ELISA Kit for Hypoxanthine Phosphoribosyltransferase 1 (HPRT1)

HGPRT; HGPRTase; HPRT; Lesch-Nyhan Syndrome; Hypoxanthine-guanine phosphoribosyltransferase
Typical Standard Curve Typical Standard Curve

Sample type Serum, plasma, tissue homogenates, cell lysates and other biological fluids. Detection range 0.156-10ng/mL The standard curve concentrations used for the ELISA's were 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.313ng/mL, 0.156ng/mL
Sensitivity The minimum detectable dose of this kit is typically less than 0.061ng/mL. Specificity This assay has high sensitivity and excellent specificity for detection of Hypoxanthine Phosphoribosyltransferase 1 (HPRT1).
No significant cross-reactivity or interference between Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) and analogues was observed.

Recovery Matrices listed below were spiked with certain level of recombinant Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) and the recovery rates were calculated by comparing the measured value to the expected amount of Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) in samples.
Matrix Recovery range (%) Average(%)
serum(n=5)97-105101
EDTA plasma(n=5)91-9994
heparin plasma(n=5)81-9592

Precision Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) 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 Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) 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)80-92%80-96%79-103%83-101%
EDTA plasma(n=5)86-94%87-95%81-99%78-99%
heparin plasma(n=5)78-96%78-92%96-105%88-95%

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.
Test principle The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Hypoxanthine Phosphoribosyltransferase 1 (HPRT1). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Hypoxanthine Phosphoribosyltransferase 1 (HPRT1). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Hypoxanthine Phosphoribosyltransferase 1 (HPRT1), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) in the samples is then determined by comparing the O.D. of the samples to the standard curve. Assay procedure summary 1. Prepare all reagents, samples and standards;
Reagent Preparation
2. Add 100µL standard or sample to each well. Incubate 2 hours at 37oC;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37oC;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37oC;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37oC;
8. Add 50µL Stop Solution. Read at 450nm immediately.

Reference
  • J Crohns Colitis.Read more(Pubmed:25518051)Response to infliximab therapy in ulcerative colitis is associated with decreased monocyte activation, reduced CCL2 expression and downregulation of Tenascin C.
Reagents and materials provided
  • Pre-coated, ready to use 96-well strip plate1
  • Plate sealer for 96 wells4
  • Standard2
  • Standard Diluent1×20mL
  • Detection Reagent A1×120µL
  • Assay Diluent A1×12mL
  • Detection Reagent B1×120µL
  • Assay Diluent B1×12mL
  • TMB Substrate1×9mL
  • Stop Solution1×6mL
  • Wash Buffer (30 × concentrate)1×20mL
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