Immunochemistry最新文献

Immunological properties of several mice hemoglobins bearing α chains 1 or 4 and β chains s, d_maj or p_min were compared using radioimmunoassays that involved inhibition by these hemoglobins of a reaction between¹²⁵I-labeled C57BL/6 hemoglobin (α chain 1, β chain s) and horse antibody against C57BL/6 hemoglobin. SJL hemoglobin (α chains 1 and 4. β chains) and C57BL/6 hemoglobin were found to have identical relative association constants,K_0(rel). The d_maj hemoglobins from DBA/2 and AU/Ss mice (α chain 1, β chain d_maj) were 3.3 to 3.6 times less reactive while the p_min hemoglobin from AU/Ss mice was found to be 17 times weaker than C57BL/6 hemoglobin. These immunological differences correlate with the amino acid sequence differences in the β chains.

IDS was prepared from supernatants of concanavalin A-stimulated rat LNC cultures in serumfree medium and partially purified as in earlier studies. Its action in inhibiting³H-thymidine incorporation by LNC stimulated with PHA was markedly enhanced in the presence of isobutyl-methylxanthine, a cAMP phosphodiesterase inhibitor, and diminished by imidazole. a phosphodiesterase activator, both used in physiologically active concentrations.

IDS acted on homogenates or partially purified membranes of PHA-stimulated LNC to stimulate synthesis of cAMP in the presence of ATP and a suitable regenerating system. This effect was observed with preparations from cells harvested 20 hr but not 3 hr after PHA stimulation. Added exogenous cAMP was not destroyed al an increased rate by 20-hr homogenates.

This evidence, together with the earlier demonstration that IDS acts only in G₁, that it produces a rise in cAMP only at this phase of the cell cycle in stimulated lymphocytes or other types of cells, and that IDS action is mimicked by elevation of cAMP, led to the conclusion that IDS action is mediated by activation of cell membrane adenylate cyclase and elevation of intracellular cAMP, in the absence of an effect on phosphodiesterase.

Glutaraldehyde treatment of rabbit albumin corresponding to the 8/1 albumin/glutaraldehyde weight ratio conferred to the molecule new immunogenic properties, by inducing two types of antigenic determinants. One of them was found on the monomeric fraction of glutaraldehyde-treated albumin, while the other on the polymeric one. A hypothesis about the possible distribution of the determinants on albumin polymers dependent on the glutaraldehyde concentration used for polymerization is presented. Glutaraldehyde treatment induced similar antigenic determinants on albumin 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 for glutaraldehyde-polymerized albumin, as they failed to react with untreated albumin as well as albumin molecules treated by other means than glutaraldehyde (heat and acid treatment).

By reacting rabbit IgG with Protein A ofStaphylococcus aureus (SpA) in a wide range of molar ratio only one type of complex was formed with a mol. wt of 350,000 daltons and a molar composition of IgG₂-SpA₁.

The IgG₂-SpA₁ complex is not able to bind SpA pointing out that the Fc region binding sites of IgG are saturated by SpA.

The inability of IgG₂-SpA₁ complex to bind rabbit IgG as compared with its ability to react with rabbit IgG when fixed to sheep erythrocytes or to Sepharose beads, and to precipitate human IgG suggests that on SpA molecule there are four combining sites with different affinities for IgG.

On the basis of these results a hypothetical model of IgG₂-SpA₁ complex is presented.

Human SLE sera with anti-nuclear activity were subjected to immunoadsorption with a singlestranded deoxyribonucleic acid (sDNA)-cellulose adsorbant. The antibodies were dissociated from the adsorbant with an equal-molar solution of 4 nucleotides (dAMP. dGMP, dCMP and TMP). Ligand-eluted antibodies were characterized for purity by immunoelectrophoresis and disc gel electrophoresis. Antibody preparations were analyzed for immunoglobulin classes by the capillary precipitin test using class-specific antisera. IgG was the predominant class in all preparations. Purified antibody preparations derived from immunoadsorption were characteristically aggregated as determined by disc-gel electrophoresis and molecular sieve chromatography. The possible sources of aggregation are discussed in detail.

Radioimmunoassays using purified and labeled anti-a3 antibodies were developed and used to study a3 allotypic determinants on normal IgGs and anti-polysaccharide antibodies of restricted heterogeneity. Our results provide additional evidence for differences in fine specificity of al anti-a3 and a2 anti-a3. Insights into the causes and nature of allotypic heterogeneity have come from the recognition that some a3 allotypic determinants on normal IgGs and restricted IgGs may bind anti-a3 antibodies with different average association constants. A purified fraction of a2 anti-a3 which cross-reacts with a1 IgG was found advantageous for assaying an a3 IgG of restricted heterogeneity. The use of this fraction (a2 anti-a3 CRI) for assays of peptides derived from the a3 heavy chain is described in a subsequent paper.

Antibodies directed against human haptoglobin type 1-1 and to porcine haptoglobin were isolated from rabbit antisera by means of affinity chromatography on Sepharose 4B. The obtained antibodies were characterized by the quantitative precipitin reaction, double immunodiffusion, immunoelectrophoresis and ultracentrifugation.

The amino acid sequence of the human Bence Jones cryoprotein Ver is reported. This protein is remarkable for its ability to crystallize at 4°C and for its unusual amino acid composition.

For structure determination, the protein was fully reduced and car☐ymethylated, then digested with trypsin or chymotrypsin. Eighteen tryptic peptides and 27 chymotryptic peptides were isolated by column and paper chromatography. Complete or partial sequences were obtained for all peptides covering the variable half of the chain, using traditional sequencing methods. Peptides of the constant region were submitted only to amino acid analysis.

The cryoprotein Ver seems to have many similarities with the Bence Jones proteins of the V_kIII type. although unusual substitutions occur in many places along the chain. The protein lacks a certain number of basic amino acids in the variable region and shows an increased number of hydrophobic residues. For that reason, some of the peptides of the variable region are large and poorly soluble.

The BALB/c mouse myeloma protein TEPC 15 (T 15) and sera of guinea pigs immunized with lecithin (lec) precipitate in agarose gels with mixed micelles bearing the phosphorylcholine lipids (PC-lipids) lec, lysolecithin and sphingomyelin. The T 15 IgA and the guinea pig anti-lee antibody are both totally inhibited in agarose gels containing phosphorylcholine. The advantages of defined PC-lipids in contrast to the commonly employed complex antigens for the detection of anti-PC immunoglobulins are discussed.