Development of an enzyme immunoassay using a monoclonal antibody against the psychoactive diterpenoid salvinorin A

Article abstract of DOI:10.1021/np400358n

Salvinorin A (1), the main active constituent in Salvia divinorum, is a highly selective kappa-opioid receptor agonist with hallucinogenic effects, which is regulated in several countries. In the present study, a monoclonal antibody (mAb) against 1 was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) system was developed for the detection of salvinorins. To raise mAbs against 1, salvinorin B (2) hemisuccinate was synthesized and used to prepare the immunogen 2-bovine serum albumin conjugate. This technique was used to prepare a hybridoma cell line, 3D5, which secreted a mAb that recognized 1. The mAb was shown to have specificity for 1 and other salvinorins in cross-reactivity tests. The intra-assay calibration range by icELISA using the mAb against 1 was 0.0195-0.625 μg/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the identification of S. divinorum.

Full text of DOI:10.1021/np400358n

Article
pubs.acs.org/jnp
Development of an Enzyme Immunoassay Using a Monoclonal
Antibody against the Psychoactive Diterpenoid Salvinorin A
Madan Kumar Paudel,† Osamu Shirota,‡ Kaori Sasaki-Tabata,† Hiroyuki Tanaka,*,† Setsuko Sekita,‡
and Satoshi Morimoto†
†Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan
‡Laboratory of Pharmacognosy and Natural Products Chemistry, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri
University, Sanuki, Kagawa 769-2193, Japan
ABSTRACT: Salvinorin A (1), the main active constituent in
Salvia divinorum, is a highly selective kappa-opioid receptor
agonist with hallucinogenic effects, which is regulated in several
countries. In the present study, a monoclonal antibody (mAb)
against 1 was prepared, and an indirect competitive enzyme-
linked immunosorbent assay (icELISA) system was developed
for the detection of salvinorins. To raise mAbs against 1,
salvinorin B (2) hemisuccinate was synthesized and used to
prepare the immunogen 2−bovine serum albumin conjugate.
This technique was used to prepare a hybridoma cell line, 3D5,
which secreted a mAb that recognized 1. The mAb was shown to
have specificity for 1 and other salvinorins in cross-reactivity
tests. The intra-assay calibration range by icELISA using the mAb
against 1 was 0.0195−0.625 μg/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis
of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only
simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the
identification of S. divinorum.
of physical or mental displacement, exceptionally convincing
illusions, and a loss of identity. Its potency is slightly inferior to
that of lysergic acid diethylamide, the well-known synthetic
hallucinogen.15−17 Several recent studies have suggested the
involvement of the endocannabinoid system in some of its
effects.18
́
Salvia divinorum Epling & Jativa-M. is a powerful psychoactive
herb that belongs to the Lamiaceae (mint) family and is
used in traditional spiritual and curative treatments by the
indigenous Mazatec people of southern Mexico. Recently,
this plant and its extracts have been used recreationally,
which has led to social problems in the United States,
Europe, Japan, and elsewhere. Thus, the sale of S. divinorum
has been prohibited in several countries because of its
psychoactive effects.
Analytical approaches for 1 include thin-layer chromatography
and gas chromatography−mass spectrometry (GC-MS).19−21
Additionally, high-performance liquid chromatography−mass
spectrometry (HPLC-MS) was reported to be applicable for the
quantitative analysis of plant materials and biological samples
from consumers.22,23 In contrast, immunochemical approaches
have been used largely for the analyses of natural products,
including drugs of abuse, such as morphine and cannabi-
noids.24−34 The practicality and usefulness of the enzyme-linked
immunosorbent assay (ELISA), immunochromatographic anal-
ysis, and eastern blotting have been validated in various
experiments. To facilitate the development of an immunoassay
for 1, a polyclonal antibody against 1 has been prepared and
made available by Randox Laboratories Ltd. (U.K.). However,
the preparation of a monoclonal antibody (mAb) against 1 has
not been reported previously.
Salvinorin A (1) is a neoclerodane diterpene and an extremely
potent and highly selective kappa-opioid receptor agonist.1−4
It is the main active constituent isolated from the leaves of
S. divinorum, while the structurally related salvinorins B (2), C
(3), D (4), E (5), and F (6) are present in lower quantities.5,6
At present, only 1 is known to be an effective kappa-opioid
receptor agonist. It is capable of inhibiting excess intestinal
motility (e.g., diarrhea), and its plant of origin is used in
traditional healing practices for treating pain, inflammatory
disorders, rheumatism, and headache.7 In addition, it is an
attractive substance for drug development because kappa-
opioid receptors are potential targets for obesity, depression,
anxiety, and alcoholism and can normalize cognitive and
behavioral alterations.8−14
In humans, an inhaled dosage of 200−500 μg of 1 induces
profound hallucinations that last up to 1 h, which include feelings
Received: May 1, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Thus, the aim of the present study was to prepare a mAb
against 1 and to develop an indirect competitive ELISA
(icELISA) that could be used to identify S. divinorum.
RESULTS AND DISCUSSION
■
Synthesis of 2−Bovine Serum Albumin Conjugate (2−
BSA) and Direct Determination of the Hapten−Carrier
Protein Conjugates by Matrix-Assisted Laser Desorption/
Ionization Time-of-Flight Mass Spectrometry (MALDI-
TOFMS). Salvinorin A (1) lacks immunogenicity and does not
possess ether a COOH or NH2 group in its molecule. Thus, 1
had to be modified to prepare an appropriate hapten. This
compound was converted to 2 (salvinorin B), which possesses an
OH group and is able to be modified to a hemisuccinate after its
reaction with succinic anhydride. Then, 2-hemisuccinate was
prepared successfully and conjugated with BSA as a carrier
protein using the carbodiimide method (Figure 1). A conjugate
with an adequate hapten number is essential for eliciting an
immune response. Thus, the analysis of the 2−BSA conjugate by
MALDITOFMS, which is useful for the precise determination of
the hapten number of a conjugate, was performed as previously
reported.35,36 The broad peak of 2−BSA in the MALDITOFMS
is shown in Figure 2. This revealed that there were 2−BSA
conjugates with different hapten numbers in the sample. On the
basis of the peak around m/z 75 901 and the molecular weights of
BSA and 2 (66 433 and 390, respectively), 24 molecules of 2 were
conjugated to BSA in this particular conjugate. MALDITOFMS
analysis indicated that the conjugates contained sufficient hapten
numbers and could elicit adequate immunogenicity to raise
antibodies against 1. In addition, the broad peak of 2−human
Figure 2. Direct determination of the hapten number of salvinorin B
(2)−bovine serum albumin (2−BSA) conjugates by MALDITOFMS.
serum albumin (2−HSA), which was used as a solid-phase
antigen in ELISA, was also observed in the MALDITOFMS
spectrum, and its hapten number was calculated as 25, similar to
that of 2−BSA.
Production and Characteristics of mAbs against
Salvinorin A (1). BALB/c mice were hyperimmunized with
2−BSA to generate the cell clones used for this study, and
splenocytes were obtained from these mice. These splenocytes
were fused with SP2/0 myeloma cells using a previously
published procedure.37 After fusion, the cells were cultured in
medium containing hypoxanthine, aminopterin, and thymidine
to obtain hybridomas. Next, antibodies against 1 were screened
using the supernatants from each well of the cell culture plates, by
indirect ELISA and icELISA. Finally, a hybridoma cell that
secreted a mAb against 1 was picked from a positive well and
cloned. A hybridoma cell line that produced a mAb reactive to 1
(referred to as 3D5) was obtained and classified under the IgG2b
category that possesses κ light chains.
Development of an Indirect Competitive Enzyme-Linked
Immunosorbent Assay (icELISA) Using 3D5. To develop the
Figure 1. Synthetic scheme for salvinorin B (2)−carrier protein conjugates.
B
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2-fold serial dilution of 1 was added to the microimmunoplate,
which competed with the binding of 3D5 to 2−HSA. Following
treatment with a secondary antibody and addition of ABTS to the
plate, the gradation of color development was distinguishable
and depended on an increase in the concentration of 1 to form
the standard curve, as shown in Figure 3. To assess the affinity
of 3D5, an icELISA as described by Friguet et al. was used to
estimate the dissociation constant (KD) of the mAb against that
of 1, which was 5.1 × 10−7 M according to typical Scatchard
plots.38
The specificity of the mAb was determined on the basis of its
cross-reactivity (CR) with other compounds related to 1 in the
icELISA, which was calculated using the equation developed by
Weiler and Zenk.39 Figure 4 shows the CRs of the mAb against 1
and several structurally related compounds and shows that it
recognized 1 and other salvinorins. Compared with 1 (CR =
100%), the CRs for 2−6 were <0.084%, 26.2%, 9.79%, 9.33%,
and 10.3%, respectively. In addition, four types of synthesized
salvinorin derivatives with different side chains at the C-2 posi-
tion were also examined for this characterization, as listed in
Table 1. It was found that 3D5 could react with all of the
additional compounds, except benzoyl-salvinorin (14). Consid-
ering their structural similarity to 2-hemisuccinate, which was
used as the immunogen, the specificity may be affected by the
presence of the succinyl group at C-2 in the target molecule. On
the basis of the low observed CRs of 3D5 against salvidivins, the
furan ring at C-12 plays an essential role in this antigen and its
interactions. In addition, the mAb may recognize the main
characteristic structure of salvinorins precisely, because the CRs
for C-8-epi-salvinorin A (7) and C-2-epi-salvinorin A (9) were
reduced to 0.14% and 21.5%, respectively. As explained above,
Figure 3. Standard and calibration curves for salvinorin A (1) based on
icELISA using 3D5. The concentrations of 3D5 and the salvinorin
B (2)−human serum albumin (2−HSA) coating conjugate were
333 ng/mL and 0.1 μg/mL, respectively.
icELISA, the optimal concentration of 3D5 was determined by
indirect ELISA. Various concentrations of 3D5 were examined
using a polystyrene microimmunoplate that was precoated with
2−HSA. The level of antibody that bound to the 2−HSA
conjugate was calculated by direct comparison with the level of
labeled peroxidase with the use of a secondary antibody. Color
development was observed after adding 2,2′-azinobis(3-ethyl-
benzothiazoline-6-sulfonic acid) diammonium salt (ABTS)
solution to the plate. Thus, the optimum concentration of 3D5
for icELISA was found to be 333 ng/mL, which yielded an
absorbance of approximately 0.8−1.0 at 405 nm.
Under the conditions defined, the icELISA was used to
characterize 3D5. During the initial antibody application step a
Figure 4. Structures of salvinorin A (1) derivatives for which the cross-reactivities (CRs) with 3D5 were tested. Each CR was calculated using the
equation shown in parentheses.
C
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A recovery experiment was conducted to confirm the reliability
of the icELISA procedure developed. After spiking S. divinorum
samples with 1 (10−40 μg/mg dry wt), sample extracts were
prepared and the concentration of 1 in each sample was deter-
mined using the icELISA method. Table 3 shows that good
recoveries of 1 were observed in this experiment, which ranged
from 96.15% to 104.1% with an RSD of 1.05−2.26%. The
recovery of 1 from each spiked sample was almost 100%, which
demonstrated the high reliability of this system and confirmed
that this technique is useful for the reliable determination of
salvinorin derivatives from plant samples.
Analysis of S. divinorum and Other Plants in the Family
Lamiaceae by icELISA Using 3D5. The mAb, 3D5, prepared
in this study shows the novel characteristic of reacting with
various salvinorins. Salvinorins can be isolated only from
S. divinorum; therefore, icELISA using 3D5 serves as a useful
differentiation method for menthol plant compounds. Several
plants in the family Lamiaceae including S. divinorum were
obtained, and their methanol extracts were tested using icELISA.
Table 4 shows the results of the quantitative analysis of samples
Table 1. Cross-Reactivities of 3D5 against 1 and Structurally
Related Compounds
a
compound
cross reactivity (%)
1
100
C-8-epi-salvinorin A (7)
C-8-epi-salvinorin B (8)
C-2-epi-salvinorin A (9)
C-2-epi-salvinorin B (10)
propinoyl-salvinorin (11)
butyryl-salvinorin (12)
pivaloyl-salvinorin (13)
benzoyl-salvinorin (14)
0.14
0.24
21.5
<0.084
67.2
33.8
72.8
<0.084
a
Cross-reactivities are calculated by an equation described in the
Experimental Section.
3D5 has the novel property of only reacting to salvinorins, which
is a potent characteristic that can be used in the determination of
salvinorins from S. divinorum.
Variation and Accuracy of icELISA Using 3D5. The intra-
and interassay precision levels of icELISA using 3D5 were
examined within the range 0.0195−2.5 μg/mL of 1. The intra-
assay precision was evaluated on the basis of the variability in the
determination of 1 among wells (n = 6) on the same plate,
whereas the interassay precision was obtained using different
plates (n = 3). The results in Table 2 show that the maximum
Table 4. Determination of Salvinorins in Salvia divinorum and
Other Plants in the Family of Lamiaceae by icELISA Using
a
3D5
sample
salvinorin content (μg/mg dry wt)
S. divinorum 1
12.6 0.6
9.7 0.67
14.6 0.4
16.2 0.9
16.7 0.5
Table 2. Intra- and Inter-assay Precision of the Analytical
S. divinorum 2
a
Method for Salvinorin A (1) Using icELISA
S. divinorum 3
S. divinorum 4
b
1 (μg/mL)
intra-assay (%RSD, n = 6)
interassay (%RSD, n = 3)
S. divinorum 5
b
2.50
2.56
1.52
3.05
3.19
2.28
1.64
1.05
0.88
9.65
8.62
6.82
4.98
4.85
4.98
4.79
2.78
Salvia farinacea
Salvia patens
N.D.
1.25
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.625
0.313
0.156
0.0781
0.0391
0.0195
Salvia microphylla
Ocimum basilicum
Lavandula sp.
Isodon japonicus
Monarda citriodora
Rosmarinus officinalis
N.D.
a
a
b
The values are means SD for three plates and six replicate wells for
Data are means SD from triplicate analysis of each sample. N.D.:
b
each concentration in one plate for three consecutive days. RSD:
relative standard deviation.
not detected.
where 1 was used as a standard preparation. As expected,
salvinorins were detected only in S. divinorum. Quantitative data
showed that the salvinorin content in S. divinorum ranged from
9.72 to 16.7 μg/mg dry wt, which differs from concentrations of 1
previously reported in this plant.21,40 Probably this is likely due to
the fact that the mAbs reacted with other salvinorins in the
samples and the quantitative data reflected the approximate
levels of total salvinorins.
relative standard deviation (RSD) was 3.19% in the intra-assay.
In contrast, the interassay RSD varied from 2.78% to 9.65%. The
accuracy of icELISA was not high at concentrations of 1.25 and
2.5 μg/mL of 1, as the interassay values at these concentrations
were 8.62% and 9.65%, respectively. These data were sufficient to
demonstrate the precision of the developed icELISA. The
calibration curve to determine the concentration of 1 ranged
from 0.0195 to 0.625 μg/mL, as shown in Figure 3, and the limit
of detection was 0.0195 μg/mL.
Various immunochemical methods have been developed and
used for the identification of morphine, cocaine, marijuana, and
Table 3. Recovery of Salvinorin A (1) from Spiked Samples Using icELISA
a
b
c
spiked level (μg/mg dry wt)
measured amount (μg/mg dry wt) (%RSD )
expected amount (μg/mg dry wt)
recovery (%)
0
10
20
40
12.7 1.0 (1.36)
23.0 1.2 (1.06)
32.7 4.4 (2.26)
51.1 2.7 (1.05)
12.6
22.6
32.6
52.6
104.1
100.4
96.2
a
b
c
All values are means SD from triplicate samples at each level. RSD: relative standard deviation. Recovery (%) = (measured amount − 12.69/
added amount) × 100.
D
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other drugs.24,25,41 These methods have novel features such as
their cost-effective performance and the rapidity and simplicity
with which they can be applied. In the present study, a mAb
against salvinorin A (1), 3D5, was prepared successfully, and its
characterization showed that it could recognize novel salvinorins.
This mAb was then used to develop an icELISA to determine
the concentrations of salvinorins, which are found only in
S. divinorum. The icELISA procedure was validated using plant
materials, including S. divinorum, which confirmed that this
methodology is sufficiently sensitive, accurate, and reliable as a
differentiation method. In addition, icELISA can analyze a large
number of samples derived from complex and mixed materials in
a short period of time without any requirement for a pretreat-
ment procedure.
patens Cav., KYU-2011SP1; Salvia microphylla Kunth, KYU-2011SM1;
Ocimum basilicum L., KYU-2011OB1; Lavandula sp., KYU-2011LA1;
Isoson japonicus (Burm.) Hara, KYU-2011IJ1; Monarda citriodora Cerv.
ex Lag., KYU-2011MC1; Rosmarinus officinalis L., KYU-2011RO1]
were deposited in the Herbarium of the Faculty of Pharmaceutical
Sciences, Kyushu University.
Sample Preparations. The leaf samples of S. divinorum and other
Lamiaceae plants were each cut into small pieces, dried, and ground into
a fine powder. Each sample (30 mg) was accurately weighed and
extracted with 0.5 mL of methanol using an ultrasonic bath for 10 min.
The extract was then centrifuged at 12 000 rpm for 10 min, and the
supernatant was transferred to a microtube. The residue was extracted in
the same way at least twice, and all the supernatants were mixed to
prepare the sample solution. Next, the extracted solution was diluted to
produce a 20% methanol solution, which was used for the immuno-
assays.
This is the first report of the production of a mAb against 1 and
the development of an immunochemical method to analyze
salvinorins and identify S. divinorum. Instrumental methods, such
as HPLC-MS or GC-MS, have been used to analyze 1 and were
evaluated in terms of their accuracy and reliability. However,
these procedures must be performed by experienced laboratory
staff, as they require a certain level of technical expertise to
operate such complex instruments. In contrast, immunochemical
approaches such as icELISA and immunochromatographic
analysis are convenient to perform and do not require expensive
equipment. Therefore, immunoassays can be used for the
frequent screening of samples, and they could complement
existing instrumental analytical methods for salvinorins to help
assess S. divinorum abuse.
Various pharmacological and neurobiochemical studies re-
garding salvinorin A (1) have been conducted and reported.42−49
Thus, 1 is widely recognized as a potential drug candidate.
Immunoassays that use 3D5 to recognize this compound also
have potential applications as analytical tools in the aforemen-
tioned research fields.
Synthesis of the Hapten Derivative. Salvinorin B (2) was
obtained at a yield of 64% by hydrolyzing 1 (11.2 mg, 0.026 mmol) with
sodium carbonate (3.3 mg, 0.032 mmol) in a dichloromethane−
methanol (1:1) mixed solution (1.1 mL) at room temperature for 48 h.
Next, the hapten, which had a carboxylic acid side chain at C-2 in 2, was
obtained at a yield of 96% by reacting 2 (15.1 mg, 0.039 mmol) with a
succinic anhydride (35 mg, 0.35 mmol) in the presence of N,N-
dimethyl-4-aminopyridine (catalytic amount) in dichloromethane (2 mL)
at room temperature for 4 h.
Synthesis of Antigen Conjugates. The hapten was 2-hemi-
succinate, which was conjugated to BSA and HSA to produce the
immunogen and the coating antigen, respectively. 1-Ethyl-3-(3′-
dimethylaminopropyl)carbodiimide hydrochloride (6 mg, 0.031 mmol)
and 2-hemisuccinate (5 mg, 0.023 mmol) were added to a solution of 30%
pyridine (0.5 mL). The reaction mixture was added dropwise to a H2O
solution (0.25 mL) that contained BSA (5 mg) or HSA (5 mg) before
stirring at room temperature for 6 h. Subsequently, the mixture was
dialyzed with five changes of H2O at 4 °C for a period of 2 days and then
lyophilized to yield 6.2 mg of 2−BSA conjugate and 8.4 mg of 2−HSA
conjugate.
Animal Treatment. Male BALB/c mice (5 weeks of age) were
purchased from KBT Oriental Co. (Saga, Japan). A standard diet (MF;
Oriental Yeast Co., Tokyo, Japan) and water were provided ad libitum.
The procedures and animal care were approved by the Committee on
Ethics of Animal Experiments, Graduate School of Pharmaceutical
Sciences, Kyushu University, Japan, and were conducted according to
the Guidelines for Animal Experiments of the Graduate School of
Pharmaceutical Sciences, Kyushu University (Animal Ethics Reference
No. A22-113-0, Kyushu University, Japan).
Determination of the Hapten Number in the 2−Protein
Conjugates by MALDITOFMS. The hapten numbers in the 2−BSA
and 2−HSA conjugates were determined by MALDITOFMS. A small
amount (1−10 pmol) of antigen conjugate was mixed with a 103-fold
molar excess of sinapinic acid in an aqueous solution that contained
0.10% trifluoroacetic acid. The mixture was applied to a high-
performance MALDITOFMS system (Autoflex III; Bruker Daltonics,
Bremen, Germany). Data were analyzed using FlexAnalysis 3.0.92
(Bruker Daltonics, Bremen, Germany).
Immunization, Hybridization, and Production of the mAb.
The 2−BSA conjugate was dissolved in 8 M urea and diluted with
phosphate-buffered saline (PBS) to prepare the immunogen solution.
BALB/c male mice (5 weeks old) were injected intraperitoneally with
0.5 mL of 2−BSA (100 μg/mL emulsified with an equal volume of
Freund’s complete adjuvant). In the second immunization, 50 μg of the
conjugate in Freund’s incomplete adjuvant was injected intra-
peritoneally 2 weeks after the initial injection. Subsequently, the mice
were given boosters of immunogen (100 μg) without an adjuvant into
the abdominal cavity in the third and final immunization. Mice were
bled 4 days after each booster, and the titers of specific antibodies that
recognized 2 in the sera were monitored by ELISA. On the third day
after the final immunization (100 μg of protein), splenocytes were
prepared from an isolated spleen and fused with an aminopterin-
sensitive mouse myeloma cell line, SP2/0-Ag14, using the HVJ Envelop
(Ishihara Sangyo Kaisha Ltd., Osaka, Japan).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Salvinorin A (1) and its
related compounds, i.e., 2−6, divinatorins A (15) and B (16), and
salvidivins A (17), B (18), C (19), and D (20), were isolated from plant
material.50 The purities of these isolated compounds were checked by
nuclear magnetic resonance spectometry and were >95%. Unnatural
salvinorins, i.e., propinoyl-salvinorin (11), butyryl-salvinorin (12),
pivaloyl-salvinorin (13), and benzoyl-salvinorin (14), were synthesized
according to previously reported methods.51,52 In brief, various types of
carboxylic acids were esterified to the hydroxy group at C-2 of 2 using
established methods. C-8-epi-Salvinorin B (8) was obtained by
hydrolyzing 1 with lithium hydroxide. C-8-epi-Salvinorin A (7) was
prepared by acetylating 8. Finally, C-2-epi-salvinorin A (9) was prepared
from 2 when subjected to Mitsunobu etherification conditions, and C-2-
epi-salvinorin B (10) was obtained by hydrolyzing the resulting product.
BSA and HSA were obtained from Sigma-Aldrich (Steinheim, Baden-
Wurttemberg, Germany). Freund’s complete and incomplete adjuvants
̈
were provided by Difco (Detroit, MI, USA). The peroxidase-labeled
anti-mouse IgG was purchased from MP Biomedicals (Santa Ana, CA,
USA). All other chemicals and organic solvents were standard com-
mercial products and were of analytical reagent grade.
Plant Material. Dried S. divinorum leaves were purchased in June
2005 from Ethnogens.com (Lawrence, KS, USA) and were verified by
one of the authors (O.S.). Voucher specimens were deposited at the
Medicinal Herbarium, Kagawa School of Pharmaceutical Sciences,
Tokushima Bunri University, as specimen #050601-001. Other
Lamiaceae plants were cultivated in the medicinal plant station, Faculty
of Pharmaceutical Sciences, Kyushu University. The leaves of other
Lamiaceae plants were obtained in July 2011 in Fukuoka, Japan. The
identities of plant materials were verified by one of the authors (H.T.),
and voucher specimens [Salvia farinacea Benth., KYU-2011SF1; Salvia
E
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Hybridomas that produced mAbs reactive to 1 were detected by
ELISA and cloned using the limiting dilution method. The established
hybridoma clones were cultured in an enriched RPMI 1640-Dulbecco’s-
Ham’s F12 (e-RDF) medium (Kyokuto Pharmaceutical Industrial Co.
Ltd., Tokyo, Japan), which was supplemented with 10% fetal bovine
serum. For the mAb, a serum-free medium, i.e., an e-RDF medium
supplemented with 10 μg/mL insulin, 35 μg/mL transferrin, 20 μg/mL
ethanolamine, and 25 nM selenium, was used to obtain a supernatant
that contained the mAb against 1.
Purification of mAbs. mAbs were purified using a Protein G FF
column (0.46 cm × 11 cm; GE Healthcare, Uppsala, Sweden). The
culture medium (500 mL) that contained the mAb (IgG type) was
adjusted to pH 7 with 1 M Tris-HCl solution (pH 9) and applied to the
column. The column was then washed with 10 mM phosphate buffer
(pH 7). The IgG that adsorbed to the gel was eluted with 100 mM citrate
buffer (pH 2.8) and was immediately neutralized with 1 M Tris-HCl
(pH 9), dialyzed three times against H2O, and lyophilized to yield
8.3 mg of mAb.
Indirect ELISA Using 2−HSA. A 96-well immunoplate (Maxisorb;
Nalgene Nunc, Roskilde, Denmark) was coated with 100 μL of 0.1 μg/mL
2−HSA conjugate in 50 mM carbonate buffer (pH 9.6) and incubated
at 37 °C for 1 h. The plate was washed three times with 0.05% Tween
20-containing PBS (TPBS). Next, the plate was treated with 300 μL of
PBS containing 5% skim milk (SPBS) for 1 h to reduce any nonspecific
adsorption. The plate was washed three times with TPBS and reacted
with 100 μL of test antibodies for 1 h. Next, the plate was washed again
with TPBS three times, and mAbs were then combined with 100 μL
of 1000-fold-diluted solution of peroxidase-labeled anti-mouse IgG
(MP Biomedicals; Cappel Products, Santa Ana, CA, USA) for 1 h. After
washing the plate three times with TPBS, 100 μL of the substrate
solution, 0.1 M citrate buffer (pH 4.0) containing 0.003% H2O2, and
0.3 mg/mL 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) di-
ammonium salt (Wako, Osaka, Japan) were added to each well, and the
plates were incubated for 15 min. The absorbance was measured at
405 nm using a microplate reader (ImmunoMini, Nalgene Nunc). All
the reactions were carried out at 37 °C.
icELISA Using mAb against 1. The 2−HSA conjugate (100 μL,
0.1 μg/mL) was adsorbed onto the 96-well immunoplate, which was
then treated with 300 μL of 5% SPBS for 1 h to reduce nonspecific
adsorption. Salvinorins and plant sample extracts were dissolved with
methanol and diluted with H2O to yield 20% methanol solutions.
Various concentrations of samples in 20% methanol (50 μL) were
incubated with 50 μL of the mAb solution for 1 h. The plate was washed
three times with TPBS, and the antibody was combined with 100 μL of a
1000-fold-diluted solution of peroxidase-labeled anti-mouse IgG for 1 h.
After washing the plate three times with TPBS, 100 μL of the substrate
solution was added to each well, and the plates were incubated for
15 min. The absorbance was measured at 405 nm using a microplate
reader.
by icELISA. The recovery was calculated as follows on the basis of the
concentration measured and the amount of 1 spiked in the sample.
Measured amount − 12.69
Recovery (%) =
× 100
Added amount
AUTHOR INFORMATION
Corresponding Author
*E-mail: htanaka@phar.kyushu-u.ac.jp. Tel: +81(0)92-642-
6582. Fax: +81(0)92-642-6582.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported in part by a Grant-in-Aid from the JSPS
Asian CORE Program, the Ministry of Education, Culture,
Sports, Science and Technology of Japan; a Health Labour
Sciences Research Grant from the Ministry of Health, Labour
and Welfare; and the research fund of Kyushu University
Foundation.
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