Introduction

	Standard	Test Result
Description	This immunoassay kit allows for the speciﬁc measurement of this index in serum, Plasma , Urine ,tissue homogenates and Cell culture supernates and Other biological ﬂuids..	Conform
Identiﬁcation	Colorimetric	Positive
	Pre-coated, ready to use 96-well strip plate	1
	Standard (freeze dried)	2
	Standard Diluent	1 × 20ml
	Detection Reagent A	1× 120μl
	Detection Reagent B	1× 120μl
	Assay Diluent A	1 × 12ml
Composition	Assay Diluent B	1 × 12ml	Conform
	TMB Substhumane	1 × 9ml
	Stop Solution	1 ×6ml
	Wash Buffer(30 x concenthumane)	1 ×20ml
	Plate sealer for 96 wells	2
	Instruction manual	1

Test principle

The microtiter plate provided in this kit has been pre-coated with an antibody speciﬁc to this index. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation speciﬁc for this index and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain the index, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of the index in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Recovery

Matrices listed below were spiked with certain level of recombinant the index and the recovery humanes were calculated by comparing the measured value to the expected amount of the index in samples.

Matrix	Recovery range (%)	Average(%)
serum(n=5)	81-93	86
EDTA plasma(n=5)	80-97	88
heparin plasma(n=5)	90-101	95

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concenthumanion of the index and their serial dilutions. The results were demonsthumaned by the percentage of calculated concenthumanion to the expected.

Sample	1：2	1：4	1：8	1：16
serum(n=5)	82-96%	83-98%	81-99%	93-101%
EDTA plasma(n=5)	88-101%	86-95%	90-102%	80-93%
heparin plasma(n=5)	80-91%	82-90%	95-104%	79-95%

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.

CV(%) = SD/meanX100

Intra-Assay: CV<10%

Inter-Assay: CV<12%

Stability

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra inﬂuence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Assay procedure summary

1.Prepare all reagents, samples andstandards;

2.Add 100µL standard or sample to each well. Incubate 2 hours at37oC;

3.Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at37oC;

4.Aspirate and wash 3times;

5.Add 100µL prepared Detection Reagent B. Incubate 1hour at37oC;

6.Aspirate and wash 5times;

7.Add 90µL Substrate Solution. Incubate 15-25 minutes at37oC;

8.Add 50µL Stop Solution. Read at 450nm

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