Mouse Anti-Human Antibody (MAHA) ELISA Assay Kit

$690.00

The Eagle Biosciences Mouse Anti-Human Antibody (MAHA) ELISA Assay Kit is produced for the quantitative determination of mouse anti-human antibody (MAHA) levels in mouse serum or plasma samples. It detects both MAHA-IgG and MAHA-IgM subtypes. The test is used for detection of mice with positive MAHA that may affect prescribed studies involving humanized monoclonal antibody, which is essential to avoid misleading immunoassay test results and conclusion.

Mouse Anti-Human Antibody (MAHA) ELISA Assay Kit

For Research Use Only

Size:  1×96 wells
Sensitivity:  0.1 µg/mL
Dynamic Range:  1 – 27 µg/mL
Incubation Time:  2 hours
Sample Type:  Mouse serum, Plasma*
Sample Size: 50 µL

Controls Included

Product Developed and Manufactured in the USA

*This kit can also be used for specimens collected from other animals such as rat, dog, cat, rabbit, primate, etc.


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 Principle


This MAHA ELISA Assay Kit is designed, developed and produced for the quantitative measurement of MAHA in serum and plasma samples. The assay utilizes the two-site “sandwich” technique with two selected antibodies that bind to MAHA.

Assay standards, controls and patient samples are directly added to wells of a microplate that is coated with highly purified human IgG. After the first incubation period, the MAHA binds to the human IgG on the wall of microtiter well and unbound proteins in each microtiter well are washed away. Then a horseradish peroxidase (HRP)-labeled human antibody is added to each microtiter well and a “sandwich” of “well coated human IgG – MAHA – HRP-conjugated human antibody” is formed. The unbound HRP conjugated human antibody is removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to MAHA on the wall of the microtiter well is directly proportional to the amount of MAHA in the sample. A standard curve is generated by plotting the absorbance versus the respective MAHA concentration for each standard on point-to-point or 4 parameter curve fit. The concentration of MAHA in test samples is determined directly from this standard curve.

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


Manual

Product Manual