KLH IgM ELISA 试剂盒

General Note: Studies indicate that anti-KLH IgM is present in serum from KLH immunized mice at concentrations of ~10,000 u/mL. In order to obtain values within range of the calibration curve, we suggest samples initially be diluted 500 fold using the following procedure for each sample tested:
1. Dispense 48 µL and 237.5 µL of diluent into separate tubes.
2. Pipette and mix 2 µL of the serum/plasma sample into the tube containing 48 µL of diluent. This provides a 25 fold diluted sample.
3. Mix 12.5 µL of the 25 fold diluted sample with 237.5 µL of diluent in the second tube. This provides a 500 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 diluted samples into the wells (we recommend that samples be tested in duplicate).
   3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 1 hour.
   4. Aspirate the contents of the microtiter wells and wash the wells 5 times with 1X wash solution using a plate washer (400 µL/well). The entire wash procedure should be performed as quickly as possible.
   5. Strike the wells sharply onto absorbent paper or paper towels to remove all residual wash buffer.
   6. Add 100 µL of enzyme conjugate reagent into each well.
   7. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   8. Wash as detailed in 4 to 5 above.
   9. Dispense 100 µL of TMB Reagent into each well.
   10. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 20 minutes.
   11. Stop the reaction by adding 100 µL of Stop Solution to each well.
   12. Gently mix. It is important to make sure all the blue color changes to yellow.
   13. Read the optical density at 450 nm with a microtiter plate reader within 5 minutes.

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators and diluted samples into the wells (we recommend that samples be tested in duplicate).
   3. Incubate on an orbital micro-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 concentrations on the horizontal or X-axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of anti-KLH IgM in u/mL from the calibration curve.