Accepted Manuscript Title: Development of indirect competitive ELISA for quantification of mitragynine in Kratom (Mitragyna speciosa (Roxb.) Korth.) Author: Supattra Limsuwanchote Juraithip Wungsintaweekul Niwat Keawpradub Waraporn Putalun Satoshi Morimoto Hiroyuki Tanaka PII: DOI: Reference:

S0379-0738(14)00325-9 http://dx.doi.org/doi:10.1016/j.forsciint.2014.08.011 FSI 7705

To appear in:

FSI

Received date: Revised date: Accepted date:

19-4-2014 8-8-2014 12-8-2014

Please cite this article as: S. Limsuwanchote, J. Wungsintaweekul, N. Keawpradub, W. Putalun, S. Morimoto, H. Tanaka, Development of indirect competitive ELISA for quantification of mitragynine in Kratom (Mitragyna speciosa (Roxb.) Korth.), Forensic Science International (2014), http://dx.doi.org/10.1016/j.forsciint.2014.08.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Original Research Article Title Development of indirect competitive ELISA for quantification of mitragynine in Kratom (Mitragyna speciosa (Roxb.) Korth.)

ip t

Supattra Limsuwanchotea, Juraithip Wungsintaweekula,*, Niwat Keawpraduba, Waraporn Putalunb,

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,

us

a

cr

Satoshi Morimotoc, Hiroyuki Tanakac

Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand

c

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University,

an

b

Corresponding author. [email protected] (J. Wungsintaweekul) Faculty of Pharmaceutical

d

*

M

Fukuoka 812-8582, Japan

Ac ce pt e

Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. Tel.: +6674 288887; Fax: +6674 428220.

 

Abstract Monoclonal antibody (MAb) against mitragynine (MG), an analgesic alkaloid from Kratom leaves (Mitragyna speciosa), was produced. MG was coupled to carrier proteins employing either 1-ethyl-3(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), a zero-length cross linker or a 5-carbon length glutaraldehyde cross linker. To confirm the immunogenicity, the hapten numbers

1

Page 1 of 26

were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Preparation of the MAb was accomplished by the electrofusion method. Hybridoma 1A6 that was constructed from the fusion between splenocytes of EDC/NHS conjugate immunized mice and SP2/0-Ag14 myeloma cells was selected, cloned twice and expanded. The crossreactivities (CRs) of this MAb 1A6 with a series of indole alkaloids were 30.54%, 24.83% and 8.63%

ip t

for speciogynine, paynantheine and mitraciliatine, respectively. Using this MAb, an indirect

competitive enzyme-linked immunosorbent assay (icELISA) was developed with a measurement

cr

range of 32.92-250 µg/mL. Quantitative analysis of the MG contents in plant samples by icELISA

us

correlated well with the standard high performance liquid chromatography method (R2=0.994). The

an

MAb against mitragynine provided a tool for detection of MG in Kratom preparations.

Highlights

Monoclonal antibody against mitragynine was prepared.

•

The immunogens were prepared by the glutaraldehyde and carbodiimide methods.

•

The icELISA was used to determine mitragynine in Kratom leave samples.

Ac ce pt e

d

M

•

Keywords: Electrofusion, icELISA, Kratom, Mitragynine, Monoclonal antibody

Abbreviations: ABTS, 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid; BSA, bovine serum albumin; CR, cross-reactivity; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; eRDF, enriched RPMI-1640-Dulbecco’s-Hams 12; FBS, fetal bovine serum ; GA, glutaraldehyde; icELISA, indirect competitive enzyme-linked immunosorbent assay; KLH, keyhole limpet hemocyanin; MAb, monoclonal antibody; MG, mitragynine; NHS, N-hydroxysuccinimide; OVA, ovalbumin

2

Page 2 of 26

Acknowledgements This study was supported by the Royal Golden Jubilee Ph. D Program, the Thailand Research Fund (PHD/0311/2550) and the National Research Council of Thailand (PHA560010S). We thank to Assoc. Prof. Dr. Anuchit Plubrukarn for his advices on hapten synthesis and Associate Prof. Dr.

ip t

Tripetch Kanajanaphoom for his kindness of providing mitraphylline, secoxyloganin, carbomethoxystrictosidine. We also thank Dr. Brian Hodgson for his assistance with the English.

us

cr

 

1. Introduction

an

Mitragynine (MG) is a major biologically active constituent found in the leaves of Mitragyna speciosa (Roxb.) Korth. (Rubiaceae family) or “Kratom” in Thai. The leaves have been traditionally

M

used by natives, in particular Thai and Malaysian people, to relieve tiredness and enhance tolerance for working under the strong sunlight [1]. In addition, it has been used as an opium substitute for

d

treatment of morphine addiction [2]. Since it has a history of use as a medicinal plant i.e. relief of

Ac ce pt e

muscle pain, diarrhea, cough and hypertension [3], many pharmacological studies have been carried out to confirm its therapeutic value. Kratom extract and MG have been reported to have: antinociceptive effects [4-7], anti-inflammatory effects [8,9], an anorectic effect [10], acts as an antidepressant [11], and with antioxidant and antimicrobial activities against Salmonella typhi and Bacillus subtilis [12].

Historically, its use has been claimed to have narcotic effects and after potential abuse of Kratom preparations it can cause dependency with serious side effects [3,13,14]. Its narcotic-like action was from psychomotor stimulation that produced feelings of happiness and forcefulness within 5 to 10 minutes after consumption [3]. This of course contributed to its continued use and eventually dependency. Even though Kratom use is illegal in Southeast Asia and Australia, the sale of this plant or mitragynine products has been extensively conducted on the internet [15]. Many chromatographic methods such as high performance liquid chromatography (HPLC) coupled with UV or MS detectors [16-18] and gas chromatography/mass spectrometry (GC-MS) [19]

3

Page 3 of 26

have been widely used for the detection and determination of the active alkaloids especially MG in Kratom leaves, its preparations and even biological specimens. These methods are sufficiently sensitive and accurate, but not suitable for routine assay. Nowadays, immunoassays are worldwide used for rapid screening or detection drugs in such preparations and biological fluids [20]. They are remarkable for their sensitivity, simplicity and convenience. In this study, we therefore aimed to

ip t

produce MAb against MG and use them in an indirect competitive ELISA (icELISA) method which

cr

could be applied as a fast and rapid method for quantification of MG in Kratom samples.

us

2. Materials and methods 2.1. Reagents

an

Bovine serum albumin (BSA) and ovalbumin (OVA) were from Sigma (St. Louis, MO, USA). Freund’s complete and incomplete adjuvants (FCA and FIA) were from Difco Laboratories (Detroit,

M

MI, USA). Keyhole limpet hemocyanin (KLH), 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), N-hydroxysuccinimide (NHS) and glutaraldehyde were from Wako Pure Chemical

d

(Osaka, Japan). EDC was from Nacalai tesque (Kyoto, Japan). Peroxidase-labeled anti-mouse IgG

Ac ce pt e

was from MP Biomedicals (Salon, OH, USA). All other reagents were of analytical reagent grade. 2.2. Plant materials

Kratom leaves used for the isolation of MG were collected from Satun province, Thailand. The leaves were washed, dried in a hot air oven (50°C) overnight and ground. Kratom leaves used as sample specimens were collected from the South of Thailand independently of their age and development stage. The voucher specimens were prepared, authenticated and deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Science, Prince of Songkla University, Thailand. 2.3. MG isolation MG was isolated from Kratom dried leaves (1.13 kg) using an acid-base extraction followed by silica gel column chromatography as described previously with modifications [21]. The obtained MG (2.21 g, 0.19% based on its dried weight) was identified by 1H- and 13C- NMR and compared with the published data [22]. 4

Page 4 of 26

2.4. Preparation of MG-protein conjugates 2.4.1. Glutaraldehyde reaction MG (6.2 mg) was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and then added to an aqueous solution of BSA or OVA (3 mg/mL, 2 mL). A 2.5% glutaraldehyde solution (0.6 mL) was subsequently added to the above mixture and allowed to react while stirring for 2 h. The reducing

ip t

agent, a sodium borohydride solution (4 mg/mL), was added to the reaction and continually stirred for 1 h. After that, all the reaction mixture was dialyzed against distilled water at 4 °C with five separate

cr

changes and lyophilized. The MG-GA-BSA and MG-GA-OVA were obtained and kept at -20 °C until

us

further used. 2.4.2. MG acid (MGA) derivative synthesis and EDC/NHS reaction

an

The introduction of carboxylic group into the structure of MG, that was required to allow for protein conjugation (Fig. 1), was achieved as described by Arens and coworkers [23] with a slight

M

modification. In brief, MG (100 mg) dissolved in ethanol (6 mL) was refluxed with 3 M sodium hydroxide solution (4 mL) at 80 °C. After 2.5 h, saturated ammonium chloride solution was added to

d

quench the reaction. This was then diluted with distilled water (10 mL) and extracted with chloroform

Ac ce pt e

(5 x 20 mL). The chloroform fractions were pooled, dried over anhydrous sodium sulfate and evaporated to dryness to obtain a dried reaction mixture (80 mg). This was further purified using silica gel column chromatography (2 x 40 cm) and eluted with a gradient system of chloroform: methanol (98:2 to 8:2) to yield 50 mg of a yellow amorphous solid. IR (KBr) cm-1 3400-2500, 1651, 1408, 1256, 1105. 1H-NMR (CD3OD, 500 MHz) δ 7.47 (1H, s), 6.97 (1H, dd, J=8.01, 7.78 Hz), 6.90 (1H, d, J=8.01 Hz), 6.46 (1H, d, J=7.54 Hz), 3.85 (3H, s), 3.74 (3H, s), 3.42-3.25 (3H, m), 3.16-2.98 (3H, m), 2.58-1.93 (4H, m), 1.63 (1H, m), 1.46 (1H, m), 1.26 (1H, m), 0.92 (3H, t, J=7.32). 13C-NMR (CD3OD) 175.6, 161.2, 155.8, 139.7, 123.5, 118.0, 111.2, 107.0, 105.7, 100.4, 61.7, 60.9, 57.9, 55.6, 54.7, 41.1, 40.6, 30.1, 23.2, 20.4, 12.9. This synthesized MGA was conjugated to the carrier proteins as illustrated in Fig. 1. [24]. MGA (6.3 mg) was dissolved in dried dimethyl formamide (DMF) (1 mL). EDC (6.9 mg) and NHS (4.1 mg) were then added to the MGA solution and stirred overnight at room temperature. The NHS activated MGA was subsequently coupled to the amine groups of protein by adding to BSA or OVA or KLH 5

Page 5 of 26

each suspended in 2 mL of phosphate buffer saline (PBS). The reaction was continuously stirring for 6 h in the dark. The resulting mixture was dialyzed against distilled water at 4 °C with five separate changes. After freeze-drying, the MGA-BSA, MGA-OVA and MGA-KLH were made available and

Ac ce pt e

d

M

an

us

cr

ip t

kept at -20 °C until further used.

Fig. 1. Synthesis of mitragynine acid derivative (MGA) and its conjugation to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) and ovalbumin (OVA) through N-hydroxysuccinimide ester method.

2.5. Analysis of hapten number

6

Page 6 of 26

The hapten numbers of all conjugates were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) [25]. A small amount of each conjugate was mixed with a 103 fold molar excess of sinapinic acid in 1:2 acetonitrile/water containing 0.1% trifluoroacetic acid. The mixture was deposited on the MALDI plate (Bruker Daltonics, MA, USA) and allowed to crystallize at room temperature. The sample plate was subjected

ip t

to the Bruker AutoFlex III MALDI-TOF MS spectrometer (Daltonics, MA, USA) and the data were analyzed using FlexAnalysis 3.0.92 (Bruker AutoFlex III, MA, USA). The hapten number was

2.6. MAb production

M

an

us

cr

calculated using the molecular weight of the native protein as reference as follow:

d

For production of MAb, protein-conjugates: MG-GA-BSA, MGA-BSA and MGA-KLH were used

Ac ce pt e

as immunogens. The 150 µg of each conjugate was intraperitoneally injected into BALB/c mice (Ref. 11/2011; MOE 0521.11/777; The Animal Ethic Committee, Prince of Songkla University) for each at 10 day intervals. The conjugates were emulsified with FCA and FIA (1:1 v/v) for the first and second injections, respectively. The booster injections were performed without adjuvant. Antibody responses were assayed by bleeding 3 days after each administration. Following the last immunization (3 days), the spleen of the positive responder was removed and dispersed in enriched RPMI 1640-Dulbecco’sHam F12 (eRDF) medium. The splenocytes were fused with SP2/0-Ag14 myeloma cells (RCB0209) by the electrofusion method. Cell fusion was conducted by following the published protocol [26]. Briefly, the splenocytes and myeloma cells were mixed in a ratio of 10:1, washed twice with fusion medium (0.28 M mannitol, 0.1 mM CaCl2 and 0.1 mM MgCl2) and produced a total number of cells of 5 x 107 per mL before being introduced in the fusion chamber. In the pre-fusion step, cells were attracted into pearl chains by applying an alternate electric field first at 30 V/cm for 20 s then 3 electric pulses of 350 V/cm, for 30 s at intervals of 0.5 s was applied to promote cell membrane

7

Page 7 of 26

breakdown and fusion. In the post-fusion step, the alternate electric field was re-applied for 7 s to hold the fused cells together. The fused cells were incubated at 37 °C for 20 min, then the fusion medium was replaced with eRDF medium containing 20% FBS and 1% HybridoMax®. After 2 weeks, the supernatants of the wells that contained a cluster of cells were tested for the presence of antibody by an indirect ELISA. The positive wells were cloned twice by the limiting dilution method. The MAb

ip t

were produced from these established hybridomas cultured in a serum-free medium. 2.7. Purification and classification of MAb

cr

The culture medium (500 mL) was loaded onto a Protein G Sepharose 4 Fast Flow column (GE

us

Healthcare, OH, USA) which had been pre-equilibrated with 10 mM phosphate buffer (pH 7). The unbound substances were washed out with the same buffer. Bound IgG was eluted with 100 mM

an

citrate buffer (pH 2.7) and neutralized with 1 M Tris-HCl (pH 9). The pooled IgG fraction was dialyzed against distilled water at 4 °C and concentrated using an ultracentrifugal filter (Amicon®

(Sigma-Aldrich, MO, USA).

M

Ultra-15, MWCO 10,000 Da). The obtained MAb was isotyped using an Isoquick® IgG strip test

d

2.8. ELISA procedures using a homologous hapten

Ac ce pt e

Screening of the antibody titer or hybridoma supernatant was performed by an indirect ELISA using coating antigen that had been synthesized by the same method for each immunogen. Polystyrene, 96-well microtiter plates were coated with MG-GA-OVA and MGA-OVA (1 µg/mL, 100 µL/well) in 50 mM carbonate buffer (pH 9.6). After 1 h incubation, the plates were washed three times with PBS containing 0.05% Tween 20 (TPBS). The unbound surfaces on the plates were blocked with 5% w/v skim milk in PBS (300 µL/well) for 1 h to reduce non-specific binding in subsequent steps. After the washing step, the serial dilution of tested sera or hybridoma supernatants were added to the plates (100 µL/well) and allowed to react for 1 h. These plates were washed again and peroxidase-labeled anti-mouse IgG, diluted 1:1000 in TPBS was added (100 µL/well) to detect the antibody reactions. The plates were incubated for 1 h, then washed with TPBS three times. The enzyme substrate, ABTS (0.3 mg/mL) in 0.1 M citrate buffer (pH 4.0) containing 0.003% H2O2, was added to each well (100 µL/well) and further incubated for 20 min. The plates were measured at 405

8

Page 8 of 26

and 490 nm using a Multiskan FC microplate photometer (Thermo Scientific, MA, USA). All reactions were carried out at 37 °C. The icELISA was developed similar to those of the non-competitive format and used for determination of the MG content. After the blocking step, MG or other compounds were dissolved in 20% methanol (50 µL), to act as inhibitors, were incubated with a fixed dilution of MAb (50 µL) for 1

ip t

h. The following steps were the application of the peroxidase-labeled anti-mouse IgG and a signal was

cr

developed using ABTS as an enzyme substrate. The samples were analyzed in triplicate.

us

2.9. Method validation of the icELISA

The reactivity of MAb to immobilized MG was determined by an indirect ELISA. It indicated the

an

optimum concentration of MAb for the icELISA. The icELISA was validated with regard to sensitivity, specificity, precision and accuracy. Competition with various concentrations of standard

M

MG (14.6-375 µg/mL) revealed the sensitivity of the MAb. A sigmoidal standard curve was plotted between A/A0 and the logarithm of the MG concentration, where A is the absorbance in the presence

d

of MG and A0 is the absorbance value of the blank. The limit of detection (LOD) was defined as the

Ac ce pt e

smallest concentration of analyte that can be distinguished from the absorbance value of the blank by three standard deviations (SD). The specificity of the assay was determined by cross-reactivity studies using standard MG and other structurally related and non-related compounds. The cross-reactivity values were calculated following the Weiler and Zenk’s equation [27]. The precision of the assay was measured either within the same run or between runs. Within the run the precision was performed from well to well (n=5) in the same plate whereas between- the run was the result of comparing plate to plate (n=4). The accuracy was estimated by the recovery experiment. Kratom extracts that contained 650.03 µg of MG were spiked with six amounts of standard MG (50, 75, 100, 150, 200 and 400 µg) and used to determine the recovered amount by the icELISA. Each spiked level was performed in triplicate. 2.10. Determination of the MG content 2.10.1. Sample preparation

9

Page 9 of 26

The dried powders of Kratom leaves (100 mg) were extracted with 1 mL of methanol using sonication for 30 min. After repeating the extraction 5 times, all methanol extracts were pooled and evaporated to dryness under reduced pressure. The solid residue was re-dissolved with methanol (1 mL) and diluted appropriately to be a 20% (v/v) methanol solution before analysis by ELISA and

ip t

HPLC. 2.10.2. HPLC analysis

cr

An Agilent 1100 series HPLC apparatus equipped with a quaternary pump, autosampler and DAD

us

detector was used. The samples were subjected to a Vertisep™ UPS C18 (4.6 x 250 mm, 5 µm) at a volume of 20 µL and isocratic elution with 5 mM phosphate buffer (pH 6.0)/acetonitrile (35:65) was

an

employed. The flow rate was kept at 1 mL/min and analytes detected at 225 nm [28]. The calibration curve was performed with five concentrations of standard MG (1.56-25 µg/mL) and used to determine

M

the MG content in each sample. 3. Results

d

3.1. Preparation of MG-protein conjugates

Ac ce pt e

The haptens were designed to differ in the length and point of attachment of the spacer arms. A typical approach to couple small molecules containing the indole nucleus to protein was through the Mannich reaction using formaldehyde or glutaraldehyde as a cross-linker [29-31]. The 5-carbon length of glutaraldehyde was selected for linkage of the indole ring and the amine groups of the lysines of the protein molecule. In addition, we also prepared a zero-cross bridge immunogen using the EDC/NHS method. The carboxylic group was firstly introduced to the MG structure by alkaline hydrolysis of the ester at the C-22 position. A broad band of carboxylic acid O-H stretching in the region 3400-2500 cm-1 and the absence of a carbonyl ester (22-OCH3) signal both in the 1H and 13CNMR confirmed the success of the MG derivatization. It was then chemically activated and coupled to protein via amide bonds. To evaluate the immunogenicity of these immunogens, the hapten numbers were determined by the MALDI-TOF-MS (Fig. 2). Using the molecular weight of the peak centroid of 74,534.54 for MG-GA-BSA, 70,651.49 for MGA-BSA and 66,493.88 for native BSA, the

10

Page 10 of 26

hapten numbers were calculated as 17 and 10 molecules for the MG-GA-BSA and MGA-BSA, respectively. The results indicated that the hapten numbers were high enough to use as immunogens [32]. Because of the limitation of the MALDI-TOF-MS instrument, the immunogenicity of the MGAKLH conjugate could not be analyzed directly, it was assessed by immunizing mice and monitoring

74534.54 [M+H]+

cr

MG-GA-BSA

40

us

Counts

60

ip t

for the presence of antibody by an indirect ELISA.

an

20

20000

30000

M

0 40000

MGA-BSA

50000 60000 m/Z

70000

90000

70651.49 [M+H]+

Ac ce pt e

d

600

80000

Counts

400

200

0

20000

30000

40000

50000 60000 70000 m/Z

80000

90000

Fig. 2. The MALDI-TOF-MS spectra of MG-GA-BSA and MGA-BSA.

2. Antibody responses to immunogens All mice immunized with MG-GA-BSA, MGA-BSA and MGA-KLH were measured for their antibody responses after the second round of injections. The MG-GA-BSA was a much better immune inducer than the MGA-BSA and MGA-KLH, respectively (Fig 3). The antibody titers of the MG-GA-

11

Page 11 of 26

BSA and MGA-BSA were greater than 1:800 after six rounds of immunization. Because KLH had a large molecular weight, it was expected to be a more potent immunogen than BSA. However, the conjugation of MGA to KLH induced poor water solubility leading to a lower immune stimulation.

ip t

Therefore, immunizations with MGA-KLH were discontinued.

1.80 1.60

cr

1.20 1.00

MGA-BSA

0.80

us

OD 405 nm

1.40

MG-GA-BSA

0.60 0.40

MGA-KLH

an

0.20 0.00 2

3

4 5 Injection times

6

7

M

1

d

Fig. 3. Immune responses of mice immunized with MG-GA-BSA, MGA-BSA and MGA-KLH were

Ac ce pt e

detected using ELISA with the respective MG-GA-OVA, MGA-OVA and MGA-BSA as a coating antigen. The serum dilution of 1:800 in 0.05% TPBS was used for determining the antibody levels.

3. MAb production and characterization

Splenocytes obtained from mice immunized with MG-GA-BSA and MGA-BSA were used for hybridization. Forty nine clones of the MG-GA-BSA hybridoma were screened by the icELISA. The results indicated that free MG had no influence on the binding of antibody. Thus, there were no antibodies specific to free MG. However, for the MGA-BSA, 88 hybridoma clones were established and then expanded into a 24-well tissue culture plates. The second screening offered 18 positive clones that were transferred to 10-mL culture plates. The subsequent screenings with icELISA narrowed the positive clones to 5. Following the cloning by the limit dilution method, 2 clones designated as 1A6 and 2B1 which had a low cross-reactivity to OVA were obtained. Finally, the antiMG MAb was obtained from hybridoma 1A6 and classified as IgG2a with a kappa light chain.

12

Page 12 of 26

4. Sensitivity and specificity The specificity and sensitivity are the most important factors to determine the value of the obtained MAb. MAb produced from hybridoma 1A6 were characterized for these properties by icELISA as mentioned above. Fig. 4 shows the inhibition curve of MG with an LOD value of 32.47 µg/mL. The

cr

ip t

measurement range was 32.92-250 µg/mL.

1.20

1.20

y = -0.324ln(x) + 2.0677 R² = 0.9957

1.00

us

0.80

1.00

0.60

0.80

0.20

an

0.00

10

0.60 0.40

100

1000

M

A/A0 at 405 nm

0.40

0.20 0.00

100

d

10

1000

Ac ce pt e

MG conc (µg/mL)

Fig. 4. Inhibition curve of the icELISA using anti-MG MAb. The concentration of anti-MG MAb was 336.66 ng/mL. The inset indicates the linear range of the icELISA.

The specificity was tested against other structural related and non-related compounds and the results are shown in Table 1. The experiment revealed that the anti-MG MAb had a medium crossreactivity to speciogynine (30.54%), paynantheine (24.84%) and mitraciliatine (8.63%) which had a high similarity of structure to MG. It also showed a little cross-reactivity to tryptamine (2.79%) which is a primary precursor in the MG biosynthesis pathway. MG possesses the three stereocenters (3S, 15S and 20S). Among those CR compounds, speciogynine and paynantheine differed from MG at the configuration of C-20 and the latter was replaced with methylene at C-18 position, therefore, the %CR of speciogynine was slightly higher than paynantheine. The more different configurations of

13

Page 13 of 26

mitraciliatine at the position of C-3 and C-20 resulted in the lower %CR (Fig. 5). These results

M

an

us

cr

ip t

indicated that the configuration might be an epitope for MAb recognition.

Ac ce pt e

d

Fig. 5. The chemical structures of MG diastereomers.

14

Page 14 of 26

Table 1 Cross-reactivity of MAb 1A6 with MG and various compounds. % Cross reactivitya (CR)

Mitragynine

100

Speciogynine

30.54

Paynantheine

24.83

Mitraciliatine

8.63

Tryptamine

2.79

Hirsutine

1.57

Ajmaline

0.77

Ajmalicine

0.42

Irinotecan

0.28

cr

0.15

0.08

Ac ce pt e

d

Isomitraphylline

a

us

an

M

Tryptophan

Mitraphylline

ip t

Compound

0.05

Yohimbine

0.05

Secologanin

0.04

Ursolic acid

0.01

Carbomethoxystrictosidine