Immunochemical analysis of sulfonamide drug allergy: Identification of sulfamethoxazole-substituted human serum proteins,☆☆,,★★

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Abstract

Background: Sulfonamides undergo oxidative metabolism to yield reactive metabolites that haptenate proteins readily. Although it has been shown that sulfonamide metabolites bind covalently to murine microsomes, sulfonamide-conjugated serum proteins have not been analyzed in the peripheral blood of treated individuals. Objective: We hypothesized that during treatment with sulfamethoxazole, intracellular proteins are haptenated by drug metabolites, and some of these are destined for secretion into the serum. Methods: Using antibodies specific for sulfamethoxazole and an alkaline phosphatase immunoblotting technique, we attempted to demonstrate the presence of sulfamethoxazole-substituted proteins in the serum of individuals during a course of treatment. Results: Five days into therapy, serum protein haptenation by sulfamethoxazole was demonstrated in two of the three individuals studied. In addition, Western blot analysis revealed that haptenation is not indiscriminate, but highly selective. A single 30 kd protein is the target of haptenation in all instances. A kinetic analysis revealed that substituted proteins can be detected early, within hours of administration. Moreover, haptenated proteins remain detectable in the serum 48 hours after discontinuation of the drug. Conclusion: The results presented here constitute the first direct evidence that sulfonamides, on being metabolized, covalently haptenate human serum proteins during a course of therapy. (J ALLERGY CLIN IMMUNOL 1994;94:1017-24.)

Section snippets

Rabbit immunizations/antibody purification

Hunter's Titermax adjuvant was obtained from CytRx Corporation (Norcross, Ga.). SMX-bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, Mo.) was prepared according to the method described previously4 and was used as the immunizing antigen. An Econo-Pak Protein A kit from Bio-Rad (Richmond, Calif.) was used for the purification of the rabbit IgG.

Enhanced ELISA

Borate-buffered saline, phosphate buffered saline (PBS), blocking solution/standard diluent (ovalbumin, 5 gm/L in PBS/Tween), PBS/Tween (Tween

Characterization of rabbit anti-SMX antibodies

SMX-specific polyclonal antibodies were generated in New Zealand white rabbits. After animals were immunized and boosted with SMX-BSA (prepared by covalent linkage of the diazonium salt of SMX to BSA), serum was obtained, partially purified, and used in inhibition ELISAs. Prebleed serum was also obtained and partially purified for use as a negative control. Before addition to microtiter plates containing antigen, antibodies diluted 1:24,000 were incubated with one of three inhibitors: BSA,

DISCUSSION

A significant number of adverse drug reactions appear to be immune-mediated, and as for any immune-mediated event involving simple haptens, induction and elicitation of the reaction requires the formation of immunogenic conjugates. Although it has been assumed that these conjugates are formed through covalent binding of the drug hapten itself or a reactive intermediate to tissue macromolecules, knowledge of the metabolic processes involved in the generation of these immunogenic conjugates is

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From aUniversity of Texas Southwestern Medical Center, Dallas; and bEmory University School of Medicine, Atlanta.

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Supported by grant AI33711-02 from the National Institutes of Health.

Reprint requests: Rebecca S. Gruchalla, MD, PhD, Department of Internal Medicine, Division of Allergy, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8859.

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