Diclofenac-KLH-Conjugate for diagnosis of type I allergy

Background: Immediate type drug hypersensitivity is a serious problem for the patient as it may cause life threatening reactions. Precise diagnosis is important as in case of true drug allergy, alternative drugs must be prescribed by the physician. The causative agents are IgE antibodies which sensitize allergy effector cells and, upon consecutive drug contact, release mediators which produce allergic symptoms. The triggering event is dependent on crosslinking of at least two IgE molecules, which occurs in vivo when the chemical compound attaches to self- proteins. Diagnosis of drug allergy today is performed with unmodified drugs, and does often give false-negative results. Our working hypothesis is that multimeric drug conjugates can improve diagnosis. Methods: As a paradigm, the analgetic and anti-inflammatory drug diclofenac was covalently coupled to keyhole limpet hemocyanin (KLH), which is approved by the FDA as a carrier for haptens and thus can be safely used in humans. The coupling efficacy was controlled with a rabbit anti-diclofenac antibody in ELISA. Sera of two diclofenac allergic patients with repeated immediate type reactions to diclofenac and of non-allergic control persons were tested in ELISA. Further, both patients with IgE towards diclofenac and two control persons were subjected skin prick testing using the conjugate, as well as uncoupled KLH or diclofenac and, furt5her, to intradermal testing with unconjugated diclofenac. Preliminary Results: IgE antibodies in patients` sera towards diclofenac could be detected in ELISA, demonstrating the diagnostic value of this improved method. In skin tests of patients the multimeric conjugate elicited positive reactions, whereas KLH alone or diclofenac alone did not elicit skin prick reactivities. Uncoupled diclofenac sodium produced positive reactions in intradermal tests only and at an approximately 100 times higher concentration. Conclusions: Crosslinking can be achieved when drugs are coupled to polyvalent carriers, allowing the presentation of several drug molecules in tight spacing to the cytophilic IgE antibodies. Thus, testing with KLH- coupled diclofenac facilitates diagnosis of immediate type diclofenac allergy and avoids intradermal testing. Aims of this project: The principal purpose of this pilot study with KLH-coupled diclofenac is to confirm in a larger population 1) that the new test substance is more effective and reliable and, therefore, superior to today`s standard skin testing, 2) that it is well applicable for reliable in vitro tests and 3) that the results of in vitro and in vivo tests correlate with the clinical reaction confirmed by drug provocations tests. Furthermore, the ideal concentration of KLH-coupled diclofenac resulting repeatedly in true positive skin prick test reactions shall be identified. An improved test method would greatly improve today`s drug allergy diagnosis and, therefore, be a great benefit for doctors and patients.