KLH IgM ELISA 试剂盒

Wash Buffer: The wash solution is provided as 20x stock. Prior to use dilute the contents of the bottle (50 mL) with 950 mL of distilled of deionized water. Diluent The diluent is provided as 10x stock. Prior to use estimate the final volume of diluent required for your assay and dilute one volume of the 10x stock with nine volumes of distilled or deionized water. Calibrator
1. Working 400-12.5 ng/mL anti-KLH IgM calibrators should be used within 1 hour of preparation.
2. The Monkey anti-KLH IgM calibrator is provided as lyophilized stock. Reconstitute as directed on vial label. The reconstituted calibrator should be frozen and stored at -20°C after reconstitution if future use is intended.
3. Label 6 polypropylene or glass tubes as 400, 200, 100, 50, 25, and 12.5 ng/mL.
4. Into the tube labeled 400 ng/mL, pipette the volume of diluent detailed on the anti-KLH IgM calibration vial label. Then add the indicated volume of anti-KLH IgM calibrator (shown on the anti-KLH IgM calibrator vial label) and mix gently. This provides the 400 ng/mL calibrator.
5. Dispense 200 µL of diluent into the tubes labelled 200, 100, 50, 25, and 12.5 ng/mL.
6. Prepare a 200 ng/mL calibrator by diluting and mixing 250 µL of the 400 ng/mL calibrator with 250 µL of diluent in the tube labelled 200 ng/mL.
7. Similarly prepare the 100, 50, 25, and 12.5 ng/mL calibrators by serial dilution.

Note: The optimal sample dilution should be determined empirically. However, studies indicate that a 500-fold dilution is a reasonable starting point. In order to achieve high dilutions we suggest that a serial dilution strategy be used. If, for example, a 500-fold sample dilution is desired the following procedure should be used. This approach minimizes diluent usage and favors accurate and precise sample dilution.
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 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.
5. Do not use dilutions lower than 100-fold.

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 45 minutes.
   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 and 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 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. Calculate the average absorbance values for each set of calibrators and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each 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 ng/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration for anti-KLH IgM in the serum/plasma sample.
   5. PC graphing software may be used for the above steps.
   6. If the OD values of samples fall outside the calibration curve samples should be diluted appropriately and re- tested.