Development of recombinant-based mass spectrometric immunoassay with application to resistin expression profiling

Kemmons A. Tubbs, Urban A. Kiernan, Eric E. Niederkofler, Dobrin Nedelkov, Allan L. Bieber, Randall W. Nelson

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

This report addresses the need for additional assays for human resistin (hRES) by developing a rational progression of the mass spectrometric immunoassay to incorporate recombinant proteins. The recombinant-based hRES mass spectrometric immunoassay (RES-MSIA) was initially developed for the qualitative analysis of the human resistin homodimer from normal (healthy) plasma samples. The method involved selective extraction and detection of both endogenous and recombinant resistant proteins. RES-MSIA was then applied to the rigorous quantification of resistin. The resistin standard addition curve was constructed from serially diluted concentrations of rhRES using endogenous hRES, inherent in the human plasma, as the internal reference standard (IRS). The roles of endogenous and recombinant resistin were subsequently reversed, using rhRES as the 1RS during RES-MSIA quantification. Concurrently, the relative ratio of hRES to rhRES was used as an ancillary technique to rapidly determine the relative concentration of hRES in each of plasma samples. Overall, normal hRES levels determined by RES-MSIA were found to be comparable to those selected and determined by ELISA. With regard to gender, female donor samples were slightly elevated over males. Four single cardiac samples were analyzed and found to have hRES concentrations approximately three times that of the normal. The recombinant-based RES-MSIA is rapid and is amendable to parallel high-throughput robotic processing of resistin related disease cohorts.

Original languageEnglish (US)
Pages (from-to)3271-3276
Number of pages6
JournalAnalytical chemistry
Volume78
Issue number10
DOIs
StatePublished - May 15 2006

ASJC Scopus subject areas

  • Analytical Chemistry

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    Tubbs, K. A., Kiernan, U. A., Niederkofler, E. E., Nedelkov, D., Bieber, A. L., & Nelson, R. W. (2006). Development of recombinant-based mass spectrometric immunoassay with application to resistin expression profiling. Analytical chemistry, 78(10), 3271-3276. https://doi.org/10.1021/ac060013g