96 well strep tactinxt coated microplates  (IBA Lifesciences)

Journal: NPJ Vaccines

Article Title: Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines

doi: 10.1038/s41541-024-00862-8

Figure Lengend Snippet: a Linear representation of SOSIP, single-chain (SC) and triple tandem trimer (TTT) Env constructs. The SOSIP construct presents a R6 furin cleavage motif (black triangle) between gp120 and gp140 subunits, which is replaced by SC linkers (green lines) in SC and TTT constructs. The TTT construct is composed of three SC protomers fused by TTT linkers (purple discontinuous lines). N- and C-termini of each protomer are colored in yellow and red, respectively. b Expected position of TTT linkers on the structure of a BG505.SOSIP trimer (PDB 5CEZ). The distance, in angstrom (Å), between the C ɑ atoms of N- (Glu32, HxB2 numbering) and C-termini (Asp664) of neighbor protomers is indicated. c SEC profiles of StrepTactinXT-purified BG505 SOSIP.v8, SOSIP.v8 + furin, SC and TTT protein preparations on a Superdex 200 Increase 10/300 GL column. d 2D class averages generated by nsEM analysis of the StrepTactinXT-purified SOSIP.v8, SC and TTT protein preparations. Percentages of native-like trimers (green), malformed trimers (yellow) and monomers/dimers (red) are indicated. 2D class averages representing non-native-like particles are shaded in the corresponding yellow or red colors. e StrepTactinXT ELISA assay with StrepTactinXT-purified SOSIP.v8, SC and TTT protein preparations against a panel of bNAbs (2G12, VRC01, PGT121, PGT145, VRC26.25, PGT151) and non-NAbs (F105). Heatmap values correspond to the ratio between the areas under the curve (AUC) of each Ab and the one of 2G12, calculated using the binding curves in Supplementary Fig. . f Crystal structure of 2G12-purified BG505 TTT (blue) in complex with PGT124 Fabs (dark pink) and 35O22 scFvs (dark yellow) at 5.8 Å resolution.

Article Snippet: StrepTactinXT ELISA assays were performed following the same procedures as lectin-capture assays, with the difference that StrepTactinXT coated microplates (IBA, cat. no. 2-5101-001) did not require any functionalization or blocking steps prior to protein immobilization.

Techniques: Construct, Purification, Generated, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: NPJ Vaccines

Article Title: Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines

doi: 10.1038/s41541-024-00862-8

Figure Lengend Snippet: a Linear representation of standard (HA), GCN4-trimerized (HA GCN4), and triple tandem trimer (HA TTT) HA constructs. The TTT construct is composed of three HA protomers fused by TTT linkers (discontinuous purple lines). The N- and C-termini of each protomer are colored in yellow and red, respectively. b Expected position of TTT linkers on the structure of a HK05 trimer (A/Hong Kong/4443/2005(H3N2), PDB 2YP7). The distance, in angstrom (Å), between the C ɑ atoms of N- (Asn8, PDB numbering) and C-termini (Lys503) of neighbor protomers are indicated. c 2D class averages generated by nsEM analysis of the StrepTactinXT-purified H3 HA, HA GCN4, and HA TTT protein preparations, both unliganded and in complex with a CR9114 Fab (+ CR9114 Fab). d nsEM-generated 3D model of the purified HA TTT protein in complex with CR9114 Fab, independently (up) and superimposed with a previously determined structure of a similar HK68 trimer (A/Hong Kong/1/1968(H3N2)) + CR9114 complex (PDB 4FQY) (down). e StrepTactinXT ELISA with StrepTactinXT-purified HA, HA GCN4, and HA TTT protein preparations against a panel of head- (C05, FluA-20) and stem-specific (CR8020, CR9114, FI6v3) antibodies. Dots and error bars represent the average and standard deviation of values obtained in two independent experiments.

Article Snippet: StrepTactinXT ELISA assays were performed following the same procedures as lectin-capture assays, with the difference that StrepTactinXT coated microplates (IBA, cat. no. 2-5101-001) did not require any functionalization or blocking steps prior to protein immobilization.

Techniques: Construct, Generated, Purification, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: NPJ Vaccines

Article Title: Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines

doi: 10.1038/s41541-024-00862-8

Figure Lengend Snippet: a , e Linear representations of chimeric cH125 TTT ( a ) and cH125/111 TTT ( e ) constructs, and the corresponding monomeric and GCN4-trimerized controls. The cH125 TTT construct is composed of NL09 (A/Netherlands/602/2009(H1N1)), SG57 (A/Singapore/1/1957(H2N2)) and IN05 (A/Indonesia/5/2005(H5N1)) protomers connected by TTT linkers (purple discontinuous lines). The cH125/111 TTT construct is composed of protomers with NL09, SG57 and IN05 heads and PR8 (A/Puerto Rico/8/1934/Mount Sinai (H1N1)) stalks. N- and C-termini of each protomer are colored in yellow and red, respectively, and the GCN4 trimerization motifs are colored in dark gray. b , f AlphaFold2-predicted structure of cH125 TTT ( b ) and cH125/111 TTT ( f ) (Supplementary Fig. , ). c , g StrepTactinXT ELISA with StrepTactinXT-purified cH125 TTT ( c ) and cH125/111 TTT ( g ) proteins against a panel of HA head- (5J8, FluA-20, C05) and stalk-specific (CR8020, CR6261, CR9114, FI6v3) antibodies. Heatmap values correspond to the areas under the curve (AUC) of the binding curves in Supplementary Fig. . d , h nsEM-generated 3D models (C3 symmetry) of the purified cH125 TTT ( d ) and cH125/111 TTT ( h ) proteins in complex with CR9114 Fabs. i nsEM-generated 2D class averages of the homotrimeric H1 GCN4, H2 GCN4 and H5 GCN4 proteins and the chimeric cH125/111 TTT protein in complex with Fabs of the H1-specific 2B05 antibody (colored in purple).

Article Snippet: StrepTactinXT ELISA assays were performed following the same procedures as lectin-capture assays, with the difference that StrepTactinXT coated microplates (IBA, cat. no. 2-5101-001) did not require any functionalization or blocking steps prior to protein immobilization.

Techniques: Construct, Enzyme-linked Immunosorbent Assay, Purification, Binding Assay, Generated