Human TNF-α Kit (HICA)

Test Principle:

This kit employs a homogeneous immuno chemiluminescence assay (HICA) for the detection of cytokine concentrations. The operation is simple and requires no washing steps.

The detection system consists of two types of microspheres: acceptor microspheres conjugated with antibody 1 targeting the protein of interest, and donor microspheres conjugated with streptavidin, along with biotin-labeled antibody 2. During the reaction, the target protein binds with the antibodies and microspheres to form an immune complex, bringing the two types of microspheres into close proximity. When the distance between the donor and acceptor microspheres is less than 200 nm, singlet oxygen generated upon light excitation can transfer to the acceptor microspheres, triggering chemiluminescence. Conversely, if the target protein is absent, the microspheres remain too far apart, and no signal is produced.

By measuring the intensity of the chemiluminescent signal, the target protein can be quantitatively analyzed. This method features simplicity of operation, rapid reaction, and high sensitivity.

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Note: The recommended plates are microplates (384 or 96 wells, white, shallow well)

Reagent R3 should be protected from light during use, and sample addition and incubation are recommended to be performed under green light (<100 LUX).

Each test requires recalibration, with at least duplicate wells for each concentration point of the standard. A four-parameter (weighting 1/Y²) fitting method should be used for calculation.

Temperature and time should be carefully controlled during incubation. Microplates should be covered with film, and a microplate reader with ALPHA function is recommended.

The dilution matrix for calibrators should match the sample matrix, and reconstituted calibrators should be used within 2 hours.

Components from different reagent kit lots must not be mixed.