Pg (Progesterone) ELISA Kit (UNES00066)

This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with Pg. During the reaction, Pg in the sample or standard competes with a fixed amount of Pg on the solid phase supporter for sites on the Biotinylated Detection Ab specific to Pg. Excess conjugate and unbound sample or standard are washed away, and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns from blue to yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Pg in tested samples can be calculated by comparing the OD of the samples to the standard curve.

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Pg were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Pg were tested on 3 different plates, 20 replicates in each plate.

Recovery

The recovery of Pg spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

1. Set standard, test sample and control (zero) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Note: add all solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensure solutions do not foam when adding to the wells.
2. Add 50µL of Standard, Blank or Sample to their respective wells. The blank well is added with Sample / Standard dilution buffer.
3. Immediately add 50 µL of Biotin-detection antibody working solution to each well.
4. Cover with a plate seal and gently tap the plate to ensure thorough mixing. Incubate for 45minutes at 37°C.
5. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
6. Add 100µL of HRP Conjugate working solution to each well and over with a plate seal. Incubate for 30 minutes at 37°C.
7. Repeat the aspiration/wash process 5 times according to step 5.
8. Add 90µL of the Substrate reagent to each well and cover with a new plate seal. Incubatefor approximately 15 minutes at 37°C and protect from light. The reaction time can beshortened or extended according to the colour change, but not by more than 30 minutes. Whenapparent gradient appears in standard wells, terminate the reaction.
9. Stop: Add 50µL of Stop Solution to each well (wells will develop a yellow color immediately). Note: Adding the stop solution should be done in the same order as the substrate solution.
10. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm. In advance, preheat the instrument and set the testing parameters.