Method Description

Primary method:

The LIAISON QuantiFERON-TB Gold Plus detects the analyte interferon-gamma (IFN-gamma) by direct, sandwich chemiluminescence immunoassay. Anti IFN- gamma monoclonal (mouse) antibodies are used for coating magnetic particles (solid phase) and anti IFN-gamma monoclonal (mouse) antibodies are linked to an isoluminol derivative forming an isoluminol-antibody conjugate. Binding between the monoclonal antibodies and isoluminol in the conjugate is mediated by a biotin-streptavidin immunocomplex.

During the first incubation, analyte IFN-gamma present in calibrators, samples, or controls will bind to the solid phase and conjugated monoclonal antibodies and form a sandwich. Any unbound material in the test sample is removed with a wash cycle. During the second incubation, assay buffer W is added to reduce nonspecific binding, followed by a second wash cycle.

Next, the starter reagents are added and a flash chemiluminescence reaction is induced. The light signal, and hence the amount of isoluminol-antibody conjugate, is measured by a photomultiplier as relative light units (RLU) and is indicative of the amount of analyte IFN-gamma present in calibrators, samples, or controls.(Package insert: LIAISON QuantiFERON-TB Gold Plus. DiaSorin; EN-200/077-002, 01/2022)

As needed:

The QuantiFERON-TB Gold Plus test uses specialized blood collection tubes, which are used to collect whole blood via venipuncture. Two of the 4 collection tubes contain antigens representing certain Mycobacterium tuberculosis proteins and 2 of the tubes are controls (Nil and mitogen). TB Antigen Tube 1 (TB1) and TB Antigen Tube 2 (TB2) both contain peptide antigens from the Mycobacterium tuberculosis (MTB)-complex-associated antigens ESAT-6 and CFP-10 and are designed to elicit a cell-mediated immunity (CMI) response from CD4+ T-helper lymphocytes. The TB2 tube contains an additional set of peptides targeted to the induction of a CMI response from CD8+ cytotoxic T lymphocytes. The tubes are immediately shaken after collection to mix antigen with the blood and then incubated at 37° C + or - 1 degree C within 16 hours from collection. The tubes are incubated for 16 to 24 hours, and then plasma is tested for the presence of IFN-gamma produced in response to the peptide antigens. The amount of IFN-gamma is measured by enzyme-linked immunosorbent assay in IU/mL.

A test is considered positive for an IFN-gamma response to the TB antigen cocktail when results are significantly above the negative control value. The Nil tube adjusts for background, heterophile antibody effects, or nonspecific IFN-gamma in blood samples. The IFN-gamma level of the Nil tube is subtracted from the IFN-gamma level for the TB1 and TB2 antigen tubes and Mitogen tube. The mitogen-stimulated plasma sample serves as an IFN-gamma positive control for each specimen tested. A low response to mitogen (<0.5 IU/mL) indicates an indeterminate result when a blood sample also has a negative response to the TB Antigens. This pattern may occur with insufficient lymphocytes, reduced activity due to prolonged specimen transport, or improper specimen handling, including filling/mixing of blood tubes or inability of the patient's lymphocytes to generate IFN-gamma. Elevated levels of IFN-gamma in the Nil tube may occur with the presence of heterophile antibodies or due to intrinsic IFN-gamma secretion.(Package insert: QuantiFERON-TB Gold Plus (QFT-Plus). Qiagen; 06/2022)