Human igg elisa quantitation set

The local Ethical Committee for Laboratory Animal Experiments of the University of Ghent approved all described mice experiments. Briefly, human PBMC are isolated from a buffy coat (Blood Transfusion Centre - Ghent) by isopycnic density gradient centrifugation. Hu-PBL-SCID mice are produced essentially as described before [35 (link)]. Briefly, one day before transplantation, SCID mice (Prkdc^scid/Prkdc^scid) receive total body irradiation (300 rad) and are injected intraperitoneally with 1 mg of TM-β1, a rat monoclonal antibody that targets the β-chain of the murine IL-2 receptor. It was previously shown that TM-β1 pretreatment efficiently depletes mouse NK cells in vivo [36 (link)]. In the spleen of the SCID mice, either 5 × 10⁶ PBMC, 1 × 10⁶ SKP, 1 × 10⁶ SKP+INFL or a mixture of 5 × 10⁶ PBMC and 1 × 10⁶ SKP or SKP + INFL (5:1 cell ratio) is injected. One, two, three and four weeks after transplantation, mice are weighed and mouse EDTA plasma is collected. The concentration of human IgG is measured with a human IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, AL, USA) according to the protocol provided by the manufacturer.

Splenic cells (2.4 × 10⁵) were stimulated with Pokeweed mitogen (PWM, 40 μg/ml, Sigma, St Louis, MO), human IL-2 (15 U/ml), IL-4 (100 U/ml) (Life Technologies), and human CD3 and CD28 Abs (1 μg/ml each, BD Biosciences). At day 7 of culture, half of the medium was replaced by fresh medium. Immunoglobulin secretion in supernatants at day 16 of culture was measured by an enzyme-linked immunosorbent assay (ELISA) (human IgG ELISA quantitation set; Bethyl Laboratories,). For co-cultures, negatively isolated human CD4 T cells (7.5 × 10⁴) from DRAG mice (CD4 dynabeads, Invitrogen) were added to the splenic cells of A2 mice and stimulated as above.

Each CD4⁺ T cell subset (1 × 10⁵ cells/well) was cocultured with B cells (1 × 10⁵ cells/well) and T_FH cells (5 × 10⁴ cells/well) in round-bottomed 96-well plates. Recombinant staphylococcal enterotoxin B (SEB) (2 μg/ml; Toxin Technology, Sarasota, FL, USA) was added to stimulate B cells. Blocking antihuman IL-10 mAb (25209; R&D Systems), blocking antihuman Fas ligand mAb (100410; R&D Systems), or mouse immunoglobulin G1κ (IgG1κ) isotype control (eBioscience) was added at a concentration of 10 μg/ml on the first day of the coculture in the same experimental system as above. To assess cell-cell contact, a coculture experiment was performed using a Transwell plate (HTS Transwell-96 System [3381]; Corning Life Sciences, Acton, MA, USA) with the following cells: B cells, 3 × 10⁵ cells/well; T_FH cells, 2 × 10⁵ cells/well; and CD4⁺CD25⁻LAG3⁺CD45RA⁻ T cells, 1 × 10⁵ cells/well. The concentrations of IgG, IgA, and IgM in the 12-day culture supernatant were measured by ELISA (Human IgG ELISA Quantitation Set, Human IgA ELISA Quantitation Set, and Human IgM ELISA Quantitation Set; Bethyl Laboratories, Montgomery, TX, USA).

A human IgG ELISA Quantitation set (Bethyl Laboratories, Inc.) was used, with slight modification. K1-18 Ab for detection of IgG using V_H 1-69 germline gene and affinity-purified Human IgG Coating Antibody for standard curve were coated onto a 96-well Maxisorp immuonoplate. Serum or human reference serum (for standard curve) was added to the assigned well. After incubation with HRP-conjugated Human IgG Detection Antibody, TMB substrate was added to each well. The peroxidase reaction was stopped by adding H₂SO₄, and the absorbance of sample at 450 nm was measured. The concentration of IgG bound to K1-18 Ab was calculated from the standard curve of human reference serum.

To detect the total amount of IgG in HIV⁻ and HIV⁺ plasma samples, we used a human IgG ELISA quantitation set (Bethyl Laboratories, Montgomery, TX) per the manufacturer’s instructions.

Pullulan was purchased from Meihua Group (Hebei, China). PAA (average MW of 450,000) was purchased from Aladdin Industrial Corporation Chemical (Shanghai). N-(3-Dimethylaminopropyl)-N’-Ethylcarbodiimide Hydrochloride (EDC) was purchased from Heowns Chemical (Tianjin, China). 4-Dimethylaminopyridine (DMAP) and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd. Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc. aCD47 (Catalog no. BE0270) used in vivo was purchased from BioX Cell. AF790-labeled goat anti-mouse IgG (H&L) (Catalog no. 115-655-146) was purchased from Jackson ImmunoResear. Anti-CD3-PerCP-Cy5.5 (Catalog no. 551163), anti-CD4-APC (Catalog no. 553051), anti-CD8-FITC (Catalog no. 553030), anti-CD86-PE (Catalog no. 553692), anti-CD80-APC (Catalog no. 560016), anti-Foxp3-PE (Catalog no. 563101), anti-CD11c-FITC (Catalog no. 557400), anti-CD62L-BV421 (Catalog no. 562910), anti-CD44-APC (Catalog no. 559250) and anti-CD11b-PE-Cy7 (Catalog no. 552850) were purchased from BD Biosciences. Anti-mouse CD206-PE (Catalog no. 141705) and anti-mouse F4/80-FITC (Catalog no. 123107) were purchased from Biolegend. Human IgG ELISA Quantitation Set (Catalog no. E80-104) was purchased from Bethyl Laboratories, Inc.