Human TNF-β Kit (HICA)

Test Principle:

This kit employs a homogeneous immuno chemiluminescence assay (HICA) for the detection of cytokine concentrations, featuring a simple operation process with no washing steps required.

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 upon light excitation can transfer to the receptor microsphere, triggering chemiluminescence. Conversely, if the target protein is absent, the microspheres remain too far apart, resulting in no signal.

By measuring the intensity of the chemiluminescent signal, quantitative analysis of the target protein can be achieved. This method is characterized by its simplicity, rapid reaction, and high sensitivity.

Note: A microplate (384 or 96-well plate, white, shallow well) is recommended.

I. Sample Requirements
1. To remove impurities from the samples, centrifugation is required before testing (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, standards should be diluted using negative serum with a background value below the detection limit, and a standard curve should be established to calculate the actual concentration.
3. If the measured concentration of a sample exceeds the highest value of the standard curve, appropriate dilution and retesting are required to ensure the results fall within the valid measurement range.

II. Testing Procedure

2.1 Preparation of Standard Gradient Samples Reconstitute lyophilized standards with 50μL of deionized water, then dilute with standard buffer. The recommended dilution scheme is as follows:

2.2 Testing Process: 

Here is the translated content with preserved HTML structure: ```html 
 

Note:

* Before testing, pre-mix R1A, R1F, and R2 in a volume ratio of 1:1:1. After mixing, use within 1 hour.

* 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 a complete standard curve:

3.2 Performance parameter validation:

LOD: Repeat testing of 20 replicates of standard C1, calculate the mean signal and SD, then use the standard curve to determine the concentration corresponding to mean signal+2×SD, which is the limit of detection (LOD).

```
 

•Detection range: 0~100,000 pg/mL.

•Quantitative range: 0.35~3000 pg/mL.

Precision (Precision): 
Intra-assay Precision (Intra-Precision): On the same microplate, low, medium, and high concentration samples were each tested 10 times to evaluate the intra-assay precision. Both standards and samples were detected using different procedures. The coefficient of variation (%CV) for all concentration samples was below 10%, indicating good reproducibility within the same batch.

Inter-assay Precision: Known samples with low, medium, and high concentrations were tested in 5 independent experiments. Both standards and samples were analyzed using Assay Procedure 1 with 5 replicates. The results for samples at each concentration showed low coefficient of variation (%CV <10%), indicating good reproducibility across different assay batches.

Accuracy (Recovery):

High and low concentration quality control materials were mixed at a 1:9 ratio, and their recovery rates were tested, all within 80%-120%, indicating good accuracy of the method.

Specificity: Dilute the following proteins to the corresponding concentrations using standard buffer solution to test the cross-interference rate.

•Traceability: Testing reference material NIBSC 87/640, 1IU/mL≈12pg/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) conditions.

Recalibration is required for each test, with at least duplicate wells for each concentration point of the standard. A four-parameter (weighted 1/Y²) curve fitting method should be used for calculation.

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

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

Components from different reagent kit lots must not be mixed.