Microarray of recombinant antibodies using a streptavidin sensor surface self-assembled onto a gold layer.
We have developed a sensitive method for the detection of recombinant antibody-antigen interactions in a microarray format. The biochip sensor platform used in this study is based on an oriented streptavidin monolayer that provides a biological interface with well-defined surface architecture that dramatically reduces nonspecific binding interactions. All the antibody or antigen probes were biotinylated and coupled onto streptavidin-coated biochip surfaces (1 microL total volume). The detection limits for the immobilized probes on the microarray surface were 0.5 microgram/mL (200 fmol/spot) for the peptide antigen and 0.1 microgram/mL (3 fmol/spot) for the recombinant antibodies. Optimal concentrations for the detection of the Cy5-labeled protein target were in the range of 20 micrograms/mL. Protein microchips were used to measure antibody-antigen kinetics, to find optimal temperature conditions, and to establish the shelf life of recombinant antibodies immobilized on the streptavidin surface. For recombinant antibody fragments with a kDa of 10-100 nM, we have established an easy and direct immunoassay. In addition, we developed an indirect method for antibody detection with no need for expensive and time-consuming antibody purifications and modifications. Such a method was shown to be useful for large-scale screening of recombinant antibody fragments directly after their functional expression in bacteria. Our data demonstrate that recombinant antibody fragments are suitable components in the construction of antibody chips.
Pavlickova
P
P
University of Ulm, Ulm, Germany.
Knappik
A
A
Kambhampati
D
D
Ortigao
F
F
Hug
H
H
D000906:Antibodies / Q000737:chemistry*; D000936:Antigen-Antibody Complex / Q000032:analysis / Q000737:chemistry; D000941:Antigens / Q000032:analysis* / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D019921:Biotinylation / Q000379:methods; D002232:Carbocyanines; D020099:Coated Materials, Biocompatible; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D006046:Gold; D007118:Immunoassay / Q000379:methods; D007128:Immunoglobulin Fragments / Q000737:chemistry; D015335:Molecular Probes / Q000737:chemistry; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000941:Antigens; D002232:Carbocyanines; D020099:Coated Materials, Biocompatible; D007128:Immunoglobulin Fragments; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; C085321:cyanine dye 5; D006046:Gold; D019809:Streptavidin