Mouse IFN-γ Kit (HICA)

Principle of the Assay:

This kit utilizes 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 includes 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 by photoexcitation of the donor microspheres can transfer to the acceptor microspheres and trigger 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 offers advantages such as simplicity, rapid reaction, and high sensitivity.

Note: It is recommended to use microplates (384or96well plates, white, with shallow wells)

I. Sample Requirements
1. To remove impurities from the sample, centrifuge the sample before testing (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, the standard should be diluted using negative serum with a background value below the lower limit of detection, 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 must be appropriately diluted and retested to ensure the results fall within the valid measurement range.

II. Testing Procedure

2.1 Preparation of Standard Gradient Samples Reconstitute the lyophilized standard with 100μL of deionized water, then dilute it 50-fold with standard diluent. Subsequently, perform serial dilutions according to the table below (adjustments may be made based on specific conditions):

2.2 Testing Procedure:

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

* If testing RPMI samples, you may additionally add RPMI enhancer solution (Catalog No.: UA086145). Pre-mix 50 μL of R3 with 100 μL of RPMI enhancer solution before adding, which significantly improves reagent performance.

III. Performance Testing

3.1 Example of Complete Standard Curve:

3.2 Performance Parameter Validation:

LOD: Detect standard C1 repeatedly for 20 times, calculate the mean signal and SD. Use the standard curve to calculate the concentration corresponding to Mean Signal+2×SD, which is the Limit of Detection (LOD).

Precision:
Intra-Assay Precision: On the same plate in a single experiment, samples with known low, medium, and high concentrations are each tested repeatedly for 10 times to evaluate the intra-assay precision. Both standards and samples are tested using different procedures. The coefficient of variation (%CV) for all concentration samples is below 10%, indicating good reproducibility of this method within the same batch.

Inter-Assay Precision: Samples with known low, medium, and high concentrations are tested in 5 independent experiments. Both standards and samples are tested using Testing Procedure 1 with 5 replicates. The test results for all concentration samples show a low coefficient of variation (%CV < 10%), indicating good reproducibility of this method across different batches.

Accuracy (Recovery):

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

Specificity: The following proteins are diluted to 0.3 μg/mL using standard diluent to test the cross-reactivity rate.

Reagent R3 must be protected from light. Sample addition and incubation should be performed under green light (<100 LUX).

Recalibration is required for each test, with at least duplicate wells for each standard concentration point. A four-parameter (weighting: 1/Y²) fitting calculation is recommended.

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

The dilution matrix for calibrators should match the sample matrix. Reconstituted calibrators must be used within 2 hours.

Components from different reagent kit lots must not be mixed.