Elsevier

Analytical Biochemistry

Volume 599, 15 June 2020, 113733
Analytical Biochemistry

Development of an ELISA for detection of mitragynine and its metabolites in human urine

https://doi.org/10.1016/j.ab.2020.113733Get rights and content

Highlights

  • An immunogenic antigen and enzyme conjugates were synthesized using Mannich reaction.

  • Specific antibodies produced highly cross-reacted to its metabolites.

  • High concentration levels of mitragynine in μg/mL was found in positive human urine of drug addicts.

Abstract

An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC50 of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.

Introduction

Mitragyna speciosa is an herbal plant indigenous to Southeast Asian countries, such as Thailand, Malaysia and Myanmar [[1], [2], [3]]. It is a member of the Rubiaceae family which is the same as the coffee tree [4]. The plant is known by various names such as ‘Kratom’ in Thailand; ‘biak-biak’ and ‘ketum’ in Malaysia [2,5,6]. This traditional medicinal plant is used by local communities to treat human diseases including fever, cough, diarrhea, hypertension, counter fatigue, relieve pain, and diabetes mellitus [3,5,7,8]. It is also used to prevent withdrawal symptoms in opiate-dependent individuals and as a substitute for opium [5,9,10]. The leaves are consumed by chewing, brewing, and smoking [11]. Effects appear within 5–20 min and last for 60–90 min depending on the amount consumed [5].

More than 40 alkaloids have been isolated from Kratom. Mitragynine is the major alkaloid (up to 66% of the total alkaloid extract) in Kratom [12]. The methoxy group at position C9 is the key for pharmacophore binding to opioid receptors which controls the intrinsic activities on opioid receptors [13]. 7α-Hydroxy-7H-mitragynine accounts for 2% of the total alkaloid extract. Although it is present in a minor amount, it is 46-fold more potent as an analgesic compared to mitragynine [14]. 7α-Hydroxy-7H-mitragynine is a potential candidate for pain management as an opioid alternative. It is believed to be the most pharmacologically active alkaloid from Mitragyna speciosa.

Nelsen et al. [15] reported a case of seizure and coma. The patient had a medical history of managing chronic pain and depression with amitriptyline, oxycodone, and Kratom. A death involving abuse of propylhexedrine and mitragynine was reported by Holler et al. [16]. From 2009 to 2010, nine fatal cases of intoxication associated with the use of ‘Krypton’ – a blend containing Kratom and O-desmethyltramadol were reported in Sweden [17]. Furthermore, Neerman et al. [18] reported a fatal case possibly related to Kratom toxicity in United States. According to Domingo et al. [19], there were 2 cases of fatalities reported which confirmed the use of Kratom. The results showed mitragynine was not the primary cause of death in both cases reported. Olsen et al. [20] reported 152 Kratom-positive fatalities during July 2016–December 2017 in United States. Among the 152 Kratom-positive decedents, Kratom was determined to be a cause of death although the presence of additional substances cannot be ruled out.

Using Kratom regularly can have many long-term effects. Kratom users can experience dryness of mouth, darkening of the skin, stomach distension, withdrawal syndrome, sweating, itching, nausea, loss of appetite, anorexia, weight loss, insomnia, constipation, and increase urination [10,11,21,22]. According to Chan et al. [21], this plant has been prohibited by Thai law since 1943 due to its narcotic-like effects. It is also controlled in other countries like Malaysia, Myanmar, Australia, Vietnam, and Denmark [7]. However, it is uncontrolled in Europe and the United States and it is widely available through the internet [10]. Cases of Kratom abuse in Malaysia have raised significant concerns among the public and Law enforcement authorities. Therefore, the Malaysia government is in the final process of scheduling Kratom under the Dangerous Drug Act 1952 in order to strengthen control of Kratom misuse [23].

Detection of mitragynine in biological fluids is necessary to monitor Kratom abusers. Immunoassays are traditionally useful methods for the rapid detection of drugs of abuse as they are cheap, fast, easy, and sample pre-treatment is not necessary [24]. Limsuwanchote et al. [25] have reported the production of monoclonal antibody against mitragynine for quantification of mitragynine in Kratom samples using an indirect competitive Enzyme-linked Immunosorbent assay (ELISA). Currently, there is no study reported using polyclonal antibody. Polyclonal antibody has the ability to detect multiple epitopes and gives more robust detection as well as offer greater sensitivity for detection. This paper describes the preparation of mitragynine-protein conjugate, enzyme conjugate and the generation of polyclonal antibodies against mitragynine. The antibodies are characterized by cross-reactivity study. Competitive inhibition ELISA for the detection of mitragynine, other closely related Kratom alkaloids and metabolites in human urine are described.

Section snippets

Reagents and standards

Bovine serum albumin (BSA) in lyophilized form, complete and incomplete Freund's adjuvants, sodium acetate anhydrous, anti-rabbit IgG, Tween® 20, ProClin® 300, ethylenediamine dihydrochloride (EDA.2HCl), formaldehyde, trifluoroacetic acid, sinapinic acid, hexane, ammonium formate, formic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), protein A from Staphylococcus Aureus, acetonitrile, caffeine, and methadone were purchased from Sigma Aldrich Corporation, St. Louis,

Preparation of antibody

Mitragynine is not sufficiently immunogenic to induce antibody production because of low molecular mass, therefore it has to be conjugated to a carrier protein, which has high molecular mass and immunogenic to elicit an immune response when introduced into animals. Both the cBSA-MG and HRP-MG conjugates were synthesized using the same approach - Mannich reaction. In the Mannich reaction, mitragynine was coupled through the indole nitrogen via formaldehyde treatment with sodium acetate to cBSA.

Conclusions

A sensitive ELISA assay has been developed to screen the presence of mitragynine in urine samples. This is a useful method for rapid screening of biological samples where no sample preparation is required. The antibodies produced were specific against mitragynine and metabolites as well as other closely related Kratom alkaloids. The results obtained were confirmed using LC-MS/MS method which proved the reliability and sensitivity of the ELISA assay. The future study is to investigate the

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

CRediT authorship contribution statement

Mei Jin Lee: Methodology, Investigation, Writing - original draft, Writing - review & editing, Funding acquisition. Surash Ramanathan: Conceptualization, Investigation, Writing - review & editing, Resources, Supervision. Sharif Mahsufi Mansor: Investigation, Resources. Soo Choon Tan: Conceptualization, Methodology, Writing - original draft, Funding acquisition, Supervision.

Declaration of competing interest

There are no financial or other relations that could lead to a conflict of interest.

Acknowledgements

This study was funded by the Higher Institution Centre of Excellence, HICoE (grant number 311.CIPPM:4401005, 311/CDADAH/4401009) which provided by Ministry of Higher Education (MOHE), Malaysia; and the Ministry of Education (Malaysia) MYBRAIN15 scholarship.

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