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 1M 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 microg/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 microg/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 microg/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.
Vikholm-Lundin
Inger
I
Information Technology, Technical Research Centre of Finland, PO Box 14021, FIN-33101 Tampere, Finland. inger.vikholm@vtt.fi
Albers
Willem M
WM
D000327:Adsorption; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000097:blood; D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001774:Blood Chemical Analysis / Q000295:instrumentation / Q000379:methods*; D002097:C-Reactive Protein / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003460:Crystallization / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D020099:Coated Materials, Biocompatible; D002097:C-Reactive Protein