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Optimization of Spotting Buffer for Polystyrene Based ELISA Arrays

[ Vol. 13 , Issue. 6 ]


Yong-Jin Li*   Pages 488 - 492 ( 5 )


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.


ELISA array, spotting buffer, microarray, protein chip, spot morphology, multiplex ELISA.


College of Life Science, Huzhou University, 313000 Huzhou

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