Fusion of selected regions of mycobacterial antigens for enhancing sensitivity in serodiagnosis of tuberculosis.

ABSTRACT

Serodiagnosis of tuberculosis requires detection of antibodies against multiple antigens of Mycobacterium tuberculosis, because antibody profiles differ among the patients. Using fusion proteins with epitopes from two or more antigens would facilitate in the detection of multiple antibodies. Fusion constructs tn1FbpC1-tnPstS1 and tn2FbpC1-tnPstS1 were produced by linking truncated regions of variable lengths from FbpC1 to the N-terminus of the truncated PstS1. Similarly a truncated fragment of HSP was linked to the N-terminus of a truncated fragment from FbpC1 to produce tnHSP-tn1FbpC1. ELISA analysis of the plasma samples of TB patients against tn2FbpC1-tnPstS1 showed 72.2% sensitivity which is nearly the same as the expected combined value for the two individual antigens. However, the sensitivity of tn1FbpC1-tnPstS1 was lowered to 60%. tnHSP-tn1FbpC1 showed 67.7% sensitivity which is slightly less than the expected combined value for the two individual antigens, but still significantly higher than that of each of the individual antigen. Data for secondary structure analysis by CD spectrometry was in reasonable agreement with the X-ray crystallographic data of the native proteins and the predicted structure of the fusion proteins. Comparative molecular modeling suggests that the epitopes of the constituent proteins are better exposed in tn2FbpC1-tnPstS1 as compared to those in tn1FbpC1-tnPstS1. Therefore, removal of the N-terminal non-epitopic region of FbpC1 from 34-96 amino acids seems to have unmasked at least some of the epitopes, resulting in greater sensitivity. The high level of sensitivity of tn2FbpC1-tnPstS1 and tnHSP-tn1FbpC1, not reported before, shows that these fusion proteins have great potential for use in serodiagnosis of tuberculosis.