Karl Himmelspach

Immunochemical properties of oligosaccharide-protein conjugates with Klebsiella-K2 specificity. I. Specificity and crossreactivity of anti-conjugate versus anti-bacterial antibodies.

A series of repeating unit oligomers ceGlcUA IJ~3 was prepared by depolymerization of Klebsiella pneumoniae B5055 (01 :K2) capsular polysaccharide (K2-PS) as catalyzed by a bacteriophage-associated gly- canase. The monomeric repeating unit (tetrasaccharide, (TS)) and its di- and tri- mer (octa- and dodecasaccharide (OS and DS)) were conjugated to edestin via reductive aminophenylation and azo coupling at the reducing sugar end group. Rabbit anti-conjugate and anti-bacterial antibodies raised in rabbits were com- pared with respect to specificity and crossreactivity towards the oligomers of the various molecular sizes, towards parent PS, and towards whole bacteria. Antibodies able to bind specifically to the PS and the bacteria were elicited by all three conjugates. However, the anti-TS conjugate antibodies, in contrast to those obtained with OS and DS conjugate, proved to be practically unable to effect bacterial agglutination. Correspondingly, the TS played an exceptional role in binding to anti-bacterial antibodies. In contrast to the OS and DS it could not fully inhibit precipitation of these antibodies with the bacterial PS. Moreover, the inhibition of the binding of the PS to antibacterial antibodies produced by TS was about 50-fold weaker than that produced by OS, DS, and higher mem- bers of the series, all of which were about equally potent inhibitors (on a molar Address for offprint requests: Dr. Hildegard Geyer, Zentrum fiir Biochemie am Klinikum der Justus-Liebig-Universit/it, Friedrichstr. 24, D-6300 Labn-Giessen, Federal Republic of GermanyAbstractA series of repeating unit oligomers

Isolation and dynamic hapten binding properties of 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium (THP-) specific antibodies from bovine colostrum--I. 7S and 19S anti-THP antibodies. Preparation, Characterization and analytical application of immunoadsorption.

\begin{gathered} \alpha - GlcUA \hfill \\ 1 \downarrow 3 \hfill \\ [ \to 4) - \beta - Man - (1 \to 4) - \alpha - Glc - (1 \to 3) - \beta - Glc - (1 \to ]_n , 1 \leqslant n \leqslant 7 , \hfill \\ \end{gathered}

Isolation and dynamic hapten binding properties of 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium (THP-) specific antibodies from bovine colostrum--II. Characteristic differences in hapten binding kinetics of 7S and 19S anti-THP antibodies.

was prepared by depolymerization ofKlebsiella pneumoniae B5055 (01∶K2) capsular polysaccharide (K2-PS) as catalyzed by a bacteriophage-associated glycanase. The monomeric repeating unit [tetrasaccharide, (TS)] and its di- and trimer [octa- and dodecasaccharide (OS and DS)] were conjugated to edestin via reductive aminophenylation and azo coupling at the reducing sugar end group. Rabbit anti-conjugate and anti-bacterial antibodies raised in rabbits were compared with respect to specificity and crossreactivity towards the oligomers of the various molecular sizes, towards parent PS, and towards whole bacteria. Antibodies able to bind specifically to the PS and the bacteria were elicited by all three conjugates. However, the anti-TS conjugate antibodies, in contrast to those obtained with OS and DS conjugate, proved to be practically unable to effect bacterial agglutination. Correspondingly, the TS played an exceptional role in binding to anti-bacterial antibodies. In contrast to the OS and DS it could not fully inhibit precipitation of these antibodies with the bacterial PS. Moreover, the inhibition of the binding of the PS to antibacterial antibodies produced by TS was about 50-fold weaker than that produced by OS, DS, and higher members of the series, all of which were about equally potent inhibitors (on a molar basis). The results show that two repeating units are the minimum requirement for a substantial representation of the PSs serologic specificity. The exceptional behavior of the TS correlates with its lack of theβ-Glc-(1–4)-Man linkage present in all higher members of the series.

MOPC 104E IgM/anti-idiotype solid-phase inhibition assay as a model for screening myeloma proteins for ligand binding specificity

The K3-antigenic capsular polysaccharide (K3 antigen) of Escherichia coli contains L-rhamnose, a 4-deoxy-2-hexulosonic acid, and an O-acetyl group in the molar ratio of 3:1:1. The backbone consists of a ----2)-O-alpha-L-rhamnopyranosyl-(1----3)-O-alpha-L-rhamnopyranosyl-(1----3)-O-alpha-L-rhamnopyranosyl-(1---- repeating unit. Either one of the 3-linked L-rhamnopyranosyl residues of each repeating unit may be substituted at O-2 with a 4-deoxy-2-hexulosonic acid, an isomer of the furanosyl form of KDO, about 90% of which is acetylated at 0-6. The 4-deoxy-2-hexulosonic acid residue is linked to the L-rhamnan backbone in a very labile linkage which is split by 1% acetic acid (30 min, 100 degrees). The K3 polysaccharide has a molecular weight of approximately 38,000, corresponding to approximately 60 repeating units.

Chemistry and Immunochemistry of Bacterial Lipopolysaccharides as Cell Wall Antigens and Endotoxins

Abstract Antibodies against the solvatochromic dye 4-(3-aminophenyl)-2,6-diphenyl- N -(4-hydroxy-phenyl)-pyridinium betain (ATHP ∗ ) we produced by immunization of pregnants cows with an THP-edestin conjugate. Large amounts of 7S and 19S anti-THP antibodies were isolated from the colostrum of one of the immunized animals. The specific isolation was performed with the aid of an immunoadsorbent prepared by coupling ATHP to bovine γ-globulin, previously cross-linked with glutaraldehyde. Various conditions for antibody binding to, and elution from, the adsorbent were studied and compared. Using the adsorbent a test was developed which enabled quantitative determination of anti-THP antibodies, including the non-precipitating ones, which were found to be present in the colostrum in large quantities. The isolated anti-THP antibodies were fractionated by chromatography on Sephadex G-200 into 7S and 19S antibodies. The 7S anti-THP antibodies proved to belong the type IgG s whereas the 19S anti-THP antibodies were composed of 19S IgG s and IgM.

Conformational Changes of Apigenin 7‐O‐(6‐O‐malonylglucoside), a Vacuolar Pigment from Parsley, with Solvent Composition and Proton Concentration

Oligosaccharide subunits from Klebsiella pneumoniae B5055 capsular polysaccharide, covalently linked to protein carriers, the native polysaccharide, and killed whole bacteria, were compared with respect to their immunogenicity in mice and their capacity to protect mice against bacterial infection. The protection was studied (a) by active immunization and (b) by passive administration of antisera raised in rabbits using the different immunogens. The results were as follows: in the active protection experiments the octa- and dodecasaccharide conjugates were as effective as the polysaccharide in increasing the LD50 by a factor of 10(5) as compared to nonimmunized animals. Thus, these antigens were only slightly less effective than killed bacteria. In contrast the tetrasaccharide conjugate was at least 1000-fold less effective than all other antigens used. In the passive protection experiments these results were paralleled in that the antiserum against the tetrasaccharide conjugate also showed the lowest degree of protection, though, for methodical reasons, the differences in the LD50s were less pronounced. The drastically lower efficiency of the tetrasaccharide in comparision to its higher oligomers is in agreement with serological findings previously published (see first communication), which showed that two repeating units are the minimum requirement for a substantial representation of polysaccharide serological specificity.Oligosaccharide subunits fromKlebsiella pneumoniae B5055 capsular polysaccharide, covalently linked to protein carriers, the native polysaccharide, and killed whole bacteria, were compared with respect to their immunogenicity in mice and their capacity to protect mice against bacterial infection. The protection was studied (a) by active immunization and (b) by passive administration of antisera raised in rabbits using the different immunogens. The results were as follows: in the active protection experiments the octa- and dodecasaccharide conjugates were as effective as the polysaccharide in increasing the LD50 by a factor of 105 as compared to nonimmunized animals. Thus, these antigens were only slightly less effective than killed bacteria. In contrast the tetrasaccharide conjugate was at least 1000-fold less effective than all other antigens used. In the passive protection experiments these results were paralleled in that the antiserum against the tetrasaccharide conjugate also showed the lowest degree of protection, though, for methodical reasons, the differences in the LD50s were less pronounced.The drastically lower efficiency of the tetrasaccharide in comparison to its higher oligomers is in agreement with serological findings previously published (see first communication), which showed that two repeating units are the minimum requirement for a substantial representation of polysaccharide serological specificity.

Use of 1-(m-aminophenyl)flavazoles for the preparation of immunogens with oligosaccharide determinant groups. A method is described for the preparation of immunogens with oligosaccharide determinant groups

Abstract The hapten binding properties of purified 7S and 19S 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium (THP∗-) specific antibodies were studied by temperature-jumped spectrometry. In both cases a nearly diffusion-controlled hapten-antibody recombination process in the μsec range was observed. In the case of the 19S immunoglobulins an additional relaxation process in the msec range was found to be independent of reactant concentrations. This slow first-order process, suggesting structural rearrangements of the antibodies, is discussed with respect to hapten-dependent conformational changes of the immunoglobulins.

Degradation of bacterial surface carbohydrates by virus-associated enzymes

The inhibition of binding of 125I-labeled MOPC 104E IgM anti-idiotype to the controlled pore glass-bound IgM by various oligo- and polysaccharides was investigated as a model for the screening of myeloma proteins with unknown hapten binding specificity. The inhibitory efficiencies of the different haptens in this system were found to correlate well with their known abilities to bind to MOPC 104E IgM. Thus B1355S dextran, nigerosyl-alpha (1-3)-nigerose, and nigerosyl-alpha (1-3)-glucose, all known for specific binding to MOPC 104E IgM, were efficient inhibitors in the assay, in contrast to a series of more or less unrelated carbohydrate compounds, which showed only weak inhibitory capacity. According to these results there seems to be no obstacle to applying the anti-idiotype assay system to screening myeloma proteins whose binding specificity has not yet been determined.