ENHANCED IMMUNOREACTIVITY AND PREFERENTIAL HETERODIMER FORMATION OF REASSOCIATED FEL-DI RECOMBINANT CHAINS

In this study we have addressed the question of whether reassociating the two recombinant protein chains that comprise the major cat dander allergen, Fel d I, would change the overall IgE and allergic patient T cell immunoreactivity compared to the native molecule. To accomplish this, the chains were combined under reducing and denaturing condition s, then allowed to reassociate by dilution and extensive dialysis agai nst a physiological buffer. An initial examination of the reaction pro ducts using quantitative capture ELISA demonstrated comparable reactiv ity to Fel d I. Further analysis, using a pool of cat allergic patient plasma, showed that the products of the reassociation reaction (rFel dI) also possessed an enhanced IgE binding capacity. Depletion ELISA r esults gave only a 5% difference in reactivity between rFeldI and the native protein versus a 20% difference with the mixture of the two cha ins. Comparative secondary T cell stimulation assays were subsequently performed using cat allergic patient peripheral blood lymphocytes. He re the results demonstrated no loss of reactivity with the reassociate d chains as compared to Fel dI or the two mixed recombinant chains. To biochemically characterize the products of the reassociation reaction we have performed reverse phase HPLC and then analysed the isolated f ractions by mass spectrometry. It was clear from these results that li ke the native Fel dI, the products of the reassociation reaction favor ed heterodimer formation, with no homodimer being detected. This impli es that the reassociated protein chains had preferentially adopted a n ative-like conformation.