The use of enzyme immunoassays for the detection of abzymatic activities. Application to an enantioselective thioacetal hydrolysis activity.
Relying on the particularly high specificity displayed by antibodies, enzyme immunoassays have proved to be one of the most efficient tools for early detection of the catalytic activities displayed by antibodies. We took advantage of such an assay, namely the Cat-enzyme-linked immunoassay (EIA) approach developed in our laboratories, both to exhibit and characterise an antibody-catalysed thioacetal hydrolysis. Monoclonal antibody (mAb) H3-32 was thus identified to accelerate the hydrolysis reaction of thioacetal substrate (NC9) to vanillylmandelic acid (VMA), with a k(cat) of 0.148 h(-1) (k(uncat) = 6.85 x 10(-5) h(-1)), and a K(M) of 720 microM. Taking advantage of the enantiomeric discrimination between (R)- and (S)-VMA displayed by some of the anti-H3 monoclonal antibodies, we were also able to determine that (S)-VMA was preferentially formed during this abzymatic hydrolysis with a 47% enantiomeric excess. All these EIA measurements were confirmed through HPLC analyses.
Gauchet
Cécile
C
Service des Molécules Marquées, DBCM/DSV CEA Saclay 91191 Gif sur Yvette cedex, France.
Taran
Frédéric
F
Renard
Pierre-Yves
PY
Créminon
Christophe
C
Grassi
Jacques
J
Pradelles
Philippe
P
Mioskowski
Charles
C
D000085:Acetates / Q000378:metabolism*; D000110:Acetylcholinesterase / Q000378:metabolism*; D017151:Antibodies, Catalytic / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002384:Catalysis; D006868:Hydrolysis; D007124:Immunoenzyme Techniques / Q000379:methods*; D008333:Mandelic Acids / Q000378:metabolism; D006180:Proton-Translocating ATPases; D014642:Vanilmandelic Acid / Q000378:metabolism
D000085:Acetates; D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D008333:Mandelic Acids; C469301:hydroxy-(3-methoxy-4-methylsulfanylmethoxyphenyl)acetic acid; D014642:Vanilmandelic Acid; D000110:Acetylcholinesterase; D006180:Proton-Translocating ATPases; C037938:mandelic acid