Recombinant Protein Coupling to Fluorescent Beads

Extracellular domain of recombinant human MOG_1-125 (rMOG) (Eurogentec, France) expressed in E. coli, human albumin (LFB, France), and nontoxic tetanus toxin C-fragment (Sigma, France) proteins were coupled to fluorescent Bio-Plex COOH beads (Bio-Rad, France) as described [21 (link)]. The carboxyl groups of fluorescent COOH beads were activated by EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (Fisher Scientific, France) and S-NHS (sulfo-N-hydroxysulfosuccinimide) (Fisher Scientific); then the Bio-Plex amine coupling kit (Bio-Rad) was used to couple proteins to the activated COOH beads. The coupling reaction was systematically checked through flow cytometry using the appropriate antibodies. For MOG, we checked that the anti-MOG 8.18C5 mouse antibody, shown to react with the folded MOG pattern [24 (link)], effectively recognizes the MOG_1-125 covalently coated beads. In addition, we checked that these MOG_1-125 coated beads are also recognized by B cells from transgenic 8.18C5 antibody mice [21 (link)].

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Elong Ngono A., Lepetit M., Reindl M., Garcia A., Guillot F., Genty A., Chesneau M., Salou M., Michel L., Lefrere F., Schanda K., Imbert-Marcille B.M., Degauque N., Nicot A., Brouard S., Laplaud D.A, & Soulillou J.P. (2015). Decreased Frequency of Circulating Myelin Oligodendrocyte Glycoprotein B Lymphocytes in Patients with Relapsing-Remitting Multiple Sclerosis. Journal of Immunology Research, 2015, 673503.

Publication 2015

Bio-Plex COOH beads Bio-Plex amine coupling kit

Amine Anti antibody Antibodies Antibody B cells Carbodiimide E coli Flow cytometry Human Human albumin Mouse Proteins Tetanus toxin Transgenic mice

Corresponding Organization : Nantes Université

Other organizations : Centre Hospitalier Universitaire de Nantes, Innsbruck Medical University

Top 5 similar protocols

Variable analysis

independent variables

Extracellular domain of recombinant human MOG_1-125 (rMOG) (Eurogentec, France) expressed in E. coli
Human albumin (LFB, France)
Nontoxic tetanus toxin C-fragment (Sigma, France)

dependent variables

Recognition of MOG_1-125 covalently coated beads by anti-MOG 8.18C5 mouse antibody
Recognition of MOG_1-125 coated beads by B cells from transgenic 8.18C5 antibody mice

control variables

Fluorescent Bio-Plex COOH beads (Bio-Rad, France)
EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (Fisher Scientific, France)
S-NHS (sulfo-N-hydroxysulfosuccinimide) (Fisher Scientific, France)
Bio-Plex amine coupling kit (Bio-Rad)

positive controls

Anti-MOG 8.18C5 mouse antibody, shown to react with the folded MOG pattern
B cells from transgenic 8.18C5 antibody mice

negative controls

Not explicitly mentioned

Annotations

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