Cardiac Tn-I ELISA 试剂盒

Serum should be prepared as quickly as possible after blood collection and stored at 4°C. All samples should be similarly processed (i.e., storage times and temperatures should be the same). If serum samples cannot be assayed immediately they should be frozen at -70°C and thawed only once prior to use.

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of cTnI HRP Conjugate into each well.
   3. Dispense 100 µL of calibrators and samples into the appropriate wells.
   4. Thoroughly mix for 10-15 seconds. It is very important to mix completely.
   5. Incubate at room temperature (18-25°C) on a plate shaker (150 rpm) for one hour.
   6. Remove the incubation mixture either with a plate washer or by flicking plate contents into a bio-waste container.
   7. Wash and empty the microtiter wells 5 times with 1X wash solution. This may be performed using either a plate washer (400 µL/well) or with a squirt bottle. The entire wash procedure should be performed as quickly as possible
   8. Strike the wells sharply onto adsorbent paper or paper towels to remove all residual droplets.
   9. Dispense 100 µL of TMB Reagent solution into each well. Gently mix for 5 seconds.
   10. Incubate on a plate shaker (150 rpm) at room temperature for 20 minutes.
   11. Stop the reaction by adding 100 µL of Stop Solution to each well.
   12. Gently mix until all the blue color changes to yellow.
   13. Read absorbance at 450 nm with a microtiter plate reader within 15 minutes. Please note: Due to plate reader differences, the high calibrator absorbance values may be out of range occasionally. If this occurs, absorbance values may be determined at 405 nm instead.
   14. If absorbance values exceed the high calibrator, the samples should be appropriately diluted with sample diluent and re-determined. Samples with absorbance values below those of the lowest calibrator should be assigned a zero troponin-I value.

1. Calculate the mean absorbance values (A450) for each set of reference calibrators and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each reference calibrator against its concentration in ng/mL on graph paper, with absorbance values on the vertical or Y-axis and concentration on the horizontal or X-axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of cTnI in ng/mL from the calibration curve.
   4. If available, graphing software may be used to analyze the data. Depending on the range of the calibration curve used, we find that good fits of the data may be obtained with a second order polynomial or using a two-site binding model. Alternatively, calibration curves may be generated using a point-to-point fit.