Production of recombinant antigens of Ureaplasma parvum serotypes 3 and 6 for development of a serological assay

Abstract

Recombinant antigens of Ureaplasma parvum serotypes 3 and 6 were produced in order to develop a serological assay for Ureaplasma antibody detection. The genes of the multiple banded antigen (MBA) were amplified by PCR and cloned in a pTrcHis TOPO plasmid. Purified recombinant proteins were evaluated in Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies and human sera. Our approach was successful in the production of the recombinant MBAs (rMBAs) for serotypes 3 and 6. The antigens tested positive with serotype-specific monoclonal antibodies in Western blotting and in ELISA. Prominent reactions were detected with the rMBAs and their homologous monoclonal antibodies. Strong cross-reactions were visible in ELISA between rMBA 3 and the monoclonal antibodies from the other U. parvum serotypes. A weak cross-reaction was seen with rMBA 3 and the monoclonal antibody from serotype 4. rMBA 6 showed cross-reaction only with the monoclonal antibody from U. parvum serotype 1. Fifty-one percent of the sera obtained from culture-positive women reacted with one or both rMBAs. Only three (15%) of the sera from culture-negative women reacted with the rMBA. The positive reactions were observed only with rMBA 6. These preliminary tests showed the potential usefulness of the rMBAs produced for detecting an antibody response against Ureaplasma antigens.

FIG. 1.

Western blot showing the reaction patterns of rMBA 3 with all the serotype-specific MAbs. MM, molecular mass ladder (97.4, 66.2, 45, 31, 21.5, and 14.4 kDa). Lanes 1 to 14 contain rMBA 3 reactions with all 14 serotype-specific MAbs of Ureaplasma spp.

FIG. 2.

Western blot showing the reaction patterns of rMBA 6 with all the serotype-specific MAbs. MM, molecular mass ladder (97.4, 66.2, 45, 31, 21.5, and 14.4 kDa). Lanes 1 to 14 contain rMBA 6 reactions with all 14 serotype-specific MAbs of Ureaplasma spp.