Pepsin Digestion of Antibodies to Produce Functional Antigen-Binding Fragments (Fab): A Scientific Fantasy?

reportActive / Technical Report | Accession Number: ADA394374 | Open PDF

Abstract:

A pepsin antibody fragmentation procedure was trialed and optimised. The procedure used was a modified version of that described in Current Protocols in Immunology. Pilot digestions using a range of pepsin concentrations and incubation times were completed at pH 4.0 and pH 4.5. The digestion products were examined by Phastgel electrophoresis, and the combination of pepsin concentration, incubation time and pH that provided the most efficient digestion of the antibody into dimeric antigen-binding fragments Pab2 was then used in the large-scale digestion of the antibody. Monomeric antigen binding fragments Fab were obtained through reduction of Fab2 using 2-mercaptoethanol. In this study Fab from a mouse polyclonal anti-ricin antibody and a sheep polyclonal anti-Bacillus anthracis antibody generated with the optimised procedure were comparable to the whole molecule when used as the capture antibody in an ELISA. Nevertheless, it was noted that, in spite of meticulous optimisation of the digestion process it can be fraught with problems.

Author(s):
Gauci, Penelope J. ; Alderton, Malcolm R.
Author Organization(s):
DEFENCE SCIENCE AND TECHNOLOGY ORGANISATION VICTORIA (AUSTRALIA) AERONAUTICAL AND MARITIME RESEARCH LAB
Descriptive Note:
Technical rept.
Supplementary Note:
--Original contains color plates: All DTIC reproductions will be in black and white.
Pagination:
0025

Security Markings

DOCUMENT & CONTEXTUAL SUMMARY

Distribution:
Approved For Public Release

RECORD

Collection: TR
Identifying Numbers
Report Number(s):
DSTO-TR-1189, DODA-AR-011-961
Monitor Series:
AR-011-961, X5
 Subject Terms
Joint Capability Areas:
JCA_5_Command and Control; JCA_5.2_Understand; JCA_5.2.3_Share Knowledge and Situational Awareness; JCA_6_Net Centric; JCA_1.2_Force Preparation; JCA_1_Force Support; JCA_5.5_Direct; JCA_1.2.3_Educating; JCA_6.1.3_Switching and Routing; JCA_3.2_Engagement; JCA_3_Force Application; JCA_5.5.1_Communicate Intent and Guidance; JCA_6.1_Information Transport; JCA_6.4.1_Secure Information Exchange; JCA_3.2.1_Kinetic Means; JCA_6.2.1_Information Sharing; JCA_6.2.2_Computing Services; JCA_6.2.3_Core Enterprise Services; JCA_1.2.5_Lessons Learned; JCA_5.5.2_Task; JCA_6.2_Enterprise Services
Modernization Areas:
Biotechnology
Communities of Interest:
No COI(s) Identified
Descriptor(s):
*ANTIBODIES, *DIGESTION(BIOLOGY), *PEPSINS, AUSTRALIA, IMMUNOASSAY, RICIN, ANTIGENS, BACILLUS ANTHRACIS
Field(s)/Group(s):
Biochemistry, Anatomy and Physiology
Keyword(s):
BIOSENSORS, ANTIGEN ANTIBODY REACTIONS, FOREIGN REPORTS, AQ F01 12 2583
Report Date:
2001 Jul 01
Creation Date:
2001 Oct 17