Development of Two Multiplex Immunoassays and Preliminary Application in Determining Protein Depletion:
For Seppro® IgY14/SuperMix Depletion Columns

By: Jiaxin Dong, Chris Melm, Holly Chapman, Farrah Fan, Dian Er Chen and Henry Duewel, Protein Technology and Assay, Research Biotechnology, Sigma-Aldrich Corporation

Abstract

Depletion of Highly Abundant Protein (HAP) and Moderately Abundant Protein (MAP) from plasma or serum samples can be achieved through Sigma Seppro® Tandem IgY14/ SuperMix columns. A range of 77–129 MAPs has been identified from the bound fraction of Seppro SuperMix resin by LC/MS-MS method. However, the efficiency and lot-to-lot consistency of MAP depletion using SuperMix resin has not been determined and requires a robust method to monitor. By taking advantage of the Luminex® technology, we have developed two multiplex immunoassays to monitor protein targets of depletion or retention. The MAP-10PLEX assay can simultaneously quantify 10 MAPs (Ceruloplasmin, C4, Plasminogen, IgD, C1q, Antithrombin III, Hemopexin, α-Antichymo-trypsin, CRP and Prealbumin) from plasma or serum samples depleted utilizing IgY14/Supermix columns. The LAP4PLEX assay, which is used to track loss of low-abundant proteins (LAP) during depletion, is able to simultaneously quantify Adiponectin, soluble L-selectin, soluble ICAM-1 and TIMP-1. The preliminary assay data on Seppro IgY-SuperMix depleted samples has suggested the two assays to be accurate, fast and cost-saving methods to monitor depletion consistency from lot-to-lot of the SuperMix resin in the R & D, Operations and QC departments.

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