Phytochemrstry,Vol. 30, No. 10, pp. 3273-3276, 1991 Printedin Great Britain.
0031-9422/91 $3.00+0.00 Q 1991 PergamonPress plc
A MONOCLONAL ANTIBODY TO SCOPOLAMINE AND ITS USE FOR COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY YUTAKA KIKUCHI, MASACHIKA IRIE, KAYO YOSHIMATSU,* KANJI ISHIMARU,* KOICHIRO SHIMOMURA,* MOTOYOSHI SATAKE,* SHOKO SUEYOSHI,~ MASAYUKI TANNO,~ SHOZO KAMIYA,~ JUN-ICHI SAWADA,~~ and TADAO TERA@
Department of Microbiology, Hoshi College of Pharmacy, 2-4-41, Ebara, Shinagawa, Tokyo 142, Japan; *Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan; TDivision of Organic Chemistry, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158, Japan; $Division of Biochemistry and Immunochemistry, National Institute of Hygienic Sciences, l-18-1, Kamiyoga, Setagaya, Tokyo 158, Japan (Received in revised form 14 March 1991)
Key Word Index-Solanuceae; Duboisia hybrid; monoclonal antibody; anti-scopolamine; radioimmunoassay; root culture; scopolamine; tropane alkaloids.
competitive ELISA;
Abstract-A hybridoma clone producing a monoclonal antibody (SC78.H81) against scopolamine was established. The monoclonal antibody was an IgGl (k) antibody with high affinity (1.6 x 10’ M- ’ for methylscopolamine). The monoclonal antibody was cross-reactive with methylscopolamine and butylscopolamine, and showed weak crossreactivity with 68- and 7/%hydroxyhyoscyamine. The cross-reaction with L-hyoscyamine, atropine, scopine and m-tropic acid was very weak. A competitive enzyme-linked immunosorbent assay using SC78.HSl was established to quantify scopolamine. The sensitivity of the assay allowed detection of 20 pgassay-’ (0.2 ngml-‘) of scopolamine. The assay was applied to the estimation of scopolamine content in hairy root cultures of a Duboisia hybrid. INTRODUCTION The tropane alkaloid L-scopolamine is a medicinally important, secondary metabolite having parasympatholytic, anti-cholinergic, anti-emetic and sedative actions. Scopolamine is usually extracted from leaves or roots of solanaceous plants such as Datura, Duboisia, Atropa,
Scopolia and Hyoscyamus. Recently, a number of groups
[l] have attempted to establish root cultures of solanaceous plants to produce scopolamine and other alkaloids in greater quantities. For screening a large number of plant cell cultures grown in vitro, it would be useful to develop a simple, rapid and reproducible assay technique to quantify scopolamine of relatively low concentrations in a small volume of sample. Immunoassays including radioimmunoassay (RIA) are well suited for this purpose. So far several RIA systems using polyclonal antibodies have been employed to quantify tropane alkaloids [Z-7]. For immunoassays, monoclonal antibodies are superior to polyclonal antibodies in terms of availability and reproducibility once a monoclonal antibody of high affinity and good specificity is obtained. We report here the production and characterization of a monoclonal anti-scopolamine antibody suitable for immunoassay for scopolamine to select high-yielding plant cultures.
bridoma clones were obtained. Among these, the clone SC78.H81.CSl.A9 was stable and found to be positive in a radioimmunoassay (RIA). The monoclonal antibody (named SC78.H81) was obtained in the form of ascites and serum by propagating the cloned hybridoma cells in uivo. The titre of the mixture of the ascites and serum obtained was ca 40000 as assessed by ELISA. It was shown by isotyping ELISA that the monoclonal antibody SC78.H81 used yl and K chains as heavy and light chains, respectively (data not shown). The association constant of SC78.H81 for methylscopolamine was 1.6 x 10’ M- ‘. To further characterize the monoclonal antibody, the cross-reactivity of the antibody with various scopolamine-related compounds was determined by competitive ELISA and RIA. The data obtained by the competitive ELBA are summarized ,in Table 1. The cross-reactivity in the competitive RIA was almost the same as that in the competitive ELISA (data not shown). SC78.H81 was cross-reactive with methylscopolamine and butylscopolamine. However, the reactivity of the antibody with 6/I-hydroxyhyoscyamine and 7/?-hydroxyhyoscyamine was low, and that with L-hyoscyamine, atropine (DL-hyoscyamine) and methylatropine was even lower. Scopine, tropine, and DL-tropic acid did not bind to the monoclonal antibody under the conditions used.
RESULTS
Production and characterization of the monoclonal antiscopolamine antibody
From two independent fusion experiments, five enzyme-linked immunosorbent assay (ELISA)-positive hyIAuthor to whom correspondence should be addressed. 3273
Competitive ELISA for scopolamine
A competitive ELISA system using the SC78.H81 antibody was established. A standard curve in the competitive ELISA for scopolamine is shown in Fig. 1. of scopolamine in this assay was ca The I&, 110 pg well-’ (ca 4 nM). The assay could quantify scopolamine within the range of 2(rlooO pg well-‘. In order to
Y. KIKUCHI et al.
3214 10
0.4
G
.& 03
b-"
. . . . . m / -
051
5
I
0
3 0 02 8
2
50
100
Scopolamme
500
1
( pg!well )
0
Fig. 1. Standard curve for scopolamine in competitive ELISA.
validate our competitive ELISA, the scopolamine contents (% dry wt) in roots and leaves of solanaceous plants were determined by both HPLC and competitive ELISA; a good correlation (r = 0.957) was obtained (Fig. 2). Using Duboisia hybrid root extracts, we determined intra- and interassay variations. The intra- and interassay CV values at 30,60, 120, 180,300 and 600 pg well - ’ (n = 5) were less than 8.8 and 13.8%, respectively. When scopolamine (0.01&0.5X of dry weight) was added to the extracts, the recovery was between 84 and 121%. The competitive ELISA was used to compare the scopolamine content in roots of a Duboisia hybrid [8] cultured under different hormone conditions. The amount of scopolamine determined by competitive ELISA highly correlated with that determined by HPLC (Table 2).
Table 1. Cross-reactivity of SC78.H81 antibody with scopolamine-related compounds in competitive enzyme-linked immunosorbent assay (ELISA) Inhibitor
I&*
(nM)
(% Cross-reaction?)
Scopolamine Methylscopolamine Butylscopolamine
3.6 13 6.5
6j3-Hydroxyhyoscyamine 7/I-Hydroxyhyoscyamine
39 44
(9.2) (8.2)
200 360 1380
(1.8) (1.0) (0.3)
L-Hyoscyamine Atropine Methylatropine
(lfw (28) (55)
Cocaine
71000000 71000000
( < 0.ooo4) (lOOOOOO 71000000 71000000
(
0031-9422/91 $3.00+0.00 Q 1991 PergamonPress plc
A MONOCLONAL ANTIBODY TO SCOPOLAMINE AND ITS USE FOR COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY YUTAKA KIKUCHI, MASACHIKA IRIE, KAYO YOSHIMATSU,* KANJI ISHIMARU,* KOICHIRO SHIMOMURA,* MOTOYOSHI SATAKE,* SHOKO SUEYOSHI,~ MASAYUKI TANNO,~ SHOZO KAMIYA,~ JUN-ICHI SAWADA,~~ and TADAO TERA@
Department of Microbiology, Hoshi College of Pharmacy, 2-4-41, Ebara, Shinagawa, Tokyo 142, Japan; *Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan; TDivision of Organic Chemistry, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158, Japan; $Division of Biochemistry and Immunochemistry, National Institute of Hygienic Sciences, l-18-1, Kamiyoga, Setagaya, Tokyo 158, Japan (Received in revised form 14 March 1991)
Key Word Index-Solanuceae; Duboisia hybrid; monoclonal antibody; anti-scopolamine; radioimmunoassay; root culture; scopolamine; tropane alkaloids.
competitive ELISA;
Abstract-A hybridoma clone producing a monoclonal antibody (SC78.H81) against scopolamine was established. The monoclonal antibody was an IgGl (k) antibody with high affinity (1.6 x 10’ M- ’ for methylscopolamine). The monoclonal antibody was cross-reactive with methylscopolamine and butylscopolamine, and showed weak crossreactivity with 68- and 7/%hydroxyhyoscyamine. The cross-reaction with L-hyoscyamine, atropine, scopine and m-tropic acid was very weak. A competitive enzyme-linked immunosorbent assay using SC78.HSl was established to quantify scopolamine. The sensitivity of the assay allowed detection of 20 pgassay-’ (0.2 ngml-‘) of scopolamine. The assay was applied to the estimation of scopolamine content in hairy root cultures of a Duboisia hybrid. INTRODUCTION The tropane alkaloid L-scopolamine is a medicinally important, secondary metabolite having parasympatholytic, anti-cholinergic, anti-emetic and sedative actions. Scopolamine is usually extracted from leaves or roots of solanaceous plants such as Datura, Duboisia, Atropa,
Scopolia and Hyoscyamus. Recently, a number of groups
[l] have attempted to establish root cultures of solanaceous plants to produce scopolamine and other alkaloids in greater quantities. For screening a large number of plant cell cultures grown in vitro, it would be useful to develop a simple, rapid and reproducible assay technique to quantify scopolamine of relatively low concentrations in a small volume of sample. Immunoassays including radioimmunoassay (RIA) are well suited for this purpose. So far several RIA systems using polyclonal antibodies have been employed to quantify tropane alkaloids [Z-7]. For immunoassays, monoclonal antibodies are superior to polyclonal antibodies in terms of availability and reproducibility once a monoclonal antibody of high affinity and good specificity is obtained. We report here the production and characterization of a monoclonal anti-scopolamine antibody suitable for immunoassay for scopolamine to select high-yielding plant cultures.
bridoma clones were obtained. Among these, the clone SC78.H81.CSl.A9 was stable and found to be positive in a radioimmunoassay (RIA). The monoclonal antibody (named SC78.H81) was obtained in the form of ascites and serum by propagating the cloned hybridoma cells in uivo. The titre of the mixture of the ascites and serum obtained was ca 40000 as assessed by ELISA. It was shown by isotyping ELISA that the monoclonal antibody SC78.H81 used yl and K chains as heavy and light chains, respectively (data not shown). The association constant of SC78.H81 for methylscopolamine was 1.6 x 10’ M- ‘. To further characterize the monoclonal antibody, the cross-reactivity of the antibody with various scopolamine-related compounds was determined by competitive ELISA and RIA. The data obtained by the competitive ELBA are summarized ,in Table 1. The cross-reactivity in the competitive RIA was almost the same as that in the competitive ELISA (data not shown). SC78.H81 was cross-reactive with methylscopolamine and butylscopolamine. However, the reactivity of the antibody with 6/I-hydroxyhyoscyamine and 7/?-hydroxyhyoscyamine was low, and that with L-hyoscyamine, atropine (DL-hyoscyamine) and methylatropine was even lower. Scopine, tropine, and DL-tropic acid did not bind to the monoclonal antibody under the conditions used.
RESULTS
Production and characterization of the monoclonal antiscopolamine antibody
From two independent fusion experiments, five enzyme-linked immunosorbent assay (ELISA)-positive hyIAuthor to whom correspondence should be addressed. 3273
Competitive ELISA for scopolamine
A competitive ELISA system using the SC78.H81 antibody was established. A standard curve in the competitive ELISA for scopolamine is shown in Fig. 1. of scopolamine in this assay was ca The I&, 110 pg well-’ (ca 4 nM). The assay could quantify scopolamine within the range of 2(rlooO pg well-‘. In order to
Y. KIKUCHI et al.
3214 10
0.4
G
.& 03
b-"
. . . . . m / -
051
5
I
0
3 0 02 8
2
50
100
Scopolamme
500
1
( pg!well )
0
Fig. 1. Standard curve for scopolamine in competitive ELISA.
validate our competitive ELISA, the scopolamine contents (% dry wt) in roots and leaves of solanaceous plants were determined by both HPLC and competitive ELISA; a good correlation (r = 0.957) was obtained (Fig. 2). Using Duboisia hybrid root extracts, we determined intra- and interassay variations. The intra- and interassay CV values at 30,60, 120, 180,300 and 600 pg well - ’ (n = 5) were less than 8.8 and 13.8%, respectively. When scopolamine (0.01&0.5X of dry weight) was added to the extracts, the recovery was between 84 and 121%. The competitive ELISA was used to compare the scopolamine content in roots of a Duboisia hybrid [8] cultured under different hormone conditions. The amount of scopolamine determined by competitive ELISA highly correlated with that determined by HPLC (Table 2).
Table 1. Cross-reactivity of SC78.H81 antibody with scopolamine-related compounds in competitive enzyme-linked immunosorbent assay (ELISA) Inhibitor
I&*
(nM)
(% Cross-reaction?)
Scopolamine Methylscopolamine Butylscopolamine
3.6 13 6.5
6j3-Hydroxyhyoscyamine 7/I-Hydroxyhyoscyamine
39 44
(9.2) (8.2)
200 360 1380
(1.8) (1.0) (0.3)
L-Hyoscyamine Atropine Methylatropine
(lfw (28) (55)
Cocaine
71000000 71000000
( < 0.ooo4) (lOOOOOO 71000000 71000000
(
