Journal: Biochemical Journal

Article Title: Crystal structure and nanobodies against domain 3 of the malaria parasite fusogen Plasmodium falciparum HAP2

doi: 10.1042/BCJ20250297

Figure Lengend Snippet: ( A ) Schematic of full-length PfHAP2 and the recombinant construct of domain 3 ( D3 ). Signal peptide (SP), transmembrane domain (TM) and three domains of PfHAP2 are indicated ( D1, D2, D3 ). (B ) Overview of enrichment in the 30 anti-PfHAP2 D3 clonal groups identified by biopanning (top). PfHAP2-specific nanobodies are highlighted in colour. The number of clones per group in brackets and the CDR3 amino acid sequence are indicated. ( C ) Alluvial plots showing NGS analyses of abundance of clones over four rounds of biopanning in both PBS and carbonate-bicarbonate (bicarb) buffer conditions. PfHAP2-specific clones are highlighted in colour, all other clones are coloured in grey. R0, prior to panning; R1-R4, panning rounds 1–4. ( D ) ELISA of nanobody-Fcs against PfHAP2 D3 (teal) and negative control antigen Pfs48/45 D3 (hashed bars). Microtiter wells were coated with recombinant PfHAP2 D3 or Pfs48/45 D3 and probed with anti-PfHAP2 nanobody-Fcs, WNb7-Fc or PBS. Bound nanobodies were detected with HRP-conjugated anti-human IgG. Error bars represent standard deviation of the mean. ( E ) Affinity curves for nanobody-Fc binding to PfHAP2 D3. Representative binding curves of one experiment from two independent experiments. Different concentrations of PfHAP2 D3 to immobilised nanobody-Fcs are plotted. Binding curves were generated by bio-layer interferometry (BLI) and curves were fitted using a 1:1 Langmuir binding model. Binding affinities ( Kd ) for the single experiment are indicated above binding curves. ( F ) Epitope competition experiments using BLI. Nanobodies in the top row were pre-mixed with PfHAP2 D3 at a 10:1 molar ratio. Nanobodies in the left column were immobilised on the sensor and then dipped into wells with the nanobody-antigen pre-mixes. The percentage binding of the antigen-nanobody complexes was calculated relative to antigen binding alone, which was assigned to 100%. ( G ) affinities of anti-PfHAP2 nanobody-Fcs to PbHAP2 and PvHAP2 D3 by BLI. Mean affinities ( Kd ) are given with the standard deviation of two independent experiments. pf, Plasmodium falciparum; pb, Plasmodium berghei; pv, plasmodium vivax; NB, no binding.

Article Snippet: Plates were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (Jackson ImmunoResearch, Cat # 109–035-088) diluted 1:5000.

Techniques: Recombinant, Construct, Clone Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation, Binding Assay, Generated