Preparation of monoclonal antibodies reactive to the endogenous small molecule, anandamide.
The development of an easy and inexpensive immunoassay to measure the limited quantities of endogenous cannabinoids found in the body would be beneficial for both cannabinoid researchers and clinicians. This report describes the hapten design and carrier molecule strategy that we used to generate a panel of monoclonal antibodies (mAB) to the endogenous cannabinoid anandamide (N-arachidonylethanolamide, AEA). We designed and successfully prepared a hapten, N-arachidonyl-7-amino-6-hydroxy-heptanoic acid (AHA), which retained the basic characteristic features of anandamide--the carboxamide, the hydroxyl and the lipophilic arachidonyl moiety with its skipped double bond system, while still allowing attachment to protein. In addition, a secondary alcohol structure was added to reduce the potential for biological hydrolysis of the hapten. Because of the diverse responses obtained after coupling this hapten to four different carriers, we determined that the type of carrier molecule used was particularly important for generating anti-anandamide antibodies. Described in this report are the characteristics of a panel of 11 mAB, generated from four separate fusions, with a range of relative affinities and cross reactivities. Excellent selectivity for anandamide vs. two other endogenous cannabinoids and arachidonic acid was achieved this strategy (cross-reactivities <5%). In addition, at least one mAB maintained specificity for anandamide compared to two very closely related fatty acid amide molecules. However, the IC50 values in a standard enzyme-linked immunosorbent assay (ELISA) format (ca. 2-3 microM) indicate that improvement in antibody affinities or assay format will be required for an immunoassay to measure endogenous levels. Such work is underway.