Adrenocorticotropic Hormone Porcine ELISA Assay Kit For Accurate Quantitative Detection
Size: 96 wells
Storage: Store the reagents at 2-8°C. For over 6-month storage refer to the expiration date keep it at -20°C. Avoid repeated thaw cycles. If individual reagents are opened it is recommended that the kit be used within 1 month.
*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.
This Sandwich ELISA Kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Porcine ACTH antibody. ACTH present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Porcine ACTH Antibody is added and binds to ACTH in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated ACTH antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Porcine ACTH. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.
|Standard Solution (80ng/L)||0.5ml x1|
|Pre-coated ELISA Plate||12 * 8 well strips x1|
|Standard Diluent||3ml x1|
|Stop Solution||6ml x1|
|Substrate Solution A||6ml x1|
|Substrate Solution B||6ml x1|
|Wash Buffer Concentrate (30x)||20ml x1|
|Biotinylated Porcine ACTH Antibody||1ml x1|
|Plate Sealer||2 pics|
|Zipper bag||1 pic|
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (80ng/L) with 120μl of standard diluent to generate a 40ng/L standard stock solution. Allow the standard to sit for 15 mins with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the standard stock solution (40ng/L) 1:2 with standard diluent to produce 20ng/L, 10ng/L, 5ng/L and 2.5ng/L solutions. Standard diluent serves as the zero standard(0 ng/L). Any remaining solution should be frozen at -20°C and used within one month. Dilution of standard solutions suggested are as follows:
|40ng/L||Standard No.5||120μl Original Standard + 120μl Standard Diluent|
|20ng/L||Standard No.4||120μl Standard No.5 + 120μl Standard Diluent|
|10ng/L||Standard No.3||120μl Standard No.4 + 120μl Standard Diluent|
|5ng/L||Standard No.2||120μl Standard No.3 + 120μl Standard Diluent|
|2.5ng/L||Standard No.1||120μl Standard No.2 + 120μl Standard Diluent|
|Standard Concentration||Standard No.5||Standard No.4||Standard No.3||Standard No.2||Standard No.1|
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or distilled water to yield 600 ml of 1x Wash Buffer. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved.
1. Prepare all reagents, standard solutions and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature.
2. Determine the number of strips required for the assay. Insert the strips in the frames for use. The unused strips should be stored at 2-8°C.
3. Add 50μl standard to standard well. Note: Don’t add antibody to standard well because the standard solution contains biotinylated antibody.
4. Add 40μl sample to sample wells and then add 10μl anti-ACTH antibody to sample wells, then add 50μl streptavidin-HRP to sample wells and standard wells (Not blank control well). Mix well. Cover the plate with a sealer. Incubate 60 minutes at 37°C.
5. Remove the sealer and wash the plate 5 times with wash buffer. Soak wells with at least 0.35 ml wash buffer for 30 seconds to 1 minute for each wash. For automated washing, aspirate all wells and wash 5 times with wash buffer, overfilling wells with wash buffer. Blot the plate onto paper towels or other absorbent material.
6. Add 50μl substrate solution A to each well and then add 50μl substrate solution B to each well. Incubate plate covered with a new sealer for 10 minutes at 37°C in the dark.
7. Add 50μl Stop Solution to each well, the blue color will change into yellow immediately.
8. Determine the optical density (OD value) of each well immediately using a microplate reader set to 450 nm within 10 minuets after adding the stop solution.
Calculation of Result
Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.