BMN701 Assay Concentration %Matrix in assay

Determine, in pooled serum, activity of endogenous a-d-glucosidases with activity buffer at pH 4.2 and activity buffer at pH 7.5 (using conditions from QC analytical) Controls needed: Control for the quality of activity of the assay. Standards will show how much PNP was created Before you work with activity, make sure you can detect the PNP in matrix, and determine what MRD detects the best Determine which MRD of matrix is best for optimal activity at saturating PNPG Find a buffer for pH 6.0 to 7.5 as activity buffer diluents Activity buffers and Diluents buffer need to be at the same pH. (Each assay with different pHs will have their own activity/diluents buffers.) Spike BMN701 in pooled serum, and then determine activity of spiked serum in an activity buffer at pH 4.2 and an activity buffer at pH 7.5 Test antibodies that we have for anti-BMN701 to see if any of them neutralize activity at pH 7.5 down to 4.2

Procedure Warm the PNP-a-Glucoside Working Substrate in a water bath at 37 C while preparing controls/samples dilutions. (30 to 45 minutes) Dilute the BMN701 in Dilution Buffer (determine concentration for analysis). Dilute controls/samples (find MRD)

(Diluted working standard 1:2 to determine activity range)

Load the microtiter plate Using a multichannel pipette, dispense 50 L/well of Activity Buffer. Transfer 50 L of the diluted controls/samples in to wells Pipette up and down after each addition to mix.

Note: Cover the plate during incubations to prevent evaporative losses. Std = 1 mM PNP Working Standard Ref = reference material diluted to 14 g/mL Blank = plate blank (50 L BMN701 Activity Buffer) Equilibrate the loaded plate to 37 C by placing in the incubator for 10 - 15 minutes. 8.6 Enzyme Reaction: 8.6.1 Remove the plate from the incubator. 8.6.2 Begin the 30-minute incubation time with simultaneous addition of 50 L of the PNP--Glucoside Working Substrate (preheated to 37 C) to each well of the first row of the plate using the multichannel pipette. 8.6.3 Working at a uniform rate, add 50 L of the PNP--Glucoside Working Substrate to each remaining row. 8.6.4 Immediately cover and return the plate to the incubator. Note: It is critical to work quickly and uniformly to prevent excessive cooling of the plate and to assure consistent incubation time for all rows. Take care not to contact the contents of each well with the tips to avoid cross-contamination between rows. 8.7 Stopping Reaction. 8.7.1 Stop the reaction by adding 200 L of Stop Buffer to each well in the first row with a multichannel pipette at 30 minutes. 8.7.2 Working at a uniform rate, add 200 L of Stop Buffer to each remaining row, working in the same sequence as for addition of the PNPGlucoside Working Substrate. Note: It is critical to work quickly and uniformly to assure a consistent incubation time for all rows. Take care not to contact the contents of each well with the tips to avoid cross-contamination between rows. 8.8 Blank the spectrophotometer with the contents of wells H1 to H12. 8.9 Read absorbance for each well at 405 nm.