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.
Table 1.
LC-MS/MS analysis of major gel bands from Fab-purified P. alecto.
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.
Table 2.
Detection of immunoglobulin heavy chains by LC-MS/MS of gel-purified tissue lavages, extracts and secretions.
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 IgGH and IgMH 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 IgGH (lanes 1 and 2), IgMH (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 IgMH (panel C) and IgGH (panel D).
Figure 7.
Cross-species reactivity of rabbit anti-P. alecto IgGH and IgMH.
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-IgGH (lanes same as panel A). Panel C; Immunoblot of serum samples from panel A using P. alecto anti-IgMH (lanes same as panel A).