BIOCHEMISTRY, BIOMEDICINE & PHARMACEUTICS

 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 : i) Heat Denaturation for Dissociation for 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 ...

            Solid Phase with Extraction  Acid Dissociation (SPEAD) is a plate-based method for reducing drug interference in the ADA assay. This method utilizes weak organic acids (Glycine, Acetic acid) to dissociate the drug-ADA complex. The sample is neutralized and allowed the bind to a biotinylated drug and captured on the streptavidin-coated 96 well plate.  After the ADA is captured, the plate is washed to remove any unbound drug present in the sample. The second acid treatment is used to elute the ADA bound to the plate. The ADA sample is transferred, neutralized with Tris-HCl base, and processed in the ADA detection assay.  Procedure: 1) Coating of the plate with a drug: Streptavidin-precoated 96 well high binding plate is widely used for capturing the biotinylated drug / ADA complex. For the optimization of drug concentration,  Test different concentrations of biotinylated drug ( 0.5, 1.0, 5.0 ug / ml, and 10 ug/ml ) and ch...