Analytical and Bioanalytical Chemistry

, Volume 410, Issue 4, pp 1389–1396 | Cite as

Development of a sandwich ELISA with potential for selective quantification of human lactoferrin protein nitrated through disease or environmental exposure

  • Amani Y. Alhalwani
  • John E. Repine
  • Michelle K. Knowles
  • J. Alex Huffman
Research Paper


Lactoferrin (LF) is an important multifunctional protein that comprises a large fraction of the protein mass in certain human fluids and tissues, and its concentration is often used to assess health and disease. LF can be nitrated by multiple routes, leading to changes in protein structure, and nitrated proteins can negatively impact physiological health via nitrosative stress. Despite an awareness of the detrimental effects of nitrated proteins and the importance of LF within the body, cost-effective methods for detecting and quantifying nitrated lactoferrin (NLF) are lacking. We developed a procedure to selectively quantify NLF using sandwich enzyme-linked immunosorbent assay (ELISA), utilizing a polyclonal anti-LF capture antibody paired with a monoclonal anti-nitrotyrosine detector antibody. The assay was applied to quantify NLF in samples of pure LF nitrated via two separate reactions at molar ratios of excess nitrating agent to the total number of tyrosine residues between 10/1 and 100/1. Tetranitromethane (TNM) was used as a laboratory surrogate for an environmental pathway selective for production of 3-nitrotyrosine, and sodium peroxynitrite (ONOO) was used as a surrogate for an endogenous nitration pathway. UV-vis spectroscopy (increased absorbance at 350 nm) and fluorescence spectroscopy (emission decreased by > 96%) for each reaction indicate the production of NLF. A lower limit of NLF detection using the ELISA method introduced here was calculated to be 0.065 μg mL−1, which will enable the detection of human-physiologically relevant concentrations of NLF. Our approach provides a relatively inexpensive and practical way to assess NLF in a variety of systems.

Graphical abstract

We developed a procedure to selectively quantify nitrated lactoferrin (NLF) protein using a sandwich enzyme-linked immunosorbent assay (ELISA) and verified results against several spectroscopic techniques. Our approach provides an inexpensive and practical way to assess NLF in a variety of systems.


Protein nitration Nitrotyrosine Ocular disease Atmospheric pollution Peroxynitrite Tetranitromethane 



Amani Alhalwani acknowledges scholarship support from King Saud bin Abdulaziz University for Health Sciences (KSAU-HS).

Funding information

This study received funding support through a grant from the University of Denver Knoebel Institute for Healthy Aging (KIHA).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_779_MOESM1_ESM.pdf (251 kb)
ESM 1 (PDF 250 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Amani Y. Alhalwani
    • 1
  • John E. Repine
    • 2
  • Michelle K. Knowles
    • 1
  • J. Alex Huffman
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of DenverDenverUSA
  2. 2.Webb-Waring Center, School of MedicineUniversity of ColoradoAuroraUSA

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