RESUMO
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.
Assuntos
Anticorpos/sangue , Metanefrina/imunologia , Normetanefrina/imunologia , Grupos de População Animal , Animais , Antígenos/química , Antígenos/imunologia , Ligação Competitiva , Reagentes de Ligações Cruzadas/química , Ensaio de Imunoadsorção Enzimática , Etildimetilaminopropil Carbodi-Imida/química , Feminino , Glutaral/química , Haptenos/imunologia , Metanefrina/química , Camundongos , Camundongos Endogâmicos BALB C , Normetanefrina/química , Ácido Salicílico/química , Soroalbumina Bovina/química , Soroalbumina Bovina/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Succinimidas/químicaRESUMO
Sensitive and specific radioimmunoassays of metanephrine and normetanephrine were developed by use of 125I-labeled synephrine and specific metanephrine antibody, and 125I-labeled octopamine and specific normetanephrine antibody. Specific antibody for both metanephrine and normetanephrine was raised in rabbits by immunization with bovine serum albumin conjugated with the corresponding hapten, prepared by the method of Grota and Brown (Endocrinology 1976;98:615). The detection limits of the metanephrine and the normetanephrine radioimmunoassays were 2 and 6 pg/tube, respectively. Mean plasma metanephrine and normetanephrine values for 24 normal subjects were 62 (SD 14) and 100 (SD 40) ng/L, respectively. Mean urinary metanephrine and normetanephrine values for 22 normal subjects were 154 (SD 74) and 217 (SD 109) micrograms/day. For 14 pheochromocytoma patients, plasma metanephrine and normetanephrine values ranged from 29 to 683 and from 28 to 7850 ng/L, and urinary metanephrine and normetanephrine values were 606 to 6630 and 296 to 4800 micrograms/day, respectively. The present methods are simple and suitable for routine tests or for mass screening for pheochromocytoma.
