Site-directed immobilisation of antibody fragments for detection of C-reactive protein

C-reactive protein, CRP antibody Fab′-fragments have been attached on pre-cleaned gold slides and protein repellent polymers have been used to block the remaining free space in between the antibody fragments. At optimal conditions the antibody fragments are site-directly immobilised on the surface and non-specific binding is reduced. The amount of Fab′-fragments in the polymer host monolayer has been optimised for various buffers. Binding of CRP to Fab′-fragment/polymer layers produced in phosphate buffered saline decreased with NaCl salt concentration. In a 1 M NaCl phosphate buffer, the antibodies seem to be randomly oriented on the surface with a similar response to CRP as that of an antibody F(ab) 2-fragment layer. In a 150 mM NaCl phosphate buffer, on the other hand, the fragments seem to be site-directly oriented and the response to CRP was fivefold. The highest response to CRP was obtained to a layer with a Fab′-fragment concentration of 60 μg/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 μg/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 μg/ml from serum/PBS. CRP was, moreover, successfully detected in patient samples with good reproducibility. The layer would thus be sensitive enough to analyse the CRP concentration in human serum for predicting cardiovascular disease.