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. The Rat anti-KLH IgM calibrator is provided as lyophilized stock. Reconstitute with 1 mL of distilled or deionized water. The reconstituted calibrator is stable at 4°C for one week but should be aliquoted and stored frozen at - 20°C after reconstitution if future use is intended.
2. Label 6 polypropylene or glass tubes as 250, 125, 62.5, 31.2, 15.6, and 7.8 ng/mL.
3. Into the tube labeled 250 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 250 ng/mL calibrator.
4. Dispense 300 µL of diluent into the tubes labeled 125, 62.5, 31.2, 15.6, and 7.8 ng/mL.
5. Prepare a 125 ng/mL calibrator by diluting and mixing 250 µL of the 250 ng/mL calibrator with 250 µL of diluent in the tube labelled 125 ng/mL.
6. Similarly prepare the 62.5, 31.25, 15.6, and 7.8 ng/mL calibrators by serial dilution.

Note: Studies indicate that anti-KLH IgM is present in rat serum or plasma at concentrations ranging from approximately 20 to 200 µg/mL. In order to obtain values within range of the calibration curve, we suggest samples initially be diluted 1,000 fold using the following procedure for each sample tested.
1. Dispense 196 µL and 285 µL of diluent into separate tubes.
2. Pipette and mix 4 µL of the serum/plasma sample into the tube containing 196 µL of diluent. This provides a 50 fold diluted sample.
3. Mix 15 µL of the diluted sample with 285 µL of diluent in the second tube. This provides a 1,000 fold dilution of the sample.
4. Repeat this procedure for each sample to be tested.
5. Do not use dilutions lower than 500 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 u/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 when tested at a dilution of 1,000, samples should be diluted appropriately and re-tested. Do not use dilutions lower than 500 fold.