Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG.

A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin.

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.

Use of recombinant human antibody fragments for detection of cytomegalovirus antigenemia.

The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomegalovirus (HCMV) antigens is widely employed in clinical virology for rapid diagnosis of HCMV-related infections. We describe how CMV antigenemia may be accurately detected by means of human recombinant monoclonal Fab fragments rescued from a combinatorial phage display library prepared from an HCMV-infected donor. Fourteen recombinant Fabs were tested against HCMV-positive PBLs from a patient with ongoing HCMV infection. Three clones were found to react specifically with the nuclei of these cells. These three recombinant Fabs were subsequently tested, individually and pooled together, against 60 PBL samples taken from immunosuppressed patients. The reactivity observed was comparable to that obtained with mouse monoclonal antibodies commercially available for this purpose. The three recombinant Fabs were shown to react specifically with the 65-kDa viral tegument phosphoprotein encoded by UL83 (pUL83), which is the most abundant viral antigen in HCMV-infected PBLs.

Construction of a polyepitope fusion antigen of human cytomegalovirus ppUL32 and detection of specific antibodies by ELISA.

We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides can be valuable diagnostic material in the serology of HCMV infection (5, 6, 13). In this work we fused sequences expressing two different epitopes (aa 1005-1048 and aa 595-614) contained in the basic phosphoprotein of 150 KD coded by UL32 (1, 2), (ppUL32), which has repeatedly been shown to be the strongest immunogen present in the viral particle. The fusion protein was tested by ELISA with HCMV-positive human sera in comparison with other fusion proteins of ppUL32. We found that the double epitope fusion protein was recognised by IgM present in a larger number of sera and with more intense reactions than all the other ppUL32 fusion proteins. The double epitope reacted positively with 81.3% and, when denatured, with 94.7% of IgM-positive sera respectively. IgG reactivity was also high, reaching a percentage of 90.7.

Enzyme-linked immunoadsorbent assay for the detection of cytomegalovirus-IgM: comparison between eight commercial kits, immunofluorescence, and immunoblotting.

Eight commercially available enzyme-linked immunoadsorbent assays (ELISA) for the detection of cytomegalovirus (CMV)-specific IgM were used in parallel to determine the presence of CMV-IgM in 123 serum samples from pregnant women. The results obtained with the eight kits were compared. Based on concordance of six or more of the eight kits, we assessed sensitivity, specificity, and overall agreement, as well as incidence of false-positive and -negative results for each kit. The results obtained by ELISA were then compared with those obtained by immunofluorescence (IF) and immunoblotting (IB). Our study did not single out one outstanding ELISA kit among the eight evaluated, nor did it suggest that IF or IB are better than ELISA. Furthermore our results indicate that IB might be useful in several cases as, beside its good sensitivity, most IB-false-positive sera are easily recognized as reacting exclusively with pp150, the unique reactivity to pp150 not being among the IB profiles of IB-true-positive sera. Nevertheless 14.6% of sera remained CMV-IgM-indeterminate.