Thrombomodulin

The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of dog TM in plasma. For research use only. Not for use in diagnostic procedures.

1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination. 2. When performing the assay slowly bring samples to room temperature. 3. Sample hemolysis will influence the result, so hemolytic specimen can not be detected.

REAGENT PREPARATION
Bring all kit components and samples to room temperature (18-25°C) before use.

Calibrator
Reconstitute the Calibrator with 1.0 mL of Calibrator Diluent, kept for 10 minutes at room temperature, shake gently (not to foam). The concentration of the calibrator in the stock solution is 5,000 pg/mL. Please firstly dilute the stock solution to 1,000 pg/mL and the diluted calibrator serves as the highest calibrator (1,000 pg/mL). Then prepare 7 tubes containing 0.5 mL Calibrator Diluent and use the diluted calibrator to produce a double dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted calibrator such as 1,000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.2 pg/mL, 15.6 pg/mL, and the last EP tubes with Calibrator Diluent is the blank as 0 pg/mL.

Detection Reagent A and B
Briefly spin or centrifuge the stock Detection Reagent A and Detection Reagent B before use. Dilute to the working concentration with Assay Diluent A or B, respectively (1:100).

Wash Solution
Dilute 20 mL of Wash Solution Concentrate (30X) with 580 mL of de-ionized or distilled water to prepare 600 mL of Wash Solution (1X).

TMB substrate
Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again. It is recommended to suck more than 10 µL for once pipetting. 4. The reconstituted Calibrators, Detection Reagent A and Detection Reagent B can be used only once. 5. If crystals have formed in the Wash Solution concentrate (30X), warm to room temperature and mix gently until the crystals have completely dissolved. 6. Contaminated water or container for reagent preparation will influence the detection result.

SAMPLE PREPARATION
1. Kamiya Biomedical Company is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
2. 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. 4. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
5. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals.
6. Owing to the possibility of mismatching between antigen from other resource and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.

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 -20°C until the kit expiration date. 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 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 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
Average the duplicate readings for each calibrator, control, and samples and subtract the average zero calibrator optical density. Construct a calibration curve by plotting the mean O.D. and concentration for each calibrator and draw a best fit curve through the points on the graph or create a calibration curve on log-log graph paper with TM concentration on the y-axis and absorbance on the x-axis. 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.