효능검색정보센터 외국 효능검색기술

Protocol for Metcombi Radioimmunoassay

Protocol Code : FR01010
Screening target : Urinary catecholamine metanephrine
Screening method : Metcombi Radioimmunoassay
Sources : Immuno-Biological Laboratories (IBL)

I. Introduction

Assessment of catecholamine production and excretion is important in the laboratory detection of pheochromocytoma, a rare but curable cuse of hypertension. Advances in catecholamine and metabolite methodologies have enhanced the diagnostic acumen by increasing analytical sensitivity and eliminating many of the interferences observed with earlier methods. Estimation of urinary catecholamine metanephrine is routinely used in the biochemical detection of pheochromocytoma and in monitoring the completeness of tumor excision as well as the possibility of recurrence.

2. Principle of the Test

The METCOMBI Radioimmunoassay provides material for the quantitative measurement of derivatized normetanephrine and metanephrine in urine. The sample preparation is performed in two steps. First, conjugated normetanephrine and metanephrine is hydrolyzed and then derivatized to N-acylnormetanephrine and N-acylmetanephrine. The assay procedure follows the basic principle of radioimmunoassays: the competition between a radioactive and a non-radioactive antigen for a fixed number of antibody-binding sites. The amount of 125I-labeled antigen bound to the antibody is inversely proportional to the analyte concentration of the sample. When the system is in equilibrium, the antibody-bound radioactivity is precipitated with a second antibody in the presence of polyethylene glycol. The precipitate is counted in a gamma counter. Quantification of unknowns is achieved by comparing their activity with a response curve prepared by using known standards.

3. Test Methods

3.1 Summary
  A. Hydrolyzation
    1) Pipet 50㎕ of standards, controls and patient samples into Hydolyzation Tubes.
    2) Pipet 1.0 ml of 0.1 N HCl into all tubes and hydolyze for 1 h at 90℃.
    3) Dilute 50㎕ of each hydrolyzed sample with 1.0ml of Assay Buffer.

  B. Normetanephrine RIA
    1) Pipet 100㎕ of hydolyzed Standard A into NSB Tubes.
    2) Pipet 50㎕ of hydrolyzed Standard A into Bo tubes.
    3) Pipet 50㎕ of hydrolyzed Standards B-E and 50㎕ of hydrolyzed patient samples.
    4) Incubate for 15 min. at room temperature.
    5) Add 50㎕ of 125I-Tracer Normetanephrine.
    6) Add 50㎕ of Normetanephrine Antiserum to all tubes except T and NSB, vortex mix,
        centrifuge (1 min at 500 x g).
    7) Incubate over night (15-20h) at 2-8℃.
    8) Add 1000㎕ of Pecipitating Antiserum.
    9) Incubate for 15 min. at room temperature.
    10) Centrifuge for 15 min. at 1500-3000 x g.
    11) Aspirate all tubes except T.

  C. Metanephrine RIA
    1) Pipet 150㎕ of hydolyzed Standard A into NSB Tubes.
    2) Pipet 100㎕ of hydrolyzed Standard A into Bo tubes.
    3) Pipet 100㎕ of hydolyzed Standards B-E and 100㎕ of hydolyzed patient samples.
    4) Vortex mix and incubate for 15 min. at room temperature.
    5) Add 50㎕ of 125I-Tracer Metanephrine.
    6) Add 50㎕ Metanephrine Antiserum to all tubes except T and NSB, vortex mix,
         centrifuge (1 min. at 500 x g).
    7) Incubate over night (15-20h) at 2-8℃.
    8) Add 1000㎕ of Pecipitating Antiserum.
    9) Incubate for 15 min. at room temperature.
    10) Centrifuge for 15 min. at 1500-3000 x g.
    11) Aspirate all tubes except T.

3.2 Detailed Instructions for Use

    After removing assay reagents from the refrigerator, allow them to reach room temperature before pipetting. Unused reagents should be stored at 2-8℃. Standards, controls and unknowns should be assayed in duplicate.

  A. Sample Preparation

  A-1. Hydrolyzation of standards, controls and urine samples for determination of total
        Normetanephrine and total Metanephrine.
  For the hydrolyzation of standards, controls and urine samples use the supplied Hydrolyzation Tubes. Label Hydrolyzation Tubes accordingly and proceed to

    1) Pipet 50㎕ of standards, patient urine samples and controls into respective
        hydrolyzation tubes.
    2) Pipet 1.0ml of 0.1N HCl into all tubes, close tubes with stopper, vortex and hydrolyze
        for 1 h at 90℃ Allow to cool to room temperature afterwards.
    3) Dilute 50㎕ of each hydrolyzed sample in new disposable tubes with 1.0 ml of Assay
        Buffer and mix well.

  A-2. Determination of free Normetanephrine and Metanephrine in urine
    1) Pipet 50㎕ of standards, patient urine samples and controls into disposable tubes.
    2) Pipet 1.0 ml of bidistilled water to all tubes and mix well.
    3) Dilute 50㎕ of each sample in new disposable tubes with 1.0 ml of Assay Buffer and
        mix well.

  B. Acylation and Radioimmunoassays Standards, controls and samples should be
      assayed in duplicates.

  B-1. Normetanephrine
    1) Pipet 100㎕ of hydrolyzed Standard A into NSB tubes.
    2) Pipet 50㎕ of hydrolyzed Standard A into Bo-tubes.
    3) Pipet 50㎕ of hydrolyzed Standards B-E, 50㎕ Controls and 50㎕ of hydrolyzed patient
        urine samples into the respective tubes.
    4) Vortex mix and incubate at room temperature for 15 min.
    5) Add 50㎕ of 125I-Tracer Normetanephrine to all tubes.
    6) Add 50㎕ of Normetanephrine Antiserum to all tubes, except T and NSB, vortex mix and
        centrifuge briefly (1 min. at 500 x g).
    7) Incubate overnight (15-20 h) at 2 - 8 ℃
     8) Add 1ml of Precipitating Antiserum to all tubes, except T and vortex mix.
    9) Incubate at room temperature for 15 min.
    10) Centrifuge all tubes except T for 15 min. at 3000 x g*.
    11) Aspirate all tubes except T.
    12) Count all tubes in a gamma counter for one minute.

  B-2. Metanephrine
    1) Pipet 150㎕ of hydrolyzed Standard A into NSB tubes.
    2) Pipet 100㎕ of hydrolyzed Standard A into Bo tubes.
    3) Pipet 100㎕ of hydrolyzed Standards B-E, 100㎕ Controls and 100㎕ of hydrolyzed
        patient urine samples into the respective tubes.
    4) Vortex mix and incubate for 15 min at room temperature.
    5) Add 50㎕ of 125I- Tracer Metanephrine to all tubes.
    6) Add 50㎕ of Metanephrine Antiserum to all tubes except T and NSB, vortex mix and
        centrifuge briefly (1 min at 500 x g).
    7) Incubate overnight (15-20 h) at 2 - 8 ℃
     8) Add 1 ml of Precipitating Antiserum to all tubes except T and vortex mix.
    9) Incubate for 15 min at room temperature.
    10) Centrifuge all tubes except T for 15 min at 3000 x g*.
    11) Aspirate all tubes except T.
    12) Count all tubes in a gamma counter for one minute.

4. References

Kagedal, B. (1988). Catecholamines and their metabolites (review). J.Chromatogr. 429: 177-233.
Benowitz, N.L.(1990). Pheochromocytoma. Adv. Intern. Med. 35: 195-220
Rosano TG, Swift TA, Hayes LW (review), Andvances in Catecholamine and Metabolite Measurements for Diagnosis of Pheochromocytoma, Clin. Chem. 37/10(B), 1854-1867, (1991).
Gerlo EAM,Sevens C, Urinary and Plasma Catecholamines Metabolites in Pheochromocytoma: Diagnostic Value in 19 Cases, Clin. Chem. 40/2, 250-256, (1994).
Graham PE, Smythe GA, Edwards GA, Lazaruz L, Laboratory Diagnosis of Pheochromocytoma: which analytes should we measure?, Ann. Clin. Biochem. 30, 129-134, (1993).
Bouloux PMG, Fakeeh M, Investigation of Pheochromocytoma, Clinical Endocrinology, 43, 657-664, (1995).