Human IFN-γ Kit (HICA)

Test Principle:

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 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 microspheres into close proximity. When the distance between the donor and acceptor microspheres is less than 200 nm, singlet oxygen generated by light excitation can transfer to the acceptor 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 offers advantages such as simplicity, rapid reaction, and high sensitivity.

Name	Specification	Component Specification
Detection Reagent R1	500T	2mL/bottle×1
	2000T	8mL/bottle×1
	10000T	40mL/bottle×1
Detection Reagent R2	500T	2mL/bottle×1
	2000T	8mL/bottle×1
	10000T	40mL/bottle×1
Detection Reagent R3	500T	5mL/bottle×1
	2000T	20mL/bottle×1
	10000T	100mL/bottle×1
Standard	500T	0.05μg lyophilized product×1
	2000T	0.05μg lyophilized product×2
	10000T	0.05μg lyophilized product×5
Standard Buffer	500T	6mL/bottle×1
	2000T	12mL/bottle×1
	10000T	30mL/bottle×1

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

I. Sample Requirements
1. To remove impurities from the sample, it must be centrifuged prior to detection (1000 ×g, 10 minutes).
2. When detecting target proteins in serum samples, the Standard should be diluted with negative serum that has a background value 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 must be appropriately diluted and retested to ensure the results fall within the valid measurement range.

II. Detection Protocol

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

Gradient	Concentration (pg/mL)	Standard(μL)	Diluent (μL)
C12	100000	20μLCalibrator	180
C11	30000	60μL C12	140
C10	10000	60μL C11	120
C9	3000	60μL C10	140
C8	1000	60μL C9	120
C7	300	60μL C8	140
C6	100	60μL C7	120
C5	30	60μL C6	140
C4	10	60μL C5	120
C3	3	60μL C4	140
C2	1	60μL C3	120
C1	0	—	120

 

 

2.2 Detection Protocol:

Detection Protocol	Protocol 1 (37℃ Rapid Detection)	Protocol 2 (Room Temperature Detection)
​Step 1:	Add 2µL of sample to 8µL of pre-mixed R1+R2*	Add 2µL of sample to 8µL of pre-mixed R1+R2*
Incubation	​Shake at 400 rpm for 1 min, incubate at 37℃ for 15 minutes	​Shake at 400 rpm for 1 min, ​incubate at room temperature for 60 minutes
​Step 2:	Add 10µL R3, protect from light/green light	Add 10µL R3, protect from light/green light
Incubation	​Shake at 400 rpm for 1 min, incubate at 37℃ for 10 minutes, protect from light/green light	​Shake at 400 rpm for 1 min, ​incubate at room temperature for 30 minutes, protect from light/green light
Read​	Instrument reading, protect from light/green light	Instrument reading, protect from light/green light

 

* Note: Prior to testing, pre-mix R1 and R2 at a volume ratio of 1:1, and use the mixture 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 Complete Standard Curve:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.2 Validation of Performance Parameters:

LOD:Repeatedly detect 20 times the Standard C1, calculate the mean signal and SDmean signal + 2×SDLimit of Detection (LOD)

Detection Protocol

Matrix

LOD (pg/mL)

Quantitative Range (pg/mL)

Dynamic Range (pg/mL)

Protocol 1

Buffer

1.07

1.1~10000

0~100000

DMEM

1.40

1.4~10000

RPMI

16.80

16.8~10000

HumanSerum

0.28

0.3~10000

Protocol2

Buffer

2.40

2.4~10000

 

Precision (Precision):
Intra-assay Precision (Intra-Precision): On the same plate10detected using different protocols

Detection Protocol

Matrix

Precision

Low Concentration

High Concentration

Protocol 1

Buffer

1.9%

2.5%

DMEM

3.3%

1.2%

RPMI

1.2%

1.1%

HumanSerum

2.8%

2.9%

Protocol 2

Buffer

3.7%

3.7%

 

Inter-assay Precision (Inter-Precision): Known low, medium, and high concentration samples were detected in 5Protocol 15

Detection Protocol

Matrix

Inter-assayPrecision

Low Concentration

High Concentration

Protocol 1

Buffer

2.0%

1.7%

 

Accuracy (Recovery):

High and low concentration quality control materials were mixed at a ratio of 1:9recovery rates were tested80%-120%accuracy

Detection Protocol

Matrix

Recovery Rate

Protocol 1

Buffer

89.6%

DMEM

95.9%

RPMI

92.0%

HumanSerum

90.8%

Protocol 2

Buffer

93.2%

 

Specificity: The following proteins were diluted using Standard0.03μg/mL

Detection Protocol

Tested Substance

Cross-reactivity Rate

Protocol 1

Mouse IFN-γ

0.00%

Rat IFN-γ

0.00%

Canine IFN-γ

0.00%

Rhesus Macaque IFN-γ

0.00%

 

Traceability: Certified Reference Material NIBSC 82/587.

 

 

 

 

 

Reagent R3 must be protected from light. It is recommended to perform sample addition and incubation under green light (<100 LUX).

Each test requires recalibration. At least duplicate wells should be set for each concentration of the standard, and a four-parameter (weighting 1/Y²) fitting calculation should be applied.

Temperature and time must be controlled during incubation. The microplate 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. Reconstituted calibrators should be used within 2 hours.

Components from different reagent kit lots must not be mixed.