PHGDH ELISA 试剂盒

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine preg- nanediol-3-glucuronide concentrations.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water.
Once diluted this is stable at 4 °C for 3 months.
Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water.
Once diluted this is stable at room temperature for 3 months.
Standard Preparation Label eight test tubes as #1 through #8.
Pipet 450 μL of Assay Buffer into tube #1 and 200 μL into tubes #2 to #8.
The Pregnanediol-3-Glucuronide stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 50 μL of the preg- nanediol-3-glucuronide stock solution to tube #1 and vortex completely.
Take 200 μL of the preg- nanediol-3-glucuronide solution in tube #1 and add it to tube #2 and vortex completely.
Repeat the serial dilutions for tubes #3 through #8.
The concentration of pregnanediol-3-glucuronide in tubes 1 through 8 will be 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781 and 0.391 ng/mL.
Use all Standards within 2 hours of preparation.

Serum and Plasma Samples. We would recommend the following protocol for serum and plasma. 1. Add diethyl ether to serum or plasma samples at a 5:1 (v/v) ether:sample ratio. 2. Mix solutions by vortexing for 2 minutes. Allow ether layer to separate for 5 minutes. 3. Freeze samples in a dry ice/ethanol bath and pipet off the ether solution from the top of the sample into a clean tube. Repeat steps 1-3 for maximum extraction efficiency, combining top layer of ether solutions. 4. Dry pooled ether samples down in a speedvac for 2-3 hrs. If samples need to be stored they should be kept at -20 °C. 5. Redissolve samples at room temperature in the Assay Buffer. A minimum of 125 μL of the Assay Buffer is recommended for reconstitution to allow for duplicate sample measurement. Dried Fecal Samples We have a detailed Extraction Protocol available on our website at: http://www.ArborAssays.com/ resources/lit.asp. The ethanol concentration in the final Assay Buffer dilution added to the well should be ≤1 % . Urine Samples Urine samples should be diluted at least 1:5 with the provided Assay Buffer. For comparison to creatinine as a urine volume marker please see our NIST-calibrated 2 plate and 10 plate Urinary Creatinine Detection kits, K002-H1 and K002-H5.

1. Use the plate layout sheet on the back page to aid in proper sample and standard identification. We suggest you run samples and standards vertically down the plate columns so that unused wells can be easily kept for further experiments. Determine the number of wells to be used and return unused wells to the foil pouch with desiccant. Ensure the desiccant is blue. Seal the ziploc plate bag and store at 4ºC.
   2. Pipet 50 μL of samples or standards into wells in the plate.
   3. Pipet 50 μL of Assay Buffer into maximum binding wells (B0 or 0 pg/mL).
   4. Pipet 75 μL of Assay Buffer into the non-specific binding (NSB) wells.
   5. Add 25 μL of the DetectX® Pregnanediol-3-Glucuronide Conjugate to each well using a repeater pipet.
   6. Add 25 μL of the DetectX® Pregnanediol-3-Glucuronide Antibody to each well, except the NSB wells, using a repeater pipet.
   7. Gently tap the sides of the plate to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and shake at room temperature for 2 hours. If the plate is not shaken signals bound will be approximately 35 % lower.
   8. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
   9. Add 100 μL of the TMB Substrate to each well, using a repeater pipet. 10. Incubate the plate at room temperature for 30 minutes without shaking. 11. Add 50 μL of the Stop Solution to each well, using a repeater pipet. 12. Read the optical density generated from each well in a plate reader capable of reading at 450 nm. 13. Use the plate reader's built-in 4PLC software capabilities to calculate pregnanediol-3- glucuronide concentration for each sample. NOTE: If you have any unused wells left, please retain the plate frame to holds these wells for the next experiment.

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean OD's for the NSB.
The sample concentrations obtained, calculated from the %B/B0 curve, should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from www.myassays.com/arbor-assays-pregnanediol-3-glucuronide-(pdg)-eia-kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data Sample Mean OD Net OD % B/B0 PDG Conc. (ng/mL) NSB 0.048 0 - - Standard 1 0.355 0.307 13.2 50 Standard 2 0.555 0.507 21.8 25 Standard 3 0.794 0.746 32.0 12.5 Standard 4 1.106 1.058 45.4 6.25 Standard 5 1.391 1.343 57.6 3.125 Standard 6 1.706 1.658 71.1 1.563 Standard 7 1.958 1.910 81.9 0.781 Standard 8 2.129 2.081 89.3 0.391 B0 2.379 2.331 100 0 Sample 1 1.307 1.259 54.0 3.9 Sample 2 1.766 1.718 73.7 1.3 Always run your own standard curve for calculation of results.
Do not use this data.
Conversion Factor: 100 pg/mL of PDG is equivalent to 201.4 pM.