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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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.
The authors declare no competing financial interests.
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)
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)
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)
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)
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).
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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