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Multiplex Assay Kit for Riboflavin Kinase (RFK) ,etc. by FLIA (Flow Luminescence Immunoassay)

RIFK; Flavokinase; ATP:riboflavin 5'-phosphotransferase

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

Specificity

This assay has high sensitivity and excellent specificity for detection of Riboflavin Kinase (RFK).
No significant cross-reactivity or interference between Riboflavin Kinase (RFK) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Riboflavin Kinase (RFK) and the recovery rates were calculated by comparing the measured value to the expected amount of Riboflavin Kinase (RFK) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 80-97 93
EDTA plasma(n=5) 78-101 96
heparin plasma(n=5) 82-91 85
sodium citrate plasma(n=5) 82-99 95

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Riboflavin Kinase (RFK) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Riboflavin Kinase (RFK) 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 Riboflavin Kinase (RFK) 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) 84-98% 96-103% 96-105% 85-102%
EDTA plasma(n=5) 90-97% 97-105% 84-93% 83-97%
heparin plasma(n=5) 91-105% 80-96% 95-102% 89-103%
sodium citrate plasma(n=5) 95-104% 78-104% 89-96% 87-97%

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#:RFK) 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. Add 200μL analysis buffer solution to each well of the plate for pre-wetting;
2. Preparation of standards, reagents and samples before the experiment;
3. Add 100μL standard or sample to each well, add 10μL magnetic beads, and incubate 120 minutes protect from light at 25°C on shaker;
4. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent A. Incubate 60 minutes protect from light at 25°C on shaker;
5. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent B, and incubate 30 minutes protect from light at 25°C on shaker;
6. Wash plate once on magnetic frame;
7. Add 100μL sheath fluid, swirl for 10 minutes, read on the machine.

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