Published: Vol 9, Iss 5, Mar 5, 2019 DOI: 10.21769/BioProtoc.3184 Views: 5433
Reviewed by: Lokesh KalekarVishal NehruPorkodi Panneerselvam
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Abstract
Murine models of allergic airway disease are frequently used as a tool to elucidate the cellular and molecular mechanisms of tissue-specific asthmatic disease pathogenesis. Paramount to the success of these models is the induction of experimental antigen sensitization, as indicated by the presence of antigen-specific serum immunoglobulin E. The quantification of antigen-specific serum IgE is routinely performed via enzyme-linked immunosorbent assay. However, the reproducibility of these in vitro assays can vary dramatically in our experience. Furthermore, quantifying IgE via in vitro methodologies does not enable the functional relevance of circulating IgE levels to be considered. As a biologically appropriate alternative method, we describe herein a highly reproducible in vivo passive cutaneous anaphylaxis assay using Sprague Dawley rats for the quantification of ovalbumin-specific IgE in serum samples from ovalbumin-sensitized murine models. Briefly, this in vivo assay involves subcutaneous injections of serum samples on the back of a Sprague Dawley rat, followed 24 h later by intravenous injection of ovalbumin and a blue detection dye. The subsequent result of antigen-IgE mediated inflammation and leakage of blue dye into the initial injection site indicates the presence of ovalbumin-specific IgE within the corresponding serum sample.
Keywords: AsthmaBackground
Allergic asthma is a complex inflammatory disease, the development of which involves multiple intricate gene-environment interactions. A defining factor of the genetic component for disease inception is the predisposition to developing allergen-specific immunoglobulin (Ig) E to common allergens, otherwise known as atopy (Sly et al., 2008). As such, allergic (atopic) sensitization to innocuous perennial aeroallergens during early childhood is now recognized as a fundamental risk factor in the establishment of potentially life-long allergic asthmatic disease (Illi et al., 2006; Sly et al., 2008), particularly when sensitization occurs to multiple aeroallergens (Stoltz et al., 2013).
Murine models have long provided a means to investigate the cellular and molecular mechanisms that drive onset and progression of allergic airway diseases such as allergic asthma (Kumar et al., 2016). In this regard, mice are typically experimentally sensitized to a model antigen, resulting in CD4+ T-helper (Th) type 2-driven inflammation and the production of antigen-specific IgE (Mincham et al., 2018); the hallmark feature of allergic sensitization. Quantification of circulating serum antigen-specific IgE from antigen sensitized murine models is routinely performed by enzyme-linked immunosorbent assay (ELISA). However, in our experience, these in vitro assays lack the necessary reproducibility for accurate detection of ovalbumin (OVA)-specific serum IgE in both Brown Norway rat and BALB/c mouse models of allergic airway disease. Furthermore, the use of in vitro techniques disregards the functional activity of antigen-specific IgE in a biologically relevant setting.
To combat the disadvantages of in vitro IgE quantification assays, we herein detail a highly reproducible in vivo passive cutaneous anaphylaxis (PCA) assay which enables both the quantification and assessment of biological activity of OVA-specific IgE within serum samples from OVA-sensitized murine models. In this regard, the localized cutaneous anaphylaxis response observed from an OVA-specific IgE positive sample is indicative of mast cell degranulation driven via OVA-induced cross-linking of OVA-specific IgE (Kawakami and Galli, 2002). The resultant release of mast cell granule-derived proinflammatory mediators exemplified by histamine, prostaglandin, leukotriene and tryptase, ultimately stimulates vascular permeability and the subsequent extravasation of the blue detection dye (Bradding and Arthur, 2016). While this protocol has been described for quantifying OVA-specific IgE in murine serum samples, it could be adapted to assess alternative model antigens.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Plot data on a log2 scale to account for the serial dilutions (i.e., neat = 1, 1:2 = 2, 1:4 = 3, 1:8 = 4, 1:16 = 5, 1:32 = 6, 1:64 = 7, 1:128 = 8) using a graphing program such as GraphPad prism. Depending on the number of experimental groups, statistical significance can be determined by Student’s t-test or one-way ANOVA. As detailed in the original research article studying BALB/c mice (Mincham et al., 2018; Figure 1A), by additional research conducted within our laboratory studying Brown Norway rats (Leffler et al., 2018) and in Figure 3 below comparing gender-specific responses following early life OVA sensitization and aerosol challenge (Protocol ID: 1802387), OVA-specific serum IgE titres are significantly greater in OVA sensitized and aerosol challenged animals compared to naïve control animals.
Figure 3. Enhanced serum OVA-specific IgE titers in early life OVA sensitized and aerosol challenge BALB/c mice. Comparison of male and female serum OVA-specific IgE titers as measured by passive cutaneous anaphylaxis assay. Data are presented from individual animals comparing PBS control versus OVA sensitized and aerosol challenged male (shaded) and female (white) BALB/c mice and displayed as box and whisker plots showing minimum to maximum of n ≥ 6 independent experiments. Statistical significance was determined using Student’s t-test and presented as **P < 0.01, ****P < 0.0001.
Recipes
Acknowledgments
The authors would like to acknowledge the animal technicians at the Telethon Kids Institute Bioresources facility. The original research article (Mincham et al., 2018) utilizing this protocol was funded by the National Health and Medical Research Council of Australia.
Competing interests
The authors declare no financial or non-financial competing interests related to this work.
Ethics
All animal experiments relating to this protocol were formally approved by the Telethon Kids Institute Animal Ethics Committee, which operates under guidelines developed by the National Health and Medical Research Council of Australia for the care and use of animals in scientific research.
References
Article Information
Copyright
© 2019 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Mincham, K. T., Leffler, J., Scott, N. M., Lauzon-Joset, J., Stumbles, P. A., Holt, P. G. and Strickland, D. H. (2019). Quantification of Serum Ovalbumin-specific Immunoglobulin E Titre via in vivo Passive Cutaneous Anaphylaxis Assay. Bio-protocol 9(5): e3184. DOI: 10.21769/BioProtoc.3184.
Category
Immunology > Antibody analysis > Antibody detection
Cell Biology > Cell-based analysis
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