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Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays

Nature Protocols volume 6, pages 439–445 (2011)Cite this article

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Abstract

This protocol describes an improved and optimized approach to develop rapid and high-sensitivity ELISAs by covalently immobilizing antibody on chemically modified polymeric surfaces. The method involves initial surface activation with KOH and an O2 plasma, and then amine functionalization with 3-aminopropyltriethoxysilane. The second step requires covalent antibody immobilization on the aminated surface, followed by ELISA. The ELISA procedure developed is 16-fold more sensitive than established methods. This protocol could be used generally as a quantitative analytical approach to perform high-sensitivity and rapid assays in clinical situations, and would provide a faster approach to screen phage-displayed libraries in antibody development facilities. The antibody immobilization procedure is of ∼3 h duration and facilitates rapid ELISAs. This method can be used to perform assays on a wide range of commercially relevant solid support matrices (including those that are chemically inert) with various biosensor formats.

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Figure 1: Schematic representation of the optimized surface modification and antibody immobilization strategy.
Figure 2: Assay performance with the developed and conventional ELISA procedure.
Figure 3: The performance of the developed procedure on a range of polymeric solid support matrices.

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References

  1. Jia, C.-P. et al. Nano-ELISA for highly sensitive protein detection. Biosens. Bioelectron. 24, 2836–2841 (2009).

    Article  CAS  Google Scholar 

  2. Andrade, G., Barbosa-Stancioli, E.F., Mansur, A.A.P., Vasconcelos, W.L. & Mansur, H.S. Design of novel hybrid organic–inorganic nanostructured biomaterials for immunoassay applications. Biomed. Mater. 1, 221–234 (2006).

    Article  CAS  Google Scholar 

  3. Ball, V., Huetz, P., Elaissari, A., Cazenava, J.P., Voegel, J.C. & Schaaf, P. Kinetics of exchange processes in the adsorption of proteins on solid surfaces. Proc. Natl Acad. Sci. USA 91, 7330–7334 (1994).

    Article  CAS  Google Scholar 

  4. Huetz, P., Ball, V., Voegel, J.C. & Schaaf, P. Exchange kinetics for a heterogeneous protein system on a solid surface. Langmuir 11, 3145–3152 (1995).

    Article  CAS  Google Scholar 

  5. Essa, H., Magner, E., Cooney, J. & Hodnett, B.K. Influence of pH and ionic strength on the adsorption, leaching and activity of myoglobin immobilized onto ordered mesoporous silicates. J. Mol. Catal. B 49, 61–68 (2007).

    Article  CAS  Google Scholar 

  6. Wang, X., Wang, Y., Xu, H., Shan, H. & Lu, J.R. Dynamic adsorption of monoclonal antibody layers on hydrophilic silica surface: a combined study by spectroscopic ellipsometry and AFM. J. Colloid Interface Sci. 323, 18–25 (2008).

    Article  CAS  Google Scholar 

  7. Kaur, J., Boro, R.C., Wangoo, N., Singh, K.R. & Suri, C.R. Direct hapten-coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides. Anal. Chim. Acta. 607, 92–99 (2008).

    Article  CAS  Google Scholar 

  8. Mansur, H., Palhares, R., Andrade, G., Piscitelli-Mansur, A. & Barbosa-Stancioli, E. Improvement of viral recombinant protein-based immunoassays using nanostructured hybrids as solid support. J. Mater. Sci. Mater. Med. 20, 513–519 (2009).

    Article  CAS  Google Scholar 

  9. Gomez-Serrano, V., Acedo-Ramos, M., Valenzuela-Calahorro, C. & Lopez-Peinado, A.J. Regeneration of activated carbon after contact with sulfuric acid solution. J. Chem. Technol. Biotechnol. 75, 835–839 (2000).

    Article  CAS  Google Scholar 

  10. Vasconcellos, A.S., Oliveira, J.A.P. & Baumhardt-Neto, R. Adhesion of polypropylene treated with nitric and sulfuric acid. Eur. Polym. J. 33, 1731–1734 (1997).

    Article  CAS  Google Scholar 

  11. Dixit, C.K., Vashist, S.K., O'Neill, F.T., O'Reilly, B., MacCraith, B.D. & O'Kennedy, R. Development of a high sensitivity rapid sandwich ELISA procedure and its comparison with the conventional approach. Anal. Chem. 82, 7049–7052 (2010).

    Article  CAS  Google Scholar 

  12. Patel, G.N. & Bolikal, D. Single step pre-swelling and etching of plastics for plating. US patent no. 5,049,230 (1991).

  13. Patel, G.N. & Patel, S.H. Etching plastics with nitrosyls. US patent no. 5,591,354 (1997).

  14. Park, S. & Jung, W. Effect of KOH activation on the formation of oxygen structure in activated carbons synthesized from polymeric precursor. J. Colloid Interface. Sci. 250, 93–98 (2002).

    Article  CAS  Google Scholar 

  15. Vijayendran, R.A. & Leckband, D.E. A quantitative assessment of heterogeneity for surface-immobilized proteins. Anal. Chem. 73, 471–480 (2001).

    Article  CAS  Google Scholar 

  16. Park, S., Seo, M., Ma, T. & Lee, D. Effect of chemical treatment of Kevlar fibers on mechanical interfacial properties of composites. J. Colloid Interface Sci. 252, 249–255 (2002).

    Article  CAS  Google Scholar 

  17. Svarnas, P., Spyrou, N. & Held, B. Polystyrene thin films treatment under DC point-to-plane low-pressure discharge in nitrogen for improving wettability. Eur. Phys. J. Appl. Phys. 28, 105–112 (2004).

    Article  CAS  Google Scholar 

  18. Laib, S. & MacCraith, B.D. Immobilization of biomolecules on cycloolefin polymer supports. Anal. Chem. 79, 6264–6270 (2007).

    Article  CAS  Google Scholar 

  19. Raj, J. et al. Surface immobilisation of antibody on cyclic olefin copolymer for sandwich immunoassay. Biosen. Bioelectron. 24, 2654–2658 (2009).

    Article  CAS  Google Scholar 

  20. Boulares-Pender, A., Prager-Duschke, A., Elsner, C. & Buchmeiser, M.R. Surface-functionalization of plasma-treated polystyrene by hyperbranched polymers and use in biological applications. J. Appl. Polym. Sci. 112, 2701–2709 (2009).

    Article  CAS  Google Scholar 

  21. North, S.H., Lock, E.H., Cooper, C.J., Franek, J.B., Taitt, C.R. & Walton, S.G. Plasma-based surface modification of polystyrene microtitre plates for covalent immobilization of biomolecules. Appl. Mater. Interface 2, 2884–2891 (2010).

    Article  CAS  Google Scholar 

  22. Kaur, J., Singh, K.V., Raje, M., Varshney, G.C. & Suri, C.R. Strategies for direct attachment of hapten to a polystyrene support for applications in enzyme-linked immunosorbent assay (ELISA). Anal. Chim. Acta. 506, 133–135 (2004).

    Article  CAS  Google Scholar 

  23. Lacy, A. et al. Rapid analysis of coumarins using surface plasmon resonance. J. AOAC Int. 89, 884–892 (2006).

    CAS  PubMed  Google Scholar 

  24. Vashist, S.K., O'Sullivan, S.A., O'Neill, F., Holthofer, H., O'Reilly, B. & Dixit, C.K. A multiwell plate for biological assays. WIPO, Publication no. WO2010/044083 (2010).

  25. Song, Y., Hildebrand, H. & Schmuki, P. Optimized monolayer grafting of 3-aminopropyltriethoxysilane onto amorphous, anatase and rutile TiO2 . Surf. Sci. 604, 346–353 (2010).

    Article  CAS  Google Scholar 

  26. Cass, T. & Ligler, F.S. Immobilized Biomolecules in Analysis: A Practical Approach (Oxford University Press, 1998).

  27. Armbruster, D.A., Schwarzhoff, R.H., Hubster, E.C. & Liserio, M.K. Enzyme immunoassay, kinetic microparticle immunoassay, radioimmunoassay, and fluorescence polarization immunoassay compared for drugs-of-abuse screening. Clin. Chem. 39, 2137–2146 (1993).

    CAS  PubMed  Google Scholar 

  28. Armbruster, D.A., Tillman, M.D. & Hubbs, L.M. Limit of detection (LOD)/limit of quantitation (LOQ): comparison of the empirical and the statistical methods exemplified with GC-MS assays of abused drugs. Clin. Chem. 40, 1233–1238 (1994).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge Bristol-Myers Squibb (BMS), Syracuse, USA and Industrial Development Agency, Ireland for the financial support under the Centre for Bioanalytical Sciences (CBAS) project code 116294. This material is based on work supported in part by the Science Foundation Ireland under grant 05/CE3/B754.

Author information

Author notes

  1. Sandeep Kumar Vashist

    Present address: Present address: NUS Nanoscience and Nanotechnology Initiative Nanocore, National University of Singapore, Singapore.,

  2. Chandra Kumar Dixit and Sandeep Kumar Vashist: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Bioanalytical Sciences, National Centre for Sensor Research, Dublin City University, Dublin, Ireland

    Chandra Kumar Dixit, Sandeep Kumar Vashist, Brian D MacCraith & Richard O'Kennedy

  2. Applied Biochemistry Group, School of Biotechnology, Dublin City University, Dublin, Ireland

    Chandra Kumar Dixit & Richard O'Kennedy

  3. Swords Laboratories, Bristol-Myers Squibb, Dublin, Ireland

    Sandeep Kumar Vashist

  4. Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland

    Brian D MacCraith & Richard O'Kennedy

Authors
  1. Chandra Kumar Dixit
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  3. Brian D MacCraith
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  4. Richard O'Kennedy
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Contributions

C.K.D., S.K.V. and R.O. conceived, designed and refined the method; C.K.D., S.K.V. and R.O. wrote this manuscript; S.K.V., R.O. and B.D.M. supervised the work.

Corresponding author

Correspondence to Richard O'Kennedy.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Optimization of silanization duration and APTES concentration for the developed protocol. Optimization of silane concentration (b) and silanization time with optimum concentration (a). The selection of optimum concentration and time was based upon the corresponding optimum signals obtained by specific set. Figure 'a' demonstrates that 1hr is the optimum duration for silanization using 2% (v/v) APTES. The figure 'b' shows that 2% (v/v) APTES is the optimum conc. If conc. greater than 2% (v/v) APTES are used, there is a gradual reduction in the assay signal. This may be attributed to the formation of multilayers, which causes decrease in the signal as additional molecules are not correctly oriented and thus their amino groups are not accessible for covalent binding. (DOC 67 kb)

Supplementary Fig. 2

Characterization of amino silane-functionalization of the polystyrene surface. FTIR analysis of APTES-based amine-functionalized polystyrene surface. The Perkin Elmer FTIR instrument was employed to analyze the functionalization. Characteristic N-H stretch and vibrations are visible in the spectra. A ninhydrin test was also performed in addition to contact angle measurement in order to characterize the appearance of amine groups (data not shown). (DOC 235 kb)

Supplementary Fig. 3

Relevant process controls employed to develop the reported procedure. To optimize the developed procedure various process controls were employed. These process controls help in analyzing the cross-reactivity of the various assay components against each other. Since the antibodies and other proteins are developed in animal models, therefore, the probability of cross-reactivity among the biomolecules of animal origin is always a significant consideration. Various process controls employed to monitor the efficacy of each process step. NH2 corresponds the amine-functionalized microtiter plate. Whereas, all the components in the process controls 1 to 8 were adsorbed on the amine-functionalized plate. Process controls 1-8 were employed to analyze the cross-reactivity of the kit components that is non-specific interaction of these components on plate. Significantly negligible absorbance obtained for controls 1-7 suggests that there is minimum or no non-specific adsorption. Whereas, control 8 suggests high functional efficiency of the detection system that is streptavidin-HRP. In case of control 9, the biotin-conjugated detection antibody was covalently immobilized on the functionalized plate that serves as a positive control and determines the efficacy of the immobilization procedure on the amine-functionalized surface. ‘NH2-BSA’ controls were employed to analyze the blocking potential of BSA. Since BSA is used as surface blocker in this study this control also checks the cross-reactivity of kit components to BSA. ‘Ab’ corresponds to antibody and ‘SA/HRP’ to streptavidin-HRP conjugate. (DOC 26 kb)

Supplementary Fig. 4

Standard curve analysis based on a four-parameter logistic in Sigmaplot 11 software. The standard curve plotted using Sigmaplot 11.0 software. An ideal standard curve as represented here is sigmoidal 'S' shaped. The dotted line represents the linear working range. Various analytical parameters such as EC50 and Hill slope can be determined using mathematical calculations for this curve (Supplementary Methods). (DOC 48 kb)

Supplementary Fig. 5

Procedure for assembling the modified microtitre plate for performing assays on various polymeric substrates using the developed protocol. (DOC 164 kb)

Step 1. The bottomless microtitre plates were preblocked with 1% (v/v) BSA by putting the plate in a container for 30 min.

Step 2. The preblocked bottomless microtiter plates were attached to the double-sided pressure sensitive adhesive, which has the same patterns of holes as the microtiter plate wells.

Step 3. The other side of the double-sided pressure sensitive adhesive attached to the bottomless microtiter plate was then attached to the polymeric substrate(s) in the form of slides or sheets.

Step 4. The assembled modified microtiter plate was then pressed together to form strong attachment of the bottomless microtiter plate and substrate by the pressure sensitive adhesive.

Specific details of the modified microtiter plate are provided in a patent application1.

1. Vashist, S.K., O'Sullivan, S.A., O'Neill, F., Holthofer, H., O'Reilly, B. & Dixit, C.K. A multiwell plate for biological assays WIPO, Publication number WO2010/044083 (2010).

Supplementary Methods

The data described in this section supplements the main text. It provides information about the determination of analytical information for the modified ELISA protocol using standard curve analysis based on a four-parameter logistic in SigmaPlot 11 software; calculation of LOD; and analytical comparison of the developed protocol with the conventional protocol. (DOC 47 kb)

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Dixit, C., Vashist, S., MacCraith, B. et al. Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays. Nat Protoc 6, 439–445 (2011). https://doi.org/10.1038/nprot.2011.304

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