alphalisa immunoassay  (Revvity)

Journal: Cell reports

Article Title: HIV-1 neutralizing antibodies elicited in humans by a prefusion-stabilized envelope trimer form a reproducible class targeting fusion peptide

doi: 10.1016/j.celrep.2023.112755

Figure Lengend Snippet: (A) VRC 018 clinical regimen and a flowchart for B cell sorting and antibody screening. (B) Single B cell sorting with glycan-base BG505 trimer probes reveals only a small fraction of memory B cells from VRC 018 clinical trial to be directed to the glycan-dense surface of the Env trimer versus its glycan-free base. (C) AlphaLISA screening of RATP-Ig supernatants from sorted B cells for antibodies that bind both BG505 DS-SOSIP and glycan-base BG505. Data were measured in triplicates; error bars represent standard error of the mean (SEM). (D) Apparent affinity measurement of top antibodies from AlphaLISA screening for binding to BG505 DS-SOSIP and glycan-base BG505 trimers. Antibody IgGs were expressed and purified, and their binding to trimers was measured by Carterra. 2G12 and VRC01 were used as positive controls. Motavizumab was used as negative control, and no binding was observed. (E) Antibodies 2C06 and 2C09 neutralize BG505, but no other tested strains. Asterisk denotes tier status for BI369.9A unknown but resistant to antibodies 17b, 48d, F105, 3074, and 447-52D that neutralize only laboratory-adapted strains. Neutralization for other antibodies from (D) that bind glycan-base BG505 are shown in . (F) Affinity of antibody Fabs binding to BG505 DS-SOSIP trimer, measured by SPR. Data in (D) and (F) were measured once and the curves fitted with a simple 1:1 Langmuir binding model for the reported mean ± SEM. See also and .

Article Snippet: RATP-Ig supernatants from glycan-base BG505-positive B cells were diluted into AlphaLISA buffer (PBS +0.05% Tween 20 + 0.5 mg/mL BSA), and 5 μL of each were transferred to an OptiPlate-384 assay plate (white opaque, PerkinElmer, Waltham, MA).

Techniques: FACS, Glycoproteomics, Binding Assay, Purification, Negative Control, Neutralization

Journal: Pharmaceutics

Article Title: Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery

doi: 10.3390/pharmaceutics15071841

Figure Lengend Snippet: Smaller antibody formats more effectively penetrate the human corneal epithelium and remain functional following penetration. ( A ) Schematic depiction of the 3D human epithelial corneal tissue model grown in cell culture inserts. IF8-Fc, db V2D7, and scFv V2D7 were applied topically on the corneal tissue layer at a concentration of 500 nM and left to penetrate for 6 h. ( B ) H&E-stained cross-section of 3D human corneal epithelial tissue model (upper image) and human corneal tissue (bottom image). AL: apical layer; WC: wing cells; BL: basal layer; M: microporous membrane; S: stroma ( C ) Percentage (%) of the antibody compared to the initial input or ( D ) antibody concentration (nM) that crossed the corneal epithelial layer after 6 h determined with an AlphaLISA assay. Representative plots of one out of two experiments performed. Mean of n = 3–8 replicates. ( E ) Human corneal epithelial cells express ALCAM, as assessed by flow cytometry (n = 1). ( F ) Transepithelial electrical resistance (TEER) of the 3D corneal tissue model measured after the penetration assay. Representative plot of one out of two experiments performed. Mean of n = 4 replicates. ( G ) Schematic depiction of the dendritic cell (DC) transmigration assay performed in transwells containing a lymphatic endothelial cell (LEC) monolayer. The medium from the penetration assay containing mAbs that crossed the corneal epithelial layer was applied to the top of the transwell to assess its ability to block DC transmigration towards the chemokine CCL21. ( H ) DC transmigration performed in the presence of the medium collected from the corneal tissue penetration assays (see A – D ) with scFv, db and IF8-Fc (one technical replicate out of two shown) or negative control PBS (n = four transwells per condition). IF8-Fc at 1 nM was used as a positive control, and PBS was used as the negative control (control). Representative plot of one out of two experiments. Statistics: One-way ANOVA, Holm–Sidak multiple comparison test ( C , D ) or Dunnett’s multiple comparison test ( H ).

Article Snippet: In brief, 10 μL of 10 nM hALCAM V1 diluted in 1X AlphaLISA buffer (AL000C, PerkinElmer) was added to wells of a 96-well plate (6002350, PerkinElmer).

Techniques: Functional Assay, Cell Culture, Concentration Assay, Staining, Membrane, Flow Cytometry, Transmigration Assay, Blocking Assay, Negative Control, Positive Control, Control, Comparison