Initial steps for antibody fragment cloning from hybridoma cells

Technical Report · Thu May 13 00:00:00 EDT 1993

OSTI ID:7113644

Schlager, J J; Clark, J H; Legere, R H; Courtney, B C; Brecht, K M

Our present research has focused on the production, isolation and cloning of analytically active mouse antibody fragments (Fab) which are capable of accelerating organophosphorus acid fluoride hydrolysis. As an initial part of this effort, mouse hybridoma cells were produced to isolate a catalytic immunoglobulin for incorporation as a positive control in Fab cloning experiments. Mice were inoculated biweekly with the organophosphorus transition state analog (TSA) conjugated to porcine thyroglobulin (PTG), N-4(PTG-succinyl) 2(4-amino-3,3-dimethyl-2-butoxy)2-menthoxy(l,3,2-dioxa-4,5(di-tert- butyl)) benzophosphol, until exhibiting hyperimmune polyclonal binding sera. A 50% polyethylene glycol fusion was performed between splenocytes from a PTG-menthoxy inoculated Balb/c mouse and op2 myeloma cells. From the most successful fusion to date, 850/1344 wells produced visible cell clones and supernatants from 88 wells exhibited binding activity in ELISA to bovine albumin-menthoxy TSA. Supernatants from 10 of these same parental wells exhibited catalytic activity toward the soman derivative 4-nitrophenyl (1,2,2-trimethyl)propyl methyl-phosphonate (4-NPMP). The expressed catalytic immunoglobulin mRNA will be cloned into either the lambda or M13 vector systems for optimizing the binding and catalytic activity screening of mouse splenic Fab immunoglobulin libraries.

• Research Organization: Army Medical Research Inst. of Chemical Defense, Aberdeen Proving Ground, MD (United States)
• OSTI ID: 7113644
• Report Number(s): AD-P-008863/3/XAB
• Resource Relation: Other Information: This article is from 'Proceedings of the Medical Defense Bioscience Review (1993) Held in Baltimore, Maryland on 10-13 May 1993. Volume 3', AD-A275 669, 1087-1096
• Country of Publication: United States
• Language: English

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