RPLs do not possess disulphide bonds and so are compatible with buffers containing reducing agents.
Prior to analysis the MAb was denatured, to exposed sterically inaccessible Fc glycans, simply by adding dithiothreitol (DTT) and heating to 60C for 1 hour.
Analysis of MAb Fc Glycans Using an RPL Functionalised Biosensor Tip
Use of an RPL-Gal1 functionalised BLI biosensor tip for simple, rapid and label free detection and quantification of the Fc glycans of a CHO produced human IgG1 MAb.
Readings were performed on a forte Blitz instrument. Measurement cycles consisted of the following steps: (1) Baseline acquisition in Sample Buffer for 30 seconds (2) Target binding performed for 120 seconds (3) Target elution in Sample Buffer supplemented with 0.5M galactose for 120 seconds and (4) Biosensor regeneration in Sample Buffer for 300 sec.
Serial 1:1 dilution performed using Sample Buffer from an initial concentration of 500ug/mL to a final concentration of 15ug/mL.
Sample Conc. Range:
Human IgG1 MAb was prepared in Sample Buffer: TBST - Tris Buffered Saline, pH7.6. Each of the metal ions CaCl2, MnCl2 and MgCl2 was added to a final concentration of 1 mM. DTT was added to final concentration of 2 mM. Samples were heated to 60C for 1 hour and then cooled to room temperature prior to measurements being taken
Ni-NTA BLI Biosensor Tip Funtionalised with Galactophilic RPL-Gal1 which exhibits specificity for terminal beta-linked galactose & lactosamine.
Biosensor Type Used: