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ELISA Kit for N-Acylsphingosine Amidohydrolase 2 (ASAH2)

HNAC1; non-lysosomal ceramidase; Neutral ceramidase

Specificity

This assay has high sensitivity and excellent specificity for detection of N-Acylsphingosine Amidohydrolase 2 (ASAH2).
No significant cross-reactivity or interference between N-Acylsphingosine Amidohydrolase 2 (ASAH2) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant N-Acylsphingosine Amidohydrolase 2 (ASAH2) and the recovery rates were calculated by comparing the measured value to the expected amount of N-Acylsphingosine Amidohydrolase 2 (ASAH2) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 97-105 101
EDTA plasma(n=5) 82-89 85
heparin plasma(n=5) 80-101 85

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level N-Acylsphingosine Amidohydrolase 2 (ASAH2) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level N-Acylsphingosine Amidohydrolase 2 (ASAH2) 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 N-Acylsphingosine Amidohydrolase 2 (ASAH2) 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) 90-102% 96-105% 79-89% 81-94%
EDTA plasma(n=5) 90-98% 81-95% 92-99% 78-93%
heparin plasma(n=5) 94-105% 80-88% 98-105% 81-104%

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
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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