Analysis of allelic cross-reactivity of monoclonal IgG antibodies by a multiplexed reverse FluoroSpot assay
- Centre for Medical Parasitology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
- West African Centre for Cell Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Ghana
- Department of Immunology, Noguchi Memorial Institute for Medical Research, University of Ghana, Ghana
- Mabtech AB, Sweden
- Department of Molecular Biosciences, The Wenner-Gren Institute, Sweden
- Department of Infectious Diseases, Rigshospitalet, Denmark
Figures
Figure 1 with 4 supplements
Cross-reactivity in the VAR2CSA-specific response present in serum.
(A) Schematic representing ‘true’ cross-reactivity where an epitope shared among different VAR2CSA variants (V1–V4) is recognized by a single monoclonal antibody (colored in black) present in the …
Figure 1—figure supplement 1
Instant blue-stained gels of purified, recombinant peptide-tagged VAR2CSA domains under reducing (+DTT) and non-reducing conditions (−DTT).
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Figure 1—figure supplement 1—source data 1
Uncropped images used to build Figure 1—figure supplement 1.
The dashed lines represent the portions of the images used for the final figure.
- https://cdn.elifesciences.org/articles/79245/elife-79245-fig1-figsupp1-data1-v1.jpg
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Figure 1—figure supplement 1—source data 2
Original picture file for the instant blue-stained gel of the purified recombinant GAL-tagged VAR2CSA domains.
- https://cdn.elifesciences.org/articles/79245/elife-79245-fig1-figsupp1-data2-v1.tif
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Figure 1—figure supplement 1—source data 3
Original picture file for the instant blue-stained gel of the purified recombinant TRAP-tagged VAR2CSA domains.
- https://cdn.elifesciences.org/articles/79245/elife-79245-fig1-figsupp1-data3-v1.tif
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Figure 1—figure supplement 1—source data 4
Original picture file for the instant blue-stained gel of the purified recombinant TWIN-tagged VAR2CSA domains.
- https://cdn.elifesciences.org/articles/79245/elife-79245-fig1-figsupp1-data4-v1.tif
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Figure 1—figure supplement 1—source data 5
Original picture file for the instant blue-stained gel of the purified recombinant WASP-tagged VAR2CSA domains.
- https://cdn.elifesciences.org/articles/79245/elife-79245-fig1-figsupp1-data5-v1.tif
Figure 1—figure supplement 2
IgG-binding to recombinant VAR2CSA domains by ELISA.
IgG binding of malaria-exposed (female and male pool) and malaria-naïve (Danish pool) donors to immobilized recombinant proteins: (A) ID1-ID2, (B) DBL3X, (C) DBL5ε, and (D) control antigens. Means …
Figure 1—figure supplement 3
Antibody binding to recombinant VAR2CSA domains by ELISA.
(A) 6E2 binding to the four ID1-ID2 variants (NF54, M. Camp, IT4, and M920) and to full-length IT4 VAR2CSA. (B) PAM2.8, PAM8.1, PAM3.10, and PAM1.4 (control) binding to the four DBL3X and DBL5ε …
Figure 1—figure supplement 4
FluoroSpot detection reagents binding to recombinant VAR2CSA domains by ELISA.
Means and standard deviations of data from three independent experiments are shown. ELISA, enzyme-linked immunosorbent assay.
Figure 2 with 2 supplements
FluoroSpot analysis of the 6E2 monoclonal antibody against the IT4 ID1-ID2 domain.
Captured antibodies secreted by 6E2 cells were incubated with peptide-tagged IT4 ID1-ID2 domains and detected using peptide-specific detection reagents. (A) Different antigen×detection reagent …
Figure 2—figure supplement 1
Schematic showing the different antigen×detection reagent combinations tested, corresponding to the data presented in Figure 2.
(A) 1×1 (B) 1×4, (C) 4×1, and (D) 4×4.
Figure 2—figure supplement 2
Well images for the different antigen×detection reagent combinations tested, corresponding to the schematic presented in Figure 2—figure supplement 1 and the data presented in Figure 2.
(A) 1×1, (B) 1×4, and (C) 4×1.
Figure 3 with 2 supplements
FluoroSpot analysis of the 6E2 monoclonal antibody against four peptide-tagged ID1-ID2 domain variants (NF54, M.Camp, IT4, and M920).
(A) Captured 6E2 antibodies secreted by 6E2 cells were incubated with all four peptide-tagged ID1-ID2 domain variants and detected using the four peptide-specific detection reagents (multiplex 4×4). …
Figure 3—figure supplement 1
Schematic showing the different antigen×detection reagent combinations tested, corresponding to the data presented in Figure 3.
(A) 4×4, (B) 1×1, (C) 1×4, and (D) 4×1.
Figure 3—figure supplement 2
Well images for the different antigen×detection reagent combinations tested, corresponding to the schematic presented in Figure 3—figure supplement 1 and the data presented in Figure 3.
(A) 1×1, (B) 1×4, and (C) 4×1.
Figure 4 with 5 supplements
FluoroSpot analysis of the VAR2CSA specific antibodies PAM2.8, PAM8.1, and PAM3.10 against four peptide-tagged DBL3X and DBL5ε domain variants (IT4, NF54, M920, and M.Camp).
Captured antibodies secreted by the corresponding EBV-immortalized cell lines were incubated with all four peptide-tagged DBL3X (PAM2.8 and PAM8.1) or DBL5ε (PAM3.10) domain variants and detected …
Figure 4—figure supplement 1
Schematic showing the different antigen×detection reagent combinations tested (A) 4×4 (corresponding to the data presented in Figure 4), (B) 1×1, (C) 1×4, and (D) 4×1.
Figure 4—figure supplement 2
Well images for the different antigen×detection reagent combinations tested for PAM2.8, corresponding to the schematic presented in Figure 4—figure supplement 1.
(A) 1×1, (B) 1×4, and (C) 4×1.
Figure 4—figure supplement 3
Well images for the different antigen×detection reagent combinations tested for PAM8.1, corresponding to the schematic presented in Figure 4—figure supplement 1.
(A) 1×1, (B) 1×4, and (C) 4×1.
Figure 4—figure supplement 4
Well images for the different antigen×detection reagent combinations tested for PAM3.10, corresponding to the schematic presented in Figure 4—figure supplement 1.
(A) 1×1, (B) 1×4, and (C) 4×1.
Figure 4—figure supplement 5
Comparison of the average relative spot volume (RSV) for all the positive spots per antigen between the assay combinations for (A) PAM 2.8, (B) PAM8.1, and (C) PAM3.10.
Figure 5 with 2 supplements
VAR2CSA specific responses in malaria-exposed Ghanaian donors against four ID1-ID2 (A), DBL3X (B), and DBL5ε (C) variants tested simultaneously.
The scatter dot plots depict the percentages of antigen specific spots/total IgG secreting cells for each individual donor tested discriminating variant specific (single-coloured) and cross-reactive …
Figure 5—figure supplement 1
Images acquired in each detection channel are presented (LED490, LED550, LED380, and LED640) for the two representative donors highlighted in Figure 5.
DC19: malaria exposed male donor and MK601: malaria exposed female donor.
Figure 5—figure supplement 2
Comparison of number spots/well (A) and average RSV (B) for each of the four VAR2CSA variants and three domains tested, either in a 4×4 (circles) or a 1×1 (triangles) configuration.
RSV, relative spot volume.
Additional files
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Supplementary file 1
List of primers used for VAR2CSA domain cloning.
- https://cdn.elifesciences.org/articles/79245/elife-79245-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79245/elife-79245-mdarchecklist1-v1.docx
