The following protocol is a sample protocol for the human CORO1C ELISA kit (Coronin-1C) using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting the absorbance of known concentrations against known standard concentrations. This allows the investigator to calculate the amount of human CORO1C (Coronin-1C) present in his sample.
Before adding to the wells, equilibrate the SABC Working Solution and TMB Substrate for at least 30 min at 37 ° C. When diluting samples and reagents, they must be thoroughly and evenly mixed. It is recommended to draw a standard curve for each test.
Sandwich ELISA protocol
1. Place the standard, test sample, and control (zero) wells on the precoated plate, respectively, and then record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample, and control (zero) wells!
2. Place 0.1 ml aliquots of standard solutions into standard wells.
3. Add 0.1 ml of Standard / Sample Dilution Buffer to the control (zero) well.
4. Add 0.1 ml of appropriately diluted sample (human serum, plasma, tissue homogenates, and other biological fluids) to the test sample wells.
5. Seal the plate with a lid and incubate at 37 ° C for 90 min.
6. Remove the lid and discard the contents of the plate, place the plate on absorbent filter papers or other absorbent material. DO NOT let the wells dry completely at any time. Wash plate X2.
7. Add 0.1 ml of Biotin Detection Antibody Working Solution to the previous wells (standard, test sample, and zero wells). Add the solution to the bottom of each well without touching the side wall.
8. Seal the plate with a lid and incubate at 37 ° C for 60 min.
9. Remove the cap and wash the plate 3 times with wash buffer. Allow Wash Buffer to stand in wells for 1 minute between each wash.
10. Add 0.1 ml of SABC Working Solution to each well, cover the plate and incubate at 37 ° C for 30 min.
11. Remove the cap and wash the plate 5 times with wash buffer, each time allow the wash buffer to remain in the wells for 1-2 minutes.
12. Add 90 µl TMB Substrate to each well, cover the plate and incubate at 37 ° C in the dark for 10-20 min. (Note: This incubation time is for reference use only, the optimal time must be determined by the end user.) And shades of blue can be seen in the first 3-4 wells (with the more concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µl of Stop Solution to each well and mix well. The color changes to yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.