116

ASSAYS

[ 13]

gram range (using standard 4 to 5-mm diameter columns). Nevertheless, reversed-phase HPLC offers major advantages for lipoxygenase product analysis, since it provides a profile of compounds, as opposed to measurements of a single component, thereby supplying more information on the composition of the samples. In addition, sample preparation procedures are usually very simple, thus minimizing the possibility of sample loss and chemical alterations; this is a point of major concern considering that lipoxygenase products are polyunsaturated compounds susceptible to oxidation.

[13] P r e p a r a t i o n o f A n t i b o d i e s D i r e c t e d a g a i n s t L e u k o t r i e n e s

By EDWARD C. HAYES Introduction The leukotrienes (LTC4, LTD4, LTE4, and LTB4) must be coupled to high-molecular weight carrier proteins such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) to be immunogenic. Such coupling is possible because these leukotrienes contain a number of groups that can react directly with the carrier protein or with a conjugating reagent serving as a multicarbon spacer arm (see below and Table I). The use of such spacers will often lead to the generation of antibodies that are specific for the hapten rather than a determinant that is shared between the hapten and the cartier molecule. The site on the hapten that is used for coupling, the number of haptens per cartier (5-20), and the configuration of the hapten (whether it is the naturally occurring leukotriene or an analog) may have a profound effect on the antibody specificity. The influence of the carrier protein must also be considered. Although KLH is difficult to work with, it is one of the most immunogenic carriers and offers the additional advantage that antibodies directed against it do not cross-react with proteins found in most biological samples. A number of coupling procedures will be presented and the individual investigator must decide which are to be employed based on the titer and specificity of the antibody required as well as availability of chemical expertise. METHODS IN ENZYMOLOGY, VOL. 187

Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.

[13]

PREPARATION OF ANTIBODIES AGAINST L T s

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