Catecholamine ELISA and RIA Immunoassays

Introduction

Adrenaline (epinephrine) and noradrenaline (norepinephrine) together with dopamine constitute the group of catecholamines, which play an important role as hormones and neurotransmitters. Catecholamines are biogenic amines which are synthesized from the amino acid tyrosine via the intermediate product dopa.

catecholamine-metabolism

After they have exerted their effects, adrenaline and noradrenaline are broken down via the intermediate products metanephrine and normetanephrine to the end product of metabolism vanillylmandelic acid and excreted in urine. Therefore urine as well as plasma is used as a test material for determining catecholamine concentrations.

Catecholamines are synthesized predominantly in the adrenal medulla and in the nerve ends of the sympthetic nerve system. Here they are stored in granula and released following a specific stimulus. The catecholamines then exert their effects by binding to specific membrane receptors on the target cells. Afterwards, they are broken down rapidly by various enzymes. The main role of catecholamines is to help the body adapt to acute and chronic stress. Adrenaline affects mainly the heart muscles and metabolism, while noradrenaline acts as a vasoconstrictor on the peripheral arteries. A special application for determination of catecholamine concentrations in the body lies in the area of stress research and sports medicine.

Diseases which are associated with altered levels of catecholamines include hypertonia, degenerative heart diseases, schizophrenia, manic-depressive disorders, and catecholamine- producing tumours (neuroblastoma, ganglioneuroma, and phaeochromocytoma). Of these, phaeochromocytoma has the greatest significance. Phaeochromocytomas are excessively catecholamine-secreting tumors, which originate in the adrenal medula and can, in combination with operative procedures and anaesthesia, lead to life-threatening situations. Around 10% of all phaeochromocytomas show malignant growth. In most cases, however, they can be cured by operative therapy combined with medication therapy using ß-adrenergen antagonists. An unidentified phaeochromocytoma on the other hand poses a serious risk to the patient.

The determination of adrenaline and noradrenaline concentrations is therefore essential in patients with suspected phaeochromocytoma. These tests are also frequently used in the initial diagnosis of hypertonia.
The measurement of dopamine and its derivatives is of special diagnostic value with children who are suspected to have a neuroblastoma. Adrenaline, noradrenaline and dopamine are extracted using a cis-diolspecific affinity gel and acylated to N-acyladrenaline, N-acylnoradrenaline and N-acyldopamine and then converted enzymatically during the detection procedure into N-acylmetanephrine, N-acylnormetanephrine and N-acyl-3-methoxytyramine.

RIA- The assay procedure follows the basic principle of radioimmunoassays, involving 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 unknown samples is achieved by comparing their activity with a reference curve prepared with known standards.

ELISA- The competitive EIA kit uses the microtiter plate format. Adrenaline, noradrenaline and dopamine, respectively, are bound to the solid phase of the microtiter plate. Acylated catecholamines from the sample and solid phase bound catecholamines compete for a fixed number of antiserum binding sites. When the system is in equilibrium, free antigen and free antigen-antiserum complexes are removed by washing. The antibody bound to the solid phase catecholamine is detected by an anti-rabbit IgG-peroxidase conjugate using TMB as a substrate.  The reaction is monitored at 450 nm with the amount of antibody bound to the solid phase catecholamine being inversely proportional to the catecholamine concentration of the sample.

RIA vs. HPLC Comparison- Plasma and Urine:

The adrenaline, noradrenaline and dopamine concentrations in 21 urine samples were assessed using both the LDN RIA (x) and the HPLC method (y) (external QC samples from UK NEQAS).  The Radio Immunoassays measure equally well when compared to the UK NEQAS HPLC data. The UK control values are the mean of about 40 different HPLC users, and contain always one pathological sample per distribution. The results of linear regression analysis showed the following correlation characteristics for adrenaline: y=0.95x – 0.03, r=0.99, for noradrenaline: y=1.23x – 0.12, r=0.99 and for dopamine: y=1.07x + 0.01, r=0.98.

The adrenaline, noradrenaline and dopamine concentrations in 20 plasma samples were assessed using both the LDN RIA (x) and the HPLC method (y). The results of linear regression analysis showed the following correlation characteristics for adrenaline: y=0.8x – 0.03, r=0.96, for noradrenaline: y=1.27x – 0.14, r=0.99 and for dopamine: y=x + 0.003, r=0.96.

Bottom Line:

The RIA method is comparable for adrenaline, noradrenaline and dopamine in urine and plasma.

 

ELISA vs. HPLC Comparison - Plasma and Urine:

The adrenaline, noradrenaline and dopamine concentrations in 30 urine samples were assessed using both the LDN EIA (x) and the HPLC method (y) (external QC samples from UK NEQAS). The correlation between EIA and HPLC is excellent.  The Enzyme Immunoassays measure equally well when compared to the UK NEQAS HPLC data. The UK control values are the mean of about 40 different HPLC users, and contain always one pathological sample per evaluation. The results of linear regression analysis showed the following correlation characteristics: adrenaline y=1.17x + 0.06, r=0.99, for noradrenaline y=1.27x - 0.04, r=0.96 and for dopamine y=0.98x – 0.08, r=0.95. The two assay methods are comparable.

The adrenaline, noradrenaline and dopamine concentrations in 20 plasma samples were assessed using both the LDN ELISA (x) and the HPLC method (y). The results of linear regression analysis showed the following correlation characteristics for adrenaline: y=1.04x – 0.05, r=0.98, for noradrenaline: y=1.26x – 0.05, r=0.97 and for dopamine: y=x + 0.003, r=0.96.


Bottom Line:

 

The ELISA assay method is comparable for adrenaline, noradrenaline and dopamine in urine and plasma.

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