Homogeneous antibodies demonstration

A portion of this chapter will be concerned with antibodies to bacterial vaccines, particularly killed streptococci and pneumococci. Such antigens are of unique value for the production of homogeneous antibodies because of the very high concentrations of precipitable antibody which can be induced in experimental animals, as well as the frequent appearance of uniform subpopulations. Serum concentrations exceeding 15 mg/ml are common and values as high as 60 mg/ml have been reported (e.g., 4). Not all such sera possess a readily demonstrable homogeneous component when the latter is present it may account for a small or a large fraction of the antibody. 

Studies on idiotypes of homogeneous antibodies in rabbit families suggested that idiotypes were genetically transmitted. Further work on the idiotypes of rabbit antibodies showed linkage of group a allotypes to idiotypes The association of an idiotype to an infrequently occurring subgroup has also been demonstrated in these same rabbit families . ... 

In competitive homogeneous immunoassay, separation and quantitation of free and bound labeled antigen (cortisol) were carried out in a fused silica chip. Since the antibody-antigen complex was not detected, an internal standard (fluorescein) was added to aid quantitation. In addition, since most of the total cortisol was bound in the serum, a releasing agent, 8-anilino-l-naphthalenesulfonic acid (ANS), should be added [1006]. In other reports, competitive immunoassay for BSA was demonstrated after performing a CE separation on-chip [105,1005]. 

In order to fully evaluate urease as a potential label for EIA in general, we felt that it was necessary to demonstrate that urease could be covalently coupled to an antigen and still maintain sufficiently high activity for sensitive immunoassay. In addition, the effect of antibody binding to the labeled antigen should be tested to see whether enzyme activity can be homogeneously inhibited by the antibody. 

LCAT activity can be demonstrated in plasma by incubating freshly obtained blood plasma for 30-45 min at 37°C and measuring changes in the concentrations of the reaction substrates or products. Alternatively, radioactive UC, PC, or UC-PC vesicles can be incubated with plasma, and the formation of radioactive CE can be measured. Because LCAT has been purified essentially to homogeneity [53-55] and antibodies to it have been developed [56], it is now possible to measure the concentration of the enzyme protein in plasma by immunoassay [56]. 

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