Partial characterization of Ia antigens from murine lymphoid cells

Congenic anti-Ia antisera were used to bind radiolabelled Ia antigens from cells of various strains of mice of knownH-2 haplotype. The results indicate that Ia antigens are proteins of molecular weight 30,000 to 35,000 daltons. The Ia antigens are distinct from known H-2 antigens as judged by independent immunoprecipitation as well as by molecular weight. Ia antigens are synthesized by, and are present on the surface of lymphoid cells as evidenced by incorporation studies using³H-leucine and enzymatic radioiodination of cells, respectively. Tissue distribution of cell surface Ia suggests that Ia antigens are on B cells. Ia antigens were detected in the incubation media of³H-leucine labeled splenocytes suggesting that antigens may be secreted.     

Ontogeny of human Ia antigens

Indirect immunofluorescence (IIP) staining of tissues from human fetuses (ages ranging from 8 to 32 weeks of intrauterine life) with monoclonal antibodies (MoAb) to monomorphic determinants of Ia antigens and HLA-A,B,C antigens has shown that both types of antigens are already detectable in tissues of 8-week-old fetuses. Ia antigens and HLA-A,B,C antigens reach their almost-complete tissue distribution after 32 and 24 weeks of intrauterine life, respectively. The structure of Ia antigens synthesized by fetal thymus cells is similar to that of B-lymphoid cell-derived Ia antigens. Ia antigen-bearing thymic fetal cells can stimulate allogeneic lymphocytes in mixed lymphocyte reactions (MLRs). These reactions are blocked by monoclonal antibodies to monomorphic determinants of human Ia antigens and of HLA-A,B, antigens.     

Dirofilaria immitis: identification and partial characterization of parasite antigens in the serum of infected dogs

We have recently developed a sensitive and specific immunodiagnostic test for canine Dirofilaria immitis infection based on detection of soluble parasite antigens in dog sera by monoclonal antibody-based enzyme immunoassay. In addition to their importance as markers of infection, these antigens may contribute to the pathogenesis of heartworm disease in dogs. In the present study, a variety of methods were used to identify and characterize circulating D. immitis antigens. Two antigens were identified in infected dog sera that formed lines of identity in rocket-line immunoelectrophoresis with soluble antigens extracted from adult D. immitis. Circulating D. immitis antigens were also demonstrated in infected dog sera by immunoblot analysis with polyclonal and monoclonal antibodies. These antigens had apparent molecular weights that ranged from 50 to 250 kDa. Most of the circulating D. immitis antigens contained the epitope defined by monoclonal antibody 1418BF2.1 which is used in our enzyme immunoassay for circulating D. immitis antigen. Studies of parasite antigens released during in vitro culture indicated that the circulating D. immitis antigens in dog sera that are detected by our enzyme immunoassay are primarily derived from adult female worms.     

Association of mouse adenovirus-induced cell surface antigen(s) with histocompatibility antigens

The topological relationship on the mouse adenovirus (M-Ad)-infected cell surface between virus-induced specific cell surface(s) antigens and serologically defined major histocompatibility antigens (H-2) was analyzed by the cap formation technique. Rhodamine-isothiocyanate (RITC)-labeled anti-S serum failed to stain the surface of virus-infected lymphoid cells which were pretreated with anti-H-2 serum and fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin serum (anti-M-Ig) to cap the appropriate H-2 antigens. Conversely, the capping of the S antigens by pretreatment with anti-S followed by FITC anti-M-Ig serum induced cocapping of H-2 antigens. The β₂ microglobulins (β₂m) were also shown to be cocapped with S antigens by anti-β₂m or by anti-S serum. The S antigens, however, did not cocap with mouse-immunoglobulins or Thyl. 2 antigens on virus-infected B or T lymphocytes, respectively. To further elucidate the molecular relationship between S and H-2 antigens, radio-iodinated virus-infected cells were solubilized with Nonidet P40 (NP40) and S antigens were precipitated with anti-S serum. When the precipitates were analysed with sodium dodecyl sulfate Polyacrylamide gel electrophoresis, two major peaks were seen at positions of molecules of about 45,000 and 12,000 daltons both of which corresponded with molecules which were observed when NP40 extracts of virus-infected or uninfected cells were precipitated with anti-H-2 serum. Sequential immunoprecipitation analysis of infected cell extracts showed that S antigens were coprecipitated with either H-2K or H-2D antigens. These results suggest that the S antigens are somehow associated with H-2K or H-2D antigens separately.     

Immunochemical and functional analysis of HLA class II antigens induced by recombinant immune interferon on normal epidermal melanocytes

The effect of recombinant immune interferon (IFN-gamma) on the expression and shedding of HLA antigens and of melanoma-associated antigens (MAA) by epidermal melanocytes was investigated by using serologic and immunochemical techniques. IFN-gamma enhances the expression and/or shedding of HLA class I antigens and of the cytoplasmic MAA defined by monoclonal antibody (MoAb) 465.12S and induces a slight reduction in the expression of the high m.w. melanoma-associated antigen (HMW-MAA). In agreement with the data in the literature, melanocytes incubated with IFN-gamma acquire HLA-DR, -DQ, and -DP antigens. Contrary to previous information in the literature, the effect is not restricted to HLA class II antigens, since IFN-gamma also induces the expression of the 96-kDa MAA recognized by MoAb CL203. The effect of IFN-gamma on HLA class II antigens and 96-kDa MAA is dose and time dependent and is specific, because recombinant leukocyte interferon affects the expression of neither type of antigen. In spite of the expression of HLA class II antigens, IFN-gamma-treated melanocytes do not acquire the ability to stimulate the proliferation of allogeneic lymphocytes. HLA-DR antigens are more susceptible to induction by IFN-gamma than HLA-DQ and -DP antigens, since the percentage of melanocytes acquiring HLA-DQ and -DP antigens is lower than that acquiring HLA-DR antigens. Furthermore, the dose of IFN-gamma is higher and the time of incubation is longer to induce HLA-DQ and -DP antigens than to induce HLA-DR antigens. The differential susceptibility of HLA-DR, -DQ, and -DP antigens as well as of melanocytes from various donors to the modulating effect of IFN-gamma may provide an explanation for the more frequent detection of HLA-DR than of HLA-DQ and -DP antigens in melanoma lesions and for the expression of HLA class II antigens by some, but not all, melanoma lesions.     

The reverse proteomics for identification of tumor antigens

The identification of tumor antigens is essential for the development of anticancer therapeutic vaccines and clinical diagnosis of cancer. SEREX (serological analysis of recombinant cDNA expression libraries) has been used to identify such tumor antigens by screening sera of patients with cDNA expression libraries. SEREX-defined antigens provide markers for the diagnosis of cancers. Potential diagnostic values of these SEREX-defined antigens have been evaluated. SEREX is also a powerful method for the development of anticancer therapeutics. The development of anticancer vaccines requires that tumor antigens can elicit antigen-specific antibodies or T lymphocytes. More than 2000 antigens have been discovered by SEREX. Peptides derived from some of these antigens have been evaluated in clinical trials. This review provides information on the application of SEREX for identification of tumor-associated antigens (TAA) for the development of cancer diagnostics and anticancer therapeutics.     

Erythrocyte diagnostica made from salmonellal phagolysates]

For the first time O antigens obtained from phagolysates were proved to be suitable for use as material for the production of highly specific erythrocyte diagnostic preparations. O antigens obtained from Salmonella by two methods, i.e. phage disintegration and Grasset's method, were subjected to comparative chemical analysis and found to have no essential difference. Nevertheless, the sensitizing potency of O antigens obtained from phagolysates were experimentally shown to be 3 times greater than that of O antigens obtained by Grasset's method. The optimum sensitizing doses established in the passive hemagglutination test for O antigens obtained by both methods indicated that these antigens were highly sensitive and specific.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

A serologic comparison of Moloney lymphoma cell surface and Moloney oncornavirus antigens.

Moloney lymphomas and Moloney sarcomas share strong tumor antigens. In this report we analyze the cell-surface antigens on a Balb/c Moloney lymphoma, LSTRA, using hyperimmune sarcoma regressor sera (alphaMo) as a primary reagent. We also use heterologous anti-viral p30 and gp70 sera for a direct analysis of virion protein antigens on the LSTRA surface. Using radiolabeled alphaMo-binding assays, we demonstrate that LSTRA tumor antigens detected by these sera are all Moloney viral antigens; approximately 1/3 of these antigenic determinants are expressed on the intact virus, and the other determinants are revealed by detergent lysis of the virus. The major viral antigens expressed on the LSTRA cell surface are viral env gene products, whereas gag gene products are only sparsely represented. We conclude that alphaMo sera detect almost exclusively viral antigens on LSTRA cells, and these antigens are almost exclusively virion env gene products.     

Characterizion of Mycoplasma pulmonis variants isolated from rabbits. II. Basis for differentiation of antigenic subtypes

The antigen composition of Mycoplasma pulmonis variants was studied by complement-fixation, agar-gel diffusion, and growth-inhibition tests. Two classes of complement-fixing antigens were demonstrated for M. pulmonis strains 47 and 63: (i) cross-related, heat-labile, water-soluble antigens, and (ii) high-titered, subtype-specific, heat-stable, water-soluble antigens. Lipid antigens prepared by organic solvent fractionation were low-titered antigens and showed little specificity. With the aid of agar-gel double-diffusion plates, the subtype-specific antigens were found to be precipitated by trichloroacetic acid and to be stable to periodate, but they were inactivated by pronase. Pronase-stable, periodate-labile precipitating antigens were observed as common components between the two variants. Antisera prepared with boiled antigens were found to be serologically active on gel diffusion but lacked neutralizing ability in growth-inhibition tests. Each of three strains of M. pulmonis (47, 63, ATCC 14267) could be identified as a variant because each strain possessed immunologically distinct heat-stable subtype-specific antigen(s).     

Separation and comparison of human TL-like antigens and HLA(A,B, C) antigens expressed on Cultured T cells

Beta₂-microglobulin-bound T-cell membrane components containing both human TL-like antigens and HLA(A, B, C) antigens were partially purified from Renex 30-solubilized membrane material of cells of a human T-cell-type leukemia cell line, HPB-ALL. The radioiodinated preparation was subjected to limited papain digestion; the HLA(A, B, C) antigens split, whereas a large portion of the human TL-like antigens remained intact. The antigen molecules were recovered by lentil-lectin affinity chromatography and separated by gel filtration on the basis of the induced difference in molecular size. The human TL-like-antigen preparation thus obtained was essentially free of HLA(A, B, C) antigens. The human TL-like antigens were immunospecifically precipitated and the component polypeptide, heavy and light, chains were separated by acid dissociation followed by gel filtration. The component chains were compared with the corresponding chains of HLA(A, B, C) antigens obtained similarly from the same HPB-ALL cells with respect to their fragmentation patterns on chemical or enzymatic cleavage. The results provided convincing evidence for the identity of the light chains of human TL-like antigens and HLA(A, B, C) antigens, and also evidence suggesting the presence of substantial differences in the fundamental structure of the heavy chains of human TL-like antigens and HLA(A, B, C) antigens.A unit of the New York State Department of Health.     

Thrombocyte antigens of dogs]

Four platelet-specific antigens were identified in dogs by means of the platelet agglutination test; these antigens were not connected with the erythrocyte or leukocyte antigens, they were inherited by the mendelian type and controlled by several recessive genes.     

Comparison of Various Methods for Preparation of Viral Serological Antigens from Infected Cell Cultures

In efforts to prepare more potent and sensitive viral serological antigens, several aspects of the production of antigens from infected cell cultures were studied. Antigens derived from whole, infected culture material and from the cellular and fluid phases were compared. Freezing and thawing, sonication, and alkaline buffer extraction were compared for effectiveness in releasing antigen from host cells. The effect of the multiplicity of infection on titers of viral antigens produced in cell cultures was studied. Generally, higher titered antigens were derived from the infected cells than from the culture fluids, but for certain viruses complement-fixing (CF) antigens derived from the culture fluids gave higher antibody titers than did cell-associated antigens. With each virus-host cell system studied, treatment with alkaline buffers extracted appreciable amounts of CF antigen from the host cells, but in some instances more antigen was released by freezing and thawing or by sonication. Extraction of infected cells with alkaline buffers was not a satisfactory method for preparation of hemagglutinating (HA) antigens for any of the viruses studied. The highest-titered HA antigens were produced from infected cells disrupted by freezing and thawing or sonication. The highest titered CF and HA antigens were produced from cell cultures infected at multiplicities of one or greater. Complement-fixing antigens produced by infecting cells in suspension and then planting had lower titers than antigens produced in parallel by infecting developed monolayers. Optimal methods are summarized for preparation of serological antigens to a variety of viruses of man.     

Partial purification and characterization of mold antigens commonly found in foods.

Rapid methods are needed for detection of molds in foods; therefore, an enzyme-linked immunosorbent assay was developed. The extracellular and mycelial antigens for Mucor, Aspergillus, Cladosporium, and Geotrichum species were partially purified and characterized. The molecular masses of the mycelial and extracellular antigens, as determined by size exclusion chromatography, ranged from 4.5 x 10(5) to 6.7 x 10(5) Da. There was only one main antigenic peak separated by Sepharose CL-4B and concanavalin A-Sepharose columns for Mucor, Cladosporium, and Geotrichum mycelial and extracellular antigens, but there were two for Aspergillus mycelial antigens and three for Aspergillus extracellular antigens. These antigens contained 10 to 50% protein which was part of the active site since protease digestion significantly decreased antigenic activity. Neutral sugars, ranging from 13 to 75%, made up the rest of the active site, and < 1% phosphate was detected in mycelial antigens. Geotrichum, Cladosporium, and Aspergillus antigens contained mainly glucose, galactose, and mannose. Mucor antigens contained these sugars plus fucose. The percentage of sugars differed between the mycelia and extracellular antigens. Enzymatic digestion and competitive inhibition tests using different sugar derivatives showed that galactosyl residues with beta linkages were immunodominant for Aspergillus, Geotrichum, and Cladosporium antigens and mannosyl residues with alpha linkages were immunodominant for Mucor antigens.     

Antigenic analysis of sequential erythrocytic stages of Plasmodium knowlesi.

The antigenic composition of sequential erythrocytic stages of Plasmodium knowlesi has been compared by crossed immuno-electrophoresis using a pool of immune rhesus monkey antiserum. Eleven major parasite antigens have been identified; 9 are stage-independent, and 2 stage-dependent. Differences in the relative amount of the stage-independent antigens have been demonstrated and quantified. The distribution of antigens between parasites and schizont and infected red cell membranes has been examined. Only 6 of the 11 parasite antigens were exhibited by a schizont membrane preparation, all these antigens were also expressed by the intracellular parasite. Antigens exclusive to the schizont membrane were not demonstrated.     

Schistosoma mansoni: Complement activation by antigenic preparations

The ability of antigens prepared from adult worms and eggs of Schistosoma mansoni to activate complement in vitro in normal, human serum in the absence of specific antibodies was investigated. It was demonstrated that whole viable eggs activated the complement system; this was shown to be effected by egg antigens released into the medium. Egg-hatching fluid induced a high degree of complement consumption, whereas purified egg shells gave almost no complement consumption. The complement-activating antigens of the eggs are possibly of polysaccharide nature as indicated by an almost complete complement activation by trichloroacetic acid-soluble egg antigens. No detectable complement consumption occurred upon incubation of living adult worms, but antigens extracted from adult worms did give complement consumption. Circulating cathodic antigens and excretory and secretory antigens proved to be quite capable of inducing complement activation; tegumental antigens gave lower, but still significant levels of complement consumption.     

The response of lymphocytes to H-2D and H-2K region antigens: I. Differential effect of cortisone acetate

Cell surface antigens controlled by separate portions of the H-2 region differentially stimulate lymphocytes. Cells responding to antigens controlled by loci in or near the H-2D region transform later (5 days) than cells responding to antigens controlled by loci in or near the H-2K region (3 days). Treatment of lymphocyte donors with cortisone acetate shows that lymphocytes responding to some H-2K-associated antigens are cortisone resistant and that lymphocytes responding to H-2D-associated antigens are cortisone sensitive. Parallels are drawn between these characteristics and the lymphocytes responsible for cellular and humoral immunity.     

A study of human-type ABH antigens on erythrocytes and in saliva of sheep

The results of serological investigations of human type ABH antigens and antibodies of 116 sheep are presented. Traces of ABH antigens on sheep erythrocytes are recognized by elution. Agglutinins with anti-AA1, anti-B and sometimes anti-H specificity besides of heteroagglutinins were differentiated by cross-absorption experiments. It was established that the sheep saliva also contains H antigens and sometimes A and/or A1 antigens. On the basis of their serological characteristics the sheep divided into 11 serological groups. In order to explain some serological peculiarities in sheep the existence of genes for regulation, the production of ABH antigens in glucolipid form and genes for regulation of the same antigens in glucoprotein form were postulated.     

Immunodiffusion analysis of water-soluble rat bone marrow antigens

Antisera were obtained against six electrophoretic fractions of the rat bone marrow extract. With their help, 18 tissular antigens and 11 antigens immunologically similar to the blood serum proteins were revealed in the rat bone marrow. All tissular antigens are divided in five groups by the degree of organ specificity: 1) bone marrow organospecific antigens (4 antigens), 2) antigens present in the bone marrow, spleen and lung extracts (2), 3) "granulocytic" antigens (4), 4) antigens common for many rat organs, but not found in the extracts of blood formed elements, skeletal muscle, heart, brain, eyes (3), 5) antigens present in all the organs studied (5). The bone marrow organospecific antigens may be specific antigens of hemopoietic cell precursors. The possibility of utilization of antisera against the bone marrow water soluble proteins for labelling hemopoietic cells of different lines of differentiation is discussed.     

Tissue antigens from the third instar larva of Drosophila melanogaster

Antibodies were prepared against the soluble proteins from six tissues of Drosophila larvae. These were used to analyse the antigens in different tissues and at different developmental stages. The results suggest (1) the pattern of antigens determines the characteristics of a tissue, (2) salivary gland antigens are sequestered by the imaginal disks, (3) not all pupal glue antigens are synthesized in the salivary glands, and (4) most larval serum antigens are synthesized by the fat body.