Improved ELISA with immunoabsorbent-purified mycobacterial antigen for serodiagnosis of tuberculosis

Abstract

An antigen was purified from Mycobacterium tuberculosis H37Ra culture filtrate by immunoabsorbent affinity chromatography with CNBr-activated sepharose 4B column coupled with pooled gamma-globulin fraction from patients with active tuberculosis. The column was washed extensively with PBS and eluted with 3M sodium thiocyanate. Peak fractions were pooled and used as coating antigen in an ELISA. Sera from 86 normal subjects and 54 patients with active tuberculosis were tested against the immunoabsorbed antigen by ELISA with biotin-conjugated anti-human globulin and avidin-peroxidase reagents. At a selected "cut-off" dilution of 320, 49 (91%) of 54 sera from active cases and 8 (9.3%) of 86 sera from normal subjects gave positive test results--a sensitivity and specificity each of 91%, compared with our previous results of sensitivity 75% and specificity 83% with PPD as antigen.