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Validation Parameters for Quantitating Specific Proteins Using ELISA or LC-MS/MS: Survey Results

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Abstract

Genetically modified organisms (GMOs) express one or more exogenous genes inserted through genetic engineering resulting in the production of novel protein(s). Agencies responsible for deregulating GMOs require that levels of the newly expressed protein(s) be characterized. Ideally, the quantitation methods used will be validated according to Good Laboratory Practices (GLPs) that are accepted by these global regulatory agencies. Targeted protein quantitation may be performed using enzyme-linked immunosorbent assays (ELISAs) with commercial kits or specially produced and validated antibodies. Proteins may also be quantitated by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Although some scientific guidance exists for validation of ELISA, the need remains for a harmonized set of protocols for protein quantitation in testing of GMOs, particularly for LC-MS/MS. We surveyed various industry experts to determine current practices for assay linearity, accuracy, and precision; assay specificity; detection limits; and methods to assess analyte stability for both ELISA and LC-MS/MS. We compared the survey results with assay criteria suggested in the literature and governmental guidance documents.

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Acknowledgments

The authors thank the Analytical Excellence through Industry Collaboration (AEIC) organization for providing a venue for discussion and a helpful review of the manuscript.

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Authors and Affiliations

  1. Critical Path Services, Garnett Valley, PA, USA

    Sharon B. Settlage & Julie E. Eble

  2. AMAR Immunodiagnostics, Hyderabad, India

    Jayant K. Bhanushali

  3. Bayer, Morrisville, NC, USA

    Matthew L. Cheever

  4. Monsanto, Chesterfield, MO, USA

    Ai-Guo Gao

  5. Pyxant Labs, Colorado Springs, CO, USA

    David A. Goldstrohm

  6. Dow AgroSciences, Indianapolis, IN, USA

    Ryan Hill & Guomin Shan

  7. Pioneer Hi-Bred International, Inc., Johnston, IA, USA

    Tiger X. Hu & Anita Unger

  8. E. I. DuPont de Nemours and Company, Wilmington, DE, USA

    Charles R. Powley

Authors
  1. Sharon B. Settlage
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  2. Julie E. Eble
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  3. Jayant K. Bhanushali
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  4. Matthew L. Cheever
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  5. Ai-Guo Gao
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  6. David A. Goldstrohm
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  7. Ryan Hill
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  8. Tiger X. Hu
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  9. Charles R. Powley
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  10. Anita Unger
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  11. Guomin Shan
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Corresponding author

Correspondence to Sharon B. Settlage.

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Funding

No outside funding was received for this study.

Conflict of Interest

Sharon B. Settlage declares she has no conflicts of interest. Julie E. Eble declares she has no conflicts of interest. Jayant K. Bhanushali declares he has no conflicts of interest. Matthew L. Cheever declares he has no conflicts of interest. Ai-Guo Gao declares he has no conflicts of interest. David A. Goldstrohm declares he has no conflicts of interest. Ryan Hill declares he has no conflicts of interest. Tiger X. Hu declares he has no conflicts of interest. Charles R. Powley declares he has no conflicts of interest. Anita Unger declares she has no conflicts of interest. Guomin Shan declares he has no conflicts of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Settlage, S.B., Eble, J.E., Bhanushali, J.K. et al. Validation Parameters for Quantitating Specific Proteins Using ELISA or LC-MS/MS: Survey Results. Food Anal. Methods 10, 1339–1348 (2017). https://doi.org/10.1007/s12161-016-0689-x

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  • DOI: https://doi.org/10.1007/s12161-016-0689-x

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