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Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody - PubMed

Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody

William T Lee et al. Dev Comp Immunol. 2016 Dec.

Abstract

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease, White Nose Syndrome, while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study, we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence, the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most, if not all, secreted immunoglobulins utilized lambda light chains. Finally, mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence, this reagent may have both basic and practical applications for studying immune process.

Keywords: B lymphocytes; Bats; Immunoglobulins; Microchiroptera; Monoclonal antibody.

Copyright © 2016 Elsevier Ltd. All rights reserved.

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Figures

Fig. 1
Fig. 1

mAb BT1-4F10 recognizes a lymphocyte surface and secreted antigen. (A) E. fuscus spleen cells were collected, depleted of RBC and incubated with RGG (10 μg/mL) prior to staining with culture supernatant (100 μl) from BT1-4F10 hybridoma cells followed by Cy5-ramIg and analysis by flow cytometry. The marked region indicates positive staining as compared to cells stained only with RGG and Cy5-RamIg. Data are gated to show staining only on lymphocytes as determined by forward and side light scatter. (B) A BiaCore sensor chip was coated with a 50% SAS precipitate of E. fuscus serum. Culture supernatants from hybridomas BT1-4F10 and BT1-4H2 (non-bat reactive negative control) were passaged over the chip. Shown are overlayed sensorgrams from the two samples.

Fig. 2
Fig. 2

The target antigen for mAb BT1-4F10 is E. fuscus Ig light chain. (A) After precipitation with a 50% SAS solution, E. fuscus serum was either directly immunoprecipitated or first adsorbed to Protein G magnetic beads and the Protein G-bound and non-bound fractions were then immunoprecipitated using mAb BT1-4F10 magnetic beads. The starting SAS precipitate (1), non-protein G passaged immunoprecipitate (2) and the immunoprecipitated protein G-bound (3) and non-bound (4) samples were resolved on 4–12% SDS-PAGE gels followed by Coomassie staining. All samples are from the same (non-reducing or reducing) gels; lanes were cropped to remove irrelevant samples. The portions of the gels marked with rectangles represent areas of excision for mass spectrometry analyses. (B) Varying amounts of whole E. fuscus serum were resolved on SDS-PAGE gels and immunoblotted with mAb BT1-4F10.

Fig. 3
Fig. 3

mAb BT1-4F10 cross-reacts with bat and swine Ig light chain. A 50% SAS precipitate of E. fuscus serum or purified swine IgG were resolved by SDS-PAGE and immunoblotted with mAB 4F10. Reduced and non-reduced samples were run on the same gel; lanes were cropped to remove irrelevant lanes.

Fig. 4
Fig. 4

Serum Ig from different microchiropteran species is identified by Ab BT1-4F10. Unreduced heart extracts from (1) E. fuscus, (2) M. lucifugus, (3) L. cinereus, (4) L. noctivagans, and (5) L. borealis bats were resolved by SDS-PAGE followed by immunoblotting with mAb BT1-4F10. All samples were assessed in the same experiment; shown are lanes cropped from samples of different volumes and exposures to permit normalized display of similar IgG band intensities. Similar sample volumes showed staining intensities of the order indicated by Table 2. Data are representative of analysis of at least 5 individual bats per indicated species. All bats of a given species showed the same staining pattern.

Fig. 5
Fig. 5

Some portion of E. fuscus Ig contains non-covalently associated H and L chains. (A) Varying amounts of unreduced E. fuscus or BALB/c sera were resolved by SDS-PAGE and immunoblotted with mAb BT1-4F10 (bat only) and HRP-goat anti-mouse Ig (H+L). The blot was developed using a chemiluminescent substrate. (B) E. fuscus sera was fractionated by centrifugation through a 100 kDa filter and the unfractionated serum (Se), retentate (>100), and filtrate (<100) were resolved by SDS and either directly stained (left) or immunoblotted using mAb BT1-4F10 with colorimetric development (right). (C) E. fuscus serum was fractionated using Protein G-magnetic microbeads and the non-protein G-binding (−) and bound (+) material was detected by immunoblotting with mAB BT1-4F10 followed by chemiluminescent visualization.

Fig. 6
Fig. 6

E. fuscus Ig preferentially use lambda light chains. (Upper) Varying volumes of E. fuscus serum (expressed in μl) and swine IgG (expressed in μg) or (Lower) 0.03 μl of E. fuscus, BALB/c, or horse serum, as indicated, were fractionated using protein L-magnetic microbeads. The unbound (λ) and protein-L-binding (κ) material was reduced and then resolved using SDS-PAGE, followed by visualization using silver stain. Boxed areas represent individual IgH and IgL chains.

Fig. 7
Fig. 7

Secreted Ig from E. fuscus is identified by Ab BT1-4F10. (A) Serum (SE), fecal extracts (FE), or Saliva (SA) from E. fuscus bats were resolved by SDS-PAGE followed by immunoblotting with mAb BT1-4F10. (B) E. fuscus fecal extracts were fractionated using Protein G or Protein A-magnetic microbeads, as indicated, and the non-binding (−) and bound (+) material were resolved by SDS-PAGE followed by immunoblotting with mAb BT1-4F10.

Fig. 8
Fig. 8

Bats and mice display distinct follicular structure in the spleen. Cryosections of spleens from E. fuscus or M. lucifugus bats or BALB/c mice were stained with biotin-mAb BT1-4F10 (bats) or biotin mAb 187.1 (mice), followed by HRP-streptavidin. Similar results were obtained in 3 independent experiments.

Fig. 9
Fig. 9

Visualization of bat and mouse splenic follicles using immunofluorescence. Cryosections of spleens from E. fuscus and BALB/c mice were stained with Alexa 488-conjugated mAb BT1-4F10 or Alexa 488-conjugated mouse CD19, respectively. The images show representative images of B cells identified from several randomly selected cryosections. Similar results were obtained in 3 independent experiments.

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