Anti human igg fc apc antibody

The activity of Nbs was determined by using indirect ELISA (iELISA), indirect immunofluorescent assay (IFA), and flow cytometry (FCM). For iELISA, BCMA-mFc or CD47-mFc recombinant proteins were coated overnight at 4℃ in the 96-well Immunoplates, washed, blocked with 5% skimmed milk, and then incubated with a dilution series of humanized nanobodies-hFc (huNbs-hFc) for 1 h at 37℃, and HRP-conjugated goat anti-human IgG (cat# 109-035-088, Jackson) was added and incubated for another hour. After washing, 3,3’,5,5’-tetramethylbenzidine (TMB) soluble reagent (cat# PR1200, Solarbio) was added and then the reaction was stopped with 2M H₂SO₄. The absorbance at 450 nm was measured using an automatic ELISA plate reader. Finally, the binding activity was evaluated using a four-parameter nonlinear regression curve fit (Graphpad Prism 9.0).
For IFA and FCM identification, the expression levels of BCMA on human MM cell lines were detected using APC anti-human BCMA antibody (cat# 357506, Biolegend). Additionally, Alexa Fluor® 594 rabbit anti-human IgG (cat# 309-585-003, Jackson) or APC anti-human IgG Fc antibody (cat# 410712, Biolegend) were incubated with MM cell lines. The z-stack images were acquired using an Olympus SpinSR10 instrument with 100 × objective lenses and analyzed using Olympus OlyVIA analysis software.

Env expression was analysed on the surface of vero cells 2 days after infection with 50 PFU/cell of either Ad5 vaccine. Cells were stained with the ITS52, ITS03, and ITS40 mAbs [37 (link)] (kindly provided by Dr. Mario Roederer, VRC, NIAID, NIH). Binding of the mAbs was detected using anti-human IgG Fc-APC antibody (BioLegend), and the cells were acquired using an LSRII instrument (BD Biosciences) and analysed with FlowJo software (Tree Star, Ashland, OR).

Env surface expression on Vero cells was analyzed 2 days post infection with 50 IFU/cell Ad-HIVB. The cells were stained with the monoclonal antibody clones VRC01 [NIH AIDS Reagent Program (NARP)], PGT145 [International AIDS Vaccine Initiative (IAVI)] and PGT151 (IAVI). Binding of the antibodies was detected using anti-human IgG Fc-APC antibody (BioLegend, 409305) and the fluorescence of the cells acquired in an LSRII instrument (BD Biosciences). The data was analyzed with FlowJo 10 software (Tree Star, Ashland, OR).

Groups of 5 to 9-week-old female NSG mice (NOD-Prkdcscid Il2rgtm1/Bcgen) (Biocytogen, Beijing, China) were injected 1 × 106 luciferase+ Raji cells via tail vein on day 0. Tumor burden was measured using XenogenIVIS-200 Spectrum camera in mice that have been anesthetized and injected intraperitoneally with 3 mg D-luciferin (YEASEN, Shanghai, China) using Xenogen IVIS-200 Spectrum system (Caliper Life Sciences, USA). The bioluminescent signals were analyzed using Living Image Version 4.1 software (Caliper Life Sciences, Hopkinton, MA) as photons/sec/cm2/sr. CD19 CAR-T cells in peripheral blood of the transplanted mice were detected using a recombinant human CD19-Fc chimera protein (R&D Systems, Minneapolis, USA) and a secondary anti-human IgG Fc-APC antibody (BioLegend, San Diego, USA).

CD19 CAR T cells were obtained by frozen donor‐derived CD19 CAR T cells. CAR T cells were manufactured with T cells, obtained through RosetteSep™ Human T Cell Enrichment Cocktail (STEMCELL, Vancouver, CA) and transduced with the lentiviral vector expressing pCDH empty vector (#72266, addgene, MA, USA) which were used as negative control. Dexamethasone (DEX) and Ruxolitinib were purchased from MCE (HY‐50856, MCE USA). Nalm 6 cell line (CD19 positive) was purchased from (CRL‐3273, ATCC, Virginia, USA) and cultured in RPMI 1640 (Gibco, NY, USA) supplemented with 10% FCS. CD19 expression on Nalm 6 cells was confirmed by flow cytometry. Monocytes were isolated from donor peripheral blood mononuclear cells (PBMCs) by multi‐analyte flow assay kit (Human CD8/NK Panel, 740267, BioLegend, San Diego, USA). Anti‐human CD3/CD28 monoclonal antibodies (mAb) were purchased from Stemcell (10971, Stemcell, Vancouver, Canada). Flow cytometry was performed using allophycocyanin (APC)‐anti‐human CD19 antibody (392504, BioLegend, San Diego, USA), APC‐anti‐human CD14 antibody (367117, BioLegend, San Diego, USA), APC‐anti‐human CD3 antibody (300311, BioLegend, San Diego, USA), cell surface expression of CD19 CAR was detected by a recombinant human CD19‐Fc chimera protein (789006, BioLegend, San Diego, USA) and a secondary staining of anti‐human IgG Fc‐APC antibody (409603, BioLegend, San Diego, USA ).