ELISA Kit for Hydroxyproline (Hyp)

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

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

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Hydroxyproline (Hyp) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Hydroxyproline (Hyp) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Hydroxyproline (Hyp) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

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.

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°C;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
7. Add 50µL Stop Solution. Read at 450 nm immediately.

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