Optimizing immobilization conditions on a two dimensional carboxylbiosensor surface: pH dependence of antibody orientation andantigen binding capacity

Andersson, HenrikAttana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden/The Ångström Laboratory, Solid State Electronics, Uppsala University, P.O. Box 534, SE-751 21 Uppsala, Sweden (författare)

Optimizing immobilization conditions on a two dimensional carboxylbiosensor surface : pH dependence of antibody orientation andantigen 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 have shown that successful immobilization is highly dependent on surface pKa, antibody pI and pH of immobilization buffer. By use of EDC/sulfo-NHS activation reagents, the effect of the intrinsic surface pKa is avoided and immobilization also at very low pH has been made possible which is of importance for immobilization of acidic proteins. Generic immobilization conditions were demonstrated on a panel of antibodies which resulted in an average coefficient of variation of 4% for the immobilization of these antibodies. 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 antibodies on different antibody surfaces showed that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

Myrskog, Annika,1980-Linköpings universitet,Sensorvetenskap och Molekylfysik,Tekniska högskolan(Swepub:liu)annmy34 (författare)
Ingemarsson, BjörnAttana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden (författare)
Pei, ZhichaoAttana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden (författare)
Attana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden/The Ångström Laboratory, Solid State Electronics, Uppsala University, P.O. Box 534, SE-751 21 Uppsala, SwedenSensorvetenskap och Molekylfysik (creator_code:org_t)

Av författaren/redakt...: Andersson, Henri ...; Myrskog, Annika, ...; Ingemarsson, Bjö ...; Pei, Zhichao

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