Research paperMarsupial and monotreme serum immunoglobulin binding by proteins A, G and L and anti-kangaroo antibody
Graphical abstract
Introduction
Early comparative studies on mammalian immunoglobulins have suggested that marsupials are evolutionarily closer to eutherian mammals than they are to monotremes (De Château et al., 1993, Erntell et al., 1986, Kirsch, 1968, Kronvall et al., 1970). This is consistent with evolutionary studies based on morphological data (e.g. fossil records, as reviewed in Clemens, 1970) and, more recently, on molecular phylogenetic studies (Bininda-Emonds et al., 2007, van Rheede et al., 2006). Monotremes are considered to have diverged from therian (marsupial and placental) mammals in the late Jurassic to early Cretaceous periods, at a time when mammalian immunoglobulin (Ig) classes and subclasses may not have fully emerged (Bininda-Emonds et al., 2007, Clemens, 1970). All vertebrate species examined to date, including members of the Monotremata and Marsupialia, possess molecules resembling the immunoglobulins from at least four of the five major immunoglobulin classes; IgM, IgG, IgA and IgE, with further subclasses detected in some species (Bell et al., 1974a, Bell et al., 1974b, Belov and Hellman, 2003, Marchalonis, 1969, Marchalonis, 1971, Marchalonis and Edelman, 1968, Wang et al., 2009, Zhao et al., 2009). To date, IgD has not been described in any marsupial species examined, although a putative IgD homologue has been identified in the duck-billed platypus (Ornithorhynchus anatinus) genome. This putative IgD molecule appears to share greater structural homology with IgD from amphibians, reptiles and fish than IgD from eutherian mammals (Belov and Hellman, 2003, Zhao et al., 2009). Additionally, the platypus genome encodes a novel Ig isotype (IgO) that appears to be a structural intermediate between mammalian IgG and IgY from birds and reptiles (Zhao et al., 2009). All marsupials and monotreme species examined to date possess both κ and λ light chain (IgL) homologues, and in the grey short-tailed South American opossum (Monodelphis domestica) and common brush-tailed possum (Trichosurus vulpecula) the genomic organisation of the heavy (IgH) and IgL loci is very similar to that of eutherian mammals (Baker et al., 2005, Belov et al., 1999, Wang et al., 2009).
Microbially-derived proteins A, G and L have immunoglobulin-binding properties, although their binding affinities vary between animal species (De Château et al., 1993, Erntell et al., 1986, Kronvall et al., 1970). Protein A and G bind primarily to the Fc region of IgG (Akerström and Björck, 1986, Björck and Kronvall, 1984), while protein L binds to some κ isotype subgroups of IgL and as a result has a broader immunoglobulin binding range (Akerström and Björck, 1989, De Château et al., 1993, Nilson et al., 1992). Early immunoprecipitation and precipitation inhibition studies measuring the immunoglobulin binding affinities of these proteins were performed on sera from a range of mammalian species. Sera from representative members of the class Mammalia, including monotremes, showed some reactivity with staphylococcal protein A, with the exception of the common opossum (Didelphis marsupialis), which showed no reactivity (neither precipitation nor inhibition). Only one other marsupial species (the red kangaroo, Macropus rufus) was included in this study, and sera from the red kangaroo inhibited precipitation. Geographical and, subsequently, evolutionary distance were proposed as factors that could account for the differences in reactivity of these two animal species, but only one marsupial species from each continent was included in the study (Kronvall et al., 1970). Similar studies have examined reactivity with protein L (including sera from the common opossum and the red kangaroo) and protein G (using sera from unknown marsupial species) and identified some reactivity with both proteins. No monotreme species were included in these studies (De Château et al., 1993, Erntell et al., 1986). Occasional species-specific studies conducted in other marsupial species have reported immunoglobulin-binding, such as binding of proteins A and G to immunoglobulin in koala sera (Wilkinson et al., 1991).
Studies in monotreme and marsupial species are frequently undertaken to examine exposure to infectious agents in individual animals in zoos and wild animal populations. These studies often incorporate serological techniques such as enzyme-linked immunosorbent assays (ELISAs) or neutralisation assays, with varying degrees of success (Banazis et al., 2010, Portas et al., 2014, Stalder et al., 2015). While common limitations in immunological assay development can include factors such as the selection of appropriate antigen targets, wildlife research can be further hindered by a lack of commercially available serological reagents. As a result, several published studies have utilised institutionally prepared reagents (Brown et al., 2011, Wilkinson et al., 1991), presenting a dual problem of limited availability of reagents and the potential for lack of consistency between reagents prepared for different studies. One commercially available reagent that has been successfully applied in multiple macropodid sero-prevalence studies is a polyclonal anti-kangaroo whole serum antibody (Bethyl Laboratories Inc.) (Banazis et al., 2010, Bennett et al., 2011, Potter et al., 2011). However, there is limited published data available describing the ability of this reagent to detect antibodies present in sera from other (non-macropodid) marsupial species.
This study aimed to examine the capacity of four commercially available immunoglobulin-binding reagents to bind serum immunoglobulins from a range of members of the Marsupialia and Monotremata. Serum samples were tested for binding to three recombinant microbially-derived Ig-binding proteins — staphylococcal protein A, streptococcal protein G and peptostreptococcal protein L. Additionally, the commercially available polyclonal anti-kangaroo whole serum antibody was included for comparison.
Section snippets
Animal sera and immunoglobulin-binding reagents
The serum samples used in this study were available in our laboratory archives or in those of Melbourne Zoo. Serum samples from 17 species of Australian marsupials and monotremes were used to test immunoglobulin-binding by polyclonal rabbit anti-kangaroo whole serum antibody (Bethyl Laboratories Inc., #BETHA140-105) and three recombinant horseradish peroxidase (HRP) conjugated Ig-binding proteins — protein A (Life Technologies, #10-1123), protein G (Life Technologies, #101223) and protein L
Immunoblots
All marsupial serum samples tested reacted strongly with the anti-kangaroo antibody at all dilutions tested (Fig. 1). Binding to monotreme sera, and to each of the control sera (eutherian and avian), was either weak or undetectable at the dilutions tested.
Binding to protein G was poor across the marsupial species tested, with only weak binding detected to sera from a small number of species at the lowest dilution of sera tested (long footed potoroo, Tasmanian devil and koala; 1:50 dilution).
Discussion
Sera from fifteen marsupial and two monotreme species were assessed for binding to four commercially available immunoglobulin-binding reagents using immunoblot and ELISA formats. Variation was observed within and between families, as well as between assay methods.
For marsupial species, the results indicated that the anti-kangaroo antibody, protein L or protein A would be suitable for most serological assays, although protein A and L may be preferred for immunoblotting as they can yield cleaner
Acknowledgements
We thank Zoos Victoria, Michael Lynch, Pam Whitely, Kathryn Stalder and Jemima Amery-Gale for providing sera used in this study. Paola Vaz and Joanne Devlin are supported by the Australian Research Council.
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