Immunochemistry最新文献

Rabbit antibodies were prepared against C-phycoerythrin (PE) from a newPseudanabaena species (strain W 1173) and cross-reactivity of this antiserum with phycoerythrins from various blue-green algae was studied. Qualitative immunochemical analysis indicated similarity between the PE's isolated from the species listed in Table 1. No cross-reaction could be detected with phycocynnin. For quantitative immunological studies a viroimmunoassay was developed. Bacteriophage T4 was chemically modified by covalent attachment of PE fromPseudanabaena W 1173. The immunospecific inactivation of this bacteriophage phycoerythrin conjugate can be inhibited by free phycoerythrin, the degree of inhibition being dependent on the immunochemical similarity between the bound and the free antigen. By means of this technique, substantial differences in inhibitory capacity were found between the various PE's, thus showing that the highly sensitive phage technique is able to discriminate between antigenic differences which cannot be detected by immunodiffusion. The degree of antigenic correspondence was also studied by the method of cross-precipitation, which unlike the phage assay requires the heterologous antigen to be multivalent. As expected, the ranges of cross-reactivity obtained by the two procedures were not identical. This result indicates that theamountof cross-reaction observed and the capacity to distinguish between a series of heterologous antigens is affected by the immunochemical method applied. The potential usefulness of the results for taxonomic purposes is discussed.

It was recently shown that complement can dissociate BSA-anti-BSA immune complexes (Miller, 1977). In the present study we have formed specific complexes of idiotype and anti-idiotype. The idiotype used was the phosphorylchotine-binding Balb/c myeloma protein, HOPC-8, and the anti-idiotype was raised against purified HOPC-8 protein in A/J mice. Idiotypic complexes were prepared in either of 2 ways: (1) HOPC-8 protein was immobilized in test tubes and allowed to bind purified radiolabeled anti-idiotype. (2) Anti-idiotype was immobilized in test tubes and allowed to bind purified labeled HOPC-8 protein. Then both types of immobilized idiotypic complex were incubated with fresh or heat-inactivated rabbit serum at 37°C. The dissociation of either labeled idiotype or anti-idiotype was measured as a function of time. Active complement increased the rate und extent of dissociation of the idiotypic complex regardless of whether the idiotype or the anti-idiotype was immobilized.

The physicochemical and antigenic properties of a γ1 heavy chain disease protein (γl HCD BAZ) are described. Protein BAZ is positive for the Glm(1) determinant and gives a reaction of identity with Fc fragment, whether derived from Cohn Fraction II or from an IgG1 myeloma protein. It also gives a reaction of complete identity with the γHCD proteins CRA and ZUC. The mol. wt of the native protein was determined from (1) sedimentation-diffusion, (2) sedimentation-viscosity, and (3) sedimentation-equilibrium measurements to be approx 60,000 daltons. The mol. wt of the unreduced protein in 6M guanidine hydrochloride was found to be 30,000 daltons, which was identical to that of the reduced-alkylated form in 6M guanidine. These results established that γ1 HCD BAZ is a noncovalently linked dimer and suggested the possibility of a missing hinge region.

Studies by Wilderet al. (1975b) of the binding of Fab' fragments to small haptens have shown that the cross-linking constant (the equilibrium constant for binding an additional Fab' fragment to a hapten-Fab' complex) is strongly dependent on the length of the hapten. We present a simple model for predicting the relationship between the intermolecular cross-linking constant and the monovalent hapten-antibody binding constant. In particular we use the model to obtain the dependence of the cross-linking constant on the length of the hapten, the depth to which the hapten fills the Fab' binding site, and the size of the Fab' fragment.

To make contact with experiment, we devise expressions which allow us to analyze the data of Wilderet al. (1975b). From their data we determine the values of two parameters which we take to be unknown in the theory, the size of the Fab' fragment and the depth to which the hapten fills the Fab' binding site. The values arrived at in this way agree well with published measurements of these parameters.

A newly developed passive hemagglutination inhibition technique was used to show that at least 2 families of antibodies are generated after immunization of rabbits with ovine submaxillary mucin (OSM)§ in homogeneous form. OSM-specific antibodies were non-precipitating in agar and liquid phase, easily detectable by a passive hemagglutination technique, and directed primarily to the carbohydrate side chain. In the case of subcutaneous immunization with OSM emulsified in complete Freund's adjuvant, the immunodominant determinants were the disaccharide prosthetic group (NAN-galNAc-protein) and a desialized moiety (galNAc-protein) which comprises about 14% of total prosthetic groups in OSM as isolated. Modest changes in the terminal sialic acid were associated with reduced interaction with anti-OSM sera. By contrast, intravenous immunization with OSM coupled to red blood cells elicited an additional family of antibodies with specificity for apo-protein. Explanations for this switch in specificity are considered.

The complete amino acid sequence of the variable region of a kappa light chain derived from a canine myeloma immunoglobulin is presented. The sequence is more than 80% homologous to the human kappa variable region Cum the prototype of the human V_KII subgroup and is the first example of a non-human kappa chain which can be easily classified as a subgroup other than V_KI. The finding confirms a previous prediction that genes encoding kappa subgroups predated mammalian speciation.

By comparison with the amino acid sequence of the sperm whale myoglobin molecule five antigenicaliy reactive regions on the surface of the soybean leghemoglobin a molecule were predicted. The reactivity of these five regions was substantiated using the approach of Smithet al. (1977) in which peptides corresponding to these regions of the molecule were synthesized by a solid-phase procedure on a macroporous resin. At each step of the synthesis the peptides were tested for antigenicity using¹²⁵I-labelled anti-(leghemoglobin a) antibodies whilst still bound to the solid support.

Complexes, consisting of IgE and its receptor from rat basophilic leukemia (RBL) cells, were isolated by allowing RBL cells to interact with DNP-IgE, solubilizing the cells with Nonidet P-40 and by allowing the extract to bind to anti-DNP-Sepharose. Of the RBL surface material eluted with 2,4-dinitrophenolate, 51–65% was precipitable by anti-IgE. Polyacrylamide gel analysis of eluates in SDS. using 10% gels, revealed a single surface component with an apparent molecular weight of about 45.000 daltons.

A theory is proposed suggesting that one exposed region of the myelin basic proteinin vivo might be the immunological target for cells which have been stimulated by several different encephalitogenic determinants.

A chemiluminescent immunoassay was developed using peroxidase labeled antigen. The high sensitivity obtained permitted the determination of 1 ng staphylococcal enterotoxin B used as test substance.