A Subgroup of Latently Mycobacterium tuberculosis Infected Individuals Is Characterized by Consistently Elevated IgA Responses to Several Mycobacterial Antigens

Elevated antibody responses to Mycobacterium tuberculosis antigens in individuals with latent infection (LTBI) have previously been linked to an increased risk for progression to active disease. Studies in the field focussed mainly on IgG antibodies. In the present study, IgA and/or IgG responses to the mycobacterial protein antigens AlaDH, NarL, 19 kDa, PstS3, and MPT83 were determined in a blinded fashion in sera from 53 LTBI controls, 14 healthy controls, and 42 active TB subjects. Among controls, we found that elevated IgA levels against all investigated antigens were not randomly distributed but concentrated on a subgroup of <30%—with particular high levels in a small subgroup of ~5% comprising one progressor to active TB. Based on a specificity of 100%, anti-NarL IgA antibodies achieved with 78.6% sensitivity the highest accuracy for the detection of active TB compared to healthy controls. In conclusion, the consistently elevated IgA levels in a subgroup of controls suggest higher mycobacterial load, a risk factor for progression to active TB, and together with high IgG levels may have prognostic potential and should be investigated in future large scale studies. The novel antigen NarL may also be promising for the antibody-based diagnosis of active TB cases.

containing pure NarL protein as analysed by SDS PAGE were pooled. Densitometric SDS-PAGE analysis showed a purity of > 95 % for the final protein preparation.
19 kDa: The cells were grown in LB medium and 50 μg/mL kanamycin at 37 °C overnight. The protein expression was induced with 1 mM IPTG. The cells were harvested by centrifugation, suspended in 20 mM tris/HCl, 1 % Triton X-100, 100 mM NaCl pH 8.0, and disrupted using a LABSONIC 2000 sonicator. After centrifugation, the supernatant was applied to a metal chelate affinity column. The protein was eluted in fractions in a gradient of imidazole. The fractions containing pure 19 kDa protein as analysed by SDS PAGE were pooled. Purity analysis by automated electrophoresis with fluorimetric detection and densitometric purity measurement showed a purity of > 95 % for the final protein preparation.
PstS3: For production of PstS3, cells were grown in LB medium at 37° C, induced with 1 mM IPTG and incubated at 37° C for further 4 h. After centrifugation, the cells were suspended in 50 mM potassium phosphate buffer, pH 7.3. The cells were sonicated using a Bandelin GM70 sonifyer. After centrifugation, the pellet was solubilized in buffer containing 8 M urea. The protein was purified on a metal chelate affinity column. The protein solution was refolded by buffer exchange on a gel filtration column. The protein containing high molecular weight fraction of this gel filtration step was collected and subjected to another affinity purification step on a metal chelate affinity column.
Elution was done using 500 mM imidazole. The fractions containing pure PstS3 protein were pooled.
Purity analysis by automated electrophoresis with fluorimetric detection and densitometric purity measurement showed a purity of > 95 % for the final protein preparation.
MPT83: The cells were grown in Terrific Broth medium containing 400 μg/mL ampicillin and 34 μg/mL chloramphenicol overnight. The protein expression was induced with 0.2 mM IPTG. After further 4.5 h of incubation, the cells were harvested by centrifugation and suspended in BugBusterTM Protein Extraction Reagent (Novagen). After centrifugation, the pellet was resuspended in 20 mM tris/HCl, 150 mM NaCl, pH 8.0 containing 1 % Triton X-100. The suspension was homogenized using a Miccra D-8 homogenizer. After a washing step, the pellet was solubilized using 8 M urea as denaturation agent. MPB83 was refolded on a gel filtration column. The refolded protein was bound to an anion exchange column and eluted in a salt gradient from 0-500 mM NaCl.
The fractions containing pure MPB83 protein as analysed by SDS PAGE were pooled. Purity analysis by automated electrophoresis with fluorimetric detection and densitometric purity measurement showed a purity of > 95 % for the final protein preparation. §1.2 Enzyme-linked immunosorbent assay The antigens were diluted in 0.15 M PBS, pH 7.5 (concentration (measured by Lowry based assay) = 1µg antigen/ml coating buffer). 100 µl of the coating solution (antigen in coating buffer) was pipetted into each well of the microtiter plates (Greiner, Maxisorb). The microtiter plates were incubated over night at 2-8°C. The following day the plates were washed 3x with wash solution (0.15 3 M PBS, pH 7.5 + 0.05 % Tween-20, 300 µl/well). 300 µl of blocking solution (0.15 M PBS, pH 7.5 + 1 % BSA) were pipetted into each well of the coated plates and the plates were incubated for 2 h at 37°C.     anti-PstS3 IgA a Spearman's coefficient r of rank correlation (95% CI for r)