Definition of the fine specificity of the monoclonal antibody 81D4: its reactivity with lysine and polyamine isopeptide cross-links.
The 81D1C2 monoclonal antibody (Mab) directed against the Nepsilon-(gamma-L-glutamyl)-L-lysine isopeptide was found to cross-react on Enzyme Immuno Assay (EIA) with acylated lysines. Using a differential screening EIA procedure, a new Mab 81D4 was selected, which did not cross-react with acylated lysines but exhibited strong reactivity with Nepsilon-(gamma-L-glutamyl)-L-lysine formed by covalently coupling the gamma-carboxyl of NalphaCBZ OtBu glutamic acid to epsilon-NH2 derivatized microtiter plates. When Nepsilon-(gamma-L-glutamyl)-L-lysine isopeptides were generated on gamma-carboxyl derivatized plates, only lysine isopeptides with blocked alpha-amines were reactive, regardless of whether the bond formed by the amine blocking agent was a carbamate with carbobenzyloxychloride or an amide with acetic anhydride. The 81D4 Mab showed little or no affinity for free Nepsilon-(gamma-L-glutamyl)-L-lysine (IC50>5 mM), for N1 or N4 mono(gamma-Poly L-glutamyl)putrescine, and for N1 mono(gamma-Poly L-glutamyl)spermidine (IC50>5 mM). However, when these same isopeptides were synthesized as cross-links between two protein chains--Nepsilon-(gamma-L-glutamyl)-L-lysine between Poly L-glutamate and Poly L-lysine; N1N4 -bis(gamma-Poly L-glutamyl)putrescine, N1N8 -bis(gamma-Poly L-glutamyl)spermidine between Poly-L-glutamate chains--very good reactivity was observed (IC50 400 microM for lysine; 80 microM for putrescine and spermidine). In addition to the chemically synthesized isopeptide cross-links that were recognized by this Mab, the naturally occurring Nepsilon-(ã-L-glutamyl)-L-lysine isopeptide cross-links in D-dimer, which are formed by the action of plasma transglutaminase (Factor XIII) on fibrin, were also detected on immunoblots using 81D4 as the primary antibody.
Thomas
Vincent
V
Covalab, 3, allée Pierre de Coubertin, 69007 Lyon, France. vthomas@covalab.com
Fournet
Guy
G
Simonet
Frantz
F
Roch
Anne-Marie
AM
Ceylan
Ismaïl
I
El Alaouia
Saïd
S
Quash
Gerard
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D005338:Fibrin Fibrinogen Degradation Products / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D008239:Lysine / Q000276:immunology*; D051379:Mice; D011700:Putrescine / Q000276:immunology*; D013095:Spermidine / Q000276:immunology*
D000911:Antibodies, Monoclonal; D005338:Fibrin Fibrinogen Degradation Products; C036309:fibrin fragment D; D008239:Lysine; D013095:Spermidine; D011700:Putrescine