The Development and Characterization of Two Monoclonal Antibodies Against the Conjugates and Derivatives of the Immunometabolite Itaconate

We have developed two monoclonal antibodies, CPTC-2MeSC-1 and CPTC-2MeSC-2, against itaconate and its conjugates with sulfhydryl-containing biomolecules such as cysteines. Itaconate is a dicarboxylic acid metabolite that has recently gained much interest for its anti-inflammatory properties in many biological models. We have synthesized an itaconate-cysteine conjugate ITA-Cys designed to mimic in vivo Michael adducts of itaconate. Two monoclonal antibodies against ITA-Cys, CPTC-2MeSC-1 and CPTC-2MeSC, were developed and shown to have high immunoreactivity to unconjugated itaconate, itaconate-BSA conjugates, and Michael adducts of dimethyl itaconate. We found that CPTC-2MeSC-1 and CPTC-2MeSC-2 are specific and do not bind to other structurally similar cysteine Michael adducts, including those obtained from "sister" metabolites (fumarate, cis-aconitate), itaconate isomers (citraconate), and some itaconate esters. CPTC-2MeSC-2 is a useful tool in both studying biological actions of itaconate and developing therapeutic applications of itaconate and its derivatives.