Tetanus Toxoid IgG1 ELISA 试剂盒

Wash Solution: The wash solution is provided as 20X stock. Prior to use dilute the contents of the bottle (50 mL) with 950 mL of distilled or de-ionized water. Calibrator
1. The monkey anti-tetanus toxoid IgG1 reference calibrator is provided as lyophilized stock. Reconstitute with the volume of diluent indicated on the vial label.
2. Prepare a 2.5 µg/mL working calibrator of monkey anti-tetanus toxoid IgG1 as further directed on the reference calibrator vial label (the reconstituted calibrator should be frozen at or below -20°C after reconstitution if additional use is intended).
3. Label 5 polypropylene or glass tubes as 1.25, 0.625, 0.313 and 0.078 µg/mL and pipette 250 µL of diluent into each tube.
4. Into the tube labeled 1.25 µg/mL, pipette and mix 250 µL of the 2.5 µg/mL anti-tetanus toxoid IgG with 250 µL of diluent. This provides the 1.25 µg/mL calibrator.
5. Similarly prepare the 0.625, 0.313 and 0.156 and 0.078 µg/mL calibrators by serial dilution.

General Note: The level of anti-tetanus toxoid IgG1 will depend on dose, route of immunization and time of sample collection. We found that anti-tetanus toxoid IgG1 is present in monkey serum at concentrations of 50 µg/mL or greater. We suggest that samples initially be diluted 200 fold using the following procedure for each sample to be tested. Optimum dilutions must be determined empirically. Dilutions of 50 fold or lower should not be used.
1. Dispense 298.5 µL of diluent into separate polypropylene or glass 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.

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators (2.5-0.078 µg/mL) and diluted samples into the wells (calibrators and samples should be tested in duplicate).
   3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 60 minutes.
   4. Aspirate the contents of the microtiter wells and wash the wells 5-6 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 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 µg/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-tetanus toxoid IgG1 in µg/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration of anti-tetanus toxoid IgG1 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 200, samples should be diluted appropriately and re-tested.