LOC100192394 ELISA 试剂盒

General Note: alpha-macroglobulin is present in normal mouse serum at a concentration of ~2.5 mg/mL. In order to obtain values within the range of the calibration curve, we suggest that samples be diluted 50,000 fold using the following procedure for each sample to be tested.
1. Dispense 998 µL of water and 297 µL of diluent into separate tubes.
2. Pipette and mix 2 µL of the serum/plasma sample into the tube containing 998 µL of diluent. This provides a 500 fold diluted sample.
3. Mix 3 µL of the 500 fold diluted sample with the 297 µL of diluent in the second tube. This provides a 50,000 fold dilution of the sample.
4. Repeat this procedure for each sample to be tested.

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators and samples into the wells (calibrators and samples should be tested in duplicate).
   3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   4. Remove the incubation mixture by flicking plate contents into an appropriate Bio-waste container.
   5. 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 a squirt bottle. The entire wash procedure should be performed as quickly as possible.
   6. Strike the wells sharply onto adsorbent paper or paper towels to remove all residual droplets.
   7. Add 100 µL of enzyme conjugate reagent into each well.
   8. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   9. Wash as detailed in 4 to 5 above.
   10. Strike the wells sharply onto absorbent paper or paper towels to remove residual droplets.
   11. Dispense 100 µL of TMB Reagent into each well.
   12. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 20 minutes.
   13. Stop the reaction by adding 100 µL of Stop Solution to each well.
   14. Gently mix. It is important to make sure that all the blue color changes to yellow.
   15. Read the optical density at 450 nm with a microtiter plate reader within 5 minutes. Please Note: If the A450 of the high calibrator(s) exceeds the limits of the plate reader, absorbance of all wells may be determined at 405 nm instead.

1. Calculate the average 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 linear 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 -macroglobulin in ng/mL from the calibration curve.
   4. Multiply the derived concentration by the dilution factor to determine the actual concentration of -macroglobulin in the serum/plasma sample.
   5. PC graphing software may be used for the above steps.
   6. If the OD450 values of samples fall outside the calibration curve when tested at a dilution of 50,000, samples should be diluted appropriately and re-tested.