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.

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

I. Sample Requirements
1. To remove impurities from the sample, centrifuge the sample prior to detection (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, the standard should be diluted with negative serum having a background value below the detection limit, and used to establish a standard curve for calculating the actual concentration.
3. If the measured concentration of a sample exceeds the highest value of the standard curve, appropriately dilute and retest to ensure the result falls within the valid measurement range.

II. Detection Workflow

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

2.2 Detection Workflow:

* Note: Prior to testing, pre-mix R1 and R2 at a 1:1 volume ratio, and use the mixture 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 Validation of Performance Parameters:

LOD:Detect standard C1 repeatedly 20 times, calculate the mean signal and SD. Calculate the concentration corresponding to mean signal +2×SD using the standard curve; this value is defined as the Limit of Detection (LOD).

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

Inter-assay Precision (Inter-Precision): Samples of known low, medium, and high concentrations are tested across 5 independent experiments. Standards and samples are both tested using Detection Protocol 1 with 5 replicates. All concentration samples show low coefficients of variation (%CV <10%), indicating good reproducibility across different batches.

Accuracy (Recovery):

Mix high and low concentration QC samples at a 1:9 ratio and test their recovery rates. Both fall within 80%-120% , indicating that the method has good accuracy.

Specificity: Dilute the following proteins to 0.1μg/mL using the standard buffer, and test the cross-reactivity rate.

•Traceability: Tested against reference material NIBSC 12/154, where 1 IU/mL ≈ 36 pg/mL.

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.