Immunochemistry最新文献

The aqueous extract ofParietaria officinalis pollen has been fractionated by ammonium sulphate precipitation, gel filtration on Sephadex G-25, chromatography on DE 52, dialysis, and gel filtration on Sephadex G-75. The allergenic activity, determined with skin test, was recovered both in the dialyzable (D1. D2, D3, D4) and non-dialyzable (ND1, ND2, ND3, ND4) fractions. The active fractions, heterogeneous with respect to molecular weight which was in the range of 12,000–41,000 daltons, shared common properties: they were glycoproteins and precipitated rabbit antiserum: the allergenic activity was resistant to heating at 100°C for 2 hr and to digestion with neuraminidase, while it was destroyed by extensive pronase digestion. Isoelectric points were between 4.6 and 6.4. The spectra of fluorescence emission and of u.v. absorption were determined. Carbohydrate content was 4.9% and 6.7% respectively for dialyzable (D) and non-dialyzable (ND) fractions. A fraction with higher molecular weight, precipitating antiserum but inactive with skin test, was also recovered.

We suggest that these fractions are correlated biochemically, antigenically and in their allergenic properties.

A simple and rapid method for isolating pure mouse IgG₁, IgG_2a and IgG_2b immunoglobulins in nearly 100% yield is described. Mouse serum was fractionated on protein A-Sepharose 4B and the recovery of immunoglobulins was measured as a function of pH. When serum was applied to the column at pH 8.0, IgM, IgA and IgE were almost quantitatively recovered in the effluent together with non-immunoglobulin serum components. Providing the binding capacity of the column was not exceeded, essentially all IgG was retained at pH 8.0 and this could not be eluted by washing. Using buffers of decreasing pH, IgG₁, IgG_2a and IgG_2b were sequentially eluted at pH 6.0–7.0, pH 4.5–5.0 and pH 3.5–4.0, respectively.

The affinity values of antibodies to the random synthetic antigen poly-(Tyr,Glu)-poly(dl Ala)-poly(Lys) [known as (T,G)-A-L] and to the ordered (Tyr-Tyr-Glu-Glu)-poly(dlAla)-poly(Lys) [known as (T-T-G-G)-A-L] were measured. Determinations of the association constants were performed by antigen binding capacity assay, ABC, using the whole antigen, and by equilibrium dialysis, using a radioactive conjugate of the ordered peptide T-T-G-G which was found to represent the major determinant of the random (T,G)-A-L. The affinity values of antibodies elicited by high responder mice to (T,G)-A-L and to (T-T-G-G)-A-L were found to be similar. However, a difference of two orders of magnitude was found between the values obtained by the two methods. This difference is partially explained by probability analysis.

Upon immunizing low responder (H-2^k) mice with complexes of the antigens with methylated bovine serum albumin (MBSA), an increase in their antibody response was observed. The affinity values of these antibodies were found to be similar to those of high responder antibodies after immunization with either the antigens complexed to MBSA or with the antigens alone. On the basis of these data we conclude that low responder (H-2^k) B cells have the potential to produce antibodies of the same specificity and quality as the high responders to the random (T,G)-A-L and to the ordered (T-T-G-G)-A-L, and that the genetic defect in these mice should be located at the level of the B-cell acceptor for the T-cell signal.

Light chains of human immunoglobulins can be cleaved specifically by several types of endopeptidases into fragments corresponding to the amino-terminal, variant (V_L) portion, and to the car☐yl-terminal, constant (C_L) portion. We have investigated the effect of two human polymorphonuclear leucocyte-derived neutral proteases, the elastase-like protease (ELP) and the chymotrypsin-like protease (CLP), on Bence Jones proteins representative of the four structurally and immunochemically-defined groups of human kappa light chains. When κI, κII, κIII and κIV proteins were incubated with ELP or CLP, the result was an extensive proteolysis of the C_L portion of these molecules and the generation of V_L-related fragments. However, the V_L portions of certain κ-chains classified immunologically as a subgroup of κI-chains, κI-1, were also extraordinarily susceptible to proteolysis by these granulocyte-derived neutral proteases. These findings have provided new evidence relating specific structural and antigenic features of human kappa light polypeptide chains.

The ability of free secretory component of several species to hind to homologous and heterologous polymeric IgA and IgM was studied. Experiments were performed in which whole milk whey from man, cow, sheep, rabbit and rat was used as a source of free secretory component (SO to competitively inhibit the binding of¹²⁵I-human SC to either human polymeric IgA or IgM.

When competitive inhibition of¹²⁵I-human SC to human IgM was tested, greater differences were observed among the different secretory components than was observed when similar experiments were carried out with human IgA. The relative ability of each of the wheys to displace¹²⁵I-human SC from human IgM was: human > bovine > sheep > rabbit > rat. The relative ability of each of the wheys to displace¹²⁵I-human SC from human IgA dimer was: rabbit > human > rat ∼- sheep > cow.

Direct and relative binding studies with purified human and rabbit SC and purified human and rabbit polymeric IgA and IgM demonstrated that in man. SC bound to IgM with two-five-fold greater affinity than to IgA dimer, while in rabbit, SC bound to rabbit IgM with seven-fold lower affinity than to rabbit IgA polymers.

Binding of SC (either human or rabbit) to polymeric rabbit IgA was largely non-covalent. In contrast, binding of SC (either human or rabbit) to human IgA dimer involved covalent bond formation.

The relationship of the observed affinity differences to the relative proportion of polymeric IgA and IgM in the secretions as well as the nature of the SC-binding site, is discussed.

This paper describes a new method of fractionation of mouse and rat lymphoid cells using as an insoluble support polyacrylamide-agarose spherical beads in which iron oxide particles were trapped (Magnogel) and coated with purified anti-mouse or anti-rat Ig antibodies. The advantage of this method is that both non-adsorbed and adsorbed cells are easily and rapidly recovered due to the magnetic properties of the beads. The viability of the fractionated cells is unaffected and total recovery is high: between 80 and 100%. Surface immunoglobulin-bearing cells and cells containing immunoglobulins in their cytoplasm were searched in the various fractions. 99.6–99.9% of the cells not retained on anti-Ig coated Magnogel were devoid of surface immunoglobulins and were composed of small T lymphocytes and of some immunoglobulin-containing plasma cells. In this fraction, the depletion of the surface Ig-positive cells is of 117–400-fold. The cells adsorbed on the beads were recovered by mechanical stirring followed by the application of a magnet to separate the beads and the cells. The latter were composed of surface Ig-bearing B lymphocytes (62–79%) which were enriched 1.7–2.1-fold, surface Ig-negative cells (21–38%) and some immunoglobulin-containing cells.

In an effort to determine at what stage during intracellular transport the precursor to plasma membrane IgM becomes inserted in membranes, the solubility (in the absence of detergent) of radioactive IgM obtained by incubation of cells with³H-leucine was examined. A population of normal spleen cells of mice were used since a considerable proportion of the IgM biosynthesized is membrane IgM and because synthesis of other Ig classes is minimal. Detergent lysates of labeled cells were treated with Bio-Beads SM-2 to remove the detergent, Nonidet P40. Large aggregates were then separated from smaller aggregates and soluble molecules by ultracentrifugation. Between 30 and 40% of the³H-IgM was associated with the large aggregate fraction. Mouse IgG and partially-reduced MOPC 104E mouse IgM did not aggregate under these conditions. Aggregation of pentameric MOPC 104E IgM was sensitive to detergent. Fetal calf serum at 10% concentration reduced aggregation. The percentage of³H-IgM in the large aggregate fraction did not vary with the length of incubation of cells in the presence of radioactive amino acid between 15 min and 2 hr. The ability of intracellular or plasma membrane Ig (labelled with¹²⁵I) to aggregate was not sensitive to prior reduction of the inter-heavy chain bonds. The results are interpreted as supporting the view that insertion into a lipid bilayer is an early event in the biosynthesis of membrane IgM. It was also found that intracellular IgM shares with membrane IgM an anomalously slow mobility on electrophoresis in polyacrylamide gels containing dodecyl sulfate and that the anomalous mobility was no longer associated with molecules that had been partially reduced.

Antigenic regions of oxidized bovine pancreatic ribonuclease (O-RNase) in the mouse were analyzed by passive cutaneous anaphylaxis (PCA) and hemagglutination, using peptide segments encompassing nearly the entire molecule, Purified peptides. 1–20, 11–31, 40–61, 67–85, 67–98, and 105–124 were obtained by enzymatic digestion. Two smaller car☐y (C)-terminal peptides, ala¹¹⁴-124 and 118–124, were synthesized by solid phase procedures. Only the C-terminal half (residues 67 on) inhibited the hemagglutination of O-RNase with mouse antibodies. Studies on the antigenicity of the eicosapeptide 105–124 were particularly interesting. This peptide elicited PCA and was an efficient inhibitor of PCA and hemagglutination produced with the intact antigen. The two synthetic derivatives, ala¹¹⁴-124 and 118–124, reacted with antibody in the hemagglutination of sheep erythrocytes coated with peptide-protein conjugate. These peptides did not inhibit the hemagglutination of peptide 105–124-conjugate, suggesting that peptide 105–124 has an antigenic site not found on them. In addition, the 11-member peptide ala¹¹⁴-124 elicited PC A, demonstrating a valence of two or more. Thus, peptide 105–124 appears to be at least trivalent. The above observations further substantiate the multivalence of this eicosapeptide previously observed in the goat. This C-terminal peptide with its multiple antigenic sites contributes a major share of the antigenicity of O-RNase for the mouse. Species differences were observed in the antigenic map of O-RNase. Unlike rabbits and goats, mice responded to the 67–98 region but not to the 40–61 region which is a major determinant in the other species.