Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity.
The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK(a), antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK(a) is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab(2) antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.
Pei
Zhichao
Z
College of Science, Northwest A&F University, Yangling, Shaanxi, People's Republic of China. zhichao.pei@attana.com
Anderson
Henrik
H
Myrskog
Annica
A
Dunér
Gunnar
G
Ingemarsson
Björn
B
Aastrup
Teodor
T
D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology*; D015374:Biosensing Techniques; D002264:Carboxylic Acids / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry; D015203:Reproducibility of Results; D055672:Static Electricity; D013499:Surface Properties
D055910:Antibodies, Immobilized; D000941:Antigens; D002264:Carboxylic Acids; D007202:Indicators and Reagents