Development and validation of a house finch interleukin-1β (HfIL-1β) ELISA system

Thumbnail Image
Date
2017-01-01
Authors
Kim, Sungwon
Park, Myeongseon
Leon, Ariel
Hawley, Dana
Dalloul, Rami
Major Professor
Advisor
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Authors
Person
Adelman, James
Assistant Professor
Research Projects
Organizational Units
Organizational Unit
Natural Resource Ecology and Management
The Department of Natural Resource Ecology and Management is dedicated to the understanding, effective management, and sustainable use of our renewable natural resources through the land-grant missions of teaching, research, and extension.
Journal Issue
Is Version Of
Versions
Series
Abstract

Background

A unique clade of the bacterium Mycoplasma gallisepticum (MG), which causes chronic respiratory disease in poultry, has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. Interleukin-1β (IL-1β) is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. The overall goal of this study was to develop and validate a direct ELISA assay for house finch IL-1β (HfIL-1β) using a cross-reactive chicken antibody.

Methods

A direct ELISA approach was used to develop this system using two different coating methods, carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. To generate the standard curve, rHfIL-1b protein was serially diluted at 0, 3, 6, 9, 12, 15, 18, 21, and 24 ng/mL. Following blocking and washing, anti-chicken IL-1b polyclonal antibody was added, plates were later incubated with detecting antibodies, and reactions developed with tetramethylbenzidine solution.

Results

A commercially available anti-chicken IL-1β (ChIL-1β) polyclonal antibody (pAb) cross-reacted with house finch plasma IL-1β as well as bacterially expressed recombinant house finch IL-1β (rHfIL-1β) in immunoblotting assays. In a direct ELISA system, rHfIL-1β could not be detected by an anti-ChIL-1β pAb when the antigen was coated with carbonate-bicarbonate buffer at 4°C overnight. However, rHfIL-1β was detected by the anti-ChIL-1β pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1β with commercial anti-ChIL-1β pAb, we were able to measure plasma IL-1β levels from house finches.

Conclusions

Based on high amino acid sequence homology, we hypothesized and demonstrated cross-reactivity of anti-ChIL-1β pAb and HfIL-1β. Then, we developed and validated a direct ELISA system for HfIL-1β using a commercial anti-ChIL-1β pAb by measuring plasma HfIL-1β in house finches.

Comments

This article is published as Kim, Sungwon, Myeongseon Park, Ariel E. Leon, James S. Adelman, Dana M. Hawley, and Rami A. Dalloul. "Development and validation of a house finch interleukin-1β (HfIL-1β) ELISA system." BMC Veterinary Research 13, no. 1 (2017): 276. doi: 10.1186/s12917-017-1199-9. Posted with permission.

Description
Keywords
Citation
DOI
Copyright
Sun Jan 01 00:00:00 UTC 2017
Collections