Antiserum relationships

By 1945, Stacey speculated about the possibility of a structural relationship between Pneumococcus capsular polysaccharides and those produced by other organisms. With Miss Schliichterer, he had examined the capsular polysaccharide of Rhizobium radicicolum. This polysaccharide gave a precipitin reaction in high dilution, not only with Type III Pneumococcus antiserum, but also mixed with antisera from other Pneumococcus types. The chemical evidence indicated that the polysaccharide resembled the specific polysaccharides of Types I and II Pneumococcus. A decade later, the acidic capsular polysaccharide from Azoto-bacter chroococcum, a soil organism, was studied. It, too, produced serological cross-reactions with certain pneumococcal specific antisera. Although the molecular structure of the polysaccharide was not established, adequate evidence was accumulated to show a structural relationship to Type III Pneumococcus-specific polysaccharide. This was sufficiently close to account for the Type III serological cross-relationship. 

Furthermore, since one of the saponins showed strong cytotoxicity in these experiments, we also examined the cytotoxicity toward hepatocytes without antiserum. Moreover, from the standpoint of the structure-hepatoprotective and -hepatotoxic relationships for the carboxyl group at C-28, oleanolic acid 3-Oglucuronide (96) and oleanolic acid 28-Oglucoside (97) were prepared and tested. 

To produce an antiserum, the antigen for the first immunization is often prepared in an adjuvant (usually a water in oil emulsion containing heat-killed bacteria), which allows it to be released slowly and to stimulate the animal s immune system. Subsequent injections of antigen are done with incomplete adjuvant that does not contain the bacteria. The species used to raise the antibodies depends on animal facilities, amount of antigen available, and the amount of antiserum required. Another consideration is the phylogenetic relationship between antigen and immunized species. A highly conserved mammalian protein may require an avian species in order to raise... 

If the relationship between the concentration of the protein in the dilutions and the height of the precipitate does not give a straight line, it is often possible to obtain a straight line by further dilution of the samples and the use of less antiserum. But the limits of sensitivity for the rocket technique are determined by the formation of visible precipitin lines and this depends on the protein under study (for example, a weaker precipitin line is obtained for human serum aj-antichymotrypsin than for human serum approteinase inhibitor in the same concentration range [5]). 

Determination of Antibodies to AFP. Wells in a microtiter plate are coated with 1 fig of hAFP. After washing, antisera and normal sera are serially diluted in the wells, and the plate is incubated at room temperature for 3 hr. The plate is then washed three times and 0.2 ml of the conjugate, diluted 1 500 in the incubation buffer, is added to each well. The plate is again incubated for 3 hr at room temperature, washed three times, and incubated with the substrate solution for 15 min. The enzyme is inactivated with NaOH, and the content of each well is diluted in distilled water before measurement at 405 mm. Figure 5 shows the result of titration of a mouse antiserum to hAFP and of a normal mouse serum. There is a direct relationship between the amount of antibodies in the serum and the absorbance measured. This relationship is linear within a certain... 

Methanococcus voltae contains a membrane-bound vanadate-sensitive ATPase [48] that is inhibited by diethylstilbestrol, an inhibitor of eukaryotic P-type ATPases. The purified enzyme is composed of a single subunit (Mr 74 000), forms a covalent acyl-phosphate enzyme intermediate, and is not inhibited by nitrate or bafilomycin [49]. No such ATPase activity has been reported in other archaea. The presence of a second ATPase in M. voltae has been inferred since membranes react with antiserum prepared against the 3 subunit from the V-type ATPase of S. acidocaldarius [50]. Two peptides are detected whose Mr values (51 000 and 65 000) correspond to the masses for the two laigest subunits of the S. acidocaldarius ATPase [51]. There is evidence that ATP synthesis in the M. voltae enzyme is due to the operation of a sodium-translocating ATPase [50]. The relationship of the putative V-like ATPase to the sodium-translocating ATPase has not been established. 

The quantity of capsular polysaccharide is apparently of importance in a variety of pathogens. Klebsiella pneumoniae. Salmonella typhi, 5. typhi-murium, E. coli. Streptococcus pneumoniae and Staphylococcus aureus have all been reported to show a relationship between the amount of capsular material and virulence, resistance to phagocytosis, or resistance to humoral host defences. It has been suggested in the case of E. coli that the amount of K-antigen, as measured by its ability to coat red blood cells and inhibit their aggregation by haemagglutinating antiserum, is the most important factor determining resistance to the complement-mediated bactericidal activity of serum. Other studies have found evidence that the type of capsular polysaccharide may be more important. 

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