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Preparation and characterization of diethoxy- and monoethoxy phosphylated ('aged') serine haptens and use in the production of monoclonal antibodies.
- Source :
-
Chemico-biological interactions [Chem Biol Interact] 2014 Nov 05; Vol. 223, pp. 134-40. Date of Electronic Publication: 2014 Sep 26. - Publication Year :
- 2014
-
Abstract
- In this study, the first mechanism-based monoclonal antibodies have been produced that recognize and differentiate diethoxy- and monoethoxyphosphorylated serine residues. Haptens were synthesized as the stable phosphonate form of phosphoserine esters to improve the immunoresponse. Following condensation with a glutaric anhydride to link the phosphoserine moieties to carrier protein, the hapten densities attached to bovine serum albumin and keyhole limpet henocyanin were determined by partial trypsin digestion and MALDI mass spectrometry, and confirmed using a fluorescent assay (FITC) to quantify unmodified lysine residues. The conjugation reactions were pH optimized to improve hapten density. Screening of subclones led to the identification of two monoclonal antibodies: (a) N257/25.11 that specifically recognizes (EtO)2P(O)-Ser as the phosphylated or inhibited form, and (b) N262/16 that recognizes (EtO)(HO)P(O)-Ser as the 'aged' form. Analysis of blood samples treated with paraoxon (EtO)2P(O)-OPhNO2 showed a concentration dependent recognition of the phosphylated form.<br /> (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)
- Subjects :
- Animals
Cattle
Enzyme-Linked Immunosorbent Assay
Haptens isolation & purification
Hemocyanins chemistry
Hemocyanins immunology
Humans
Insecticides toxicity
Male
Mice
Organophosphates toxicity
Paraoxon chemistry
Paraoxon immunology
Paraoxon toxicity
Phosphoserine analogs & derivatives
Phosphoserine chemistry
Phosphoserine immunology
Rats
Rats, Inbred SHR
Serum Albumin, Bovine chemistry
Serum Albumin, Bovine immunology
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Antibodies, Monoclonal biosynthesis
Haptens chemistry
Insecticides chemistry
Insecticides immunology
Organophosphates chemistry
Organophosphates immunology
Subjects
Details
- Language :
- English
- ISSN :
- 1872-7786
- Volume :
- 223
- Database :
- MEDLINE
- Journal :
- Chemico-biological interactions
- Publication Type :
- Academic Journal
- Accession number :
- 25261769
- Full Text :
- https://doi.org/10.1016/j.cbi.2014.09.010