Home
Resources
Publications
Definition of the Fine Specificity of the Monoclonal Antibody 81D4: Its Reactivity With Lysine and Polyamine Isopeptide Cross-Links

Definition of the Fine Specificity of the Monoclonal Antibody 81D4: Its Reactivity With Lysine and Polyamine Isopeptide Cross-Links

Vincent Thomas, Guy Fournet, Frantz Simonet, Anne-Marie Roch, Ismaïl Ceylan, Saïd El Alaouia, Gerard Quash

J Immunol Methods. 2004 Sep;292(1-2):83-95.

PMID: 15350514

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
LS74731	Poly(dimer acid-co-alkyl polyamine)			Price

As a specialist in analytical chemical Products, Alfa Chemistry offers consumers a wealth of raw materials and a full range of technologies and services.

QUICK LINKS

HEADQUARTERS

Tel:

Fax:

Email: