title: Glutaraldehyde is an effective cross-linker for production of antibodies against advanced glycation end-products.
abstract: Immunohistochemical approaches have been widely used in the localization and quantification of advanced glycation end-products (AGEs). Traditional approaches for production of anti-AGE antibodies use cross-linkers such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to conjugate the AGE antigen to the carrier protein. However, these approaches often fail to produce antibodies that are specific to the particular AGE of interest. In the present study, Nepsilon-(carboxymethyl)lysine (CML), a major antigenic AGE structure, was conjugated to human serum albumin (HSA) using various cross-linkers, including EDC, bis(sulfosuccinimidyl)suberate (BS3) and glutaraldehyde, to compare their efficiency for the production of epitope-specific antibodies. All of the cross-linkers tested were capable of conjugating CML to HSA, and each CML-conjugated HSA was recognized by previously characterized anti-CML antibody. However, only the use of glutaraldehyde as the cross-linker resulted in the production of a CML-specific monoclonal antibody, termed 2G11. 2G11 significantly recognized CML-modified HSA and peptide, whereas it did not recognize Nepsilon-(carboxyethyl)lysine (CEL)-modified HSA and peptide, indicating that 2G11 is highly specific to CML, and can distinguish the difference of a single methyl group between the two epitopes. To further demonstrate the use of glutaraldehyde, anti-AGE antibodies against CEL, S-(2-succinyl)cysteine and S-(carboxymethyl)cysteine were obtained by conjugation with glutaraldehyde. These studies demonstrate the efficacy of glutaraldehyde as a cross-linker for the production of antibodies against small molecules.
authors: Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors: Katsumi
authors: K
authors: Mera
authors: Mime
authors: M
authors: Nagai
authors: Jonathan W C
authors: JW
authors: Brock
authors: Yukio
authors: Y
authors: Fujiwara
authors: Toshinori
authors: T
authors: Murata
authors: Toru
authors: T
authors: Maruyama
authors: John W
authors: JW
authors: Baynes
authors: Masaki
authors: M
authors: Otagiri
authors: Ryoji
authors: R
authors: Nagai
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay; D005976:Glutaral / Q000737:chemistry*; D017127:Glycation End Products, Advanced / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D012709:Serum Albumin / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D017127:Glycation End Products, Advanced; D012709:Serum Albumin; C048496:N(6)-carboxymethyllysine; D008239:Lysine; D005976:Glutaral