Human Semaphorin 4D (SEMA4D) E

Introduction

Human Semaphorin 4D (SEMA4D) ELISA Kit

Item	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
Composition	Pre-coated, ready to use 96-well strip plate Standard (freeze dried) Standard Diluent Detection Reagent A Detection Reagent B Assay Diluent A Assay Diluent B TMB Substhumane Stop Solution Wash Buffer(30 x concenthumane) Plate sealer for 96 wells Instruction manual	1 2 1 × 20ml 1× 120μl 1× 120μl 1 × 12ml 1 × 12ml 1 × 9ml 1 ×6ml 1 ×20ml 2 1	Conform

Human Semaphorin 4D (SEMA4D) ELISA Kit

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

Human Semaphorin 4D (SEMA4D) ELISA Kit

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 and standards;
2. Add 50µL standard or sample to each well.
And then add 50µL prepared Detection Reagent A immediately.
Shake and mix. Incubate 1 hour at 37℃;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37℃;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37℃;
7. Add 50µL Stop Solution. Read at 450 nm immediately.
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