Generation of a recombinant bacteriophage antibody library to mycobacterium tuberculosis.

Monoclonal antibodies to Mycobacterium tuberculosis (Mtb) may be useful for laboratory diagnosis, antigenic characterization, and studying the immune response to infection and vaccination. To investigate the potential of the recombinant phage antibody technique for the isolation of single-chain fragments (ScFv) reactive with Mtb culture proteins, we generated a bacteriophage antibody library from splenic tissue of mice immunized with heat-killed Mtb. Heavy- and light-chain immunoglobulin genes were isolated and amplified by the polymerase chain reaction (PCR), and the products were assembled into ScFv gene fragments and cloned into the pCANTAB5-E phagemid. Phagemids were introduced into E. coli TG1 by electrotransformation, followed by rescue of antibody-expressing phage using M13K07 helper-phage superinfection. Immunoselection of the Mtb phage library against Mtb cell wall extract or culture filtrate proteins selected antigen-specific and cross-reactive phage antibodies, none of which demonstrated reactivity to the immunodominant 65-kDa Mtb antigen. These results suggest that the phage antibody system has the potential to generate a diverse population of the reactive phage that may prove useful for investigating the immune response to Mtb infection and vaccination.

Differential expression of a Mycobacterium tuberculosis protein recognized by a mouse monoclonal antibody.

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.

Evaluation of a Treponema pallidum enzyme immunoassay as a screening test for syphilis.

The CAPTIA Syphilis-G enzyme immunoassay for the detection of antibodies to Treponema pallidum was evaluated as a screening test for syphilis in comparison with the standard rapid plasma reagin (RPR) test. One thousand samples were tested, and the standard fluorescent treponemal antibody absorption test and the standard microhemmaglutination test were used to confirm the presence of treponemal antibodies. Diagnosis of syphilis was based on traditional standard serology results. Clinical data used in the diagnosis of patients whose samples yielded conflicting results were provided by physicians. Initially, 7 patients whose samples were reactive in the RPR test and 14 patients whose samples yielded positive or equivocal results in the CAPTIA Syphilis-G test were diagnosed as not being infected. After discrepancies due to technical problems were reconciled, samples from six patients remained reactive in the RPR test and that from one patient remained positive in the CAPTIA Syphilis-G test. In addition, seven patients whose samples were nonreactive in the RPR test and two patients whose samples were negative in the CAPTIA Syphilis-G test were diagnosed as having untreated syphilis. After discrepancies were reconciled, samples from five patients remained nonreactive in the RPR test and none remained negative in the CAPTIA Syphilis-G test. Final results indicate that the specificities are 99.4 and 99.9%, respectively. In addition to the improved sensitivity and specificity of the CAPTIA Syphilis-G screen, other potential benefits of this assay lead us to believe that this method could serve as a better screening tool than the RPR test.