Data for: Phage display–mediated immuno-PCR to detect low-abundance secreted proteins in Drosophila
Abstract
Circulating hormones, that mediate communications across organs to maintain physiological balance, are commonly detected and quantified using enzyme-linked immunosorbent assays (ELISAs). However, while ELISA is well suited for organisms where sample blood can be readily obtained, its application is considerably more challenging in smaller organisms, particularly Drosophila, which has gained widespread use in recent years for physiological studies. Here, we present sensitive phage display–mediated immuno-PCR (PD-iPCR) to detect Drosophila hemolymph proteins via two approaches: 1) by identifying high-affinity nanobodies through phage display library screening and subsequent affinity maturation and 2) by generating a knock-in fly line producing secreted proteins tagged with tandem NanoTags composed of VHH05 and 127D01. Using these approaches, we successfully established PD-iPCR to detect insulin-binding ImpL2 protein in fly hemolymph. Notably, the tandem NanoTag-based sandwich PD-iPCR enabled highly sensitive detection of tagged antigens, allowing us to quantify elevated ImpL2 levels in the hemolymph of starved flies and those bearing Yki-induced gut tumors. Collectively, our results demonstrate that PD-iPCR enables detection of endogenous, low-abundance circulating hormones in Drosophila, providing a powerful tool for studying interorgan communication. This dataset includes the source data in this study.
Dataset DOI: 10.5061/dryad.0000000jj
Description of the data and file structure
We present phage display–mediated immuno-PCR (PD-iPCR) as a sensitive platform for quantifying secreted proteins in flies in vivo. Using ImpL2 as an example, we successfully detected nanomolar level of circulating ImpL2 and monitored its physiological changes during starvation and tumorigenesis using PD-iPCR. This approach can be readily expanded to multiplexed quantification of secreted proteins in vivo by leveraging the multiple available nanobodies and the vast collection of epitope-tagged Drosophila lines. This dataset includes the source data of this study.
Files and variables
File: data.zip
Description: This dataset includes the source data to five figures and three supplmentary figures. The folder names are given according to corresponding figures in the paper.
Figure 1-Source data for ImpL2 nanobody screening using phage-displayed nanobody library.
This folder contains four files/subfolders corresponding to the source data for the four panels in Figure 1:
(1) Figure 1B-ImpL2 polyclonal ELISA source data show ELISA signal of polyclonal phages from three rounds of selection. The rows indicate the decreased coating amount of mCherry-hIgG and ImpL2-hIgG. The columns indicate the polyclonal phages from three rounds of selection. The values indicate absorbance measured at 450 nm (OD450).
(2) Figure 1C-ImpL2 monoclonal ELISA source data show ELISA results of monoclonal phages isolated after the third round of selection. 96 individual clones were tested against the control protein (mCherry-hIgG) and the target protein (ImpL2-hIgG). The values indicate absorbance measured at 450 nm (OD450).
(3) Figure 1D-ImpL2 immunostaining with nanobody displaying phages source data are immunostaining results with phages. The picture names represent cell types (ImpL2-GPI or wildtype), phages and channels (BF, bright field; GFP; anti-M13 immunostaining).
(4) Figure 1E-ImpL2 immunostaining with recombinant nanobodies source data are immnostaining results with recombinant nanobodies. The picture names represent cell types (ImpL2-GPI or wildtype), nanobodies and channels (BF, bright field; GFP; anti-M13 immunostaining).
Figure 2-Source data for ImpL2 nanobody affinity maturation by random mutagenesis.
This folder contains four files/subfolders corresponding to the source data for the three panels in Figure 2:
(1) Figure 2A-ImpL2-2B mutagenesis monoclonal ELISA source data show ELISA results of monoclonal phages isolated after two rounds of mutagenesis and selection of NbImpL2-2B. 96 individual clones were tested against the control protein (Mip-hIgG) and the target protein (ImpL2-hIgG). The values indicate absorbance measured at 450 nm (OD450).
(2) Figure 2B-NbImpL2-2B mutagenesis sequence alignment source data show sequence alignment of NbImpL2-2B variants showing improved ELISA signals. The .PDF file contains the protein sequences of the variants. The .PNG file show the sequence alignment result.
(3) Figure 2D-Western blot source data show immunoprecipitation of ImpL2 from conditioned medium (CM) using NbImpL2-1C and NbImpL2-2B variants. Four variants from NbImpL2-1C or NbImpL2-2B are tested.
Figure 3-Source data for immuno-PCR with ImpL2 nanobodies.
This folder contains three files corresponding to the source data for the three panels in Figure 3:
(1) Figure 3A-phage ELISA and immuno-PCR source data show the comparison between phage ELISA and immuno-PCR. Purified ImpL2-hIgG was coated onto plates with a 10-fold serial dilution series. The ELISA result are shown as absorbance measured at 450 nm (OD450). The immuno-PCR results are shown as Cycle Threshold (Ct) in qPCR.
(2) Figure 3B-conditioned medium iPCR source data show the quantification of ImpL2 in CM from Drosophila S2R+ cells transfected with dsGFP or two distinct dsRNAs targeting ImpL2, measured by immuno-PCR. The immuno-PCR results are shown as Cycle Threshold (Ct) in qPCR.
(3) Figure 3C-ImpL2-OE flies iPCR source data show the quantification of ImpL2 in the hemolymph of control flies and ImpL2-OE flies using immuno-PCR. The immuno-PCR results are shown as Cycle Threshold (Ct) in qPCR.
Figure 4-Source data for establishment of phage display–mediated immuno-PCR with tandem NanoTags. AlphaFold3 prediction files show models of ternary protein complex composed of tandem NanoTags, NbVHH05, and Nb127D01 and the expected position error of the ternary protein complex in the models. There are 5 models (model 0-model 4) for the predicted ternary protein complex. The .cif files can be opened with ChimeraX (https://www.cgl.ucsf.edu/chimerax/download.html) or PyMOL (https://www.pymol.org/). We also export the the structure in .cif file into .PNG file.
Figure 5-Source data for detection of ImpL2tNTs with sandwich PD-iPCR in vivo.
This folder contains three subfolders corresponding to the source data for the three panels in Figure 5:
(1) Figure 5B-western blot source data show ImpL2tNTs proteins in the hemolymph of fed and starved ImpL2tNTs/+ flies. Immonoblot with Nb127D01-hIgG file contains the immunobloting result with Nb127D01. The stain-free contains the stain-free result as the loading control.
(2) Figure 5C-fly image source data show bloating phenotype in EGT/+; ImpL2tNTs/+ and EGT/+; ImpL2tNTs/UAS-yki3SA flies with up and side view.
(3) Figure 5G-western blot source data show ImpL2tNTs in gut lysates of control (EGT/+; ImpL2tNTs/+) and ImpL2tNTs-Yki (EGT/+; ImpL2tNTs/UAS-yki3SA) flies. The Nb127D01-hIgG file contains the immunobloting result with Nb127D01. The stain-free contains the stain-free result as the loading control.
Supplementary Figure 1-Source data for ImpL2 nanobody affinity maturation.
This folder contains 10 files/subfolders corresponding to the source data for the seven panels in Supplementary Figure 1:
(1) Supplementary Figure 1A-Western Blot source data show immunoprecipitation of ImpL2 from S2R+ cell conditioned medium using ImpL2 nanobodies.
(2) Supplementary Figure 1B-NbImpL2-1C mutagenesis monoclonal ELISA source data show ELISA results of monoclonal phages isolated after two rounds of mutagenesis and selection of NbImpL2-1C. 96 individual clones were tested against the control protein (Mip-hIgG) and the target protein (ImpL2-hIgG). The values indicate absorbance measured at 450 nm (OD450).
(3) Supplementary Figure 1C-NbImpL2-1C mutagenesis sequence alignment source data show sequence alignment of NbImpL2-1C variants showing improved ELISA signals. The .PDF file contains the protein sequences of the variants. The .PNG file show the sequence alignment result.
(4) Supplementary Figure 1D-NbImpL2-2C mutagenesis monoclonal ELISA source data show ELISA results of monoclonal phages isolated after two rounds of mutagenesis and selection of NbImpL2-2C. 96 individual clones were tested against the control protein (Mip-hIgG) and the target protein (ImpL2-hIgG). The values indicate absorbance measured at 450 nm (OD450).
(5) Supplementary Figure 1E-NbImpL2-2C mutagenesis sequence alignment source data show sequence alignment of NbImpL2-2C variants showing improved ELISA signals. The .PDF file contains the protein sequences of the variants. The .PNG file show the sequence alignment result.
(6) Supplementary Figure 1F-NbImpL2-2G mutagenesis monoclonal ELISA source data show ELISA results of monoclonal phages isolated after two rounds of mutagenesis and selection of NbImpL2-2G. 96 individual clones were tested against the control protein (Mip-hIgG) and the target protein (ImpL2-hIgG). The values indicate absorbance measured at 450 nm (OD450).
(7) Supplementary Figure 1G-NbImpL2-2G mutagenesis sequence alignment source data show sequence alignment of NbImpL2-2G variants showing improved ELISA signals. The .PDF file contains the protein sequences of the variants. The .PNG file show the sequence alignment result.
Supplementary Figure 2-Source data for quantification of ImpL2 in the hemolymph of flies bearing Ykiinduced gut tumors.
Female and male flies are colleceted after four and six days of yki induction. The immuno-PCR results are shown as Cycle Threshold (Ct) in qPCR. There are four files in this folder:
(1) D4 female: Female flies are colleceted after four days of yki induction.
(2) D4 male: Male flies are colleceted after four days of yki induction.
(3) D6 female: Female flies are colleceted after six days of yki induction.
(4) D6 male: Male flies are colleceted after six days of yki induction.
Supplementary Figure 3-Source data for generation of transgenic fly carrying knock-in of tandem NanoTags on ImpL2 locus.
This folder contains 3 subfolders corresponding to the source data for the four panels in Supplementary Figure 3:
(1) Supplementary Figure 3B-plasmid sequence source data show ImpL2-tandem-NanoTag plasmid sequence.
(2) Supplementary Figure 3C-Sanger sequencing for ImpL2-tNTs fly source data show the integration of tandem NanoTags in the C-terminal ImpL2 locus.
(3) Supplementary Figure 3D and 3E-fly weight source data show the body weights of flies with the denoted genotype.
Code/software
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