68 Planta Med. 58(1992)
Monoclonal Antibody-Based Enzyme Immunoassay for the Quantitative Determination of the Tropane Alkaloid, Scopolamine K Hagemann'. K Piek1, J. Stockigt2, andE. W. Weiler"3 1
Lehrstuhl für Pflanzenphysiologie, Fakultat für Biologie, Ruhr-Universität Bochum, Postfach 1021 48, D(W)-4630 Bochum, Federal Republic of Germany 2 Lehrstuhl für Pharmazeutische Biologie, Johannes-Gutenberg-Universitat, D(W)-6500 Mainz, Federal Republic of Germany
Address for correspondence
Abstract A monoclonal antibody (SP1-4-A2) against
scopolamine was produced, characterized, and used to develop a sensitive and selective, competitive enzyme immunoassay for the quantitation of the alkaloid. The assay uses nor-scopolamine-N-/3-propionic acid coupled
to alkaline phosphatase as tracer and is linear from lOpg to long of scopolamine. As little as lOpg of scopolamine can be quantitated in an unprocessed plant
extract or in human serum after suitable dilution,
corresponding to detection limits of 0.1 ng scopolamine/ml of plant extract or 0.5 ng/ml of serum. The assay is more selective for scopolamine (percent cross-reactions for hyoscyamine = 0.21 %, 6-hydroxyhyoscyamine = 0.17%) than previously reported immunoassays. The assay format was designed to minimize intra- and inter-plate variabilities which are, on an average, all below 3 % (coefficients of variation). The
and presumably continuing to the present day (1—3). Seeds
of Solanaceae containing tropane alkaloids, e.g., jimsonweed (Datura stramonium) may contaminate commercial grain and this poses potential health hazards, since the toxic alkaloids survive processing such as bread-baking [for a survey, see (4, 5) and references cited therein]. Tropane alkaloids can be quantitated in the
sub-microgram range by several techniques including gas-liquid chromatography (GLC) (6—10), mass spectrometry (MS) (11—13), and high-performance liquid chromatography (HPLC) (5, 14—23). Compared to these instrumental techniques, immunoassays [for a recent review,
assay reported here has been validated against an
see (24)1 may be more sensitive and less laborious. Immunological techniques for tropane alkaloids have been developed for the determination of atropine and cocaine (24—29) not only in human or other body fluids (25, 28), but also in plant extracts (26, 27, 29), following our demonstration (30) that highly selective antisera can be raised against scopolamine using conjugates of nor-scopolamine-N-fl-propionic acid with carrier proteins. These
HPLC-based technique using plant samples and was shownto correlate closely(y 0.959 x + 0.14, r =0.982).
were used to quantitate picogram amounts of scopolamine in plant samples by radioimmunoassay (RIA)
Key words
Duboisia, enzyme immunoassay, hyoscyamine, monoclonal antibody (scopolamine), scopolamine, Solanaceae.
(30—32). A non-radioactive immunoassay, such as enzyme immunoassay, and the use of monoclonal antibodies (MAB) would considerably simplify analysis and result in a higher degree of standardization, due to the uniformity of the re-
agents used. It is our experience that an enzyme immunoassay is also considerably more sensitive than an RIA
(24). We report here on a competitive, monoclonal anti-
Introduction The tropane alkaloid, scopolamine, found
in members of the Solanaceae, such as in the genus Atropa, Datura, Scopolia, Hyoscyamus, and Duboisia, has considerable therapeutic value due to its parasympatholytic, anti-cholinergic, and anti-emetic as well as
sedative action. At high dosages, scopolamine is hallucinogenic, and preparations containing Datura or Hy-
oscyamus extracts have been abused since medieval times
body-based enzyme-linked immunosorbent assay (ELISA), which quantitates picogram amounts of scopolamine and which is useful for the analysis of plant extracts as well as serum samples. This assay has been in use in our laboratory for several years now, and all reagents used have proven their high degree of stability over prolonged periods of time.
Downloaded by: Universite Laval. Copyrighted material.
Received: October 21, 1991
Planta Med. 58(1992) 69
Monoclonal Antibody-Based Enzyme Immunoassay for the Quantitative Determination
In initial experiments, BSA conjugates of hemisuccinyl-(—)-scopolamine and nor-(—)-scopolamine-
N-/3-propionic acid (30) were compared for immuno-
genicity. As earlier observed for rabbits (30), Balb/c mice
responded weakly to the ester-linked alkaloid, but the nor-(—)-scopolamine-N-/3-propionic acid-BSA conjugate proved to be highly immunogenic, allowing for short im-
less precise. For comparison, the reported detection limits
of HPLC techniques for tropane alkaloids range from 300pg to 5Ong (16, 19, 22), while GC-MS assays detect down to 200 pg of scopolamine (10). The assay reported here is, thus, the most sensitive known to date. The sample capacity of this ELISA is comparable to that of the RIA, however, data for completed assays are obtained in a few minutes rather than over the several hours of counting time required for RIA. The stability of MAB preparations and of the enzyme-linked scopolamine tracer exceeds 1 year when stored as described.
munization times. The hyperimmunized animal used to
Assay specficity
derive the cell clone described in this study yielded 85 x 106
splenocytes which were fused with X63-Ag8-653 (33) myeloma cells using procedures routinely established in
our laboratory (34—36). Two out of the 170 original clones (1 %) secreted anti-scopolamine antibodies. Due to its better performance, clone SP1-4-A2 was purified by limiting dilution cloning and was characterized further. SP1-4-A2 secretes antibodies of the IgG1 (K) subtype both in standard culture, and in serum-free media, as well as when reintroduced into Balb/c mice intraperitoneally.
A range of tropane alkaloids was tested for
cross-reactions in the assay under standard conditions. The data (Table 1) show that while scopolamine and norscopolamine react to a similar extent, all other compounds are weak or ineffective competitors. The structurally most closely related alkaloids, 6-hydroxyhyoscyamine and hy-
oscyamine, and also dehydrohyoscyamine show crossreactions already well below 1 %. This specificity is considerably higher than that of scopolamine antisera re-
ported previously (30, 32). Hyoscyamine and scopolamine
General assay characteristics For the ELISA, the immobilized-antibody format rather than the immobilized-antigen format was chosen due to its generally better and more economic performance. In this format (24), the MAB is bound to
polystyrene microtitration plates precoated with a polyclonal rabbit anti-mouse Ig antibody. This significantly
reduces intra- and inter-plate variability. The general
usually occur together in plants containing tropane alkaloids. The sharp discrimination of both alkaloids (100 %vs. 0.21 %) byMAB SP1-4-A2 suggests that no inter-
ference from hyoscyamine in the determination of scopolamine is to be expected. This assumption was corroborated by our experience from the use of this assay so far. The distribution of immunologically reactive material in crude methanolic extracts of powdered, dry leaf of Duboisia hybrids is shown in Fig. 2, for a typical example.
parameters (including pH-optimum, reagent titration, and timing of assay steps) have been optimized using general routines (see Materials and Methods for the optimized protocol). Under these conditions, the full measuring range of the assay (linearity range of logit/log-plot) extends from lOpg to lOng of scopolamine (as HBr, Fig. 1). Over this range, the average intra-plate variability of standard tnplicates was
Monoclonal Antibody-Based Enzyme Immunoassay for the Quantitative Determination of the Tropane Alkaloid, Scopolamine K Hagemann'. K Piek1, J. Stockigt2, andE. W. Weiler"3 1
Lehrstuhl für Pflanzenphysiologie, Fakultat für Biologie, Ruhr-Universität Bochum, Postfach 1021 48, D(W)-4630 Bochum, Federal Republic of Germany 2 Lehrstuhl für Pharmazeutische Biologie, Johannes-Gutenberg-Universitat, D(W)-6500 Mainz, Federal Republic of Germany
Address for correspondence
Abstract A monoclonal antibody (SP1-4-A2) against
scopolamine was produced, characterized, and used to develop a sensitive and selective, competitive enzyme immunoassay for the quantitation of the alkaloid. The assay uses nor-scopolamine-N-/3-propionic acid coupled
to alkaline phosphatase as tracer and is linear from lOpg to long of scopolamine. As little as lOpg of scopolamine can be quantitated in an unprocessed plant
extract or in human serum after suitable dilution,
corresponding to detection limits of 0.1 ng scopolamine/ml of plant extract or 0.5 ng/ml of serum. The assay is more selective for scopolamine (percent cross-reactions for hyoscyamine = 0.21 %, 6-hydroxyhyoscyamine = 0.17%) than previously reported immunoassays. The assay format was designed to minimize intra- and inter-plate variabilities which are, on an average, all below 3 % (coefficients of variation). The
and presumably continuing to the present day (1—3). Seeds
of Solanaceae containing tropane alkaloids, e.g., jimsonweed (Datura stramonium) may contaminate commercial grain and this poses potential health hazards, since the toxic alkaloids survive processing such as bread-baking [for a survey, see (4, 5) and references cited therein]. Tropane alkaloids can be quantitated in the
sub-microgram range by several techniques including gas-liquid chromatography (GLC) (6—10), mass spectrometry (MS) (11—13), and high-performance liquid chromatography (HPLC) (5, 14—23). Compared to these instrumental techniques, immunoassays [for a recent review,
assay reported here has been validated against an
see (24)1 may be more sensitive and less laborious. Immunological techniques for tropane alkaloids have been developed for the determination of atropine and cocaine (24—29) not only in human or other body fluids (25, 28), but also in plant extracts (26, 27, 29), following our demonstration (30) that highly selective antisera can be raised against scopolamine using conjugates of nor-scopolamine-N-fl-propionic acid with carrier proteins. These
HPLC-based technique using plant samples and was shownto correlate closely(y 0.959 x + 0.14, r =0.982).
were used to quantitate picogram amounts of scopolamine in plant samples by radioimmunoassay (RIA)
Key words
Duboisia, enzyme immunoassay, hyoscyamine, monoclonal antibody (scopolamine), scopolamine, Solanaceae.
(30—32). A non-radioactive immunoassay, such as enzyme immunoassay, and the use of monoclonal antibodies (MAB) would considerably simplify analysis and result in a higher degree of standardization, due to the uniformity of the re-
agents used. It is our experience that an enzyme immunoassay is also considerably more sensitive than an RIA
(24). We report here on a competitive, monoclonal anti-
Introduction The tropane alkaloid, scopolamine, found
in members of the Solanaceae, such as in the genus Atropa, Datura, Scopolia, Hyoscyamus, and Duboisia, has considerable therapeutic value due to its parasympatholytic, anti-cholinergic, and anti-emetic as well as
sedative action. At high dosages, scopolamine is hallucinogenic, and preparations containing Datura or Hy-
oscyamus extracts have been abused since medieval times
body-based enzyme-linked immunosorbent assay (ELISA), which quantitates picogram amounts of scopolamine and which is useful for the analysis of plant extracts as well as serum samples. This assay has been in use in our laboratory for several years now, and all reagents used have proven their high degree of stability over prolonged periods of time.
Downloaded by: Universite Laval. Copyrighted material.
Received: October 21, 1991
Planta Med. 58(1992) 69
Monoclonal Antibody-Based Enzyme Immunoassay for the Quantitative Determination
In initial experiments, BSA conjugates of hemisuccinyl-(—)-scopolamine and nor-(—)-scopolamine-
N-/3-propionic acid (30) were compared for immuno-
genicity. As earlier observed for rabbits (30), Balb/c mice
responded weakly to the ester-linked alkaloid, but the nor-(—)-scopolamine-N-/3-propionic acid-BSA conjugate proved to be highly immunogenic, allowing for short im-
less precise. For comparison, the reported detection limits
of HPLC techniques for tropane alkaloids range from 300pg to 5Ong (16, 19, 22), while GC-MS assays detect down to 200 pg of scopolamine (10). The assay reported here is, thus, the most sensitive known to date. The sample capacity of this ELISA is comparable to that of the RIA, however, data for completed assays are obtained in a few minutes rather than over the several hours of counting time required for RIA. The stability of MAB preparations and of the enzyme-linked scopolamine tracer exceeds 1 year when stored as described.
munization times. The hyperimmunized animal used to
Assay specficity
derive the cell clone described in this study yielded 85 x 106
splenocytes which were fused with X63-Ag8-653 (33) myeloma cells using procedures routinely established in
our laboratory (34—36). Two out of the 170 original clones (1 %) secreted anti-scopolamine antibodies. Due to its better performance, clone SP1-4-A2 was purified by limiting dilution cloning and was characterized further. SP1-4-A2 secretes antibodies of the IgG1 (K) subtype both in standard culture, and in serum-free media, as well as when reintroduced into Balb/c mice intraperitoneally.
A range of tropane alkaloids was tested for
cross-reactions in the assay under standard conditions. The data (Table 1) show that while scopolamine and norscopolamine react to a similar extent, all other compounds are weak or ineffective competitors. The structurally most closely related alkaloids, 6-hydroxyhyoscyamine and hy-
oscyamine, and also dehydrohyoscyamine show crossreactions already well below 1 %. This specificity is considerably higher than that of scopolamine antisera re-
ported previously (30, 32). Hyoscyamine and scopolamine
General assay characteristics For the ELISA, the immobilized-antibody format rather than the immobilized-antigen format was chosen due to its generally better and more economic performance. In this format (24), the MAB is bound to
polystyrene microtitration plates precoated with a polyclonal rabbit anti-mouse Ig antibody. This significantly
reduces intra- and inter-plate variability. The general
usually occur together in plants containing tropane alkaloids. The sharp discrimination of both alkaloids (100 %vs. 0.21 %) byMAB SP1-4-A2 suggests that no inter-
ference from hyoscyamine in the determination of scopolamine is to be expected. This assumption was corroborated by our experience from the use of this assay so far. The distribution of immunologically reactive material in crude methanolic extracts of powdered, dry leaf of Duboisia hybrids is shown in Fig. 2, for a typical example.
parameters (including pH-optimum, reagent titration, and timing of assay steps) have been optimized using general routines (see Materials and Methods for the optimized protocol). Under these conditions, the full measuring range of the assay (linearity range of logit/log-plot) extends from lOpg to lOng of scopolamine (as HBr, Fig. 1). Over this range, the average intra-plate variability of standard tnplicates was
