Anti-Human Antibody (HAHA) ELISA Assay Kit

Additional Information

Assay Background

Clinically, humanized monoclonal antibodies (IgG) and their fragments are used in vivo diagnosis procedure (radionuclides) and treatment for patients with various diseases.  In patients, even a single dose injection of a humanized monoclonal antibody or its fragment may induce immune response directed against this foreign protein (immunogen). Also, people with autoimmune diseases, such as rheumatoid arthritis, lupus, etc. produce autoantibody against human IgG. In the circulation, the presence of human antibody against human IgG would bind to the injected humanized antibody therapeutics or diagnosis and, therefore, diminish the efficacy of either in-vivo diagnosis or treatment. Especially, the HAHA would increase the risk of anaphylactic complications to subsequent administration of the humanized monoclonal antibody-based therapy.

Assay Procedure

1. Place a sufficient number of human IgG-coated microwell strips/wells in a holder to run HAHA.
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 HAHA 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 HRP-conjugated Human Antibody working solution 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 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 (Cat. 10020) into each of the wells.
11. Cover the plate with a plate sealer and also with an aluminum foil to avoid exposure to light.
12. Incubate plate at room temperature for 20 minutes.
13. Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
14. Read the absorbance at 450 nm within 10 minutes in a microplate reader.

Typical Standard Curve