Journal of Immunoassay
ISSN: 0197-1522 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ljii19
Enzyme-Linked Immunosorbent Assay for the Phytotoxin Cleistanthin A G. Ragupathi , P. Prabhasankar , P. Chandra Sekharan , K. S. Annapoorani & C. Damodaran To cite this article: G. Ragupathi , P. Prabhasankar , P. Chandra Sekharan , K. S. Annapoorani & C. Damodaran (1992) Enzyme-Linked Immunosorbent Assay for the Phytotoxin Cleistanthin A, Journal of Immunoassay, 13:3, 321-338, DOI: 10.1080/15321819208021236 To link to this article: http://dx.doi.org/10.1080/15321819208021236
Published online: 23 Sep 2006.
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Date: 16 June 2016, At: 10:47
JOURNAL OF IMMUNOASSAY, 1 3 ( 3 ) , 321-338 (1992)
ENPYME-LINKED IMMUNOSORBENT ASSAY FOR THE PHYTOTOXIN CLEISTANTHIN A
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0.
Ragupathi, P. Prabhasankar, P. Chandra+Sekharan, K.8. Annapoorani and c. Damodaran R &I D Division, Forensic Sciences Department, Madras - 600 0 0 4 , INDIA. ABSTRACT
Enzyme - linked immunosorbent assay is reported a major for the estimation of cleistanthin A, constituent of the toxic plant Cleistanthus collinus. Rabbit antibodies were obtained by immunisation with cleistanthin A hemisuccinate-BSA conjugate and the ELISA developed thereupon could detect cleistanthin A at as low a concertration as 3 ng/ml. Cross-reactivity studies with structural analogs as well as with other phytotDxins and drugs of common occurrence established the suitability of the ELISA to specifically monitor the C. collinus marker molecules in emergency clinical and forensic cases. The simplicity and specificity make the ELISA superior to the other available techniques. (KEY WORDS: Cleistanthin, C.collinus, Penicillinase, Toxicology).
ELISA
I
INTRODUCTION
Cleistanthin A (clei
a
A),
toxic principle of the plant
a lignan glycoside
is
Cleistanthus collinus
(1). This plant belongs to the Euphorbiaceae family and
*Author for correspondence
321 Copyright 0 1992 by Marcel Dekker, Inc,
3 22
RAGUPATHI ET AL.
is abundant in India. The poisonous nature of the plant especially its leaves has been well documented (2). addition namely
cleistanthin
present
methods
lignan
lactones
(1,3);
spectral
and
are
chromatographic
of quantifying them in biospecimens have
reported
However no immunoassay has
therefore
been
yet
been
The objectives of this investigation
were
(4-6).
developed.
protein
other
A,
B, diphyllin, and collinusin
cleistanthin
also
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to
to couple the haptenic clei A
with
carrier
using peptide bond forming agents in order
produce antibodies, characterise their specificity clei
and
A
In
develop
quantitative
an ELISA
application
in
for
for
qualitative
cases
of
to
and
C.collinus
poinoning.
MATERIALS AND METHODS Cleistanthin C.collinuq
A was isolated from the leaves
of
by a preparative chromatographic
procedure
and its purity checked through melting point
analysis,
thin-layer
data
chromatography
and
spectral
by
comparison with an authentic sample obtained from CIBA-
.
GEIGY (India) Bovine
serum
albumin
(BSA),
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride soluble
starch,
(TNBS)
and
penicillinase
2,4,6,-
Tween and
20
trinitrobenzene sulfonic were
obtained
penicillin V
were
from from
(EDC), acid Sigma:
Hindustan
323
ENZYME-LINKED IMMUNOSORBENT ASSAY
Antibiotics
(India): and
Ltd
(complete
Freund's
adjuvants
and incomplete) were the products
Laboratories,
USA.
Buffer
salts
of
of
Difco
reagent
grade
quality were used.
A-Drot~ein (BSA) humoseg
PreDaration of oleistDownloaded by [Nanyang Technological University] at 10:47 16 June 2016
Preparation
-
of cleistanthin A
hemisuccinate
(clei A-HS) and its conjugation to BSA were carried out according to the method developed earlier by us (7).
Enxvme
labellins o f cleistanthin
-
Cleistanthin A
HS derivative was labelled with
the marker enzyme penicillinase The
conjugation
aqueous
was
solution
A
in the following
initiated by adding
of EDC (15 mg/ml;
lml
of
pmoles)
80
way. an
to
a
solution of clei A-HS (4.5 mg/ml; 8.2 pmoles) in sodium phosphate
buffer
(pH 6.0; 0.01M).
incubated
at
occasional
shaking
temperature
for
and then added to
mixture min
30
a
buffer (pH 8.0; 0.2M). After
in
0.01M).
sodium
To
against sodium phosphate buffer the
dialyzed enzyme
with of
sodium
incubation
2-8OC for 16 to 18h, the reaction mixture was extensively
was
solution
(1 ml : 5mg/ml; 0.16 pmole)
penicillinase phosphate
room
The
dialyzed (pH
conjugate,
at
7.4;
BSA
and
1%
and
haDten
in
azide were added to a concentration of
0.2% and stored refrigerated in aliquots.
Evaluation coniusates
of
molar
Determination
DarticiDation of
of the number of haptenic clei
A
residues per protein or enzyme molecule was carried out
324
RAGUPATHI ET AL.
by spectrometric analysis ( 8 ) and TNBS method (9).
The
protein
content before
was
assayed
by
enzyme
and
after
conjugation
the method of Lowry et a1
activity
of
(10) and
the
measured
as
penicillinase was
described by Joshi (11).
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PreDaration of antiserum to cleistanthin New
Zealand
white
rabbits
A
were
injected
intradermally at multiple sites with 1 ml of clei A-HSBSA conjugate (corresponding to a concentration of pg of bound clei A-HS) dissolved in
saline ( P B S ; 0.1 M; pH complete adjuvant injection
of
the
7.4)
phosphate-buffered
and emulsified in
(lml). After four weeks immunogen
(40
100
fig)
Freund's
a
in
booster Freund's
incomplete adjuvant was given. The antiserum obtained after seven days of the booster dose was tested for the presence and
of
purified
clei
A
antibodies by
by ammonium
direct
ELISA
sulfate precipitation and
-
dialysis after which aliquots were stored at
6OoC
until use.
DeveloDment
of
Optimal
competitive
ELISA
for
clei
combination of the antibody and
labelled analyte for use in ELISA was
in 'Results'. Accordingly 200 pl
clei
antibody
diluted
with
enzyme
ascertained as
described A
A
of
coating
(bicarbonate buffer; 0.05M; pH 9.6) (1:1600) was
antibuffer added
to the wells of microtiter plates and incubated for at
37OC
3h
or overnight in refrigerator. After washing the
325
ENZYME-LINKED IMMUNOSORBENT A S S A Y
plates
(three times) with wash buffer
0.05%
(PBS-containing
Tween 20) the empty sites were blocked
with
solution of BSA in PBS. The plates were then for
This was followed by the
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amounts of clei like the
incubated
lh at room temperature and washed again with
buffer.
clei
A
-
A
sample
wash known
(1OOpl)
spiked urine and a specified aliquot
predetermined dilution
penicillinase
addition of
(100 pl) or analyte
1%
the clei
-
A
plates
were incubated at 37OC for 2h. Subsequently the
plates
washed
with
wash
substrate solution was
(100 pl; 1:lOO).
-
HS
The
were
conjugate
of
of
buffer added
and to
pl
200
each
of
well.
(The
substrate solution was starch-iodine-penicillin reagent obtained which
in
(5.329)
by mixing 0.2ml
of
turn was a preparation of
and
iodine
(200 mg) in 10
iodine
of
(SIP)
reagent
potassium ml
the
iodide
distilled
water, and 20 ml of 2% starch solution with 100 ml
of
penicillin V (15.2mg) solution in phosphate buffer: SIP reagent was prepared just before use).
The plates were
then left at ambient temperature for 20 min immediately following which the absorbance measurements were
made
at 620 nm with an E L I S A Reader (Biotek, EL 310 model). Evaluation of ELIBA for specificity to clei The different
competitive ELISA
was
ttanalytesltsuch as the
drugs
of
conducted
clei
analogs present in C.collinus, other
A
A
structural
phytotoxins
common occurrence in clinical
with
and
and
forensic
326
RAGUPATHI ET AL.
toxicology. below
The
cross-reactivity
according
to
the
method
was
calculated
described
by
as
Joshi
(personal communication):
-
(0.D of clei A excess) %binding at = known concentration (0.D of clei A excess)
(0.D of known concentration of clei A)
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.................................
The of
clei
binding
-
percent binding for various were calculated
A
vs
corresponding
and
XlOO
(0.D without clei A)
concentrations
plotted
as
concentration
percent
on
a
semi-
logarithmic graph. Similarly concentrations of
the
percent
binding
the cross-reacting
with
various
compounds
(CRC)
were calculated and the graph constructed. Let T50 be the concentration of clei A,and CRC50 that of the CRC; with respect to 50% binding, % Cross-reactivity of the CRC =
then
T50 ------
x 100
CRC50
RESULTS
A
hemisuccinate
derivative
the
of
cleistanthin A (clei A-HS) was prepared and
toxin
conjugated
with carrier protein/enzyme for obtaining the immunogen and
label.
molecules
The of
on
conjugate
clei A-HS per protein while
(penicillinase) haptens
BSA-coupled
a
-
contained the
enzyme
tagged product retained on average molar
basis.
26
Determination
4
of
321
ENZYME-LINKED IMPNNOSORBENT ASSAY
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ANTI
- CLEl
A ANTIBODY DILUTION
-
CLEl A PENlClLLlNASE DILUTION
FIGURE
1
Optimum combination of anti-clei A antibody and clei A-penicillinase label.
penicillinase
activity
in
the
conjugate
showed
retention of more than 85% of the original activity. Optimal
dilutions of the anti-clei
A
antibody
and the clei A-HS- penicillinase conjugate as
required
for ELISA were determined by constructing a graph the
difference
in absorbance values
of
the
from
reagent
328
RAGUPATHI ET A L .
0.8
-
E
0
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0.6IQ
w
x :
0.4
-
0.2
-
8
3
CONCENTRATION ng /ml
FIGURE 2
calibration curve for cleistanthin A .
blank and analyte wells for the various combinations of anti-clei A- antibody and clei A-HS
-
penicillinase.
The
dilution that gave an absorbance-difference
to
1.0
was
considered
as
optimum
dilution.
corresponding data are shown in Figure 1.
The
The dilution
factors were found to be 1600 and 100 for the and the enzyme conjugate respectively.
close
antibody
10.00
23.90
48.66
67.28
10
25
50
75
5.554
3.850 89.70
97.32
95.60
100.00
0.412 0.689
98.00
0.187
8.25
7.91
2.88
4.12
3.82
72.64
49.31
26.82
9.57
5.12
1.887
3.245
2.295
0.714
0.377
96.85
98.62
107.28
95.70
102.40
2.59
6.58
8.55
7.46
7.36
*Each sample (spiked urine) was assayed in quadruplicate
100 91.00 4.920 91.00 5.40 94.78 4.037 94.78 4.25 ......................................................................
4.90
5
Reproducibility of ELISA for the estimation of cleistanthin A
TABLE 1
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I
2 l-3
W
P m
0
v)
0
c z
3: 3:
H
W
m
x
z
H
r
330
RAGUPATHI ET AL.
TABLE 2
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Cross-reactivity of anti-cleistanthin A antibody during ELIGA as tested with cleistanthin A structural analogs
Cleistanthin B
84.4
Diphyllin
98.0
Collinusin
96.6
*Cleistanthin A-HS was assigned a value of 100
A
calibration
standard against range
by
graph
plotting
the
constructed
with
semilog
concentration
absorbance at 620 nm showed 3-100 ng/ml (Figure 2). The
of
clei
linearity in
A
the
reproducibility
of
the E L I S A was verified by estimating the amount of clei A
in
spiked
urine samples. The
figures
on
percent
recovery are presented in Table 1. The
possible cross-reactivity of
anti-clei
antibody with certain other C.collinus lignan that
are structurally
similar to clei A is
lactones shown
Table 2 and their structures in Figure 3. Table the
non-c.col1inus
occurrence
in
phytotoxins and
clinical and forensic
drugs
A
3
of
toxicology
in
lists common that
were tested in the E L I S A for cross-reactivity and ruled out therefor.
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ENZYME-LINKED IHMUNOSORBENT ASSAY
331
H, CO
H, CO
H, CO
-
CLEISTANTHIN A
GlPHYLLlN FIGURE
3
-
CLEISTANTHIN B
COL LINUSIN
Major lignan lactones of S.collinu S.
RAGUPATHI ET AL.
332 TABLE 3
Drugs and plant toxins tested and found negative for cross-reactivity with anti-clei A antibody during
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ELIlA
Brucine Cerberin Digitoxin Digoxin Ellagic acid Gitoxin Neriifolin Oleandrigenin Oleandrin Ouabain Peruvoside Strychnine Thevetigenin Thevetin
Amylobarbitone Butobarbiton Chlordiazepoxide Chlorpromazine hydrochloride Chlorpromazine sulphoxide Cyclobarbitone calcium Dextropropoxyphene hydrochloride Diazepam Dicoumarol Diphenhydramine hydrochloride Heptabarbitone Imipramine Indomethacin Lorazepam Nitrazepam Nortriptyline oxazepam Oxyphenbutazone Pentobarbitone sodium Phenacetin Phenobarbitone sodium Promethazine hydrochloride Quinalbarbitone sodium Temazepam Thiopentane sodium Trimipramine maleate
DISCUSSION
Identification and determination of in biospecimens are essential monitoring clinical
xenobiotics
for therapeutic
drug
and to establish the cause of poisoning and
forensic problems.
For
this
in
many
333
ENZYME-LINKED IMMUNOSORBENT ASSAY
investigators have been turning to enzyme immunoassays. The increasing incidence of C.collinus poisoning
calls
for the development of such simple, sensitive and rapid assays. Cleistanthin
A,
as shown in Figure 3 , is not
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immunogen and hence a conjugate with a carrier like
needs to be prepared.
BSA
available
for
the
Various
preparation
of
reactive
not
in
derivative of clei
A
then conjugated with (7).
clei (clei
BSA
The
A.
A-HS)
of
good
of
relevant
hemisuccinate
was prepared which was
by the mixed anhydride method
immunogen
since the
conjugate had a higher number of clei molecule
of
protein
than
carbodiimide method.
labeling
the
the
On
the
analyte with
resulting
obtained
other
enzyme
analyte molecules are required in
analyte-
enzyme
conjugate in order
retain its activity (13). The A
molecules per
BSA
4
clei
A
by
hand,
marker,
the
that
for the
since
resulting
the
enzyme
conjugate had 2 6 clei
molecule whereas the
conjugate contained only molecule.
BSA
the
residues per
A-HS
one
for
carbodiimide method of conjugation was preferred less
are
to protein is
A
This method of conjugation was preferred
preparation
the
methods
feasible due to the absence
groups
protein
hapten-protein
conjugate (12). Direct coupling of clei however
an
penicillinase
residues per
enzyme
334
RAGUPATHI ET AL.
Among the haptens, excepting steroids (14),
the
in
the
arylnaphthalene
lignan lactone glycoside used
present study is the first compound to be labelled with
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penicillinase
by
Determination
of
penicillinase
conjugate
more than fewer
the
the
carbodiimide
enzyme activity
method.
in
clei
showed the enzyme
to
A-HS
retain
85% of its activity. Besides the presence of
clei A-HS molecules in the conjugate
the
other
factors that contribute to retention of enzyme-activity are
the pH of the reaction medium
and
the
initial
concentrations of the reaction
of
clei
during
other
A-HS
with
conjugation
reactants.
The
at
EDC
pH
6
presumably permits the formation of 0-acylisourea (15), which then reacts with the nucleophilic amino group the
enzymic protein to form peptide bonds at
pH
8.0.
Self polymerisation of clei A-HS is not possible these conditions since it is devoid of an amino
in
under group.
This apart, the effect of carbodiimide on penicillinase in
initiating
raising
the
pH
self coupling is further
prevented
of the reaction medium from
during the addition of penicillinase and by
6
to
since
Penicillinase was chosen as the marker it is known to be stable at ambient
over the pH range 5 to 9 (17)
0.2
enzyme
temperature
.
The sensitivity of the ELISA developed here clei
8
increasing
the concentration of phosphate buffer from 0.01 t o
M (16).
by
A was 3ng/ml. The reproducibility of
the
for
method
335
ENZYME-LINKED IMMUNOSORBENT ASSAY
was
verified
samples.
by
assaying clei A
from
spiked
urine
The coefficients of variation in the
intra-
and inter-assays were less than 9% (Table 1). The specificity study conducted by testing other structurally similar arylnaphthalene lignan lactones of
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C.collinus
clearly indicated the ability of
anti-clei
A-HS
antibody to react with cleistanthin B,
diphyllin
and
collinusin
(Table 2).
This
is
preparing
the immunogen the conjugation of
with
was carried out through the
BSA
introduced xylose
in the
2-position of the
the
aromatic
haptenic
determinant
diphyllin
and
nucleus,
nucleus
site.
collinusin
also
However
the
be
the
major
B,
cleistanthin
have
degree
group
(7) thereby
A
the
same
the anti-clei A-HS antibody exhibited
reactivity.
A-HS
3,4-di-o-methyl-
to
Since
clei
carboxyl
(carbohydrate moiety) of clei
permitting
for
because
of
aryl
cross-
cross-reactivity
varied, with the glycoside cleistanthin B showing affinity
(Table
carbohydrate
2)
moiety,
which
might
namely
be
D-glucose
due as
less
to
its
shown
in
Figure 3. The cross-reactivity of anti-clei A-HS with
the
C.collin us user
other
three
major
lignan
antibody
lactones
is in fact an added advantage in
the
context of the developed ELISA since in cases
C.collinus
poisoning there is the presence of all
of endof of
336
RAGUPATHI ET AL.
these
four marker compounds in urine, blood and
tissues.
In addition the major
metabolite
C.collinus poisoning has been reported to be
specific detection of C.collinus
by
after
diphyllin
Thus the present method can be applied
(18).
other
for the
monitoring the
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characteristic active glycosidic principle as well
as
the metabolite. The operational promise and specificity of the proposed ELISA are further enhanced by the
fact
that other phytotoxins and drugs frequently encountered in clinical and forensic toxicology exhibit no reactivity with anti-clei A-HS antibody
(Table
cross3).
ACKNOWLEDGEMENTS
The authors thank Dr. Gowri Chandrakasan Madras)
and
Dr.U.M. Joshi (IRR, Bombay)
help/suggestions. the New
Bureau
of
for
(CLRI, their
The financial assitance provided Police Research
and
by
Development,
Delhi and Tamil Nadu State Council for Science and
Technology, Madras is acknowledged. REFERENCES
1.
Govindachari, T.R., Sathe, S.S., Viswanathan, N., Pail B.R. and Srinivasan, M. Chemical constituents of Cleistanthus collinus (Roxb.). Tetrahedron 1969; 25: 2815-21.
2.
Modi, N.J. Modi's Text Book of Medical Jurisprudence and Toxicology. N.M. Tripathi Private Limited, Bombay. 1977; pp.618-619.
3.
Lakshmi,
N.V.
A
T.G.,
Srimannarayana, G . and
.395-6.
new glycoside from
Curr. Sci. 1970; 39:
Subba Rao,
337
ENZYME-LINKED IMMUNOSORBENT A S S A Y
4.
Annapoorani, K . S . , Periakali, P.., Ilangovan, S., Damodaran, C. and Chandra Sekharan, P. Spectrofluorometric determination of the toxic constituents of Cleistanthus collinus. J. Anal. Toxicol. 1984;
Downloaded by [Nanyang Technological University] at 10:47 16 June 2016
8: 182-6.
5.
Annapoorani, K . S . , Damodaran, C. and Chandra Sekharan, P. Solid state fluorodensitometric quantitation of arylnaphthalene lignan lactones of Cleistanthus collinus. J. Chromatogr, 1984; 303: 296-305.
6.
Annapoorani, K.S., Damodaran, C. and Chandra Sekharan, P. High pressure liquid chromatographic separation of arylnaphthalide lignan lactones. J.Liq. Chromatogr. 1985; 8: 1173-94.
7.
Ragupathi, G., Prabhasankar, P., Chandra Sekharan, P., Annapoorani K.S. and Damodaran, C. Antibodies for cleistanthin A, a toxic glycoside of the plant Cleistanthus collinus. Ind. J. Environ. Toxicol. (submitted for publication).
8.
Erlanger, B.F., Borek, F., Beiser, S . M . , and Lieberman, S. Steroid - protein conjugates : 1. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. J. Biol. Chem. 1957; 228: 713-27.
9.
Habeeb, A.F.S.A. Determination of free amino groups in proteins by trinitrobenzene sulfonic acid. Anal. Biochem. 1966; 14: 328-36.
10. Lowry,
O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the Folin-phenol reagent. J.Bio1. Chem. 1951; 193: 265-75.
11. Joshi, U.M. In: Hand Book of VI Training Course on ELISA in Reproductive Medicine. Institute for Research in Reproduction, Bombay. 1989; p.22. 12. Aoki, K. and Kuroiwa, Y. Immunoassays in sciences. Eisei Kagaku 1987; 33: 227-42.
forensic
13. Williams, D.G. Conjugation procedures in immunoassay. In: Pal, S . B . ed. Immunoassay Technology, V01.2. Walter de Gruyter and Co. Berlin, New York 1986; pp.1-15. 14. Raghavan, V.P. U s e of enzyme marker for ELISA. In: Joshi,
penicillinase as a U.M., Iyer, K . S . ,
338
MGUPATHI ET AL.
Raghavan, V. and Bhandari, P. eds. ELISA letter; 1987; Issue 5, pp.1-4. Institute Research in Reproduction, Bombay.
News for
Carbodiimide K. L. and Koshland ,Jr D.E. modification of proteins. In: Hirs, C.H.W. and Timasheff, S.N. eds. Methods in Enzymology, Vo1.25, Academic Press, New York. 1972; pp.616-23.
15. Carraway ,
M.T.B. and Preston, J.F A simple modified small carbodiimide method for con uqation of molecular-weight compounds to inm;noglobulin G with minimal protein cross linking. Anal. Biochem. 1981; 116: 402-7.
Downloaded by [Nanyang Technological University] at 10:47 16 June 2016
16. Davis,
17. Davies, R.B., Abraham, E . P . and Dalgleish, D.G. Confonnational changes in the extracellular p-lactamase I from Bacillus cereus 569/H/9. Biochem. J. 1974; 143: 137-41. 18. Annapoorani, K . S . It Analytical Biochemistry in Forensic science: Toxicological Aspects of Cleistanthus collinusll. Ph.D. Thesis, University of Madras. 1985.
ISSN: 0197-1522 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ljii19
Enzyme-Linked Immunosorbent Assay for the Phytotoxin Cleistanthin A G. Ragupathi , P. Prabhasankar , P. Chandra Sekharan , K. S. Annapoorani & C. Damodaran To cite this article: G. Ragupathi , P. Prabhasankar , P. Chandra Sekharan , K. S. Annapoorani & C. Damodaran (1992) Enzyme-Linked Immunosorbent Assay for the Phytotoxin Cleistanthin A, Journal of Immunoassay, 13:3, 321-338, DOI: 10.1080/15321819208021236 To link to this article: http://dx.doi.org/10.1080/15321819208021236
Published online: 23 Sep 2006.
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Date: 16 June 2016, At: 10:47
JOURNAL OF IMMUNOASSAY, 1 3 ( 3 ) , 321-338 (1992)
ENPYME-LINKED IMMUNOSORBENT ASSAY FOR THE PHYTOTOXIN CLEISTANTHIN A
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0.
Ragupathi, P. Prabhasankar, P. Chandra+Sekharan, K.8. Annapoorani and c. Damodaran R &I D Division, Forensic Sciences Department, Madras - 600 0 0 4 , INDIA. ABSTRACT
Enzyme - linked immunosorbent assay is reported a major for the estimation of cleistanthin A, constituent of the toxic plant Cleistanthus collinus. Rabbit antibodies were obtained by immunisation with cleistanthin A hemisuccinate-BSA conjugate and the ELISA developed thereupon could detect cleistanthin A at as low a concertration as 3 ng/ml. Cross-reactivity studies with structural analogs as well as with other phytotDxins and drugs of common occurrence established the suitability of the ELISA to specifically monitor the C. collinus marker molecules in emergency clinical and forensic cases. The simplicity and specificity make the ELISA superior to the other available techniques. (KEY WORDS: Cleistanthin, C.collinus, Penicillinase, Toxicology).
ELISA
I
INTRODUCTION
Cleistanthin A (clei
a
A),
toxic principle of the plant
a lignan glycoside
is
Cleistanthus collinus
(1). This plant belongs to the Euphorbiaceae family and
*Author for correspondence
321 Copyright 0 1992 by Marcel Dekker, Inc,
3 22
RAGUPATHI ET AL.
is abundant in India. The poisonous nature of the plant especially its leaves has been well documented (2). addition namely
cleistanthin
present
methods
lignan
lactones
(1,3);
spectral
and
are
chromatographic
of quantifying them in biospecimens have
reported
However no immunoassay has
therefore
been
yet
been
The objectives of this investigation
were
(4-6).
developed.
protein
other
A,
B, diphyllin, and collinusin
cleistanthin
also
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to
to couple the haptenic clei A
with
carrier
using peptide bond forming agents in order
produce antibodies, characterise their specificity clei
and
A
In
develop
quantitative
an ELISA
application
in
for
for
qualitative
cases
of
to
and
C.collinus
poinoning.
MATERIALS AND METHODS Cleistanthin C.collinuq
A was isolated from the leaves
of
by a preparative chromatographic
procedure
and its purity checked through melting point
analysis,
thin-layer
data
chromatography
and
spectral
by
comparison with an authentic sample obtained from CIBA-
.
GEIGY (India) Bovine
serum
albumin
(BSA),
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride soluble
starch,
(TNBS)
and
penicillinase
2,4,6,-
Tween and
20
trinitrobenzene sulfonic were
obtained
penicillin V
were
from from
(EDC), acid Sigma:
Hindustan
323
ENZYME-LINKED IMMUNOSORBENT ASSAY
Antibiotics
(India): and
Ltd
(complete
Freund's
adjuvants
and incomplete) were the products
Laboratories,
USA.
Buffer
salts
of
of
Difco
reagent
grade
quality were used.
A-Drot~ein (BSA) humoseg
PreDaration of oleistDownloaded by [Nanyang Technological University] at 10:47 16 June 2016
Preparation
-
of cleistanthin A
hemisuccinate
(clei A-HS) and its conjugation to BSA were carried out according to the method developed earlier by us (7).
Enxvme
labellins o f cleistanthin
-
Cleistanthin A
HS derivative was labelled with
the marker enzyme penicillinase The
conjugation
aqueous
was
solution
A
in the following
initiated by adding
of EDC (15 mg/ml;
lml
of
pmoles)
80
way. an
to
a
solution of clei A-HS (4.5 mg/ml; 8.2 pmoles) in sodium phosphate
buffer
(pH 6.0; 0.01M).
incubated
at
occasional
shaking
temperature
for
and then added to
mixture min
30
a
buffer (pH 8.0; 0.2M). After
in
0.01M).
sodium
To
against sodium phosphate buffer the
dialyzed enzyme
with of
sodium
incubation
2-8OC for 16 to 18h, the reaction mixture was extensively
was
solution
(1 ml : 5mg/ml; 0.16 pmole)
penicillinase phosphate
room
The
dialyzed (pH
conjugate,
at
7.4;
BSA
and
1%
and
haDten
in
azide were added to a concentration of
0.2% and stored refrigerated in aliquots.
Evaluation coniusates
of
molar
Determination
DarticiDation of
of the number of haptenic clei
A
residues per protein or enzyme molecule was carried out
324
RAGUPATHI ET AL.
by spectrometric analysis ( 8 ) and TNBS method (9).
The
protein
content before
was
assayed
by
enzyme
and
after
conjugation
the method of Lowry et a1
activity
of
(10) and
the
measured
as
penicillinase was
described by Joshi (11).
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PreDaration of antiserum to cleistanthin New
Zealand
white
rabbits
A
were
injected
intradermally at multiple sites with 1 ml of clei A-HSBSA conjugate (corresponding to a concentration of pg of bound clei A-HS) dissolved in
saline ( P B S ; 0.1 M; pH complete adjuvant injection
of
the
7.4)
phosphate-buffered
and emulsified in
(lml). After four weeks immunogen
(40
100
fig)
Freund's
a
in
booster Freund's
incomplete adjuvant was given. The antiserum obtained after seven days of the booster dose was tested for the presence and
of
purified
clei
A
antibodies by
by ammonium
direct
ELISA
sulfate precipitation and
-
dialysis after which aliquots were stored at
6OoC
until use.
DeveloDment
of
Optimal
competitive
ELISA
for
clei
combination of the antibody and
labelled analyte for use in ELISA was
in 'Results'. Accordingly 200 pl
clei
antibody
diluted
with
enzyme
ascertained as
described A
A
of
coating
(bicarbonate buffer; 0.05M; pH 9.6) (1:1600) was
antibuffer added
to the wells of microtiter plates and incubated for at
37OC
3h
or overnight in refrigerator. After washing the
325
ENZYME-LINKED IMMUNOSORBENT A S S A Y
plates
(three times) with wash buffer
0.05%
(PBS-containing
Tween 20) the empty sites were blocked
with
solution of BSA in PBS. The plates were then for
This was followed by the
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amounts of clei like the
incubated
lh at room temperature and washed again with
buffer.
clei
A
-
A
sample
wash known
(1OOpl)
spiked urine and a specified aliquot
predetermined dilution
penicillinase
addition of
(100 pl) or analyte
1%
the clei
-
A
plates
were incubated at 37OC for 2h. Subsequently the
plates
washed
with
wash
substrate solution was
(100 pl; 1:lOO).
-
HS
The
were
conjugate
of
of
buffer added
and to
pl
200
each
of
well.
(The
substrate solution was starch-iodine-penicillin reagent obtained which
in
(5.329)
by mixing 0.2ml
of
turn was a preparation of
and
iodine
(200 mg) in 10
iodine
of
(SIP)
reagent
potassium ml
the
iodide
distilled
water, and 20 ml of 2% starch solution with 100 ml
of
penicillin V (15.2mg) solution in phosphate buffer: SIP reagent was prepared just before use).
The plates were
then left at ambient temperature for 20 min immediately following which the absorbance measurements were
made
at 620 nm with an E L I S A Reader (Biotek, EL 310 model). Evaluation of ELIBA for specificity to clei The different
competitive ELISA
was
ttanalytesltsuch as the
drugs
of
conducted
clei
analogs present in C.collinus, other
A
A
structural
phytotoxins
common occurrence in clinical
with
and
and
forensic
326
RAGUPATHI ET AL.
toxicology. below
The
cross-reactivity
according
to
the
method
was
calculated
described
by
as
Joshi
(personal communication):
-
(0.D of clei A excess) %binding at = known concentration (0.D of clei A excess)
(0.D of known concentration of clei A)
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.................................
The of
clei
binding
-
percent binding for various were calculated
A
vs
corresponding
and
XlOO
(0.D without clei A)
concentrations
plotted
as
concentration
percent
on
a
semi-
logarithmic graph. Similarly concentrations of
the
percent
binding
the cross-reacting
with
various
compounds
(CRC)
were calculated and the graph constructed. Let T50 be the concentration of clei A,and CRC50 that of the CRC; with respect to 50% binding, % Cross-reactivity of the CRC =
then
T50 ------
x 100
CRC50
RESULTS
A
hemisuccinate
derivative
the
of
cleistanthin A (clei A-HS) was prepared and
toxin
conjugated
with carrier protein/enzyme for obtaining the immunogen and
label.
molecules
The of
on
conjugate
clei A-HS per protein while
(penicillinase) haptens
BSA-coupled
a
-
contained the
enzyme
tagged product retained on average molar
basis.
26
Determination
4
of
321
ENZYME-LINKED IMPNNOSORBENT ASSAY
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ANTI
- CLEl
A ANTIBODY DILUTION
-
CLEl A PENlClLLlNASE DILUTION
FIGURE
1
Optimum combination of anti-clei A antibody and clei A-penicillinase label.
penicillinase
activity
in
the
conjugate
showed
retention of more than 85% of the original activity. Optimal
dilutions of the anti-clei
A
antibody
and the clei A-HS- penicillinase conjugate as
required
for ELISA were determined by constructing a graph the
difference
in absorbance values
of
the
from
reagent
328
RAGUPATHI ET A L .
0.8
-
E
0
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0.6IQ
w
x :
0.4
-
0.2
-
8
3
CONCENTRATION ng /ml
FIGURE 2
calibration curve for cleistanthin A .
blank and analyte wells for the various combinations of anti-clei A- antibody and clei A-HS
-
penicillinase.
The
dilution that gave an absorbance-difference
to
1.0
was
considered
as
optimum
dilution.
corresponding data are shown in Figure 1.
The
The dilution
factors were found to be 1600 and 100 for the and the enzyme conjugate respectively.
close
antibody
10.00
23.90
48.66
67.28
10
25
50
75
5.554
3.850 89.70
97.32
95.60
100.00
0.412 0.689
98.00
0.187
8.25
7.91
2.88
4.12
3.82
72.64
49.31
26.82
9.57
5.12
1.887
3.245
2.295
0.714
0.377
96.85
98.62
107.28
95.70
102.40
2.59
6.58
8.55
7.46
7.36
*Each sample (spiked urine) was assayed in quadruplicate
100 91.00 4.920 91.00 5.40 94.78 4.037 94.78 4.25 ......................................................................
4.90
5
Reproducibility of ELISA for the estimation of cleistanthin A
TABLE 1
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I
2 l-3
W
P m
0
v)
0
c z
3: 3:
H
W
m
x
z
H
r
330
RAGUPATHI ET AL.
TABLE 2
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Cross-reactivity of anti-cleistanthin A antibody during ELIGA as tested with cleistanthin A structural analogs
Cleistanthin B
84.4
Diphyllin
98.0
Collinusin
96.6
*Cleistanthin A-HS was assigned a value of 100
A
calibration
standard against range
by
graph
plotting
the
constructed
with
semilog
concentration
absorbance at 620 nm showed 3-100 ng/ml (Figure 2). The
of
clei
linearity in
A
the
reproducibility
of
the E L I S A was verified by estimating the amount of clei A
in
spiked
urine samples. The
figures
on
percent
recovery are presented in Table 1. The
possible cross-reactivity of
anti-clei
antibody with certain other C.collinus lignan that
are structurally
similar to clei A is
lactones shown
Table 2 and their structures in Figure 3. Table the
non-c.col1inus
occurrence
in
phytotoxins and
clinical and forensic
drugs
A
3
of
toxicology
in
lists common that
were tested in the E L I S A for cross-reactivity and ruled out therefor.
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ENZYME-LINKED IHMUNOSORBENT ASSAY
331
H, CO
H, CO
H, CO
-
CLEISTANTHIN A
GlPHYLLlN FIGURE
3
-
CLEISTANTHIN B
COL LINUSIN
Major lignan lactones of S.collinu S.
RAGUPATHI ET AL.
332 TABLE 3
Drugs and plant toxins tested and found negative for cross-reactivity with anti-clei A antibody during
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ELIlA
Brucine Cerberin Digitoxin Digoxin Ellagic acid Gitoxin Neriifolin Oleandrigenin Oleandrin Ouabain Peruvoside Strychnine Thevetigenin Thevetin
Amylobarbitone Butobarbiton Chlordiazepoxide Chlorpromazine hydrochloride Chlorpromazine sulphoxide Cyclobarbitone calcium Dextropropoxyphene hydrochloride Diazepam Dicoumarol Diphenhydramine hydrochloride Heptabarbitone Imipramine Indomethacin Lorazepam Nitrazepam Nortriptyline oxazepam Oxyphenbutazone Pentobarbitone sodium Phenacetin Phenobarbitone sodium Promethazine hydrochloride Quinalbarbitone sodium Temazepam Thiopentane sodium Trimipramine maleate
DISCUSSION
Identification and determination of in biospecimens are essential monitoring clinical
xenobiotics
for therapeutic
drug
and to establish the cause of poisoning and
forensic problems.
For
this
in
many
333
ENZYME-LINKED IMMUNOSORBENT ASSAY
investigators have been turning to enzyme immunoassays. The increasing incidence of C.collinus poisoning
calls
for the development of such simple, sensitive and rapid assays. Cleistanthin
A,
as shown in Figure 3 , is not
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immunogen and hence a conjugate with a carrier like
needs to be prepared.
BSA
available
for
the
Various
preparation
of
reactive
not
in
derivative of clei
A
then conjugated with (7).
clei (clei
BSA
The
A.
A-HS)
of
good
of
relevant
hemisuccinate
was prepared which was
by the mixed anhydride method
immunogen
since the
conjugate had a higher number of clei molecule
of
protein
than
carbodiimide method.
labeling
the
the
On
the
analyte with
resulting
obtained
other
enzyme
analyte molecules are required in
analyte-
enzyme
conjugate in order
retain its activity (13). The A
molecules per
BSA
4
clei
A
by
hand,
marker,
the
that
for the
since
resulting
the
enzyme
conjugate had 2 6 clei
molecule whereas the
conjugate contained only molecule.
BSA
the
residues per
A-HS
one
for
carbodiimide method of conjugation was preferred less
are
to protein is
A
This method of conjugation was preferred
preparation
the
methods
feasible due to the absence
groups
protein
hapten-protein
conjugate (12). Direct coupling of clei however
an
penicillinase
residues per
enzyme
334
RAGUPATHI ET AL.
Among the haptens, excepting steroids (14),
the
in
the
arylnaphthalene
lignan lactone glycoside used
present study is the first compound to be labelled with
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penicillinase
by
Determination
of
penicillinase
conjugate
more than fewer
the
the
carbodiimide
enzyme activity
method.
in
clei
showed the enzyme
to
A-HS
retain
85% of its activity. Besides the presence of
clei A-HS molecules in the conjugate
the
other
factors that contribute to retention of enzyme-activity are
the pH of the reaction medium
and
the
initial
concentrations of the reaction
of
clei
during
other
A-HS
with
conjugation
reactants.
The
at
EDC
pH
6
presumably permits the formation of 0-acylisourea (15), which then reacts with the nucleophilic amino group the
enzymic protein to form peptide bonds at
pH
8.0.
Self polymerisation of clei A-HS is not possible these conditions since it is devoid of an amino
in
under group.
This apart, the effect of carbodiimide on penicillinase in
initiating
raising
the
pH
self coupling is further
prevented
of the reaction medium from
during the addition of penicillinase and by
6
to
since
Penicillinase was chosen as the marker it is known to be stable at ambient
over the pH range 5 to 9 (17)
0.2
enzyme
temperature
.
The sensitivity of the ELISA developed here clei
8
increasing
the concentration of phosphate buffer from 0.01 t o
M (16).
by
A was 3ng/ml. The reproducibility of
the
for
method
335
ENZYME-LINKED IMMUNOSORBENT ASSAY
was
verified
samples.
by
assaying clei A
from
spiked
urine
The coefficients of variation in the
intra-
and inter-assays were less than 9% (Table 1). The specificity study conducted by testing other structurally similar arylnaphthalene lignan lactones of
Downloaded by [Nanyang Technological University] at 10:47 16 June 2016
C.collinus
clearly indicated the ability of
anti-clei
A-HS
antibody to react with cleistanthin B,
diphyllin
and
collinusin
(Table 2).
This
is
preparing
the immunogen the conjugation of
with
was carried out through the
BSA
introduced xylose
in the
2-position of the
the
aromatic
haptenic
determinant
diphyllin
and
nucleus,
nucleus
site.
collinusin
also
However
the
be
the
major
B,
cleistanthin
have
degree
group
(7) thereby
A
the
same
the anti-clei A-HS antibody exhibited
reactivity.
A-HS
3,4-di-o-methyl-
to
Since
clei
carboxyl
(carbohydrate moiety) of clei
permitting
for
because
of
aryl
cross-
cross-reactivity
varied, with the glycoside cleistanthin B showing affinity
(Table
carbohydrate
2)
moiety,
which
might
namely
be
D-glucose
due as
less
to
its
shown
in
Figure 3. The cross-reactivity of anti-clei A-HS with
the
C.collin us user
other
three
major
lignan
antibody
lactones
is in fact an added advantage in
the
context of the developed ELISA since in cases
C.collinus
poisoning there is the presence of all
of endof of
336
RAGUPATHI ET AL.
these
four marker compounds in urine, blood and
tissues.
In addition the major
metabolite
C.collinus poisoning has been reported to be
specific detection of C.collinus
by
after
diphyllin
Thus the present method can be applied
(18).
other
for the
monitoring the
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characteristic active glycosidic principle as well
as
the metabolite. The operational promise and specificity of the proposed ELISA are further enhanced by the
fact
that other phytotoxins and drugs frequently encountered in clinical and forensic toxicology exhibit no reactivity with anti-clei A-HS antibody
(Table
cross3).
ACKNOWLEDGEMENTS
The authors thank Dr. Gowri Chandrakasan Madras)
and
Dr.U.M. Joshi (IRR, Bombay)
help/suggestions. the New
Bureau
of
for
(CLRI, their
The financial assitance provided Police Research
and
by
Development,
Delhi and Tamil Nadu State Council for Science and
Technology, Madras is acknowledged. REFERENCES
1.
Govindachari, T.R., Sathe, S.S., Viswanathan, N., Pail B.R. and Srinivasan, M. Chemical constituents of Cleistanthus collinus (Roxb.). Tetrahedron 1969; 25: 2815-21.
2.
Modi, N.J. Modi's Text Book of Medical Jurisprudence and Toxicology. N.M. Tripathi Private Limited, Bombay. 1977; pp.618-619.
3.
Lakshmi,
N.V.
A
T.G.,
Srimannarayana, G . and
.395-6.
new glycoside from
Curr. Sci. 1970; 39:
Subba Rao,
337
ENZYME-LINKED IMMUNOSORBENT A S S A Y
4.
Annapoorani, K . S . , Periakali, P.., Ilangovan, S., Damodaran, C. and Chandra Sekharan, P. Spectrofluorometric determination of the toxic constituents of Cleistanthus collinus. J. Anal. Toxicol. 1984;
Downloaded by [Nanyang Technological University] at 10:47 16 June 2016
8: 182-6.
5.
Annapoorani, K . S . , Damodaran, C. and Chandra Sekharan, P. Solid state fluorodensitometric quantitation of arylnaphthalene lignan lactones of Cleistanthus collinus. J. Chromatogr, 1984; 303: 296-305.
6.
Annapoorani, K.S., Damodaran, C. and Chandra Sekharan, P. High pressure liquid chromatographic separation of arylnaphthalide lignan lactones. J.Liq. Chromatogr. 1985; 8: 1173-94.
7.
Ragupathi, G., Prabhasankar, P., Chandra Sekharan, P., Annapoorani K.S. and Damodaran, C. Antibodies for cleistanthin A, a toxic glycoside of the plant Cleistanthus collinus. Ind. J. Environ. Toxicol. (submitted for publication).
8.
Erlanger, B.F., Borek, F., Beiser, S . M . , and Lieberman, S. Steroid - protein conjugates : 1. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. J. Biol. Chem. 1957; 228: 713-27.
9.
Habeeb, A.F.S.A. Determination of free amino groups in proteins by trinitrobenzene sulfonic acid. Anal. Biochem. 1966; 14: 328-36.
10. Lowry,
O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the Folin-phenol reagent. J.Bio1. Chem. 1951; 193: 265-75.
11. Joshi, U.M. In: Hand Book of VI Training Course on ELISA in Reproductive Medicine. Institute for Research in Reproduction, Bombay. 1989; p.22. 12. Aoki, K. and Kuroiwa, Y. Immunoassays in sciences. Eisei Kagaku 1987; 33: 227-42.
forensic
13. Williams, D.G. Conjugation procedures in immunoassay. In: Pal, S . B . ed. Immunoassay Technology, V01.2. Walter de Gruyter and Co. Berlin, New York 1986; pp.1-15. 14. Raghavan, V.P. U s e of enzyme marker for ELISA. In: Joshi,
penicillinase as a U.M., Iyer, K . S . ,
338
MGUPATHI ET AL.
Raghavan, V. and Bhandari, P. eds. ELISA letter; 1987; Issue 5, pp.1-4. Institute Research in Reproduction, Bombay.
News for
Carbodiimide K. L. and Koshland ,Jr D.E. modification of proteins. In: Hirs, C.H.W. and Timasheff, S.N. eds. Methods in Enzymology, Vo1.25, Academic Press, New York. 1972; pp.616-23.
15. Carraway ,
M.T.B. and Preston, J.F A simple modified small carbodiimide method for con uqation of molecular-weight compounds to inm;noglobulin G with minimal protein cross linking. Anal. Biochem. 1981; 116: 402-7.
Downloaded by [Nanyang Technological University] at 10:47 16 June 2016
16. Davis,
17. Davies, R.B., Abraham, E . P . and Dalgleish, D.G. Confonnational changes in the extracellular p-lactamase I from Bacillus cereus 569/H/9. Biochem. J. 1974; 143: 137-41. 18. Annapoorani, K . S . It Analytical Biochemistry in Forensic science: Toxicological Aspects of Cleistanthus collinusll. Ph.D. Thesis, University of Madras. 1985.
