In vitro determination of antigen quality: biosensor analysis and fluorescence spectroscopy.
We are probing the potential of two techniques to monitor the quality of antigens in vitro. Structural and conformational differences between diphtheria toxin and toxoid are detected via biosensor analysis (BIA-core) and fluorescence spectrometry. With BIA-core the interaction kinetics between toxin and toxoid and a monoclonal antibody were established. The fluorescence properties of both antigens were determined with respect to fluorescence intensity and emission maximum as a function of guanidinium hydrochloride concentration. In all cases clear differences were found between toxin and toxoid. Antibody affinity of the toxoid was lower compared with toxin, caused by lower binding and higher release rates. Fluorescence intensity of toxoid was reduced by about 50%. Toxoid was less sensitive to guanidinium hydrochloride-induced denaturation, reflected in a diminished shift of the emission maximum.
Kersten
G F
GF
National Institute of Public Health and Environment, Laboratory for Product and Process Development, Bilthoven, The Netherlands.
Jiskoot
W
W
Hazendonk
T
T
Beuvery
E C
EC
D000818:Animals; D015374:Biosensing Techniques; D004167:Diphtheria Toxin / Q000737:chemistry / Q000276:immunology*; D004168:Diphtheria Toxoid / Q000737:chemistry / Q000276:immunology*; D007700:Kinetics; D051379:Mice; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D004167:Diphtheria Toxin; D004168:Diphtheria Toxoid