Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus)

Abstract

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.

Fig. 1

Protein electrophoresis to assess purified P. hypomelanus IgG. L, ladder. Lane 1, unpurified P. hypomelanus plasma. Lane 2, unpurified P. hypomelanus plasma. Lane 3, unreduced mouse IgG. Lane 4, reduced mouse IgG. Lane 5, unreduced P. hypomelanus IgG. Lane 6, reduced P. hypomelanus IgG.

Fig. 2

Corrected OD₄₀₅ values for an ELISA using protein G for detection. Plasma was diluted 1:2000. Vaccinated bats are represented by black lines, and control bats are represented by white lines.

Fig. 3

Corrected OD₄₀₅ values for an ELISA using polyclonal rabbit anti-bat IgG antibody for detection. Plasma was diluted 1:800. Vaccinated bats are represented by black lines, and control bats are represented by white lines.

Fig. 4

Corrected OD₄₀₅ values for an ELISA using monoclonal mouse anti-bat antibody HL1892 for detection. Plasma was diluted 1:100. Vaccinated bats are represented by black lines, and control bats are represented by white lines.

Fig. 5

Western blot reactivity of anti-P. hypomelanus IgG's on variable flying fox plasma. Lane 1, polyclonal rabbit anti-bat antibody. Lane 2, monoclonal mouse anti-bat antibody HL1892. Lane 3, preimmune rabbit serum (negative control).

Fig. 6

Reactivity of polyclonal rabbit anti-bat antibody against sera from eight different bat species. Six different species in the genus Pteropus are represented. Eidolon helvum is a megachiropteran outside the genus Pteropus. Phyllostomus hastatus is a microchiropteran.

Fig. 7

Reactivity of monoclonal mouse anti-bat antibody HL1892 against sera from eight different bat species. Six different species in the genus Pteropus are represented. Eidolon helvum is a megachiropteran outside the genus Pteropus. Phyllostomus hastatus is a microchiropteran.