The calibration curve concentrations used for the ELISA's were 1000 pg/mL, 333.33 pg/mL, 111.11 pg/mL, 37.04 pg/mL, 12.35 pg/mL.
- Determine wells for diluted calibrator, blank and sample. Prepare 5 wells for calibrator points, 1 well for blank. Add 50 µL each of dilutions of calibrator (read Reagent Preparation), blank and samples into the appropriate wells, respectively. And then add 50 µL of Detection Reagent A to each well immediately. Shake the plate gently (using a microplate shaker is recommended). Cover with a Plate sealer and stay overnight at 4° C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
2. Aspirate the solution and wash with 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 1~2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
3. Add 100 µL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37° C after covering it with the Plate sealer.
4. Repeat the aspiration/wash process for five times as conducted in step 2.
5. Add 90 µL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15
25 minutes at 37° C (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution.
6. Add 50 µL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. 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.
7. 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.
1. Assay preparation: Keep appropriate numbers of wells for 1 experiment and remove extra wells from microplate. Remaining wells should be resealed and stored at -20°C.
2. Samples or reagents addition: Please use the freshly prepared Calibrator. Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall. 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 10 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.
7. The environment humidity which is less than 60% might have some effects on the final performance, therefore, a humidifier is recommended to be used at that condition.
Calculation of Results
This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between CT concentration in the sample and the assay signal intensity. Average the duplicate readings for each calibrator, control, and samples. Create a calibration curve on log-log or semi-log graph paper, with the log of CT concentration on the y-axis and absorbance on the x- axis. Draw the best fit straight line through the calibrator points and it can be determined by regression analysis. Using some plot software is also recommended. If samples have been diluted, the concentration read from the calibration curve must be multiplied by the dilution factor.