Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences.
Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.
Eppley Institute, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address: hong.peng@unmc.edu.
Hong
H
Peng
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA. Electronic address: brimijoi@mayo.edu.
Stephen
S
Brimijoin
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojarov 10, Bratislava 83232, Slovakia. Electronic address: anna.hrabovska@gmail.com.
Anna
A
Hrabovska
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojarov 10, Bratislava 83232, Slovakia. Electronic address: mrvova.kaja@gmail.com.
Katarina
K
Targosova
Université Paris Descartes CNRS UMR 8194, 45 Rue des Saints Pères, 75006 Paris, France. Electronic address: eric.krejci@parisdescartes.fr.
Eric
E
Krejci
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA. Electronic address: fsi3@cdc.gov.
Thomas A
TA
Blake
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA. Electronic address: rmj6@cdc.gov.
Rudolph C
RC
Johnson
Eppley Institute, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address: pmasson@unmc.edu.
Patrick
P
Masson
Eppley Institute, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address: olockrid@unmc.edu.
Oksana
O
Lockridge
D000595:Amino Acid Sequence; D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002091:Butyrylcholinesterase / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002352:Carrier Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007158:Immunologic Techniques; D051379:Mice
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D002091:Butyrylcholinesterase
Biacore; Butyrylcholinesterase; Dynabeads; ELISA; Monoclonal antibody; Octet