Identification of the epitope of a monoclonal antibody which binds to several cytochromes P450 in the CYP1A subfamily.

The monoclonal antibody, 3/4/2, which was raised against purified rat cytochrome P450 isoenzyme 1A1 (CYP1A1) binds to cytochromes P4501A in many species. It was shown by immunoblotting that the antibody binds to CYP1A1 in microsomal fractions prepared from rat, mouse, rabbit, hamster and human. The antibody also binds to cytochrome P450 isoenzyme 1A2 in microsomal fractions prepared from rabbit and human, but not rat or mouse. Using purified isoenzymes in an enzyme-linked immunosorbent assay it was found that the affinity of binding to the two rabbit hydrocarbon-inducible isoenzymes is reduced compared with that for rat CYP1A1. Binding is not affected by denaturation of the antigens. The effects of chemical and enzymatic treatments on rat CYP1A1 showed that the epitope contains a trypsin-sensitive site that includes arginine, but lacks lysine. The epitope does not contain methionine, cysteine, aspartic acid or glutamic acid residues. In addition, digestion of the protein with cyanogen bromide produces a fragment of Mr 20,000 which contains the antibody binding site. By comparing the cross-reactivity of the antibody with the primary structures of CYP1A1 and 1A2 from the rat, mouse, rabbit and human, and by considering the results of the chemical and enzymatic treatments, it was possible to deduce the likely location and structure of the binding site of 3/4/2 on members of the CYP1A subfamily. It is concluded that the epitope for this antibody is Phe-Arg-His-Ser-Ser-Phe, which lies at positions 380-385 in rat CYP1A1. Further, it is predicted from a model of the tertiary structure of eukaryotic cytochrome P450 that a part of this binding site lies within a helix in the native protein.

The existence of two completely distinct antigenic sites within a decapeptide.

A decapeptide (1182-1191) derived from the bovine interphotoreceptor retinoid-binding protein (IRBP) was found to contain two completely distinct antigenic sites when tested in Lewis rats. One site, localized in sequence 1182-1191, is the core of the immunodominant and highly uveitogenic determinant of IRBP. The second site localizes within sequence 1183-1191 and becomes detectable only when tryptophan at position 1182 is deleted. Lymphocytes sensitized against the first, larger site recognized all longer peptides within sequence 1169-1191, as well as whole IRBP. In contrast, lymphocytes sensitized against the second, short epitope recognized only two peptides, 1184-1191 and (to a lesser degree) 1183-1191. The responses to both sites were restricted by the same major histocompatibility complex (MHC) product (I-A), as shown by monoclonal antibody blocking and by the finding that the lymphocyte response to 1184-1191 was competitively inhibited by peptide 1181-1191. The unique finding of two completely distinct antigenic sites within a decapeptide could be explained by the hypothesis that peptides of the two sites combine with the MHC molecule on antigen-presenting cells by different configurations, thus forming two distinct antigenicities.

Relative ability of distinct isotypes of human major histocompatibility complex class II molecules in binding staphylococcal enterotoxin A.

Relative ability of distinct isotypes of human major histocompatibility complex class II molecules to bind staphylococcal enterotoxin A (SEA) was investigated. SEA-binding was observed in L cells transfected with DR2 and DQw1 genes. By contrast, it was not detected in L cells transfected with DPw4 and DP (Cp63) genes. All the transfectants supported SEA-induced IL-2 production by human T cells. Levels of the accessory activity were low in the DPw4 and DP (Cp63) transfectants compared with the DR2 and DQw1 transfectants. In view of the observation that all the transfectants express well the transfected gene products on their surface, the results indicate that DR and DQ molecules bind SEA with high affinity, while DP molecules bind it with fairly low affinity.

Two screening methods show different antigen recognition patterns for four monoclonal antibodies to Candida albicans cell surface.

Four murine monoclonal antibodies (mAbs) showing similar reactivity with a cell-wall extract and mannan preparation obtained from Candida albicans were examined for epitope specificity. An enzyme-linked immunosorbent assay (ELISA) was used to determine total mAb binding to extracted cell-wall antigen when each mAb was reacted alone or in competition with a second mAb. This analysis suggested that three mAbs recognized the same determinant, which differed from that recognized by the fourth. The reactivity of these mAbs was also examined by indirect immunofluorescence assay with both yeast and germ tube forms of the dimorphic fungus. The three mAbs assigned the same epitope-specificity by ELISA showed two different patterns of reactivity with immunofluorescence. This discrepancy is discussed with respect to the postulated structure of mannan and the method of analysis.

Conformation of the beta subunit of deglycosylated human chorionic gonadotropin in the interaction at receptor sites.

The present study was performed to evaluate the relationship between the antagonist effect and the conformation of deglycosylated human chorionic gonadotropin (DGhCG) on the Leydig cell lutropin receptors. The maximum binding of [125I]DGhCG to anti-hCG beta antibody was decreased by 50%, while its binding profile to anti-hCG, anti-DGhCG and anti-hCG alpha antibodies remained unchanged, suggesting conformational changes in the beta subunit of DGhCG. However, the association of [125I]DGhCG to the binding sites of the receptors was much faster than that of [125I]hCG, and the ligand reached the binding equilibrium at 4 and 37 degrees C for 3 h and 15-30 min, respectively. Thus, the conformational changes in the beta subunit were not accompanied by loss of its receptor binding ability. The agonist properties of DGhCG which was bound to the receptors was fully restored by the addition of anti-hCG beta subunit antibody, while anti-hCG or anti-DGhCG restored only about 30% of the full agonist activity. This was probably due to a change of the conformation of the beta subunit to make it similar to that of the intact hormone. This restoration caused by anti-hCG beta was partially prevented by anti-hCG alpha. These facts indicate that some conformational change only in the beta subunit, not in the alpha subunit, of the deglycosylated hormone bound to the receptors is essential for the restoration of agonist properties.

Charge selective properties of the glomerular capillary wall influence antibody binding in rat membranous nephropathy.

IgG antibodies, eluted from kidneys of rats with Heymann nephritis were separated into cationic and anionic fractions, labeled with 125I and 131I, respectively, mixed in equal amounts, and then injected in incremental doses into 10 rats. Glomerular antibody binding was highly correlated with blood concentration of antibody at 24 hr, however, significantly more cationic antibody bound to glomeruli than did anionic antibody at all blood levels studied. The differences were not due to greater antibody content and/or avidity of the cationic preparation, as measured by binding to isolated glomeruli in vitro. These studies demonstrate the influence of glomerular permselectivity and antibody charge on subepithelial antibody deposition.

Differences in lectin binding in tissue sections of human and murine malignant tumors and their metastases.

Lectin binding to tumor cells in tissue sections of 16 nonmetastatic and 24 metastatic human adenocarcinomas and 5 nonmetastatic and 5 metastatic murine Lewis lung carcinomas (LLCs) was assessed with an avidin-biotin peroxidase technique. In human tumors, Ulex europaeus agglutinin I (UEA I) showed no binding; whereas concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and Dolichos biflorus agglutinin (DBA) bound equally to primaries and metastases. However, peanut agglutinin (PNA) bound to less than 5% of cells in 37 of 40 primaries but to greater than 50% of cells in 18 of 24 metastases. In LLC tumors, UEA I and DBA showed no binding; whereas Con A, RCA I, and WGA bound equally to primaries and metastases. SBA bound to greater than 50% of cells in 5 metastases but not to the 5 primaries. There was less than 5% binding of PNA to 10 primary murine tumors after neuraminidase pretreatment of tissue sections but greater than 50% binding in 3 of 5 metastases. These studies indicate, in both human adenocarcinomas and an experimental tumor system, that most tumor cells which metastasize show preferential binding of PNA and SBA.

Four-year evaluation of workers exposed to trimellitic anhydride. A brief report.

In a four-year clinical, immunologic, and environmental study of trimellitic anhydride (TMA) exposure in a single plant, 20 workers exposed to TMA powder were evaluated in 1979 and a total of 32 workers were evaluated from 1979 to 1983. Two distinct groups emerged before and after workplace control improvements were made in 1979. Seventeen of the original 20 workers were available for longitudinal study through 1983. Annual clinical evaluations and serum radioimmunoassays for total antibody binding and specific IgE binding to 125I TM-HSA (human serum albumin) were performed on all 32 workers. In 1979, six workers had antibody against TM-HSA, three had the late respiratory systemic syndrome, and two had TMA-induced allergic rhinitis or allergic rhinitis and asthma. One worker had antibody against TM-HSA without illness. Fifteen additional workers were evaluated longitudinally after institution of several workplace control measures. Four of these 15 workers had TMA exposure prior to environmental improvement and joined the study in 1982. The remaining 11 workers joined the study in 1982 and had at least two years of TMA exposure in the modified workplace. None of these 11 workers developed a TMA-induced immunologic syndrome or significant total or specific IgE antibody binding to 125I TM-HSA.

Binding of antibodies against human prealbumin to intestinal and bronchial carcinoids and to pancreatic endocrine tumours.

Antibodies against human prealbumin (HPA) give a strong immunoperoxidase staining of the A (glucagon) pancreatic cells and of glucagon-, insulin- and gastrin-producing pancreatic tumours. The majority of intestinal and bronchial carcinoids are also reactive. The staining may be related to presence in the C-terminal sequence of HPA of determinants in common with polypeptide (pro-) hormones.

The expression of a set of antibody variable regions in lipopolysaccharide-reactive B cells at various stages of ontogeny and its control by anti-idiotypic antibody.

An analysis is presented in which we measure the expression of a subset of antibody variable (V) regions in lipopolysaccharide (LPS)-reactive precursor B cells at various stages of ontogeny. The V regions were characterized by hapten (4-hydroxy-3-nitrophenyl)acetyl (NP)-binding specifity and/or expression of idiotypic determinants whose genetic basis had been explored in previous studies. Only V regions containing the V lambda 1 domain were considered: this allowed an unequivocal determination of idiotopes and reduced heterogeneity in the system essentially to the multiplicity of VH and D genes. It was found that approximately every fourth lambda 1-bearing LPS-reactive splenic B cell produces an NP-binding antibody. Approximately 1 in 40 lambda 1-bearing cells expressed an idiotope (Ac38) which is encoded by V lambda 1 and a set of related VH genes in combination with D and J elements. Of these cells, only a minority produce an NP-binding antibody and a few percent of the latter express a second idiotope (Ac146) which is known to be restricted to a subset of Ac38-positive, NP-binding humoral antibodies. All these frequencies are in good accord with previous analyses of anti-idiotope-induced antibodies in the serum. They can be easily accommodated into a simple model of random selection of VH genes in LPS-reactive B lymphocytes. The frequencies of the V regions under study were essentially the same in LPS-reactive B cells from spleens of adult and newborn animals and in LPS-reactive B cells generated from bone marrow pre-B cells in vitro. In the case of the latter cells the frequencies were independent of the absence or presence of T cells in the culture system. While we could thus detect, in naive mice, neither positive nor negative selection of the cells from the time of their generation in the bone marrow until their arrival in the periphery, negative selection is in principle possible: the presence of microgram amounts of anti-idiotope antibodies during maturation from pre-B to B cells specifically blocks the appearance of idiotope-bearing LPS-reactive cells in vitro. The potential physiological role of the latter effect in the sense of self-stabilization of the expressed antibody repertoire in ongoing immune responses and the possibility that frequency determinations in LPS-reactive B cells may be not representative for the repertoire expressed in the population of mature B cells is discussed.

Differential binding to human mammary and nonmammary tumors of monoclonal antibodies reactive with carcinoembryonic antigen.

Splenic lymphocytes of mice immunized with membrane enriched fractions of human mammary carcinomas were fused with the NS-1 nonsecretory++ myeloma cell line. The resulting hybridomas were screened for the synthesis of immunoglobulins reactive with human mammary tumor associated antigens, and two IgG monoclonal antibodies (B1.1 and F5.5) were identified as being reactive with purified carcinoembryonic antigen (CEA). These antibodies were shown to bind to different epitopes on CEA based on their differential reactivities to five different purified CEA preparations, and their differential binding to the surface of tumor cells derived from various organ sites. Monoclonal B1.1 bound equally to the surface of human breast, colon, and melanoma cell lines. Monoclonal F5.5, on the other hand, did not react with the surface of melanoma cell lines, and showed a differential binding to breast carcinoma versus colon carcinoma cells. Monoclonals F5.5 and B1.1 were both used in immunoperoxidase studies with fixed tissue sections of a variety of histologic types of human mammary carcinomas and were shown to be reactive with 55% and 66%, respectively, of tumor masses.

Helper and suppressor t-lymphocyte leukemia in ataxia telangiectasia.

We investigated the nature of the lymphoid leukemia that developed in one of two siblings with ataxia telangiectasia. The leukemic cells were shown to be T lymphocytes. Furthermore, the neoplastic cells carried a characteristic 14q+ chromosome tandem translocation. This chromosome abnormality had been identified 11 years earlier among the patient's "normal" lymphocytes. The patient's neoplastic T lymphocytes in vitro provided helper and suppressor T-lymphocyte activity equivalent to that of normal T lymphocytes. Some neoplastic T lymphocytes bore a receptor for the Fc portion of IgM (45 per cent Tmu) whereas other carried receptors for the Fc portion of IgG (10 per cent Tgamma). All the Tmu and Tgamma lymphocytes possessed the chromosome 14 abnormality. These data suggest that neoplastic transformation occurred in an uncommitted T lymphocyte that was capable of further differentiation into the distinct pathways for help and suppression, in a lymphoid analogy of chronic myelogenous leukemia.

Cold pepsin digestion: a novel method to produce the Fv fragment from human immunoglobulin M.

A strategy for the proteolytic fragmentation of human IgM has been developed. This method is called "cold pepsin digestion" because of its unique feature of achieving restricted peptic cleavages at 4 degrees and pH 4.0. Cold pepsin digestion has been applied successfully to produce an Fv fragment from 14 human IgM proteins. The Fv fragment consists of the heavy chain variable domain (VH) and the light chain variable domain (VL) held together by strong noncovalent interaction. Thus, each Fv fragment contains one intact antigen-binding site and represents the minimal active fragment derivable from an antibody molecule. A series of other structurally and functionally important fragments were also isolated and characterized. Two basic digestion pathways were recognized; these mainly reflect the relative accessibility of five sets of major interdomain cleavage sites.

The structural basis of rabbit VH allotypes: serologic studies on a1 H chains with defined amino acid sequence.

The amino acid sequences for the VH regions of three homogeneous antibodies elicited by type III pneumococcal vaccine were determined. All three antibodies had the group a allotype a1. Two of the antibody H chains (3372, 3381) had identical amino acid sequences in all framework positions that are considered correlates of the VH allotype, whereas the third H chain (3T72) differed from these at positions 15 and 16. The a1 allotypic specificities of the three homogeneous antibodies were compared by quantitative radiobinding and inhibition assays by using both insolubilized anti-a1 antisera and allotypic antiserum fractions rendered specific for the homogeneous antibody 3374. It was found that antibodies 3374 and 3381 are allotypically indistinguishable and have in common an a1 allotypic specificity that predominates in pooled a1 IgG. The allotypic specificity of the 3T72 antibody, on the other hand, was markedly deficient to those of 3374, 3381, and the a1 IgG pool. This correlation of allotypic difference with amino acid sequence variation at position 15 and 16 of the H chain indicates the involvement of these two residues in a major a1 allotypic determinant.