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Multiplex Assay Kit for Kelch Like ECH Associated Protein 1 (KEAP1) ,etc. by FLIA (Flow Luminescence Immunoassay)

INrf2; KLHL19; Cytosolic inhibitor of Nrf2; Kelch-like protein 19

(Note: Up to 8-plex in one testing reaction)

Specificity

This assay has high sensitivity and excellent specificity for detection of Kelch Like ECH Associated Protein 1 (KEAP1) ,etc. by FLIA (Flow Luminescence Immunoassay).
No significant cross-reactivity or interference between Kelch Like ECH Associated Protein 1 (KEAP1) ,etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Kelch Like ECH Associated Protein 1 (KEAP1) ,etc. by FLIA (Flow Luminescence Immunoassay) and the recovery rates were calculated by comparing the measured value to the expected amount of Kelch Like ECH Associated Protein 1 (KEAP1) ,etc. by FLIA (Flow Luminescence Immunoassay) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 91-101 97
EDTA plasma(n=5) 92-101 96
heparin plasma(n=5) 89-97 92
sodium citrate plasma(n=5) 81-102 90

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Kelch Like ECH Associated Protein 1 (KEAP1) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Kelch Like ECH Associated Protein 1 (KEAP1) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Kelch Like ECH Associated Protein 1 (KEAP1) ,etc. by FLIA (Flow Luminescence Immunoassay) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 91-101% 82-101% 92-99% 95-102%
EDTA plasma(n=5) 89-96% 88-99% 89-101% 99-105%
heparin plasma(n=5) 80-101% 82-93% 90-99% 81-92%
sodium citrate plasma(n=5) 78-99% 84-96% 79-97% 86-98%

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.

Reagents and materials provided

Reagents Quantity Reagents Quantity
96-well plate 1 Plate sealer for 96 wells 4
Pre-Mixed Standard 2 Standard Diluent 1×20mL
Pre-Mixed Magnetic beads (22#:KEAP1) 1 Analysis buffer 1×20mL
Pre-Mixed Detection Reagent A 1×120μL Assay Diluent A 1×12mL
Detection Reagent B (PE-SA) 1×120μL Assay Diluent B 1×12mL
Sheath Fluid 1×10mL Wash Buffer (30 × concentrate) 1×20mL
Instruction manual 1

Assay procedure summary

1. Preparation of standards, reagents and samples before the experiment;
2. Add 100μL standard or sample to each well,
    add 10μL magnetic beads, and incubate 90min at 37°C on shaker;
3. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent A. Incubate 60min at 37°C on shaker;
4. Wash plate on magnetic frame for three times;
5. Add 100μL prepared Detection Reagent B, and incubate 30 min at 37°C on shaker;
6. Wash plate on magnetic frame for three times;
7. Add 100μL sheath solution, swirl for 2 minutes, read on the machine.

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