Mouse Anti-Human Antibody ELISA

Additional Information

Assay Background

Development of humanized monoclonal antibodies (IgG) and their fragments as in vivo diagnosis procedure (radionuclides) and treatment for patients with various diseases has made great progress.  In animal study, even a single dose injection of a humanized monoclonal antibody or its fragment may induce immune response directed against this foreign protein (immunogen). In the circulation, the presence of mouse antibody against human antibody would bind to the injected humanized antibody therapeutics or diagnosis and diminish the efficacy of either in vivo diagnosis or treatment. The present of MAHA in mouse serum or plasma specimens may cause both false positive and false negative immunoassay test results depending on assay principles and monoclonal/polyclonal antibodies used in the assay system. Therefore, the presence of MAHA would eventually lead to unreliable pre-clinical data collection and study conclusion.

Assay Procedure

1. Place a sufficient number of human IgG-coated microwell strips/wells in a holder to run MAHA standards, controls and unknown samples in duplicate.
2. Add 25 µL of standards, controls and patient samples into the designated microwell.
3. Add 100 µL of assay buffer to each well
4. Cover the plate with one plate sealer and incubate plate at room temperature, shaking for 45 minutes.
5. Prepare MAHA tracer antibody working solution by 1:21 fold dilution of the HRP-conjugated human antibody with the tracer Antibody Diluent. For each strip, it is required to mix 1 mL of the tracer antibody diluent with 50 µL of the tracer antibody in a clean test tube.
6. Remove plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
7. Add 100 µL of above diluted MAHA tracer antibody to each of the wells.
8. Cover the plate with a plate sealer and an aluminum foil and incubate plate at room temperature, shaking for 45 minutes.
9. Remove foil and plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
10. Add 100 µL of ELISA HRP Substrate into each of the wells. Cover the plate with a plate sealer and also with an aluminum foil to avoid exposure to light.
11. Incubate plate at room temperature for 20 minutes.
12. Remove the aluminum foil and plate sealer.  Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
13. Read the absorbance at 450 nm within 10 minutes in a microplate reader.

Typical Standard Curve