Immunological communications最新文献

An isotope specific immunoassay which minimizes interference by endogenous rheumatoid factors was used to determine the specificity of IgM anti-F(ab')2 antibodies in human serum. We underscore the heterogeneity of these antibodies. While one subset of IgM anti-F(ab')2 antibodies reacts only with intact F(ab')2, another recognizes determinants present following reduction and alkylation of F(ab')2 and separation of Fd' fragments from light chains. IgM anti-F(ab')2 antibodies in sera from rheumatoid arthritis patients do not react significantly with intact pooled IgG and, therefore, probably are not anti-idiotypic antibodies. Some sera, but not all, contain elevated levels of antibodies that are crossreactive with rabbit F(ab')2. Such crossreactive antibodies may interfere with assays which utilize F(ab')2 fragments of rabbit antibodies specific for antigens of clinical relevance.

The determination in this laboratory of the complete antigenic structures of several proteins initially relied on a multi-approach complex chemical strategy and revealed that antigenic sites are surface locations which could be either 'continuous' or 'discontinuous' in architecture. More recently, we introduced a simplified comprehensive synthetic approach for the localization of the continuous antigenic sites of a protein. The approach depends on the synthesis of consecutive overlapping peptides, of uniform size and overlaps, and encompass the entire protein chain, from the beginning to the end. The latter approach is rather costly and labor-intensive, especially when applied to large protein molecules. All these studies showed, however, that protein antigenic sites occupy surface areas on a protein molecule. In order to render the determination of protein antigenic sites more feasible within a reasonable period of time, we considered that only the protein surface needs to be examined. Thus, for a protein of known three-dimensional structure, the protein surface can be readily screened for the continuous antigenic sites by the systematic synthesis and examination of immunochemical activity of all exposed segments of the protein. We have applied this approach here to influenza A virus hemagglutinin. Twelve peptides (11 reported for the first time here, and one reported previously), representing continuous surface segments of the molecule, have so far been synthesized, purified, characterized and their immunochemical activity studied. The peptides were found to bind anti-viral antibodies raised in outbred mice and antibodies in human sera from individuals that had suffered a recent influenza A infection. In one mouse strain (Balb/c; H-2d) so far examined, several of the peptides stimulated an in vitro proliferative response of T cells from virus (X-31)- primed mice. Finally, antisera to the peptides were raised in mice and, as expected, were found to bind to intact virus. In most cases, anti-peptide antibodies, did not bind disrupted virus. These studies indicate that protein 'continuous' antigenic sites can be localized by systematic synthetic scanning of the surface. It is emphasized that the approach is useful only for the localization of 'continuous' sites. The results also reveal that the antigenic structure of influenza virus hemagglutinin is more complex than has hitherto been suspected.

A spin-membrane immunoassay has been employed to examine the immune reactivity of whole serum from patients with chronic-progressive multiple sclerosis (CPMS) against liposomes containing ganglioside GM1. Exposure to serum resulted in complement-mediated lysis of the GM1-liposomes. No lysis occurred with liposomes devoid of ganglioside. The mean (+/- S.E.M.) lysis values were 52.6 (+/- 9.8)% for fifteen CPMS patients and 32.9 (+/- 7.2)% for nine controls. The difference between the means was highly significant (student's t-test, P less than 0.0001), indicating increased anti-ganglioside immunity in patients with CPMS.

The specific tumor-associated immune response of C3H/HeJ mice was determined at various times after subcutaneous injection with a transplantable mammary adenocarcinoma (H2712) using a radio (111In) leukocyte adherence inhibition (LAI) assay carried out in capillary tubes. Solubilized tumor-associated antigen prepared by a single phase 1-butanol extraction of the specific tumor and other transplantable tumors of different histological origin were used in the evaluation of LAI reactivity. The assay was found to be capable of detecting a significant antitumor response before the subcutaneous tumors became detectable by palpation. The response remained significant until the tumors were greater than 20 mm in diameter.

Measuring contact angle of water on dried cell or bacterium monolayers allowed van Oss (1) and others (2) to find a correlation between particle hydrophobicity and ingestion by phagocytic cells. The present study was undertaken to understand what was actually assayed with this method. Monolayers were prepared with different cell types at different densities, and they were dried under atmospheres with varying humidity before being studied with scanning electron microscopy and contact angle techniques. It is concluded that a) contact angles are independent of the cell density and substrate structure when more than 30% of the substrate area is covered with cells. b) Initial cell shape should not influence contact angle. c) Contact angles are markedly dependent on the nature of tested cells. d) Contact angles are substantially influenced by the cell drying procedure. e) A very small fraction of the energies we measured would be sufficient to account for cell-cell interactions. Hence these might play a role in some situations of biological interest.

Small nuclear ribonucleoprotein complexes are antigens in various autoimmune diseases. The serological pattern of high titers of circulating antibody to nuclear ribonucleoprotein (RNP) antigen is a diagnostic marker for mixed connective tissue disease (MCTD); whereas antibody to Sm is prevalent in systemic lupus erythematosus (SLE). Both calf thymus and rabbit thymus are commonly used, excellent sources for preparation of the corresponding antigens RNP and Sm in clinical and research laboratories (A. M. Boak et al., accompanying paper). Thus, biochemical and structural characterization of the minimal antigenic determinant in these preparations is important for its use in the laboratory, as well as significant for understanding MCTD, SLE, and other examples of autoimmunity. Purification and biochemical analyses of immunologically active RNP from many different preparations of calf thymus extract has revealed that the majority of antibody in monospecific MCTD patient sera recognizes an antigen composed of the 165 nucleotide RNA, U1 RNA, and five peptides. Calf thymus U1 RNA was found to be identical in sequence to that of man. A sequence of 55 nucleotides within the 165 nucleotide RNA was the minimal RNA fragment found in RNP particles that were still immunologically active. Two of the RNP peptides react with patient sera monospecific for RNP and thus, are presumably the antigenic peptides complexed with the 55 nucleotide RNA sequence.

The kinetics of the immune response to galactosylated liposomes in saline was studied in rabbits. The antibody level in the secondary immunization was found to be higher compared to that in the primary response. The antibodies were purified on an immunoadsorbent column of galactosylated bovine serum albumin. Anti-galactosyl antibodies belonged to both IgM and IgG classes, the former being the major fraction. Both failed to agglutinate normal or neuraminidase treated human or rabbit erythrocytes but they agglutinated trypsinized cells.

A 48 well chemotaxis microchamber, originally designed for use with polycarbonate filters, was used with nitrocellulose filters to quantitate chemotaxis and chemokinesis of granulocytes. Various features of the microchamber were compared to Boyden chambers. The accuracy and reproducibility of the method were found to be comparable to Boyden chambers in the variability between individual readings and superior in the variability between replicate values. Concentrations of optimal doses of chemoattractant for chemotaxis and chemokinesis were similar using both types of chamber. The data indicates that this method may be useful in studying components of the chemotactic response which require the use of cellulose filters. Advantages of this method over standard Boyden chambers include the use of a single filter rather than individual, non-identical filters and a reduction in the number of cells required, particularly for pediatric testing or in neutropenic patients.

A micromethod is described which allows subpopulation classification of proliferating (3H thymidine incorporating) cells, previously stimulated in microcultures. The technique is based on transferring 1,000 to 5,000 cells from microcultures to poly-L-lysine coated multispot slides. The cells are then stained for surface markers using the immuno-peroxidase method, combined with subsequent autoradiography.