Abstract
Background: Enzyme-linked immunosorbent assay (ELISA) array, a multiplex ELISA format, has significant advantages in comparison with classic ELISA technology, however, caution is necessary when fabricating an ELISA array for a research. Spotting buffer plays a key role in the performance of glass slide based protein microarray, however, such a buffer effect on polystyrene micro-plate has not been studied in detail. In this study, we describe the optimization of spotting buffer for the fabrication of ELISA array.
Methods: Antibody against interleukin 6 (IL-6) was selected as a model antibody for the construction of ELISA array. Different types of buffers (0.01 M phosphate buffered saline (PBS), pH7.4; 0.05 M carbonate- bicarbonate buffer saline (CBS), pH9.6) and different concentration of glycerol (2.5%, 5%, 10% and 20%) and Triton X-100 (0.001%, 0.003%, 0.006% and 0.01%) were investigated for improving the quality of spots and the immobilization efficiency on the polystyrene microplate. Different cytokines solutions (IL-1α, IL-1β, IL-10, IFN-α, TNF-α) were applied to determine the specificity. The different concentration of cytokine IL-6 solution ranging from (0.5-200 pg/ml) was applied to determine the dynamic range and sensitivity. The co-efficient of data was determined by repeated experiments.
Results: The results showed that the optimized spotting buffer, 0.01 M PBS with 10% glycerol and 0.003% Triton X-100 could produce spots with a homogeneous morphology (full and round) and significantly improve the signal intensities. The performance parameters experiments indicated that no cross reaction was observed, and the dynamic range of IL-6 was from 1 to 150 pg/ml with a sensitivity of 1 pg/ml. The co-efficient of data for repeated tests was less than 10.
Conclusion: The optimized spotting buffer could produce spots with a homogeneous morphology (full and round) and significantly improve the signal intensities. The results provide an improved approach to construct high performance ELISA array.
Keywords: ELISA array, spotting buffer, microarray, protein chip, spot morphology, multiplex ELISA.
Graphical Abstract
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