Anti-oxPTM-CII scFv Antibody Production

Anti-oxPTM-CII scFv was expressed in HB2151 bacteria as described [15 (link)]. ScFv was converted to full length antibody by cloning V_H domain into pFUSEss-CHIg-hG1e3 and V_L domain into pFUSEss-CLIg-hk (InvivoGen). Plasmid DNA was isolated using a QIAFilter Plasmid Maxi Kit according to the manufacturer’s instruction (QIAGEN). Following transient expression in Expi293F Expression System according to the manufacturer’s instructions (Thermo Fisher Scientific), supernatants were collected and purified using protein A Sepharose CL-4B (GE Healthcare). The ability to retain specific binding of anti-oxPTM-CII over native CII was assessed by ELISA as described [12 (link)].

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Gigout A., Harazin D., Topping L.M., Merciris D., Lindemann S., Brenneis C, & Nissim A. (2021). Early detection of osteoarthritis in the rat with an antibody specific to type II collagen modified by reactive oxygen species. Arthritis Research & Therapy, 23, 113.

Publication 2021

pFUSEss-CHIg-hG1e3 pFUSEss-CLIg-hk QIAFilter Plasmid Maxi Kit Expi293F Expression System protein A Sepharose CL-4B

Antibody Bacteria Elisa Plasmid Protein a Sepharose cl 4b Transient

Corresponding Organization :

Other organizations : Merck (Germany), Queen Mary University of London, Galapagos (France)

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Variable analysis

independent variables

Expression of anti-oxPTM-CII scFv in HB2151 bacteria
Cloning of V_H domain into pFUSEss-CHIg-hG1e3 and V_L domain into pFUSEss-CLIg-hk plasmids
Transient expression in Expi293F Expression System

dependent variables

Specific binding of anti-oxPTM-CII over native CII, assessed by ELISA

control variables

Plasmid DNA isolation using QIAFilter Plasmid Maxi Kit
Purification of supernatants using protein A Sepharose CL-4B

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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