KLH IgG 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. Control Reconstitute the lyophilized rat anti-KLH IgG control with the volume of distilled or deionized water indicated on the vial label. The concentration range of rat anti-KLH IgG after reconstitution is shown on the vial label. The assay value of the control should be within the specified range. Discard any remaining control after use. Calibrator
1. The Rat anti-KLH IgG calibrator is provided as lyophilized stock. Reconstitute with the volume of diluent indicated on the vial label to give a 500 ng/mL solution of rat anti-KLH IgG. 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 5 polypropylene or glass tubes as 250, 125, 62.5, 31.2, and 15.6 ng/mL and pipette 250 µL of diluent into each tube.
3. Into the tube labeled 250 ng/mL, pipette and mix 250 µL of the reconstituted anti-KLH IgG calibrator. This provides the 250 ng/mL calibrator.
4. 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.
5. Similarly prepare the 62.5, 31.25, and 15.6 ng/mL calibrators by serial dilution.

Note: Studies indicate that anti-KLH IgG is present in rat serum or plasma at concentrations up to approximately 20 µg/mL 14 days after i.v. immunization with KLH. Levels are likely higher after 14 days. In order to obtain values within range of the calibration curve, we suggest samples initially be diluted 100 fold using the following procedure for each sample tested. Optimal dilutions may need to be determined empirically.
1. Dispense 279 µL of diluent into a polypropylene or glass tube.
2. Pipette and mix 3 µL of the serum/plasma sample into the tube containing 279 µL of diluent. This provides a 100 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.

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators (500-15.6 ng/mL) 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 30 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 IgG in ng/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration for anti-KLH IgG 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 100, samples should be diluted appropriately and re-tested.