ABSTRACT
Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.
Subject(s)
Antibodies, Monoclonal/genetics , Glucose Oxidase/immunology , Immunoglobulin Fragments/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Aspergillus niger/enzymology , Base Sequence , Cloning, Molecular , DNA Primers/genetics , Escherichia coli/genetics , Hybridomas/immunology , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Mice , Molecular Sequence DataABSTRACT
Two monoclonal anti-diuron antibodies were generated that bind to diuron with an extremely low equilibrium dissociation constant. The antibodies prevented and restored in vitro and in vivo the diuron-dependent inhibition of photosynthesis. In isolated thylakoids prepared from spinach leaves (Spinacia oleracea L.) the diuron-inhibited Hill reaction was reconstituted immediately after the addition of the monoclonal antibodies. The antibodies also restored the diuron-dependent inhibition of the photosynthetic oxygen evolution of the cell wall-deficient mutant cw15 of the green alga Chlamydomonas reinhardtii Dangeard.
