Preparation and characterization of immunogens for antibody production against metanephrine and normetanephrine E.C.Y. Chan, P.C. Ho * Department of Pharmacy, The National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore Received 24 September 2001; received in revised form 21 March 2002; accepted 11 April 2002 Abstract 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. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Metanephrine; Normetanephrine; Immunoassay; MALDI-TOF-MS 1. Introduction Pheochromocytoma is a tumor arising from neural crest tissue that secretes catecholamines directly into the circulation, producing episodes of hypertension, severe headaches and sweating (Benowitz, 1990). The early diagnosis of pheochromocytoma is imperative, not only because it offers the possibility of curing the condition but also because unrecognized pheochro- mocytoma is a potentially fatal condition. Estimation of urinary-free catecholamines and total metanephr- ines (metanephrine (MN) and normetanephrine (NM)) is routinely used in the biochemical detection of pheochromocytoma and in monitoring the complete- ness of tumor excision, as well as the possibility of recurrence (Rosano et al., 1991). These compounds have been analyzed by liquid chromatography (LC) and gas chromatography (GC). These chromato- graphic methods are accurate and sensitive, but incon- venient for routine assay. Immunoassay is pertinent to measure a number of samples and is desirable for MN and NM that are 0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0022-1759(02)00143-6 * Corresponding author. Tel.: +65-6874-2651; fax: +65-6779- 1554. E-mail address: phahocl@nus.edu.sg (P.C. Ho). www.elsevier.com/locate/jim Journal of Immunological Methods 266 (2002) 143 – 154