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

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 O(2) 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.

Gold nanoparticles assisted characterization of amine functionalized polystyrene multiwell plate and glass slide surfaces

“…Covalent immobilization of biomolecules on PS and GS surfaces can be achieved using a variety of techniques including wet chemical treatments, silane monolayers, plasmas, flame, and UV irradiation (Goddard and Hotchkiss 2007). Among these, silane monolayer using 3-aminopropylyl triethoxy silane (APTES), 3-aminopropylyl trimethoxy silane (APTMS), N-[3-(trimethoxysilyl) propyl]ethylenediamine (TMSPED), and 3-glycidoxy propyl trimethoxy silane (GOPS) for the functionalization of the amine and the epoxy groups on the PS plate and the GS surfaces (Huang et al 2012;Moirangthem et al 2012;Wang and Vaughn 2008;Yang et al 2005;Lin et al 2006;Kobayashi et al 2007;Dixit et al 2011). APTES functionalized PS plates and GS are used in the immobilization of antibodies for the detection of antigens.…”

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

“…1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Thermo Scientific) and sulfo-NHS (Thermo Scientific)-based heterobifunctional crosslinking of proteins is one of the most widely used procedures for biomolecular conjugation [14][15][16] and biochipbased assay development [17][18][19]. Our results in this study show the interference of sulfo-NHS with the BCA protein assay, which can lead to inaccurate protein estimation.…”

“…There are numerous reports where proteins have been quantified by the BCA protein assay in the presence of sulfo-NHS [5][6][7][8][9][10][11][12][13]. However, the interference of sulfo-NHS in the BCA assay has not been reported.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Thermo Scientific) and sulfo-NHS (Thermo Scientific)-based heterobifunctional crosslinking of proteins is one of the most widely used procedures for biomolecular conjugation [14][15][16] and biochipbased assay development [17][18][19]. Our results in this study show the interference of sulfo-NHS with the BCA protein assay, which can lead to inaccurate protein estimation.…”

Immobilization of Antibodies and Enzymes on 3-Aminopropyltriethoxysilane-Functionalized Bioanalytical Platforms for Biosensors and Diagnostics

“…The most widely used heterobifunctional cross-linker combination is EDC and N-hydroxysuccinimide (NHS)/N-hydroxysulfosuccinimide (SNHS), where EDC of the EDC-NHS/SNHS complex first binds to the carboxyl-terminal of the Abs (see Figure 1). 60 The zerolength cross-linker EDC-NHS/SNHS leaves the activated surface after an amide bond between NH 2 -APTES and the COOH-Ab is formed. Commercial manufacturers often recommend the use of EDC-SNHS/EDC-NHS in their immobilization protocols; however, EDC alone still yields positive results.…”

“…Subsequently, these resulting polymers are incubated with 2% (v/v) APTES in deionized water for 1 h (Figure 1). 60 This simple treatment obviates the use of complicated oxygen plasma for silanization. 61 Recently, this procedure has been proven in SPRbased immunoassays (IAs) using Au-coated SPR chips.…”