Preparation and characterization of immunogens for antibody production against metanephrine and normetanephrine.
A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating metanephrine (MN) and normetanephrine (NM) to bovine serum albumin (BSA). The protein was activated with water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of N-hydroxysulfosuccimide (sulfo-NHS), leading to the formation of active N-succinimidyl esters of some glutamic and aspartic acid carboxyls. The pertinence of this reaction for the coupling of these haptens to carboxylate groups was confirmed via reaction with a model compound, 2-hydroxybenzoic acid, and subsequent characterization using atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for the quantitative assessment of the hapten/protein ratios of these conjugates. This technique of conjugate characterization demonstrated greater resolution in molecular weight determination compared to nondenaturing polyacrylamide gel electrophoresis (native PAGE). Preliminary results from enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA procedures using test antisera confirmed that the synthesized immunogens were highly antigenic and elicited specific antibody responses in BALB/c mice against the haptens.
Department of Pharmacy, The National University of Singapore, 18 Science Drive 4, Singapore, Singapore.
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D000828:Animal Population Groups; D000818:Animals; D000906:Antibodies / Q000097:blood*; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D001667:Binding, Competitive; D003432:Cross-Linking Reagents / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D005260:Female; D005976:Glutaral / Q000737:chemistry; D006241:Haptens / Q000276:immunology; D008676:Metanephrine / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009647:Normetanephrine / Q000737:chemistry / Q000276:immunology*; D020156:Salicylic Acid / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry
D000906:Antibodies; D000941:Antigens; D003432:Cross-Linking Reagents; D006241:Haptens; C465543:N-hydroxysulfosuccimide; D013388:Succinimides; D009647:Normetanephrine; D012710:Serum Albumin, Bovine; D008676:Metanephrine; D020156:Salicylic Acid; D005022:Ethyldimethylaminopropyl Carbodiimide; D005976:Glutaral