title: A fast universal immobilization of immunoglobulin G at 4 °C for the development of array-based immunoassays.
abstract: To maintain the antibody activity and enhance performance of array-based immunoassays, protein G was used to allow a shorter duration of immunoglobulin G immobilization at 4 °C, with the antibody placed in the appropriate orientation. The multiplexed detection of six pain-related message molecules (PRMMs) was used as examples for the development of array-based immunoassays: substance P, calcitonin gene-related peptide, nerve growth factor, brain-derived neurotrophic factor, tumor necrosis factor-α, and β-endorphin. Protein G- and non-protein G-coated slides were tested. Compared to non-protein G immunoassays, protein G shortened the antibody immobilization time at 4 °C from overnight to 2 hours. Only protein G-facilitated immunoassays succeeded in simultaneously detecting all six PRMMs with high specificity. Dose-response curves showed that the limits of detection of the protein G-multiplexed immunoassays for the PRMMs was approximately 164, 167, 120, 60, 80, and 92 pg/ml, respectively. Thus, protein G effectively shortens the duration of antibody immobilization at 4 °C, allowing the use of sensitive array-based immunoassays for the simultaneous detection of PRMMs.
authors.affiliation: Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli, Taiwan.
authors.forename: Shu-Lin
authors.initials: SL
authors.lastname: Guo
authors.forename: Po-Chung
authors.initials: PC
authors.lastname: Chen
authors.forename: Ming-Shuo
authors.initials: MS
authors.lastname: Chen
authors.forename: Yu-Che
authors.initials: YC
authors.lastname: Cheng
authors.forename: Jun-Mu
authors.initials: JM
authors.lastname: Lin
authors.forename: Hoong-Chien
authors.initials: HC
authors.lastname: Lee
authors.forename: Chien-Sheng
authors.initials: CS
authors.lastname: Chen
mesh_terms: D055910:Antibodies, Immobilized; D001426:Bacterial Proteins / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor; D015740:Calcitonin Gene-Related Peptide; D004305:Dose-Response Relationship, Drug; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G; D020932:Nerve Growth Factor; D013373:Substance P; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha; D001615:beta-Endorphin
chemical_list: D055910:Antibodies, Immobilized; D001426:Bacterial Proteins; D019208:Brain-Derived Neurotrophic Factor; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D014409:Tumor Necrosis Factor-alpha; D013373:Substance P; D001615:beta-Endorphin; D020932:Nerve Growth Factor; D015740:Calcitonin Gene-Related Peptide