Oxytocin 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 oxytocin 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 4 °C for 3 months.
Standard Preparation Label test tubes as #1 through #8.
Pipet 450 μL of Assay Buffer into tube #1 and 300 μL into the remaining tubes.
The oxytocin stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 50 μL of the oxytocin stock solution to tube #1 and vortex completely.
Take 200 μL of the oxytocin 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 oxytocin in tubes 1 through 8 will be 10,000, 4,000, 1,600, 640, 256, 102.4, 40.96 and 16.38 pg/mL.
Use all Standards within 2 hours of preparation.

Serum and Plasma Samples Serum and plasma samples should be extracted with the provided Extraction Solution, or with a solid phase C18 column extraction protocol prior to running in the kit. Protocol Using Extraction Solution: Mix 1 part sample with 1.5 parts of Extraction Solution. Vortex and then nutate at room temperature for 90 minutes. Centrifuge for 20 minutes at 4 °C at 1660 x g. Speedvac supernatant to dryness at 37 °C. Reconstitute sample with 250 µL of Assay Buffer. Milk Samples Milk samples should be clarified by centrifuging at 10,000 x g for 15 minutes. Pierce the top fatty layer and collect the supernatant liquid. Repeat the centrifugation and collection two more times. The collected supernatant liquid must then be diluted 1:10 with the provided Assay Buffer before using in the assay. The clarified milk sample, i.e., the supernatant liquid, can be stored at -20 °C until needed. Use all samples within 2 hour of preparation.

1. Use the plate layout sheet on the back page to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to the foil pouch with desiccant. Seal the ziploc plate bag and store at 4ºC.
   2. Pipet 100 μL of samples or standards into wells in the plate.
   3. Pipet 100 μL of Assay Buffer into wells to act as maximum binding wells (Bo or 0 pg/mL).
   4. Pipet 125 μL of Assay Buffer into the non-specific binding (NSB) wells.
   5. Add 25 μL of the DetectX® Oxytocin Conjugate to each well using a repeater pipet.
   6. Add 25 μL of the DetectX® Oxytocin Antibody to each well, except the NSB wells, using a repeater pipet.
   7. Shake the plate in a plate shaker at room temperature for 15 minutes to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and store at 4 °C for 16-18 hours.
   8. The following day remove the TMB Substrate from the refrigerator and allow to come to room temperature for at least 30 minutes. Addition of cold Substrate will cause depressed signal.
   9. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels. 10. Add 100 μL of the TMB Substrate to each well, using a repeater pipet. 11. Incubate the plate at room temperature for 30 minutes without shaking. 12. Add 50 μL of the Stop Solution to each well, using a repeater or a multichannel pipet. 13. Read the optical density generated from each well in a plate reader capable of reading at 450 nm. 14. Use the plate reader's built-in 4PLC software capabilities to calculate oxytocin concentration for each sample.

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 ODs 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-oxytocin-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 Oxytocin Conc. (pg/mL) NSB 0.066 0 - - Standard 1 0.128 0.062 10.7 % 10,000 Standard 2 0.163 0.097 16.7 % 4,000 Standard 3 0.222 0.156 26.9 % 1,600 Standard 4 0.301 0.235 40.6 % 640 Standard 5 0.399 0.333 57.5 % 256 Standard 6 0.489 0.423 73.1 % 102.4 Standard 7 0.567 0.501 86.5 % 40.96 Standard 8 0.624 0.558 96.4 % 16.38 B0 0.645 0.579 100 % 0 Sample 1 0.236 0.170 29.3 % 1,336 Sample 2 0.429 0.363 62.7 % 187.9 Always run your own standard curve for calculation of results.
Do not use this data.
Conversion Factor: 1 ng/mL of oxytocin is equivalent to 0.993 nM.
Calibrated to the 4th WHO International Standard NIBSC code: 76/575