl residue linked β 1,2. Alternatively, the terminal glucuronic acid residue may be β (1,2 linked to a mannosyl residue in the chain already hearing a singly branched β 1,4 xylopyranosyl residue, while the neighboring mannosyl residue carries a single branch of β 1.2 xylopyranoside.

Affinity chromatography with AMP-Sepharose allows us to purify malate dehydrogenase ofSchistosoma mansoni and to perform the studies about the immunochemical and biological properties of this enzyme. MDH has a mol. wt 60,000 and has two isoenzymes (pI 7.1 and 8.5), which are antigenically different. Isoenzyme (pI 7.1) is the antigen called 4, specific of the genusSchistosoma and is highly immunogenic. By immunofluorescence, MDH was localized in the cellular layer of the coecum. Mice immunized with purified MDH exhibited a significant decrease of worm burden.

A heat labile serum factor (BSF). required for the lysis of unsensitized human erythrocytes by the alternative pathway of bovine complement, has been isolated from bovine serum. The molecular weight of BSF was about 100,000 daltons. A specific antiserum was raised which was able to detect BSF in either serum or in the purified form as a beta-globulin. The antiserum was also used to demonstrate that, in cobra venom factor (CVF) treated bovine sera, the BSF was cleaved into at least two fragments. On the basis of its functional behaviour in haemolytic tests and as an integral component of a CVF-induced C3-convertase activity, BSF appeared to be the bovine counterpart of human factor B.

Lysozyme was purified from the annelid wormNephthys hombergii and re-examined for immunological cross-reactivity with antisera to human and chicken lysozymes. It was determined that the previously reported reaction between worm lysozyme and anti-human lysozyme and also the presently observed reaction between worm lysozyme and anti-chicken lysozyme were due to the presence of small amounts of vertebrate-likec-type lysozymes in the worm enzyme preparations. The cross-reactions were not attributable to the major lysozyme component in the worm preparations.