Site-directed Immobilization of Antibody onto Solid Surfaces for the Construction of Immunochip

  • Paek, Se-Hwan (Program for Bio-Microsystem Technology, Korea University, Department of Biotechnology, Korea University) ;
  • Cho, Il-Hoon (Program for Bio-Microsystem Technology, Korea University) ;
  • Paek, Eui-Hwan (BioDigit Laboratories Corp., Technical Incubation Center, Biotechnology, Building, Korea University) ;
  • Lee, Haewon (Department of Chemistry, Hanyang University) ;
  • Park, Jeong-Woo (Chemical Engineering Department, Sogang University)
  • Published : 2004.03.01

Abstract

The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity, i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidin-coated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.

Keywords

References

  1. Analyst v.121 Oriented immobilization of antibodies and its applications to immunoassays and immunosensors Lu,B.;M.J.Smyth;R.O'kennedy https://doi.org/10.1039/an996210029r
  2. Anal. Biochem. v.312 Optimizing antibody immobilizaiton strategies for the construction of protein microarrays Peluso,P.;D.S.Wilson;D.Do;H.Tran;M.Vendatasub-baiah;D.Quincy;B.Heidecker;K.Poindexter;N.Tolani;M.Phelan;K.Witte;L.S.Jung;P.Wagner;S.Nock https://doi.org/10.1016/S0003-2697(02)00442-6
  3. Curr. Opin. Chem. Biol. v.6 Functional protein microarrays Wilson,D.S.;S.Nock https://doi.org/10.1016/S1367-5931(01)00281-2
  4. Anal. Biochem. v.205 Antibody-antigen complex formation with immobilized immunoglobulin Schramm,W.;S.H.Paek https://doi.org/10.1016/0003-2697(92)90577-T
  5. Methods v.22 Synthesis of bifunctional Antibodies for iImmunoassays DeSilva,B.S.;G.S.Wilson https://doi.org/10.1006/meth.2000.1033
  6. Anal. Biochem. v.324 A protein A-based orientation-controlled immobilization strategy for antibodies using nanometer-sized gold particles and plasma-polymerized film Wang,H.;Y.Liu;Y.Yang;T.Deng;G.Shen;R.Yu https://doi.org/10.1016/j.ab.2003.09.032
  7. Anal. Chim. Acta v.496 Direct oriented immobilization of F(ab) antibody fragments on gold Brogan,K.L.;K.N.Wolfe;P.A.Jones;M.H.Schoenfisch https://doi.org/10.1016/S0003-2670(03)00991-7
  8. Anal. Chem. v.67 Oriented immobilization of Fab¹ fragments on silica surfaces Lu,B.;J.Xie;C.Lu;C.Wu;Y.Wei https://doi.org/10.1021/ac00097a014
  9. J. Chromatogr. v.424 Synthesis of high capacity immunoaffinity sorbents with oriented immobilized immunoglobulins or their Fab¹ fragments for isolation of proteins Prisyazhnoy,V.S.;M.Fusek;Y.B.Alakhov https://doi.org/10.1016/S0378-4347(00)81101-9
  10. J. Mol. Electron. v.4 Orientation-controlled immobilization of protein molecules on thin organic films deposited by the plasma technique Jimbo,Y.;M.Saito
  11. Anal. Biochem. v.72 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the priciple of protein-dye binding Bradford,M.M. https://doi.org/10.1016/0003-2697(76)90527-3
  12. Anal. Biochem. v.210 Defined analyte-enzyme conjugates as signal generators in immunoassays Paek,S.H.;L.G.Bachas;W.Schramm https://doi.org/10.1006/abio.1993.1165
  13. Method Enzymol. v.91 Reassessment of Ellman's reagent Riddles,P.W.;R.L.Blakeley;B.Zerner https://doi.org/10.1016/S0076-6879(83)91010-8
  14. Anal. Biochem. v.243 Immobilized nitro-avidin and nitro-streptavidin as reusable affinity matrices for application in avidin-biotin technology Morag,E.;E.A.Bayer;M.Wilchek https://doi.org/10.1006/abio.1996.0514
  15. Protein Recognition of Immobilized Ligands Wilchek,M.;E.A.Bayer
  16. Biochem. J. v.89 Avidin: The nature of the biotinbinding site Green,N.M. https://doi.org/10.1042/bj0890609