rev:November 3, 1997

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Biogenic Amine Assays for Pharmaceutical and Specialty Research-summary product data: please request full insert for current and more detailed information

The following Elisa kits are manufactured by IBL (Immuno Biological Laboratories, Hamburg Germany) and distributed by RDI Divison of researchd Industries Intl for in vitro research use only-not for use in or on humans or animals, not for use in diagnostics.


 Catalogue No   : RE 59171                           
 Product group  : Catecholamines                Determinations : 12 x 8      
 Porduct name   : Normetanephrine               ELISA   96
 Method         : ELISA                                                       
 Incubation time: 30min, 14-20h, 2h                                           
 Standard curve : 20-7,500 µg/l                                               
 Sample/Prep.   : 10 or 50µl                                                  
 Isotope/Substr.: PNPP 405nm                                                  


The catecholamines epinephrine, norepinephrine and dopamine are synthesized in the adrenal medulla, the sympathetic nervous system and in the brain. As catecholamines and their metabolites metanephrine and normetanephrine are secreted in increasing amounts in a number of diseases, they may be used for diagnostic purposes. In this context, diagnosis as well as the follow-up of tumor diseases of the nervous system are of special importance. This applies primarily to the pheochromocytoma, but also the neuroblastoma and the ganglioneuroma. Malignant growth is described in 10% of pheochromocytomas. Furthermore, an increase of catecholamines and their metabolites metanephrine and normetanephrine can be observed in the carcinoid

Contents of the Kit

1. Assay Buffer 1 bottle 50 ml, concentrate, phosphate buffer with BSA and stabilizer, dilute 1:10 with bidist. water. 2. Acylation Reagent 1 vial 2 ml, ready for use, contains dimethylformamide. 3. Indicator Buffer 1 bottle 6 ml, ready for use, Tris Puffer containing phenol red (colour change pH < 7.5). 4. Hydrolyzation Tubes 50 tubes 5. 0.1 M HCL 1 bottle 21 ml, ready for use. 6. Normetanephrine-Biotin 3 vials lyophilized, dissolve contents of each vial in 2 ml of assay buffer, stabilized. 7. Antiserum 1 vial 6 ml, ready for use, antiserum from rabbit in phosphate buffer with stabilizers, blue colored. 8. Microtiter Strips 12 strips each 8 wells, coated with anti-rabbit IgG from goat. 9. Standards A - G 0.35 ml each, ready for use. 7 vials The standards contain normetanephrine in the following concentrations: Standard A B C D E F G µg/l 0 77 192 480 1200 3000 7500 10. Control I and II 2 vials 0.5 ml each, ready for use, for concentration see quality control certificate. Store controls at 2-8 °C. 11. Wash Buffer, Concentrate 1 bottle 50 ml, phosphate buffer with Tween and stabilizer; dilute 1:10 with bidist. water. 12. Enzyme Conjugate, Concentrate 1 vial 250µl, anti-Biotin-alkaline phosphatase in Tris buffer with stabilizers, dilute 1:101 with Assay Buffer. 13. PNPP Substrate Tablets 9 tablets p-nitrophenyl phosphate (PNPP) 14. PNPP Substrate Buffer 1 bottle 30 ml, ready for use, contains diethanolamine. 15. PNPP Stop Solution 1 bottle 10 ml, ready for use, contains 1 N NaOH with 0.25 M EDTA 16. Adhesive Foil 3 pieces

Principle of the Test

The Normetanephrine-ELISA kit provides materials for the quantitative measurement of chemically derivatized normetanephrine in urine. The sample preparation, i.e. derivatization of normetanephrine to N-acylnormetanephrine, is part of the sample dilution and is achieved by incubation of the respective sample with the "Acylation Reagent". The assay procedure follows the basic principle of competitive ELISA whereby there is competition between a biotinylated and a non-biotinylated antigen for a fixed number of antibody binding sites. The amount of biotinylated antigen bound to the antibody is inversely proportional to the analyte concentration of the sample. When the system is in equilibrium, the free biotinylated antigen is removed by a washing step and the antibody bound biotinylated antigen is determined by use of anti-biotin alkaline phosphatase as marker and p-nitrophenyl phosphate as substrate. Quantification of unknowns is achieved by comparing the enzymatic activity of unknowns with a response curve prepared by using known standards.

Test Procedure

SHORT INSTRUCTION --------------------------------------------------------------------------- 1. Pipet 20µl each of acylated standards, acylated controls and acylated patient samples into the appropriate wells. 2. Pipet 50µl Normetanephrine-Biotin into each well, pipet 50µl Antiserum into each well, shake the plate carefully. 3. Seal the plate with the adhesive foil and incubate over night (14-20h) at 2-8°C. 4. Wash each well three times with Wash Buffer (the use of a washer is recommended). Remove the Wash Buffer carefully. Invert plate and remove any remaining liquid by tapping on clean blotting paper. Note: Sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure. 5. Pipet 150µl of diluted (1:101) Enzyme Conjugate into the wells. 6. Seal with the adhesive foil and incubate for 120 min. at room temperature on an orbital shaker (200 rpm). Prepare the PNPP substrate solution 10 min. before end of incubation period! 7. Wash each well 3 x with Wash Buffer (see above). 8. Pipet 200µl PNPP Substrate Solution into each well. 9. Incubate at room temperature for 20 min. on an orbital shaker (200 rpm). 10. Stop the substrate reaction by adding 50µl PNPP Stop Solution into each well. 11. Briefly mix contents by gently shaking the plate. --------------------------------------------------------------------------- After removing assay reagents from the refrigerator, allow them to reach room temperature before pipetting. Unused reagents should be stored at 2-8°C. Standards, controls and unknowns should be assayed in duplicate. --------------------------------------------------------------------------- Preparation of Reagents The contents of the kit can be divided into three separate runs. The volumes stated below are for one test procedure with 4 strips (32 determinations). If a larger number of strips is to be used, the volumes have to be changed accordingly. 1. Assay Buffer 15 ml of the Assay Buffer (concentrated) have to be diluted 1 : 10 with bidist. water to make up 150 ml. The Assay Buffer is now ready for use. Store at 2-8°C. 2. Wash Buffer 15 ml of the concentrate have to be diluted 1:10 with bidistilled water up to 150 ml. This gives the ready for use Wash Buffer. Store at 2-8°C. 3. Normetanephrine Biotin Add 2 ml of the ready for use Assay Buffer to one vial of lyophilized Normetanephrine-Biotin and wait for 15 min. Mix gently and use only once. 4. Enzyme Conjugate Dilute 60µl of Enzyme Conjugate (concentrated) in 6.0 ml of Assay Buffer (ready for use). Prepare freshly for each test run. Use only once. 5. PNPP Substrate Solution (should be prepared during the test procedure) Dissolve 3 PNPP substrate tablets in 8 ml of PNPP Substrate Buffer. Important: Prepare the PNPP substrate solution 10 min. prior to use and use only once. --------------------------------------------------------------------------- Sample Collection and Storage The total volume of urine excreted during a 24-hour period should be collected and mixed in a single bottle containing 10-15 ml of 6 N hydrochloric acid as preservative. Avoid exposure to direct sun light. Urin samples which are not assayed immediately may be stored at -20°C or lower for at least 6 months. Sample Preparation The sample preparation may be carried out in two different ways. 50µl urine are either hydrolyzed in the hydrolyzation tubes and thereafter transferred into glass tubes for acylation, or the complete sample preparation is carried out with 10µl urine in the hydrolyzation tube (e.g. in case of pipetting robots). A) Two step sample preparation 1. Pipet 50µl of standards, patient urine samples and controls into respective hydrolyzation tubes. 2. Pipet 200µl of 0.1 N HCl into all tubes. 3. Close tubes with stopper and hydrolyze for 1 h at 90°C. Allow to cool to room temperature afterwards. No hydrolyzation is required when assaying free normetanephrine. 4. Pipet 50µl of each hydrolyzed sample into glass test tubes and add 50 µl of Indicator Buffer to each tube. Add 20µl Acylation Reagent to each tube, vortex mix. 5. Close tubes and incubate for 30 min. at 37°C. 6. Add 1 ml ready for use Assay Buffer and vortex mix. Withdraw 20µl aliquotes for ELISA! B) Entire Sample preparation in the hydrolyzation tube 1. Pipet 10µl of standards, patient urine samples and controls into respective hydrolyzation tubes. 2. Pipet 40µl of 0.1 M HCl into all tubes. 3. Close tubes with stopper and hydrolyze for 1 h at 90°C. Allow to cool to room temperature afterwards. No hydrolyzation is required when assaying free normetanephrine. 4. Pipet 50µl Indicator Buffer into Hydrolyzation Tubes and vortex mix. 5. Add 20µl Acylation Reagent to each tube, vortex mix. 6. Incubate for 30 min. at 37°C. 7. Add 1 ml ready for use Assay Buffer and vortex mix. Withdraw 20µl aliquotes for ELISA

Performance Characteristics

1. Sensitivity The lowest detectable level that can be distinguished from the zero standard (defined as 3 x standard deviation of the zero calibrator) is: Normetanephrine: approx. 20µg/l Urine 2. Specificity The crossreactivity of the anti-N-acylnormetanephrine antiserum has been measured against various compounds which were processed like patient samples (without hydrolyzation). Compound Cross reactivity(%) Normetanephrine 100 Metanephrine 0.6 4-Hydroxy-3-Methoxy-phenylglycole 0.15 3-Methoxytyramine 0.4 4-Hydroxy-3-Methoxy-phenylpyruvat acid < 0.05 Homovanillic acid < 0.05 Epinephrine < 0.02 Vanillic mandelic acid < 0.02 Dopamine < 0.02 3-Methoxytyrosine < 0.02 L-Tyrosine < 0.02 Tryptophane < 0.02 L-Dopa < 0.02 á-(4-Hydroxy-3-Methoxy-phenyl) lactic acid < 0.02 Caffeic acid < 0.02 Norepinephrine < 0.02 Ferula acid < 0.01 3. Recovery Human urine was enriched with increasing amounts of normetanephrine. (Concentrations in µg/l) Actual Amount Amount Amount Recovery % conc. added recovered expected Urine A92 48 152 140 109 92 148 274 240 114 92 443 610 535 114 92 1330 1625 1422 114 92 4000 4162 4092 102 -------------------------------------------------------- Urine B 169 49 201 218 92 169 148 312 317 98 169 443 621 612 101 169 1330 1369 1499 91 169 4000 3864 4169 93 -------------------------------------------------------- Urine C 162 49 200 211 95 162 148 338 310 109 162 443 702 605 116 162 1330 1746 1492 117 162 4000 4048 4162 97 -------------------------------------------------------- Urine D 92 49 161 141 114 92 148 267 240 111 92 443 550 535 103 92 1330 1615 1422 114 92 4000 3890 4092 95 4. Precision INTRA-assay variation (all concentrations in µg/l) Mean Standard Deviation VK (%) n 271 19.8 7.33 10 908 61.4 6.76 10 INTER-assay variation (all concentrations in µg/l) Mean Standard Deviation VK (%) n 199 24.9 12.5 9 866 79.6 9.19 9 5. Dilution Unknown patient samples were diluted with 0.1 N HCl before sample preparation and assayed. The calculated results are shown in µg/l. Dilution 1/1 1/2 1/4 1/8 1/16 1/32 Urine 1 1836 1474 1684 1904 1904 2016 (100%) (80%) (92%) (104%) (104%) (110%) Urine 2 2060 2156 2148 2024 2256 (100%) (105%) (104%) (98%) (110%) Urine 3 2083 2190 1912 2080 2176 1984 (100%) (105%) (92%) (100%) (104%) (95%)

Expected Values

Reference values in 24 h urine samples It is recommended that each laboratory establishes its own range of normal normetanephrine values. The following values may serve as a guide line: Normetanephrine: 30-440µ/d

Alternative Applications

(More) Clinical Background

Measuring of Adrenalin/Noradrenalin and Metanephrine/Normetanephrine instead of Vanillic mandelic acid or Dopamine in Pheochromocytoma diagnosis Even today the diagnosis of Pheochromocytoma is still difficult. If only total catecholamines, total metanephrines, vanillyl mandelic acid, dopamine as well as homovanillic acid are determined, results show poor diagnostic sensitivity. For that reason, more sensitive analytical procedures and biochemical markers are required, having the lowest rate of false positive results. By employing new highly specific enzyme immunoassays, these disadvantages are overcome. The results obtained are reliable, of high precision as well as free of drug interferences. The analytes are evaluated versus HPLC as well as GC/MS. HPLC GC/MS Diagnost. Sensitivity Diagnost. Specificity Metanephrine/Normetanephrine 100% 97-98% Adrenalin/Noradrenalin 100% 97% Dopamine 10% Vanillyl mandelic acid, only 68-70% 92-96% The determination of vanillyl mandelic acid (VMA) has to be considered as obsolete because of ist lack of diagnostic sensitivity. Dopamine has shown insensitive for Noradrenalin and Adrenalin screting phenochromocytomas. (Graham, 1993). In a study carried out in 19 patients with histologically proven phenochromocytoma dopamine alone was not increased, independent of the other catecholamnies in any of the pheochromocytoma patients (Smythe, 1992). On the basic of these studies, the specific measurement of adrenalin/Noradrenalin and Metanephrine/Normetanephrine in 24 hours urine are the most suitable analytes in the diagnosis of pheochromocytoma. Literature is available on request.


Quality Control For the internal urine accuracy control we recommend the Bio-Rad Lyphochek Urine Controls. In comparison to the Bio-Rad HPLC Test for Adrenalin, Noradrenalin, Dopamine, Metanephrine and Normetanephrine they show well comparable results. Bio-Rad's Lyphochek Endocrinology Controls, for Plasma and with added ñ form, are only for limited use. Results corresponding with HPLC have only been found for Dopamine. Adrenalin and Noradrenalin show considerably lower results, therefore only level 2 results can be indicated. Experimental studies have shown antibody reactivities against both enantiomere forms of Adrenalin, Noradrenalin, Metanephrine and Normetanephrine being responsible for these differneces. Under the Conditions given in the enclosed Instructions for Use (e.g. standards used) only the physiological form (-) of the catecholamines and their derivatives is measured, while in HPLC both forms (ñ) are measured in the same way. The Bio-Rad Endocrinology Controls for the given HPLC ranges confirm this effect. These HPLC results are only partly comparable with the results obtained with the immunoassay. Depending on the Bio-Rad lot, HPLC shows considerably higher results for Adrenalin and Noradrenalin than the IBL immunoassay.
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