Monospecific polyclonal anti-anti-idiotypic antibodies to the carboxyterminal undecapeptide of the SV40 large tumour antigen.

The murine monoclonal antibody PAb1605 defines an epitope, peptide Lys(698)-Thr(708) (KT), on the carboxyterminus of the tumour(T)antigen of SV40-transformed cells. In vivo and in vitro experiments had shown that this sequence represents an epitope for both humoral and cellular immune responses. When injected into rabbits PAb1605 induces anti-idiotypic antibodies (Ab-2). Ab-2 beta (internal image type) was purified by adsorption chromatography and characterized by the ability of KT to compete with the binding of ab-2 with ab-1. Murine anti-anti-idiotypic antibodies (ab-3) were obtained by immunization of mice with ab-2 beta. Both ab-1 and ab-3 JgG showed affinities to immunoprecipitated SV40 T antigen by immunoblot analysis and to nuclear SV40 T antigen by the immunofluorescence assay. The binding of ab-3 to SV40 T antigen was completely inhibited by competition with KT. We conclude that the polyclonal ab-3 is of the ab-3 subtype and specific for only one epitope which is represented by KT and defined by ab-1. The results demonstrate that the specificity for a defined peptide epitope of an antibody was conserved even after two consecutive steps of anti-idiotypic-antibody formation in two host species. Since this postulate of network theory could be verified for a sequence of a tumour-associated antigen which represents a B- and T cell epitope, this model is of great interest for further tumour immunological studies.

Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene.

The superoxide dismutase (SOD) of Helicobacter pylori, a pathogenic bacterium which colonizes the gastric mucosa, evoking a marked inflammatory response, was purified and characterized, and the N-terminal amino acid sequence was determined. The enzyme consists of two identical subunits each with an apparent molecular weight of 24,000. Analysis of the primary structure and inhibition studies revealed that H. pylori possesses a typical procaryotic iron-containing enzyme. No other isoenzymes could be detected. Indirect gold immunostaining of H. pylori SOD with a polyclonal antibody directed against the iron-containing SOD of Escherichia coli showed a surface-associated localization of the enzyme. The H. pylori SOD gene was cloned by functional complementation of a SOD-deficient E. coli mutant. Sequencing and alignment revealed striking homology to the following facultative intracellular human pathogens: Listeria ivanovii, Listeria monocytogenes, Coxiella burnetti, Porphyromonas gingivalis, Legionella pneumophila, and Entamoeba histolytica. An open reading frame of 642 bp encoding 214 amino acids was determined. There was no leader sequence detectable. Cloning of the H. pylori SOD gene is one of the prerequisites to investigation of its pathophysiological role in the defense against antimicrobial mechanisms of polymorphonuclear granulocytes.

Evaluation of an antigen-capture enzyme immunoassay for rapid diagnosis of Mycoplasma pneumoniae infection.

A new enzyme immunoassay (EIA; Enzygost) for rapid detection of Mycoplasma pneumoniae antigen was evaluated in 51 young adults with acute respiratory infection. The EIA results using sputa and nasopharyngeal aspirates were compared with those of serological antibody tests, culture and a DNA probe. In sputum the sensitivity of the EIA ranged from 40% to 81% and the specificity from 64% to 100%, depending on the reference method. In nasopharyngeal aspirates the sensitivity was well below 20%, but the test was nearly 100% specific.

In situ localization of the 60 k protein of Helicobacter pylori, which belongs to the family of heat shock proteins, by immuno-electron microscopy.

The groEl homologue of Helicobacter pylori was isolated and characterized by means of immunoelectron microscopy, after cryosectioning. The 60 k protein was isolated from Helicobacter pylori by treatment of the cells with 2-butanol and purified by anion exchange chromatography. The native molecular weight of the 60 k protein was estimated to be 420 k by size exclusion chromatography. The purified 60 k protein showed the typical rotational symmetry of chaperonins when analyzed by electron microscopy. Ultrathin sections of Helicobacter pylori were immunostained by a polyclonal antibody directed against the hsp-65 of Mycobacterium tuberculosis. The label revealed a clustered localization of the 60 k protein on the cell surface as well as in the periplasmic space.

Serodiagnosis of Helicobacter pylori infections with an enzyme immunoassay using the chromatographically purified 120 kilodalton protein.

A membrane-associated 120 kDa protein of Helicobacter pylori with known species-specificity was isolated and used in an enzyme immunoassay (EIA) for the detection of Helicobacter pylori-specific IgG antibodies in patient sera. The EIA was compared with two other methods used for serodiagnosis of Helicobacter pylori infections: an EIA using sonicated whole Helicobacter pylori cell antigen and Western immunoblot. In a prospective study 127 unselected patients (76 patients with antrum gastritis, 51 patients without gastritis) who underwent gastroscopy were studied histologically and serologically. The EIA using the purified 120 kDa protein had the highest specificity (92%) compared with the EIA using a whole cell sonicate of a single Helicobacter pylori strain as antigen (60.7%) and the immunoblot (90.2%). The sensitivity was 96%, 100% and 92%, respectively. Sera of three control patients reacted strongly in all three methods, indicating possible Helicobacter pylori infection with negative histological findings. The EIA using the 120 kDa protein as antigen was shown to be a specific and sensitive technique for the serodiagnosis of Helicobacter pylori infections.

Immunodominant epitopes of the adhesin of Mycoplasma pneumoniae.

Overlapping octapeptides from the amino acid sequence of the adherence protein of Mycoplasma pneumoniae were synthesized and used as the antigen in an enzyme-linked immunosorbent assay with serum samples from M. pneumoniae-infected patients. Of a sequence of 338 amino acids positioned between leucine 801 and leucine 1139, only two regions with immunodominant continuous epitopes were detected. The immunoglobulin G and immunoglobulin M antibodies of child and adult patients reacted especially with the NH2-(810)-W-I-G-N-G-Y-R-Y peptide but also reacted with the NH2-(1124)-F-T-D-F-V-K-P-R peptide.

Topological mapping of the P1-adhesin of Mycoplasma pneumoniae with adherence-inhibiting monoclonal antibodies.

Five adherence-inhibiting monoclonal antibodies (mAbs) were used for topological mapping of the binding sites of the 169 kDa membrane-integrated adhesin of Mycoplasma pneumoniae. Antibody binding sites were characterized using overlapping synthetic octapeptides. Three regions of the protein seem to be involved in adherence: the N-terminal region [N-reg, epitopes beginning at amino acid (aa) 1 to aa 14 and aa 231 to aa 238, respectively]; a domain (D1) approximately in the middle of the molecule (beginning at aa 851 to aa 858 and aa 921 to aa 928); and a domain (D2) closer to the C-terminus (beginning at aa 1303 to aa 1310, aa 1391 to aa 1398 and aa 1407 to aa 1414). Each of the mAbs P1.26 and P1.62 reacted with two primary amino acid sequences. Both antibodies bound to the D1 region, but mAb P1.62 showed additional binding to a sequence (aa 231 to aa 238) near the N-terminus, and mAb P1.26 reacted with a second epitope in the D2 domain (aa 1303 to aa 1310). Such dual binding by the two antibodies suggests that in the native protein the epitopes are composed of two sequences which are located on two different sites of the molecule (D1/N-reg and D1/D2, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding sites of attachment-inhibiting monoclonal antibodies and antibodies from patients on peptide fragments of the Mycoplasma pneumoniae adhesin.

The adherence protein (P1 protein) of Mycoplasma pneumoniae was purified by electroelution and cleaved with cyanogen bromide. The resulting peptides were separated by two-dimensional electrophoresis. Spots reacting in Western immunoblots with two attachment-inhibiting monoclonal antibodies were isolated, and the amino-terminal ends of these peptides were microsequenced. The two monoclonal antibodies had different binding sites. One was associated with the amino-terminal region of the whole P1 protein beginning at amino acid position 237, and the other was associated with amino acid position 702, which was localized approximately in the middle of the P1 amino acid sequence. Serum samples from three M. pneumoniae-infected patients were tested by Western blotting against the cyanogen bromide peptide pattern. All three serum samples reacted with peptide fragments beginning at amino acid position 702, but the serum of only one patient also had antibodies against the oligopeptides beginning at amino acid position 237. These results indicate that the corresponding epitopes of the P1 protein are also immunogenic if they are presented at the surface of the infecting organism.