Combinatorial use of antibodies to secreted mycobacterial proteins in a host immune system-independent test for tuberculosis.

Laboratory diagnosis of tuberculosis is often difficult. Immunodetection of circulating Mycobacterium tuberculosis proteins shed during active infection would not depend on an intact host immune response and could take advantage of the speed and low costs afforded by antibody-based assays. We previously showed that patients with active tuberculosis had increased levels of circulating antigen 85 (Ag85) proteins independent of their tuberculin skin test status (S. I. Bentley-Hibbert, X. Quan, T. Newman, K. Huygen, and H. P. Godfrey, Infect. Immun. 67:581-588, 1999). To extend these observations to a Mycobacterium bovis BCG-vaccinated population and to another secreted mycobacterial protein, Ag85 and PstS-1 (protein antigen B, p38 antigen) were quantified in sera from 97 Chilean tuberculosis patients and healthy controls (many of whom had received BCG as children) using dot immunobinding, mouse monoclonal anti-BCG Ag85 complex antibody, and chicken antipeptide antibodies reactive with M. tuberculosis Ag85B and PstS-1. The latter antibodies had been raised to peptide-derived immunogens expressed on a novel proprietary protein carrier in Escherichia coli. Median serum Ag85 levels measured by using either anti-Ag85 antibody were significantly higher in patients with active tuberculosis than in healthy controls (P, <0.001 to 0.01); the median serum PstS-1 levels were similar in patients and controls. The sensitivity of significantly elevated circulating Ag85 levels in patients with pulmonary tuberculosis measured by anti-Ag85 complex or anti-Ag85B antibodies was 60 and 55%, respectively, but increased to 77% when results obtained with both anti-Ag85 antibodies were considered jointly (P < 0.02). The corresponding specificities for individual and joint consideration were 95, 85, and 80%, respectively. These results indicate that elevated Ag85 levels can be detected in patients with active tuberculosis even after BCG vaccination and suggest that combinatorial use of antibodies directed at different epitopes of this protein could provide a viable strategy for developing new host immune response-independent diagnostic tests for tuberculosis.

Serum antigen 85 levels in adjunct testing for active mycobacterial infections in orangutans.

Diagnosis of active mycobacterial disease in orangutans (Pongo pygmaeus) has been impeded by high levels of non-specific intradermal skin test reactivity to mycobacterial antigens. This may be due in part to cross reactivity between antigens, tuberculin concentrations used or other species-specific factors. Antigen 85 (Ag85) complex proteins are major secretory products of actively growing mycobacteria, and measurement of serum Ag85 could provide a method for determining active mycobacterial infections that was not dependent on host immunity. Serum Ag85 was measured by dot-immunobinding assay using monoclonal anti-Ag85, purified Ag85 standard and enhanced chemiluminescence technology in coded serum samples from 14 captive orangutans from a zoo in Colorado, 15 semi-captive orangutans in Malaysia, and 19 free-ranging wild orangutans in Malaysia. Orangutans from Colorado (USA) were culture negative for Mycobacterium tuberculosis and M. avium, although all had laboratory suspicion or evidence of mycobacterial infection; median serum Ag85 was 10 microU/ml (range, <0.25-630 microU/ml). Of the semi-captive orangutans, six were skin test reactive and two were culture positive for M. avium on necropsy. Median serum Ag85 for this group was 1,880 microU/ml (0.75-7,000 microU/ml), significantly higher than that of Colorado zoo or free-ranging Malaysian orangutans. Median serum Ag85 in the latter group was 125 microU/ml (range, 0.75-2,500 microU/ml). These data suggest that suggest that additional studies using more specific reagents and more samples from animals of known status are appropriate.

Pathophysiology of antigen 85 in patients with active tuberculosis: antigen 85 circulates as complexes with fibronectin and immunoglobulin G.

Antigen 85 (Ag85) complex proteins are major secretory products of Mycobacterium tuberculosis and induce strong cellular and humoral immune responses in infected experimental animals and human beings. We have previously shown that nanogram doses of these 30- to 32-kDa fibronectin-binding proteins inhibit local expression of delayed hypersensitivity by a T-cell fibronectin-dependent mechanism. Circulating levels of Ag85 might be expected to be elevated in patients with active tuberculosis and possibly to play a role in systemic anergy in these patients. To test this hypothesis, Ag85 was measured in serum and urine by a monoclonal antibody-based dot immunobinding assay in 56 patients and controls with known skin test reactivity. Median serum Ag85 levels were 50- to 150-fold higher in patients with active tuberculosis than in patients with active M. avium-intracellulare disease or other nontuberculous pulmonary disease or in healthy controls (P < 0.001). The median and range of serum Ag85 in patients with active tuberculosis was not significantly different between skin test-positive and -negative subjects. Patients with active M. avium disease could be distinguished from those with disease due to M. tuberculosis by monoclonal anti-Ag85 antibodies of appropriate specificities. No increases in urinary Ag85 were detected in any patient, regardless of the Ag85 level in serum. Chromatographic analysis and immunoprecipitation studies of serum revealed that Ag85 existed in the serum of these patients complexed to either fibronectin or immunoglobulin G (IgG). Uncomplexed circulating Ag85 was demonstrable in serum from fewer than 20% of patients with active tuberculosis. In patients with active tuberculosis, Ag85 is therefore likely to circulate primarily as complexes with plasma fibronectin and IgG rather than in unbound form. The existence of Ag85 complexes with plasma proteins would account for its lack of urinary clearance.