10th International Aerosol Conference September 2 - September 7, 2018 America's Center Convention Complex St. Louis, Missouri, USA
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Development of an Immunoassay for Detection of the Nitrated Form of Allergenic Ragweed Protein (nAmb a 1)
COURTNEY SEFFENSE, J. Alex Huffman, University of Denver, CO
Abstract Number: 1023 Working Group: Bioaerosols
Abstract The frequency of allergic conditions has risen over the past several decades, particularly in urban areas. It has been hypothesized that one cause for this rise may be links between allergenic pollen and urban pollutants. For example, secondary pollutant gases ozone and nitrogen oxides have been shown to react with tyrosine residues within certain proteins and that these chemically modified proteins can increase their allergenicity.
Thus far, relatively little investigation has been directed toward the measurement of nitrated proteins in ambient aerosol, and so the hypothesis about the urban pollen allergy phenomenon has not been well tested. One reason for the lack of measurements is the high relative cost and complexity of techniques to detect and quantify nitrated allergens in atmospheric samples. Several papers approximately 15 years ago pioneered the investigation of nitrated proteins in ambient samples [1,2], but since that point little study has been focused on ambient field samples. Laboratory work over the last decade has focused on aspects of the chemistry of protein nitration and oligomerization, using the birch pollen protein Bet v 1 as a model [i.e. 3-5] and other related work has utilized the human protein lactoferrin as a model [6].
Franze et al. [1,2] utilized an enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify nitrated protein material in ambient aerosol samples. Following this initial work, we have begun to develop a sandwich ELISA protocol for the selected detection of nitrated Amb a 1 (nAmb), which is a key allergenic protein in Ragweed pollen, a ubiquitous allergen in many areas of the United States and Europe. The assay entails sandwiching the protein to be detected (antigen) between two antibodies and detecting the change in color, which varies a function of the antigen concentration. Our initial work has focused on nitrated bovine serum albumin (nBSA) protein as an inexpensive antigen model as we optimize procedures. We have recently begun optimizing a parallel procedure to detect nitrated nAmb, toward the eventual goal of applying the assay to samples of airborne pollen collected in the field. We will discuss details of the ELISA protocols, discussing factors affecting reliability of assay calibration curves. Initial work testing both the nBSA and nAmb protocols shows encouraging results.
The work is being conducted as a part of an undergraduate research thesis by Courtney Seffense and is supported, in part, by the University of Denver Undergraduate Research Center (URC).
References:
[1] Franze, T., Weller, M. G., Niessner, R., and Poschl, U.: Enzyme immunoassays for the investigation of protein nitration by air pollutants, Analyst, 128, 824-831, 2003. [2] Franze, T., Weller, M. G., Niessner, R., and Pöschl, U.: Protein nitration by polluted air, Environ. Sci. Technol., 39, 1673-1678, 2005. [3] Yang, H., Zhang, Y., and Pöschl, U.: Quantification of nitrotyrosine in nitrated proteins, Analytical and Bioanalytical Chemistry, 397, 879-886, 2010. [4] Selzle, K., Ackaert, C., Kampf, C. J., Kunert, A. T., Duschl, A., Oostingh, G. J., and Poeschl, U.: Determination of nitration degrees for the birch pollen allergen Bet v 1, Analytical and Bioanalytical Chemistry, 405, 8945-8949, 2013. [5] Kampf, C. J., Liu, F. B., Reinmuth-Selzle, K., Berkemeier, T., Meusel, H., Shiraiwa, M., and Poschl, U.: Protein Cross-Linking and Oligomerization through Dityrosine Formation upon Exposure to Ozone, Environ. Sci. Technol., 49, 10859-10866, 2015. [6] Alhalwani, A. Y., Repine, J. E., Knowles, M. K., and Huffman, J. A.: Development of a sandwich ELISA with potential for selective quantification of human lactoferrin protein nitrated through disease or environmental exposure, Analytical and Bioanalytical Chemistry, 410, 1389-1396, 2018.