A system for the production of monoclonal antibodies, particularly of the IgG type, against weakly immunogenic bacterial polysaccharide antigens is described. This system, which is based on the autoimmune NZB mouse strain, has been used to produce a monoclonal IgG2a antibody against the meningococcus group B and Escherichia coli K1 polysaccharides, identical homopolymers of a(2-+8)-linked units ofNacetylneuraminic acid that are extremely poor immunogens.Comparison of the humoral immune responses of normal BALB/c mice and autoimmune NZB mice to hyperimmunization with group A, B, and C meningococci showed that, although both strains mounted a weak meningococcal B polysaccharide-specific IgM response, only the NZB strain mounted an IgG response. Similarily, NZB mice mounted a stronger IgG response to the more immunogenic group C meningococcal polysaccharide than did BALB/c mice, although this difference was less pronounced than that observed with meningococcal B polysaccharide. No difference between the two strains of mice was-demonstrable with the strongly antigenic group A meningococcal polysaccharide. These results indicate that the NZB system may be generally useful for the production of monoclonal antibodies against weakly antigenic bacterial determinants.

Sequences of antigenic determinants were identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonal antibody-bound peptide fragments by 252Cf plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453 [Burger, R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141,[553][554][555][556][557][558] were isolated from immobilized antigen-antibody complexes by partial trypsin digestion. A synthetic eicosapeptide comprised of the C-terminal sequence of the human complement component polypeptide des-Arg77-C3a as well as guinea pig des-Arg78-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded the antigens without inactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from the antibody with 4 M MgCl2. The antigenic peptides were purified by HPLC and identified by 252Cf plasma desorption mass spectro! retry. The epitope recognized by h453 resides on the C-terminal tryptic peptides of human (residues 70-76) and guinea pig (residues 70-77) C3a. As an estimation of accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of the antigen within a distance of two amino acid residues upstream from the epitope.A variety of methods have been applied to the study of monoclonal antibody (mAb)-antigen interactions and the characterization of their respective epitopes. Two major approaches that have been widely employed for epitope characterization are competitive binding analysis using synthetic peptides and fine specificity studies with panels of evolutionary variant or recombinant proteins (1). Although well established, these methods have major limitations; e.g., discontinuous or conformationally defined epitopes may not be detectable by using peptide probes (2). Site-directed mutagenesis experiments in epitope studies could be greatly facilitated if some information about the putative epitope is available in advance. A direct approach of epitope mapping, which seems promising in this respect, has been more recently introduced based on the finding that (i) mAbs exhibit remarkable resistance towards proteolytic enzymes, (ii) in immune complexes, antigenic determinants can be protected from proteolytic degradation, and (iii) proteolysis does not lead to dissociation of immune complexes (3-6). Limited proteolytic cleavage of immune complexes has been used for epitope characterization by means of PAGE (7) and by HPLC (6) of the respective peptide digests. However, HPLC separation of complex digest mixtures followed by amino acid analysis or peptide sequencing may not enable unambiguous epitope identification due to unresolved peptides.The feasibility of fast atom bombardment mass spectrometry (FABMS) and 252Cfplasma desorption mass spectrometry (PDMS) for accurate molecular weight determination of polypeptides has been established in several bioanalytical ...

The expression of the neural cell adhesion molecule (N-CAM) on cultured murine oligodendrocytes, their precursors, and myelin was examined by indirect immunofluorescence, biosynthetic radiolabeling followed by immunoprecipitation and Western blot analysis, using antibodies specific for various forms of the molecule. In all culture systems studied, whether the oligodendrocytes were cultured as an enriched fraction containing precursor cells or in the presence of astrocytes and neurons, a similar differentiation-stage-related expression of N-CAM was seen. At early developmental stages many tetanus toxin receptor- and A2B5 antigen-positive putative oligodendrocyte precursors with bipolar morphology were seen and found to express N-CAM in its embryonic form. Of the 04 antigen-positive immature oligodendrocytes with few slender processes most expressed N-CAM, but few the embryonic form of N-CAM. The more mature 01 or 010 antigen-positive oligodendrocytes were found to express exclusively the adult form of N-CAM. Oligodendrocytes synthesized the 120 and 140 kD forms of N-CAM (N-CAM 120 and N-CAM 140), but not N-CAM 180, although with differentiation, N-CAM 120 predominated in oligodendrocytes and also in pure myelin. N-CAM 120 could be released from oligodendrocytes and myelin by phosphatidylinositol-specific phospholipase C, suggesting that in both oligodendrocytes and myelin N-CAM 120 is inserted into the membrane by covalent linkage to phosphatidylinositol.

With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides. No homology with the laboratory strain LE392 was detected. The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production. Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages. By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites. A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production. Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process. Analysis in E. coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide.

A unique structural feature of the neural cell adhesion molecule N-CAM is the presence of homopolymers of alpha (2----8)-linked sialic acid units. We have used two specific probes for the detection of poly(sialic acid) in normal human kidney and Wilms tumor: a monoclonal antibody against meningococci group B capsular polysaccharide (homopolymers of alpha (2----8)-linked sialic acid units), which shows no crossreactivity with polynucleotides and denaturated DNA, and bacteriophage-induced endosialidases specifically hydrolyzing alpha (2----8)-linked poly(sialic acid) units. Additionally, for the detection of N-CAM, antibodies recognizing the polypeptide portion of the molecule and biotinylated antisense RNA transcribed from a cDNA clone for N-CAM were applied. Poly(sialic acid) was regionally detectable in human embryonic kidney but undetectable in normal adult kidney, as already reported for rat kidney. The malignant Wilms tumor, which is characterized by the presence of structural components resembling those found in embryonic kidney, reexpressed poly(sialic acid) units and showed positive immunostaining for the polypeptide portion of N-CAM. Immunoblot analysis of Wilms tumor as well as human embryonic kidney and brain with the monoclonal anti-poly(sialic acid) antibody revealed in each case the same high molecular mass broad band. In situ hybridization demonstrated the presence of mRNA for N-CAM in Wilms tumor. We conclude that poly(sialic acid), most probably present on N-CAM, is an oncodevelopmental antigen in human kidney.