Immunoassay of human Neisseria meningitidis serogroup A antibody.

An enzyme immunoassay is described which quantitates antibodies to Neisseria meningitidis serogroup A capsular polysaccharide in human sera. Modifications of a previously developed two-day assay by Carlone et al. were made to permit analysis in one day and to be compatible with automation. The allowable variations in assay conditions and the areas in which stringent control must be maintained for consistent assay performance are described. Antigen-coating parameters, the kinetics of primary and secondary antibody incubation steps, the buffer compositions, including detergents, serum requirements, and the need for blocking steps were examined. Our modified one-day assay showed excellent agreement with the standardized method of Carlone, with a correlation coefficient between the two methods of 0.989. This assay is adaptable within a permissible range of parameters thus facilitating the implementation of the standardized assay. This will maximize the consistency of results from serum analysis for conjugate vaccine trials related to serotype A Neisseria meningitidis.

Immunoassay of human Neisseria meningitidis serogroup A antibody.

An enzyme immunoassay is described which quantitates antibodies to Neisseria meningitidis serogroup A capsular polysaccharide in human sera. Modifications of a previously developed two-day assay by Carlone et al. were made to permit analysis in one day and to be compatible with automation. The allowable variations in assay conditions and the areas in which stringent control must be maintained for consistent assay performance are described. Antigen-coating parameters, the kinetics of primary and secondary antibody incubation steps, the buffer compositions, including detergents, serum requirements, and the need for blocking steps were examined. Our modified one-day assay showed excellent agreement with the standardized method of Carlone, with a correlation coefficient between the two methods of 0.989. This assay is adaptable within a permissible range of parameters thus facilitating the implementation of the standardized assay. This will maximize the consistency of results from serum analysis for conjugate vaccine trials related to serotype A Neisseria meningitidis.

The tumor association of a trisaccharide epitope: specificity of antiserum developed to galactose beta1->3 N-acetyl glucosamine beta1-->3 galactose.

A pentasaccharide carbohydrate epitope described by Nozawa et at (1) is expressed by 35% of the neoplastic tissue samples from patients with endometrial cancer but not by normal endometrium. This epitope was detected using a human monoclonal antibody (HMST-1) produced by fusion of lymphocytes from an endometrial cancer patient. We chemically linked a synthetically produced nonreducing terminal trisaccharide portion of this pentasaccharide to bovine serum albumin to create an effective immunogen, Galbeta1->3GlcNAcbeta1->3Gal-BSA. A rabbit polyclonal antibody was produced and tested against panels of tumor and normal tissues. In contrast to the results obtained with HMST-1, 100% of the endometrial adenocarcinomas we studied stained with this polyclonal antiserum while normal endometrium was non-reactive. The reactivity with other tyes of adenocarcinomas was approximately 80%, whereas most normal tissues were not reactive with the antiserum. Immunological specificity analysis was performed with structurally related carbohydrates and this shows the fine specificity reaction of the antiserum. This antigen may be clinically useful for immunolocalization and for immunotargeting.

Specificity analysis of antibodies formed in rabbits to a mannosyl trisaccharide: similarity with lectin binding activity. Para-aminophenyl O-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranosyl- (1-->6)-alpha-D-mannopyranoside linked to bovine serum albumin as an antigen.

The para-aminophenyl derivative of Man alpha 1-->2Man alpha 1-->6Man alpha 1-->was coupled via a diazotization reaction to bovine serum albumin, and the resulting glycoconjugate was used to immunize two rabbits. The resultant antisera were tested for reactivity with a number of related mono, di- and trisaccharides to determine the immunodominant portion of this trisaccharide. Two populations of antibody resulted, one of which required the reducing end mannose, and could react with either an N-acetylglucosamine or a mannose as the penultimate sugar. The other population reacted with the Man alpha 1-->2Man alpha 1-->6Man alpha 1-->. The aglycone moiety and its configurations play an important role in determining the specificity of antibodies to this synthetic antigen. The similarity of this reactivity to the reactivity of mannose binding lectins is discussed.

Synthetic antigens as immunogens: Part IV. Specificity of antisera developed to Gal beta 1-3(GlcNAc beta 1-6)GalNAc and GlcNAc beta 1-6GalNAc.

Antisera to the chemically synthesized trisaccharide, Gal beta 1-3(GlcNAc beta 1-6)GalNAc, and disaccharide, GlcNAc beta 1-6GalNAc, were developed using the diazophenyl bovine serum albumin derivatives. The binding specificity of the antisera were analyzed by enzyme immunoassays with structurally related, chemically synthesized oligosaccharides. The anti-trisaccharide antibody showed no reactivity to T antigenic structures which bear the Gal beta 1-3GalNAc moiety. The immunodominant area of the Gal beta 1-3(GlcNAc beta 1-6)GalNAc was contained in the GlcNAc beta 1-6GalNAc region of the molecule. The reactivity pattern of the anti-trisaccharide antibody, although directed to the disaccharide, did not exactly duplicate the reactivity pattern of the anti-disaccharide.

Antigenic studies on an enzymatically sialylated carbohydrate: NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc.

Sialic acid residues are often the end moiety of the carbohydrate chain of biologically important glycoconjugates. It is difficult to study sialylated glycoconjugates because the purification of these compounds is often laborious yielding only very small amounts of oligosaccharides for study. Chemical synthesis of sialylated compounds is complicated by the labile nature of the sialic acid bond. In both of these cases the sialylated compounds would need to be conjugated to a polypeptide to be an effective immunogen, and again, such conjugation is fraught with problems due to the instability of the sialic acid linkage. We have developed a combined enzymatic and synthetic route for obtaining quantities of sialylated carbohydrates conjugated to a protein carrier in amounts sufficient for antigenic studies. The notable novelty of this protocol is the addition of sialic acid after the carbohydrate-protein conjugation step. Antiserum to the compounds was developed and after absorption, antibodies that demonstrate a requirement for sialic acid for their binding were produced and studied. CA 125 has been shown to be a prognostically significant marker for ovarian adenocarcinoma. The nature of the epitope involved has been analyzed with conflicting results. To attempt to resolve this conflict, we initiated studies on sialylated antigens with NeuAc alpha 2-3Gal beta 1-3GalNAc. This trisaccharide occupies the terminal region in a series of complex carbohydrates which have been suggested to be involved as the epitope. Hanisch et al. reported that the neuraminic acid was important for the reaction.

Antigenic studies on an enzymatically sialylated carbohydrate: NeuAc(alpha 2-3)Gal(beta 1-3)[GlcNAc(beta 1-6)]GalNAc.

The CA 125 antigenic determinant has been shown to be elevated in the serum of ovarian cancer patients and is a useful prognostic marker. The chemical epitope which characterizes the CA 125 antigen is found on a high molecular weight glycoprotein and has been suggested to be carbohydrate in nature. This study was undertaken to establish the relationship of a carbohydrate to the epitope recognized by the monoclonal antibody, OC 125. Along this line a carbohydrate structure conjugated to bovine serum albumin (BSA), was enzymatically sialylated using a purified sialyltransferase to yield NeuAcGal[GlcNAc]GalNAc-OC6H4N = N-BSA. This sialylated product was used to immunize a goat and two rabbits for the development of polyclonal antisera. The resultant antisera were tested against related carbohydrates in EIA biased competitive inhibition assays. It was determined that the GlcNAc(beta 1-6)GalNAc residue was immunologically dominant for all antisera tested. As the immune response matured (greater than 40 days), there was an increase in the proportion of the antibodies that were directed to the NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc residue compared to the nonsialylated form. Known CA 125 molecules did not inhibit the binding of raised antisera to the sialylated product, nor did the sialylated product react with OC 125 monoclonal antibodies. It was therefore concluded that the carbohydrate structure in question is not the epitope, or is not a large enough part of the epitope to be recognized in these assays by the OC 125 monoclonal antibodies.

Synthetic antigens as immunogens: Part III. Specificity analysis of an anti-anti-idiotypic antibody to a carbohydrate tumor-associated antigen.

These investigations are centered on the development of anti-idiotypic and anti-anti-idiotypic antibodies to a structurally defined carbohydrate Ag, 3-O-alpha-L-fucopyranosyl-beta-D-galactopyranoside (Fuc alpha 1----3Gal). Biologic association of this disaccharide Ag structure had previously been found with tissues from areas of benign and malignant disease of the colon and breast. The exquisite specificity of binding of the original Ab1, with the antibody-Ag reaction requiring both fucose and galactose and the alpha-anomeric 1----3 linkage, was repeated with the anti-anti-idiotypic antibodies. This information indicates that although antigenic mimicry of anti-idiotypic for Ag is accomplished using amino acids in place of sugars, the specificity pattern can be precisely reproduced.

Carcinoembryonic antigen: a rat model.

It has been shown that a TAA termed rat CEA had the tissue distribution and physico-chemical properties similar to those of human CEA. In addition, it has been demonstrated that these two antigens shared antigenic determinants. These findings supported our contention that rat CEA and human CEA are analogous moieties. The production of CEA-specific autoantibodies and the induction of resistance to CEA-positive rat tumors after immunization of rats with extracts containing rat CEA raises the possibility that human CEA may be immunogenic in man. Treatment with heat, proteolytic enzymes and periodate oxidation revealed that rat CEA, similar to human CEA contained both carbohydrate and protein epitopes. The epitopes shared by rat and human CEA that were detectable by the monkey anti-human CEA serum appeared to be carbohydrate, whereas the epitopes on rat CEA with which the rat mAb combined appeared to be protein, and those detected by the rabbit anti-rat CEA serum appeared to be carbohydrate, as well as protein. These studies also indicated that, although the rat, rabbit and monkey produced antibodies specific for rat CEA, the epitopes detectable by antibodies from one species appeared to be distinct from those detectable by antibodies from the other two species. These observations have important implications in studies on human CEA. Its use as a reliable diagnostic marker for malignancy hinges on the detection of tumor-specific epitopes on the molecule. Such epitopes have not yet been clearly identified by antibodies produced in foreign species. Indeed, our finding that the rat mAb to rat CEA bound only to tumor extracts and not to extracts of normal tissues, including intestinal tissues, suggests that human beings would be the most likely source of a tumor-specific antibody to human CEA. In future studies, the role antibodies play in immunity against CEA-positive tumors will be explored and attempts will be made to determine whether all of the serologically detectable epitopes on rat CEA can induce tumor resistance, or whether this activity is limited to epitopes detectable only by rat antibodies. This information could have important implications in the use of human CEA in the immunotherapy of malignancies.

Synthetic antigens as immunogens: Part II: Antibodies to synthetic T antigen.

Antibody to the carbohydrate moiety of T antigen was developed. The synthetic antigen (Gal beta 1----3 GalNAc alpha 1----OC6H4N = N-BSA) was prepared by coupling the diazonium salt of the disaccharide derivative Gal beta 1----3 GalNAc alpha 1----OC6H4NH2 (o) with bovine serum albumin. Specificity of the antibody produced was examined with structurally related synthetic saccharides using the enzyme immunoassay technique. The presence of a glycosyl group at 0-6 of either the Gal or the GalNAc residue of the disaccharide Gal beta 1----3 GalNAc did not prevent binding of the antisera to the saccharide moiety. However, the antisera did not bind either the trisaccharide moiety NeuAc2----3 Gal beta 1----3 GalNAc alpha 1----OC6H4NO2 (o) or GlcNAc beta 1----3 Gal beta 1----3 GalNAc alpha OBn. These observations indicate that antibody approach to the antigen is to the 0-3 side of the terminal galactose in the disaccharide Gal beta 1----3 GalNAc. We have also observed that the antibody prefers Gal beta 1----3 GalNAc alpha 1----to Gal beta 1----3 GalNAc beta 1----disaccharide derivatives in its binding capacity. The antibody was found to bind natural T antigen present on neuraminidase-treated red blood cells and, by immunohistochemical analysis, it was found to bind to naturally occurring T antigen on breast tumor cells.

Synthetic antigens as immunogens: Part I. The combining site specificities of antibodies formed in rabbits to synthetic disaccharide alpha-L-Fuc-(1----3)-D-Gal.

Antibody to chemically synthesized Fuc alpha 1----3 Gal-1----OC6H4N = N bovine serum albumin was developed. Specificity analysis using related synthetic saccharides was performed. The antisera were shown to be specific for both sugars in the disaccharide, to the 1----3 positional linkage and to the alpha anomeric configuration. Immunohistochemical staining was performed on benign disease and adenocarcinoma tissue of the colon. 5/8 of the benign colon polyp tissue and 4/6 of the colonic adenocarcinoma tissue showed strong granular cytoplasmic staining of the epithelia cells lining the ducts. None of the six normal colon tissues tested showed any reactivity. Immunohistochemical staining was also performed on tissue from benign disease and from carcinoma of the mammary gland. 2/5 of the tissue from benign disease of the breast and 3/6 of the tissue from breast carcinoma showed strong granular cytoplasmic staining of the ductal epithelial cells.

Tumor immunity in rats immunized with rat carcinoembryonic antigen.

Active immunization of rats with an emulsion consisting of Freund's complete adjuvant (FCA) and an extract of rat tumor containing carcinoembryonic antigen (CEA) induced clear-cut protection from growth of the syngeneic CEA-positive tumor, RCA-1. No protection was observed in rats treated with FCA alone nor was there protection against a tumor that no serologically detectable CEA. The results suggested that the tumor immunity exhibited by the immunized rats was mediated by an immune response specific for rat CEA. It was shown further that multiparous rats were more resistant to growth of RCA-1 tumor than nulliparous rats. This suggested that immunization against rat CEA, which is an oncofetal antigen, may occur during pregnancy.

Cardiotoxicity of Forssman antibodies in in vitro perfusion experiments.

Guinea pig heart was perfused in vitro by xenogeneic sera. Sera from both Forssman-positive and Forssman-negative animals exerted strong cardiotoxicity. The toxic effect of rabbit sera could be very significantly decreased by absorption or neutralization of the naturally occurring Forssman antibodies, pointing to the significant role played by these antibodies in the "rejection" of a Forssman-positive organ.