|Year : 2016 | Volume
| Issue : 2 | Page : 37-42
Development of immunoassays of catecholamines and their metabolites
Department of Biomedical Analysis, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan
|Date of Submission||09-Oct-2016|
|Date of Acceptance||23-Nov-2016|
|Date of Web Publication||1-Feb-2017|
6-15 Otokoyamayoshii, Yawata, Kyoto 614-8363
Source of Support: None, Conflict of Interest: None
Catecholamines take the role of neurotransmitters and hormones and are involved in many diseases. Sensitive immunoassays for catecholamines and their metabolites were required to study the diagnoses of diseases. First, we achieved success in producing specific antibodies for catecholamines and basic metabolites using each antigen. We applied a monoclonal antibody to 3-methoxy-4-hydroxyphenylglycol to measure the concentrations to diagnose depression. The acidic homovanillic acid (HVA) and d-3-methoxy-4-hydroxymandelic acid (VMA) were useful in mass screening of neuroblastoma using monoclonal antibodies. The paper reviews these developments for future use. An antigen is important to produce the specific antibody. The amino acid residue of each catecholamine and l-3,4-dihydroxyphenylalanine protected with N-maleyl group as well as basic metabolites was reacted with bovine serum albumin using Mannich reaction in the presence of formaldehyde. The N-maleyl group of the conjugate was moderately liberated to give rise to the antigen. The antigen was injected with Freund’s Complete Adjuvant to the rabbit or mouse. The conventional or monoclonal antibody was used for radioimmunoassay or enzyme immunoassay. Each immunoassay showed high specificity in discriminating not only the fine structure of the hapten but also body ingredients. The kits of HVA and VMA were useful in screening the urine from the infant with neuroblastoma. The secret of specific antibody preparation is dependent on the synthetic method of the conjugation, which is chemically moderate. The preparation of antibody requires long time to increase the affinity. Thus, the methods of antibody preparation are established and repeatable for anyone. The mass screening of neuroblastoma with HVA and VMA was easily applied to infants. Our previous results had established immunoassays for all members of catecholamines and their metabolites for diagnosis and research.
Keywords: 3-methoxy-4-hydroxymandelic acid, 3-methoxy-4-hydroxyphenylglycol, epinephrine, homovanillic acid, immunoassay, metanephrine, norepinephrine
|How to cite this article:|
Yoshioka M. Development of immunoassays of catecholamines and their metabolites. J Arab Soc Med Res 2016;11:37-42
| Background|| |
Catecholamines take the role of neurotransmitters and hormones. They are involved in many diseases, such as Parkinsonism, depression, cancer, and dementia. Julius Axelrod established the metabolism ([Figure 1]) and working mechanism of catecholamines, and was awarded Nobel Prize as described in Nobel Lecture, 12 December 1970. Dopamine in the central nervous system decreases to give rise to Parkinsonism and a type of depression or dementia; hence, its precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), is a good drug. Catecholamines and metabolites are found increased in catecholamine-producing tumors such as neuroblastoma and phaeochromcytoma.
Sensitive immunoassays of catecholamines and their metabolites were required to be studied for the diagnoses of various related diseases. They are unstable and are present at very low concentrations in body fluids. Thus, we tried to develop the sensitive immunoassays. First, we achieved success in producing specific antibodies to epinephrine, norepinephrine, and dopamine and its precursor, l-DOPA, using each antigen coupled the hapten with albumin using Mannich reaction ,,,,,,.
In a similar manner, the antibody for the basic metabolite such as 3-methoxy-4-hydroxyphenyl-2-methylamino ethanol (metanephrine) was produced ,. With advancement in antibody production, the hybridoma technique for the monoclonal antibody, developed by Cesar Milstein, was awarded Nobel Prize in 1984. We also applied a monoclonal antibody to neutral metabolite and the concentration was measured to diagnose depression ,,,.
The acidic metabolites 3-methoxy-4-hydroxyphenylacetic acid [homovanillic acid (HVA)] and d-3-methoxy-4-hydroxymandelic acid (VMA) were useful in mass screening of neuroblastoma using monoclonal antibodies with techniques approved by the Food and Drug Administration in October 1989, and by Japanese government in November 1990 ,,. The paper reviews these developments for future use .
| Methods|| |
To give a hapten antigenecity, the catecolamine or the metabolite each must be coupled with a carrier protein. There are many ways to conjugate the hapten of fine structure. The side chain of the hapten was easy to couple with the amino groups of the carrier; the antibodies were not specific to discriminate the phenol derivatives of catecholamine or metabolite previously published ,,,,,,. The Ethical Committee on human research at Setsunan University, Faculty of Pharmaceutical Sciences, Osaka, Japan has been approved the protocol for all our previous research including in that review, and the medical consent has been approved from the parents of patients.
An antigen is important to produce the specific antibody. The amino acid residue of each catecholamine and l-DOPA protected with N-maleyl group was reacted with bovine serum albumin using Mannich reaction in the presence of formaldehyde. The N-maleyl group of the conjugate was moderately liberated to give rise to the antigen, as shown in [Figure 2] . The metabolites were not required to protect with the maleyl group. Catecholamine or metabolite was displaced in the neighbor position of phenol with aminomethyl-lysine residue of albumin. The antigen was injected with Freund’s Complete Adjuvant to the rabbit or mouse. The conventional or monoclonal antibody was used for radioimmunoassay or enzyme immunoassay.
|Figure 2: Synthesis of antigens of catecholamines. Modifying from Miwa et al. .|
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| Summary|| |
Each immunoassay showed high specificity in discriminating not only the fine structure of the hapten but also body ingredients. The antibody to natural l-epinephrine binds with l-form but not d-form, as shown in [Table 1]. It is important to bear in mind that the true conformation of the asymetric carbon of the side chain is d-form from d-mandeliic acid; however, it is conventionally denoted as l-form, because of the levorotatory property of the natural epinephrine. The provability of such high specific antibody is so low that it took 1 year and only one rabbit produced among 40 rabbits. The antibody to l-norepinephrine was also selected high specific, as shown in [Table 2]. The antibody to the basic metabolite, metanephrine, also discriminated the fine structure of the analogs ([Table 3]). The monoclonal antibody to 3-methoxy-4-hydroxyphenylglycol was specific ([Table 4]) and is applied in the diagnosis of depression. The antibody for VMA was selected specifically ([Table 5]), as well as the one for HVA. The kits for HVA and VMA were useful in screening the urine from infants with neuroblastoma. Apparently, a healthy infant was found with neuroblastoma ([Figure 3]) .
|Table 1: Relative cross-reactivity of the antibody to l-epinephrine to analogs by means of radioimmunoassay|
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|Table 2: Cross-reactivity of the antibody to l-norepinephrine to analogs by means of radioimmunoassay|
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|Table 3: Cross-reactivity of the antibody to d-l-metanephrine to analogs by radioimmunoassay|
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|Table 4: Cross-reactivity of the monoclonal antibody to d-3-methoxy-4-hydroxyphenylglycol to analogs using EIA|
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|Table 5: Cross-reactivity of the monoclonal antibody to d-vanilmandelic acid to analogs using enzyme immunoassay|
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|Figure 3: A patient with neuroblastoma found by enzyme immunoassay (EIA). Original picture of the patient modifying from Yokomori et al. .|
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We had been involved in the development of gas chromatography with mass spectrometry and high-performance liquid chromatography, which were not so convenient and needed high technology and were time consuming . Immunoassay is very simple and easy to measure many samples at once. It was very difficult to make specific antibodies for three endogenous catecholamines and their metabolites of fine structure, before our success. The secret of specific antibody preparation depended on the synthetic method of the conjugation, which was chemically moderate. The preparation of antibody took long time and many animals to increase the affinity. Once the antibodies of high affinity are obtained ([Table 1],[Table 2],[Table 3],[Table 4],[Table 5]), it is easy to make specific and sensitive immunoassays for practical measurements described in the results. Thus, the methods of antibody production are established and repeatable for anyone.
When the specific antibody was, however, not obtained with another conjugation of a hapten with a carrier protein, some pretreatment of sample was possible rough to separate catecholamines and metabolites, which were recognized with not so much specific, but common antibody .
The mass screening of neuroblastoma with HVA and VMA was easily applied to infants. Positive infants ([Figure 3]) were operated . This method was used in the USA and is commercially available worldwide . It was, however, not yet defined to estimate good or bad prognosis of the cancer. In that case, I recommend the other prognostic biomarkers, 2’-deoxycytidine, which was increased in breast cancer ([Figure 4]) , bladder cancer, hepatoma, and acute leukemia in our joint project with Prof. M. El-Merzabani, National Cancer Institute, Cairo University, for more than 30 years. Immunoassay of 2’-deoxycytidine was established for the future use ,,.
|Figure 4: 2’-deoxycytidine levels in bloods of individual breast cancer patients under chemotherapy. Modifying from Yoshioka et al. .|
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| Conclusion|| |
We have established immunoassays of all members of catecholamines and their metabolites for diagnosis and research . To the best of our knowledge, such basic methods for catecholamines and their metabolites have been rarely developed. This review is renewal to evaluate the significance.
I am grateful to Professor Karam Mahdy of National Research Center, Cairo, who gave me a chance to present this review at the 5th International conference of the Arab Society for Medical Research, Sharm El Sheikh, 28–31 October in 2016.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]