Purification and characterisation of immunoglobulins from the Australian black flying fox (Pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of IgA

Abstract

There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

Figure 1. Purification strategy of P. alecto IgG, IgM and IgA.

Light blue boxes represent those components which polyclonal antiserum was raised against. The green box represents P. alecto anti-Fab-specific antibody. Red dashed lines represent Protein G or Protein A affinity chromatography, blue dashed lines represent heavy chain gel elution, solid green lines represent specific affinity chromatography. Crosses represent unsuccessful purification of IgA. Abbreviations: SEC; size exclusion chromatography.

Figure 2. Purification of P. alecto IgG and generation of Fc and Fab fragments.

Panel A; Protein G purification. Panel B; Protein A purification. Lane 1 (all panels); Mark 12 standard. FT; flow-through, W; wash, E; elution. Panel C; papain digestion containing Fab and Fc fragments was fractionated on immobilised Protein A column. Flow through (FT) fraction contained the Fab fragment (a doublet of approximately 25 kDa).

Figure 3. Purification of P. alecto IgM from IgG depleted serum on immobilised anti-Fab-specific antibody.

Panel A; purification of IgM from IgG depleted serum. Immobilised Protein G column was used to deplete IgG from whole serum. The immobilised anti-Fab-specific antibody column was then used to purify IgM. Lane 1; See Blue plus 2 markers. FT; flow-through, E; elution. Panel B; purified IgM fractions from serum (lane 1) and plasma (lane 3). Lane 2; Mark 12 standard. Selected protein bands (labelled 1 to 8) were excised from gels for LC-MS/MS analysis.

Figure 4. Quantitative measurement of IGHG, IGHM, IGHA, IGJ and PIGR mRNA in P. alecto tissues.

mRNA transcripts were measured by SYBR Green qPCR and normalised to 18 s ribosomal RNA. Results show the mean ± standard deviation of n = 3 individual healthy wild-caught bats. Abbreviations: L.N., lymph node; S.I., small intestine; S.G., salivary gland; PBMC, peripheral blood mononuclear cells.

Figure 5. Electrophoretic characterisation of P. alecto and human IgG and IgM before/after deglycosylation.

Proteins were visualised with Coomassie blue (panel A) or silver nitrate (panel B). Bands with asterisks indicate neuraminidase and hashes indicate PNGaseF used for deglycosylation. Lane 1, See Blue plus 2 markers; lanes 2–4, P. alecto IgG; lanes 5–7, human IgG; lanes 8–10, P. alecto IgM; lanes 11–13, human IgM; lanes 3, 6, 9 and 12, PNGaseF treatment; lanes 4, 7, 10 and 13, neuraminidase treatment.

Figure 6. Reactivity of rabbit antibodies to P. alecto IgG_H and IgM_H in P. alecto serum.

Panel A. P. alecto serum samples (neat and 1∶5) were separated by reducing SDS-PAGE and probed with rabbit antiserum against P. alecto IgG_H (lanes 1 and 2), IgM_H (lanes 3 and 4) and Fab fragment (lanes 5 and 6). Panel B. 2-DE separation of P. alecto serum sample (silver stain). Panel C and D, 2-DE separation of P. alecto serum sample probed with rabbit antiserum against P. alecto IgM_H (panel C) and IgG_H (panel D).

Figure 7. Cross-species reactivity of rabbit anti-P. alecto IgG_H and IgM_H.

Panel A; Silver stained gel of serum samples from different animals. Lanes 1; P. alecto; 2, P. conspiculatus; 3, R. megaphylus; 4, human; 5, Tasmanian devil; 6, horse; 7, cow; 8, cat; 9, pig; 10, mouse; 11, chicken; 12, ferret. S, See Blue plus 2 markers. Panel B; western blot of serum samples from panel A using P. alecto anti-IgG_H (lanes same as panel A). Panel C; Immunoblot of serum samples from panel A using P. alecto anti-IgM_H (lanes same as panel A).