Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ϳ98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ϳ93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.Tuberculosis (TB) is a major disease in developing countries and an increasing problem in developed countries, with an estimated 8 million new cases each year (7,21,35). The rise in the number of infections is due to drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and to more incidences of coinfections with human immunodeficiency virus (HIV), particularly in sub-Saharan Africa (21). This has prompted the need to develop rapid, inexpensive, but clinically sensitive and specific tests that can improve upon current diagnostic tests used in TB diagnosis, such as culture and acidfast smear testing (20). PCR methods are available but are expensive, require culture, and are not applicable to field use (14, 33-34, 42). One such approach to providing simple tests has been to develop adjunctive serology assays. However, previous attempts to diagnose TB by serology have met with limited success (2-3, 6,8, 10-12, 15-16, 23-24, 37-38, 44-47). Many of the antigens to date do not have the appropriate clinical sensitivity and specificity required for an accurate diagnosis and do not always effectively discriminate Mycobacterium bovis BCG-vaccinated and purified protein derivative (PPD)-positive individuals from those with active TB (8, 36). Several notable antigens have been identified which have merit in TB diagnosis, among which is the immunodominant 38-kDa phosphate transport protein (1,5,9,19). This antigen has been used ...

Peptide epitopes of Trypanosoma cruzi have been identified through expression cloning. A tripeptide (2/D/E) containing three epitopes (TcD, TcE, PEP-2) was used in ELISA to detect antibodies to T. cruzi in 239 of 240 consensus-positive sera and 41 of 42 sera confirmed positive by radioimmunoprecipitation assay. The 1 discrepant consensus-positive serum was used to expression-clone a novel gene that contained a repeat sequence. A peptide corresponding to this sequence, TcLo1.2, was specific for T. cruzi. This antigen detected the discrepant consensus-positive serum and enhanced reactivity of low-positive sera in the tripeptide assay. A branched synthetic peptide, 2/D/E/Lo1.2, or a linear recombinant, r2/D/E/Lo1.2, realized all of the diagnostic features of the four epitopes, including the ability to boost reactivity of low-reactive sera. These studies show that peptides and recombinants containing multiple repeat epitopes are powerful tools for developing assays for T. cruzi antibody detection and have direct application in blood screening.