Corticosterone 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 corticosterone 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 deionized 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 for 3 months at room temperature.
Standard Preparation Label test tubes as #1 through #8.
Pipet 450 μL of Assay Buffer into tube #1 and 250 μL into tubes #2 to #8. the corticosterone stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 50 μL of the corticosterone stock solution to tube #1 and vortex completely.
Take 250 μL of the corticosterone 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 corticosterone in tubes 1 through 8 will be 10,000, 5,000, 2,500, 1,250, 625, 312.5, 156.25, and 78.125 pg/mL.
Use all Standards within 2 hour of preparation.
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 assay Buffer (μL) 450 250 250 250 250 250 250 250 addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Vol of addition (μL) 50 250 250 250 250 250 250 250 Final Conc (pg/ mL) 10,000 5,000 2,500 1,250 625 312.5 156.25 78.125 ® 8 EXPECT ASSAY ARTISTRY

Serum and plasma samples need to be treated with the supplied Dissociation Reagent. Addition of this reagent will yield the total corticosterone concentration in serum or plasma. Dissociation reagent is to be used only with Serum and Plasma samples. Serum and Plasma Samples Allow the Dissociation Reagent to warm completely to room Temperature before use. We suggest pipetting 5 μL of Dissociation Reagent into 1 mL Eppendorf tubes. Add 5 μL of serum or plasma to the Dissociation Reagent in the tube, vortex gently and incubate at room temperature for 5 minutes or longer. Dilute with 490 μL of diluted Assay Buffer. This 1:100 dilution can be diluted further with diluted Assay Buffer. Final serum and plasma dilutions should be ≥ 1:100. noTe: Dissociation reagent is to be used only with Serum and Plasma samples. Urine Samples Urine samples should be diluted ≥ 1:20 with the diluted Assay Buffer prior running in the assay. Please see our Urinary Creatinine Detection kits, K002-H1 and K002-H5, for assays to measure urine creatinine which can be used to allow normalization of corticosterone in a random urine specimen. Dried Fecal Samples: We have a detailed Extraction Protocol available on our website at: www.ArborAssays.com/resources/lit.asp. The ethanol concentration in the final diluted Assay Buffer dilution added to the well should be <5 % .

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 50 μL of samples or standards into wells in the plate.
   3. Pipet 75 μL of Assay Buffer into the non-specific binding (NSB) wells.
   4. Pipet 50 μL of Assay Buffer into wells to act as maximum binding wells (Bo or 0 pg/mL).
   5. Add 25 μL of the DetectX® Corticosterone Conjugate to each well using a repeater pipet.
   6. Add 25 μL of the DetectX® Corticosterone 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 1 hour. If the plate is not shaken signals bound will be approximately 45 % 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 or a multichannel 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 corticosterone 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 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.