Bead Extraction and Heat Dissociation (BEHD) For Mitigation of Drug Interference
BEHD is the method developed for mitigating drug interference. BEHD method utilizes heat denaturation for the dissociation of the ADA-drug complex present in the samples. The samples are subsequently incubated with the sample and ADA is allowed to bind with Biotin-Drug and captured using streptavidin coated magnetic beads. Next, the samples are eluted using weak acids such as acetic acid or glycine, neutralized, and assayed in the ADA or NAb assay. The heat denaturation steps become critical when the drugs that are used to capture ADAs are susceptible to harsher acid-base conditions. This BEHD is used for the detection of ADA for cytokine therapy.
Protocol For BEHD
Heat Denaturation for Dissociation fo ADA-Drug Complex
50ul to 100 ul of the serum samples containing ADAs are treated with mild heat at 62 degrees celsius for 1 hour in thermocycler and allow the dissociation of drug-ADA complex
Binding of ADA to Biotin Drug
50 ug/ml of Biotin-drug prepared in PBS are added the heat-treated samples and incubated for allowing the ADA binding to Biotin-Drug
Capture of ADA-Biotin Drug Complex
The ADA-Biotin Drug complex is captured by mixing the streptavidin-coated magnetic beads to the ADA-drug sample mixture and incubating overnight at 4 degrees celcius
Washing and Removal of Residual Drug
After incubation, the magnetic Bead is washed using a Kingfisher or magnetic plate washer to remove unbound drug
Acid Elution
Weak acid including glycine or acetic acid are widely used to dissociate ADA from Biotin drug. Thus obtained drug unbound ADAs tested in ADA/NAb detection assay.