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Protein A‐based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme‐linked immunosorbent assay
Author(s) -
Yuan Yuan,
He Hongyan,
Lee L. James
Publication year - 2008
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22136
Subject(s) - chemistry , glutaraldehyde , detection limit , immunoassay , amine gas treating , chromatography , bioconjugation , ethyleneimine , polymer , antibody , combinatorial chemistry , organic chemistry , immunology , biology
Highly efficient antibody immobilization is extremely crucial for the development of high‐performance polymeric microdevices for enzyme‐linked immunosorbent assay (ELISA). In this article, a site‐selective tyrosinase (TR)‐catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly‐(methyl methacrylate) (PMMA) microchannels for interferon‐γ (IFN‐γ) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine‐containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine‐glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR‐catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal‐to‐noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96‐well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices. Biotechnol. Bioeng. 2009; 102: 891–901. © 2008 Wiley Periodicals, Inc.