A novel application of affinity biosensor technology to detect antibodies to mycolic acid in tuberculosis patients.
Tuberculosis has re-emerged as a global health problem due to co-infection with HIV and the emergence of drug-resistant strains of Mycobacterium tuberculosis. HIV co-infection introduced a 30% underestimation in TB diagnosis based on sputum analysis, calling for a reliable and fast serodiagnostic assay to assist in the management of TB in HIV-burdened populations. Serodiagnosis with mycobacterial lipid cell wall antigens gave promising results, in particular with LAM and cord factor. Free mycolic acids have also been considered because they are unique in structure to each species of Mycobacterium and can be economically extracted and purified. In a standard immunoassay such as ELISA, however, an unacceptable number of false positive and false negative test results were obtained. Here we report a much improved biosensor method to detect antibodies to mycolic acids in patient serum as surrogate markers of active tuberculosis. Mycolic acid (MA) liposomes were immobilized on a non-derivatized twin-celled biosensor cuvette and blocked with saponin. A high dilution of serum was used to calibrate the binding signal of the two cells, followed by contact with patient serum at a lesser dilution, but pre-incubated with either antigen-carrying, or empty liposomes. The serum, or the protein A purified IgG thereof, from sputum-positive tuberculosis patients could be inhibited from binding to the MA in the biosensor by prior incubation with MA-containing liposomes. The accuracy of the inhibition test was 84% if HIV-positive patients for whom a negative TB sputum analyses could not be relied upon to serve as a reference standard were excluded. If biosensor technology could be made suitable for high throughput screening, then it may provide the solution to the serodiagnosis of tuberculosis against a background of HIV.
Thanyani
Simon T
ST
Department of Biochemistry, University of Pretoria, Pretoria, 0002, South Africa.
Roberts
Vanessa
V
Siko
D Gilbert R
DG
Vrey
Pieter
P
Verschoor
Jan A
JA
D000293:Adolescent; D000328:Adult; D000368:Aged; D000906:Antibodies / Q000097:blood* / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive / Q000276:immunology; D015374:Biosensing Techniques / Q000379:methods*; D002138:Calibration; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006678:HIV / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood* / Q000276:immunology; D008875:Middle Aged; D009171:Mycolic Acids / Q000276:immunology*; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000295:instrumentation / Q000379:methods; D013997:Time Factors; D014376:Tuberculosis / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*
D000906:Antibodies; D007074:Immunoglobulin G; D009171:Mycolic Acids