Progesterone ELISA Kit

Catalog No. ABIN955961

Quick Overview for Progesterone ELISA Kit (ABIN955961)

Target: Progesterone

Reactivity: Rat

Detection Method: Colorimetric

Application: ELISA, In vitro Assay (in vitro)

Product Details

Detection Range: 0.5-100 ng/mL

Minimum Detection Limit: 0.5 ng/mL

Purpose: This ELISA kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of rat Pg in serum, plasma and other biological fluids.

Specificity: This assay has high sensitivity and excellent specificity for detection of rat Pg.

Sensitivity: The minimum detectable dose of rat Pg is typically less than 0.22 ng/mL.
The Sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined the mean O.D. Value of 20 replicates of the zero calibrator plus three standard deviations.

Components: Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells (4x)
Calibrator-1,2,3,4,5,6 (6x)
Detection Reagent A (1 x 6 mL)
Detection Reagent B (1 x 6 mL)
TMB Substrate (1 x 9 mL)
Stop Solution (1 x 6 mL)
Wash Buffer (20X concentrate) (1 x 15 mL)

Material not included: 1. Microplate reader with 450 +/- 10 nm filter.
2. Precision single and multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting samples.
4. De-ionized or distilled water.
5. Absorbent paper for blotting the microtiter plate.
6. Container for Wash Solution.

Application Details

Comment:

The calibration curve concentrations used for the ELISA's were 100 ng/mL, 40 ng/mL, 10 ng/mL, 2 ng/mL, 0.5 ng/mL, 0 ng/mL.

Plate: Pre-coated

Assay Procedure:

Please predict the concentration before assaying. If values for these are not within the range of the calibration curve, users must determine the optimal sample dilutions for their particular experiments.
1. Determine wells for blank, diluted calibrator and sample. Prepare 6 wells for calibrator, 1 well for blank (didn't add anything) and other wells for sample. Add 50 µL each of calibrator solution and samples into the appropriate wells, respectively.
2. Add 50 µL of Detection Reagent A to calibrator and sample wells immediately, and then add 50 µL of Detection Reagent B to the above wells. Shake the plate gently. Cover with a Plate sealer. Incubate for 1 hour at 37° C.
3. Aspirate the solution and wash with about 300-350 µL of 1X Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 10 seconds. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat 3-5 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
4. Add 90 µL of TMB Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 minutes at 37° C. Protect from light.
5. Add 50 µL of Stop Solution to each well to stop the reaction. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
6. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450 nm immediately.

Note:
1. Assay preparation: Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4° C until the kit expiration date.
2. Samples or reagents addition: Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall as possible. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all calibrators and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of each calibrator level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be observed.
4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and falsely elevated absorbance reading.
5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 5 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.
6. TMB Substrate is easily contaminated. Please protect it from light.

Calculation of Results:

This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between Pg concentration in the sample and the assay signal intensity. Low levels of Pg result in a high luminescence intensity, while a high concentration of Pg results in a low signal. Average the duplicate readings for each calibrator and samples. Create a calibration curve by reducing the date using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a calibration curve by plotting the mean absorbance for each calibrator on the x-axis against the log of the concentration on the y-axis and draw a best fit curve through the points on the graph (5 points). The data may be linearized by plotting the log of the Pg concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. If samples have been diluted, the concentration read from the calibration curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: The kits should be stored at 4° C upon being received. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. Opened test kits will remain stable until the expiration date shown, provided it is stored as prescribed above.

Expiry Date: The expiry date is stated on the label.

Target Details

Target: Progesterone

Alternative Name: Progesterone (Pg)

Target Type: Hormone

Background: This assay employs the competitive inhibition enzyme immunoassay technique. A polyclonal antibody for goat anti-rabbit IgG has been pre-coated onto a microplate. Add Horseradish Peroxidase (HRP) labeled rat Pg, unlabeled rat Pg (Calibrators or samples) and a polyclonal antibody for Pg, then the competitive inhibition reaction is launched between with HRP labeled rat Pg and unlabeled rat Pg with the antibody. Next, A TMB substrate solution is added to each well and a change in color will be exhibited. The more the amount of rat Pg in samples, the less the HRP labeled rat Pg bound by the antibody. The substrate solution are added to the wells, respectively. And the color develops in opposite to the amount of rat Pg bound in the initial step. The color development is stopped and the intensity of the color is measured.