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Analysis of serum protein glycosylation by a differential lectin immunosorbant assay (dLISA).

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Clin Proteomics. 2013 Sep 10 (1).

Lectin immunosorbant assays (LISAs) have been widely used for analyzing protein glycosylation. However, the analysis of serum samples by LISAs could suffer from high sample-dependent background noise. The aim of this study is to develop a differential lectin immunosorbant assay (dLISA) with reduced background interferences.

For the analysis of protein glycosylation, dLISA establishes a dose-response curve for every serum sample. The sample is split into five aliquots. Four aliquots undergo differential removal of the glycoprotein of interest by immunoprecipitation. Then, all five aliquots are subject to two measurements: protein by immunoassay and protein glycans by LISA. A dose-response curve is established by plotting glycans signals on the y-axis and protein levels on the x-axis for all the aliquots. Slope of the curve, calculated by linear progression analysis and expressed as fluorescence per concentration of protein, is used for the measurement of protein glycosylation in the serum sample.

To demonstrate the feasibility of the dLISA approach, we used recombinant, fucosylated tissue inhibitor of metallopeptidase 1 (TIMP-1) as the target glycoprotein. Magnetic beads based TIMP1 immunoassay and TIMP-1 UEA LISA were developed for the measurement of TIMP1 protein and terminal α1, 2 fucosylated glycans on TIMP1, respectively. Serum samples supplemented with differentially fucosylated recombinant TIMP-1 were used to demonstrate that the slopes measured the TIMP-1 fucosylation, and were less prone to background interference.

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