Development of a human gamma interferon enzyme immunoassay and comparison with tuberculin skin testing for detection of Mycobacterium tuberculosis infection

Abstract

A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-gamma) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-gamma per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-gamma. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-gammas. Cross-reactions with other human cytokines or with IFN-gammas derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-gamma in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-gamma were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-gamma responsiveness in vitro. These results support the further study of the blood culture-IFN-gamma EIA system as an alternative to skin testing for the detection of human TB infection.

FIG. 1

Standard curve of the human IFN-γ EIA derived from the mean optical densities (OD) (450 nm) ± standard deviations of 16 replicates over four assays.

FIG. 2

Human IFN-γ EIA standard curves after 60, 75, 90, and 120 min of incubation of the assay.

FIG. 3

Differences between mean (± standard error of the mean) IFN-γ responses (in international units per milliliter) to human PPD (HuPPD), avian PPD (AvPPD), bovine PPD (BoPPD), and mitogen stimulation antigens of tuberculin skin test-negative (▨) and -positive (□) individuals. *, significant difference (P < 0.01).