Mouse IFN-γ Kit (HICA)

Principle of the Assay:

This kit employs a homogeneous immuno chemiluminescence assay (HICA) based on the double-antibody sandwich method for the detection of cytokine concentrations. The operation is simple and requires no washing steps.

The detection system consists of two types of microspheres: receptor 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 receptor microspheres is less than 200 nm, singlet oxygen generated by light excitation can transfer to the receptor microspheres and trigger chemiluminescence. Conversely, if the target protein is absent, the distance between the microspheres is too large, and no signal is produced.

By measuring the intensity of the chemiluminescent signal, the target protein can be quantitatively analyzed. This method offers advantages such as simplicity, rapid reaction, and high sensitivity.

Note: Microtiter plates (384- or 96-well plates, white, shallow wells)

I. Sample Requirements
1. To remove impurities from samples, samples need to be centrifuged before detection (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, standards should be diluted using negative serum with background values below the detection limit, and a standard curve should be established accordingly to calculate the actual concentration.
3. If the measured concentration of a sample exceeds the highest value of the standard curve, it needs to be appropriately diluted and re-tested to ensure the result falls within the valid measurement range.

II. Detection Procedure 

2.1 Preparation of Standard Gradient Samples Reconstitute the lyophilized standard with 50μL deionized water, then dilute the standard using standard buffer. The recommended dilution scheme is shown in the table below:

2.2 Detection Procedure:

* Note: Before testing, pre-mix R1 and R2 at a volume ratio of 1:1, and use within 1 hour after mixing.

III. Performance Testing

3.1 Example of Complete Standard Curve:

3.2 Performance Parameter Verification:

LOD: Repeat detection of standard C1 for 20 times, calculate the mean signal and SD, and use the standard curve to calculate the concentration value corresponding to mean signal + 2×SD, which is the Limit of Detection (LOD).

Precision：
Intra-Precision: On the same experimental plate, known low, medium, and high concentration samples are each tested in 10 replicates to evaluate the intra-plate precision of the assay. Standards and samples are detected using different procedures. The coefficient of variation (%CV) of detection results for samples at each concentration is below 10%, indicating good reproducibility of this method within the same batch detection.

Inter-Precision: Known low, medium, and high concentration samples are tested in 5 independent experiments. Standards and samples are all detected using Detection Procedure 1 with 5 replicates set. The detection results of samples at each concentration show low coefficients of variation (%CV < 10%), indicating good reproducibility of this method across different batch detections.

Accuracy (Recovery):

High and low concentration quality control samples are mixed at a ratio of 1:9 and tested for recovery rate, all within 80%-120%, indicating that this method has good accuracy.

Specificity: The following proteins are diluted to 0.3μg/mL using standard buffer, and cross-interference rates are tested.

Reagent R3 should be protected from light during use. It is recommended to perform sample addition and incubation under green light (<100 LUX). Each test requires recalibration, with at least duplicate wells for each standard concentration point. Use a four-parameter (weighting: 1/Y²) fitting method for calculation. During incubation, temperature and time must be controlled. It is advisable to cover the microplate with a film, and an ELISA reader with ALPHA function should be selected. The dilution matrix of the calibrator should match that of the test samples. The reconstituted calibrator should be used within 2 hours. Components from different reagent kit batches must not be mixed.