RBC IgM Kit ELISA

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. Calibrator
1. The Rat anti-SRBC IgM calibrator is provided as lyophilized stock. Reconstitute with volume of diluent indicated on the vial label. 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 100, 50, 25, 12.5, 6.25, and 3.125 u/mL, and pipette 250 µL of diluent into each tube.
3. Into the tube labelled 100 u/mL prepare a 100 u/mL stock by mixing the volume of reconstituted calibrator stock with the volume of diluent detailed on the vial label.
4. Dispense 250 µL of diluent into the tubes labelled 50, 25, 12.5, 6.25, and 3.125 u/mL.
5. Prepare a 50 u/mL calibrator by diluting and mixing 250 µL of the 100 u/mL calibrator with 250 µL of diluent in the tube labelled 50 u/mL.
6. Similarly prepare the 25, 12.5, 6.25, and 3.125 u/mL calibrators by serial dilution.

Note: Studies indicate that anti-SRBC IgM is present in rat serum or plasma at concentrations in excess of 4,000 u/mL. In order to obtain values within range of the calibration curve, we suggest samples initially be diluted 200 fold using the following procedure for each sample tested. Optimal dilutions may need to be determined empirically.
1. For each test sample dispense 298.5 µL of diluent into separate tubes.
2. Pipette and mix 1.5 µL of the serum/plasma sample into the tube containing 298.5 µL of diluent. This provides a 200 fold diluted sample.
3. Repeat this procedure for each sample to be tested.
4. Do not use dilutions lower than 25 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 u/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-SRBC IgM in u/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration for anti-SRBC 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 100, samples should be diluted appropriately and re-tested.