Construction of an antibody microarray based on agarose‐coated slides

Abstract

Construction of an antibody microarray based on agarose-coated slidesThe antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 mg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.

Specific detection of Salmonella enterica and Escherichia coli strains by using ELISA with bacteriophages as recognition agents

“…Their performance, especially affinity and avidity, strongly affects the assay sensitivity. Moreover, an additional drawback is their limited shelf life, which limits the viability of ready-to-use antibody-based sensors [2]. …”

Technological Development of Antibody Immobilization for Optical Immunoassays: Progress and Prospects

“…The analyte can be detected by the naked eye or fluorescent immunoassays after the antigen has been captured by an antibody encapsulated in the agarose gel that is coated on the silanized surface by physical adsorption. Such analytes include the monocyte chemoattractant protein 1 (Lv et al, 2007), antihepatitis B virus surface antigen, anti-hepatitis B virus antigen , Helicobacter hepaticus bacterium (Memisevic et al, 2009), viruses in aquatic animals , white spot syndrome virus, and lymphocystis disease virus . Recently, agarose-coated slides have been produced using new techniques, including electrospinning techniques, pore-forming techniques (Memisevic et al, 2009), and robotic printing technology (Lv et al, 2007).…”

“…Such analytes include the monocyte chemoattractant protein 1 (Lv et al, 2007), antihepatitis B virus surface antigen, anti-hepatitis B virus antigen , Helicobacter hepaticus bacterium (Memisevic et al, 2009), viruses in aquatic animals , white spot syndrome virus, and lymphocystis disease virus . Recently, agarose-coated slides have been produced using new techniques, including electrospinning techniques, pore-forming techniques (Memisevic et al, 2009), and robotic printing technology (Lv et al, 2007). Xu et al (2010) compared the use of agarose gel-coatings, APTES, acrylamide, poly-L-lysine, aldehyde, sulfydryl, and amino-modified glass slides for antibody immobilization and found that agarose gel-coated glass slides gave the best results, because the porous nature of the sol-gel network is suitable for antibody entrapment and antibodies immobilized in porous sol-gel are able to interact with external chemical species or analytes easily (Gupta and Chaudhury, 2007).…”

Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections

“…The slides were thoroughly washed with Milli-Q water (Millipore, Billerica, MA, USA) and dried overnight at 140 • C. The slides were then modified with amine (Duburcq et al, 2004a), aldehyde (Zammatteo et al, 2000), poly-llysine (Haab et al, 2001), agarose (Afanassiev et al, 2000;Lv et al, 2007), polyacrylamide (Angenendt et al, 2002) and nitrocellulose (Kiernan, 1999), respectively. The modified glass slides were stored in desiccators at 4 • C.…”

“…Before immobilization of protein probes, the chemically modified glass slides were activated in a 20 mM solution of NaIO 4 at room temperature for 30 min, then thoroughly washed three times in deionized water, and dried with an argon flow (Lv et al, 2007).…”