RBC IgG ELISA 试剂盒

Note: Studies indicate that anti-SRBC IgG is present in mouse serum or plasma from SRBC immunized animals at concentrations in excess of 500 u/mL. In order to obtain values within the range of the calibration curve, we suggest that samples initially be diluted 50 fold using the following procedure for each sample tested:
1. For each test sample dispense 294 µL of diluent into separate tubes.
2. Pipette and mix 6 µL of the serum/plasma sample into a tube containing 294 µL of diluent. This provides a 50 fold diluted sample.
3. Repeat this procedure for each sample to be tested. Important: Do not use dilutions lower than 15 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 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 that 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 (A450) for each set of calibrators and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each reference 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 IgG in u/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration for anti-SRBC IgG in the serum/plasma sample.
   5. PC graphing software may be used for the above steps.
   6. If the OD450 values of samples fall outside the calibration curve when tested at a dilution of 50, samples should be diluted appropriately and re-tested (do not use dilutions lower than 15 fold).