Question (23607) | Anti-RAGE antibody [MM0520-8D11] (ab89911)

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Question

1) For neutralization studies with this antibody: - Which detailed protocol was used in lab? - How was the ED50 measured - by ELISA? 2) Used the antibody in the following way, but received the opposite results of inhibition - activation! Details of experiment: sample: human blood (PBMC (white blood cells)) goal: blocking AGE receptor (RAGE) with antibody ab89911 Ab dilution series: 0.1, 1.0 and 5 ug/mL incubation of Ab with 1x10^6 cells for 1h 37 degrees, 5% CO2 Detection: measurement of cytokine TNF alpha by sandwich ELISA expected result: if RAGE blocked, TNF alpha would decrease observed result: TNF alpha increase

Answer

Thank you for contacting us. Here is the reply I received from the lab. First to answer your question about the testing protocol used by the lab for neutralization studies: The neutralization function of this antibody was tested in vitro using a functional ELISA (between two proteins on ELISAs plates): Biotinylated AGE-BSA can bind to immobilized recombinant human RAGE coated on an ELISA plate. When 4 μg/mL of RAGE (100ul) was coated on to the ELISA plate, a 2μg/mL of ab89911 can block 50% of the binding of 0.5μg/mL of Biotinylated-AGE-BSA to RAGE and at 10 μg/mL, this antibody can block 92% of the binding. (The suggested conditions are for reference only and the end users should optimize the conditions themselves). Second, some suggestions for your experiment: Our test indicates that ab89911 can block the binding between AGE and RAGE. In your experiment, no AGE is present in the system (right?). We are not sure what you should expect in this study by simply using ab89911 to block RAGE. We suggest that the readout would need to be specific for the downstream signal after AGE-RAGE binding for this blocking experiment when AGE is added into the system. Also, I am not really sure why the TNF alpha signal would increase when using this antibody. Thus, I was wondering if maybe TNF alpha is not the right signal to measure? Or maybe could the antibody cause an immune response with the PBMCs and thus increase the TNF alpha signal? I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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