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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.

A Fast Universal Immobilization of Immunoglobulin G at 4°C for the Development of Array-based Immunoassays