Note: Descriptions are shown in the official language in which they were submitted.
WO92/ll385 2 ~ n ~3 ~ 1~ 7 PCT/US91/09316
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~D~ NG CIGUATOXIN IN SERUW USI~G ~ CK E~E I~OASSAY"
Field of the Invention
The present invention relates to an immunoassay
~or ciguatoxin in afflicted indi~id~als.
Backqround of the Invention
Ciguatera poisoning is a p~rticular type of fish
poisoning which results from the ingestion of:certain
types o~ contaminated fish. Intoxication is associated
with the consumption of toxins produced by the dino-
~lagellate Gambierdiscus toxicus and is subsequently
passed along the marine food chain to man. Ciguatoxins
are polyether marine toxins. Approximately 27 d~fferent
ciguatoxins are known, approximately 23 of which are
toxic to.man.
Humans ara susceptible to ciguatera poisoning, both
~rom eating toxic horbivoreæ that ingest the dino-
flagellates while feeding on red or brown algae, and
from eating carnivoras which have eaten the toxic
herbivores.
The onset of the clinical symptoms of ciguatera
poisoning occurs within 10 minutes to 24 hours following
the consumption of toxic fish. Ciguatera poisoning
affects the digestive system~ (resulting in abdominal
pain, diarrhea, vomiting, nausea); the cardiovascular
system (resulti.ny in bradycardia, hypotension, tachycar- ~
: ,,
WO92/11385 pcr/~ /o9316
~9~ 2- ~-
l dia); and the neurological system (resulting primarily
in parasthesia and dysesthesia). Currently, the
diagnosis of ciguatera poisoning is based on the
appearance of the clinical symptoms of the syndrome.
Parasthesia and dysesthesia are considered clinical
hallmarks of the poisoning. However, since the symptoms
are numerous and are easily confused with others that
are more readily understood by and familiar to the
treating physician, diagnosis is often incorrect, and
the causative agen~ of tha symptoms is in~re~uently
identified.
Currently, there is no human diagnostic test *or
ciguatera poisoning, and treatment of afflicted in-
dividuals i5 presently limited. Therefore, there is a
need for a method of rapidly and accurately identifying
ciguatera poisoning in afflicted individuals.
Summary of the Invention
The present invention relates to method for
detecting the presence of ciguatoxin or related polyeth-
er marine toxin in human serum. The steps for detecting
the ciguatoxin comprises isolating a serum sample. The
acetone insoluble components oP serum may be precipitat-
ed and removed from the serum sample, or alternatively
the serum may be processed without the precipitation
step. The ciguatoxin present in the serum is attached
and fixed to a support. The resultant toxin-support
complex is rinsed in a buffer to remove unbound toxin
~nd to r2move any residual fixer.
The presence of ciguatoxin is detected by binding
anti-toxin, an antibody against the ciguatoxin, conj-
ugated to an enzyme, to the toxin-support complex to
form a toxin-antitoxin-enzyme complex. Excess anti-
toxin-enzyme conjugate is removed by washing with a
buffer. The enzyme of the toxin-anti-toxin-enzyme
complex is assayed by reacting the toxin-anti-toxin-
~nzyme complex with a substrate for tha enzyme. The
WO92~11385 2 ~ ~ 3 ~ J PCT/US91/09316
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1 amount of product ~ormed by the enzyme is then
c~antitated. The amount of product formed is directly
proportional to the amount of ciguatoxin present in the
serum sample.
A kit for performing the serum assays is also
described.
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WO92/11385 PCT/US91/09316
1 Detail ~ n
The detection of ciguatoxin in serum employs an
immunoassay using antibody to ciguatoxin. Serum samples
from individuals suspected of suffering from cig~atoxin
are obtained.
Acetone-insoluble materialD~ay be precipitated from
the serum samples by the addition of about five ml of
acetone to about one ml of each isolated serum sample.
The precipitate is separated by c:entrifugation at about
1500 x g ~or about 10 minutes. ~'he acetone solution is
decanted and evaporated to dryness under a stream of
air~ The residue le~t ~ehind, after the acetone is
evaporated, is redissolved in one ml of absolute
methan~l. The samples can then be assayed for
ciguatoxin. Alternatively, the acetone precipitation
step may be omitted, and the ciguatoxin assayed directly
from the serum sample. The color chang , detected by
comparison to a color chart, o~tained with the
subsequent enzyme assay step, described below, are
higher when the acetone precipitation step is used, thus
making the assay more sensitive. However, the rssults
obtained in the absence of the acetone precipitation
consistently result in readings, using a
spectrophotometer, that are above background. The
omission of the precipitation step results in a simpler
assay method that is more amenable to clinical testing.
To isolate the ciguatoxin from the serum sample,
which have been acetone precipitated or which have not
been precipitated with acetone, a support, such as a
bamboo stick, is coated with an adsorbent such as an
organic-base solvent correction fluid, such as that sold
by Pentel of America, Torrance, CA. The support with
the adsorbent coating is inserted into the serum samples
and swirled in ~he samples, about 3 times, to adsor~ any
ciguatoxin which is present in the serum sample onto the
adsorb~nt material. The support is removed, air-dried,
and any ciguatoxin present is fixed onto the adsorbent
WO9~t11385 2 ~ ~ 3 ~ Ll 7 PC~/US91/09316
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l material by dipping in 0.3~ hydrogen peroxide and 99.7%
methanol. After fixing the support is air-dried to form
a toxin-support complex. The toxin-support complex is
washed by rinsing in a buffer solution (50 mM Tris HCl,
pH 7.5; 2-amino-2-(hydroxymethyl)-l,3-propanediol
adjusted to the desired pH with dilute HCl: such as that
supplied by Sigma Chemical Co. St. Louis, M0, Cat. No.
Tl503) for about five seconds, to remove any excess or
unbound ciguatoxin, and blotted with tissue to remove
excess buf~er.
To assay for the pxesence of ciguatoxin, the fixed
support is immersed in a solution of anti-ciguatoxin-
antibody-horseradish peroxidase conjugate, to form an
antitoxin-bound support. After about a minute, the
fixed supports are rinsed twice in 50 mM Tris HCl, pH
7.5, and blotted to remove excess and unbound anti-toxin
antibody. The antibody may be a monoclonal or a
polyclonal antibody, which is produced by conventional
techniques. The monoclonal antibody is preferred.
If ciguatoxin was fixed onto the supports, the
antibody binds to the ciguatoxin. The amount of
antibody bound to the support is proportional to the
amount of ciguatoxin fixed to support. Since the
antibody is conjugated to horseradish peroxidase, any
antibody bound to the ciguatoxin will also carry
horseradish peroxidase, and the amount of horseradish
peroxldase present is also proportional to the amount
of ciguatoxin present. The presence of horseradish
peroxidase can then be detected ~y enzyme assay.
The horseradish peroxidase is assayed by placing
the antitoxin-bound supports into a substrate solution
prepared just prior to use. The solution is prepared
by mixing 25 ml of 0.3~ hydrogen peroxide (such as that
supplied by Sigma Chemical Co. St. Louis, M0, Cat. No.
HlO09) in 0.05 M Tris HCl, pH 7.5, with lO mg of 4-
chloro-l-naphthol (such as that supplied by Sigma
Chemical Co. St. Louis, M0, Cat. No. C8890) dissolved
: . ., 1.' ~ .
W~92/l~385 ~S~ ~ PCT/US91/09316
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1 in 0.125 ml o~ absolute ethanol filtersd through Whatman
#l filter paper to remove any insoluble material. The
reaction of the horseradish peroxidase with the
substrate results in a color change of the reaction
mixture. The intensity of the color change is directly
proportional to the amoun~ of ~lorseradi5h peroxidase
present, which in turn is directly proportional to the
amount of ciguatoxin present in the initial serum
sample.
Alternatively, more quantitative results may be
obtained by using a spectrophotometer. A convenient
means for measuring multiple sampl s is by pipetting the
reacted substrate solutions into 96 well microtiter
plat~s and deter~ining the optical density (O.D.) of
the samples at 405 nanometers (nm) in a microplate
spectrophotometer. ~n optical density reading above
about 0.1 at 405 nm, or which is above the optical
density readings ~btained with the serum of normal
indi~iduals, is considered to be a positive reaction
indicating the presence of ciguatoxin in the serum
samples.
The method for detecting ciguatoxin is also
appropriate ~or use in a kit form. The kit consists of
supports, six reagent vials, a medicine dropper, test
tubes, a blotter, and filter paper. The supports are
preferably made of bamboo and have one end coated with
correction fluid.
Vial A contain~ a fixation reagent, and it is
pre~erred that this reagent consist of 99.7~ methyl
alcohol and 0.3% hydrogen peroxide. Vial B contains a
buffer known as Tris. This is a weak basic compound
extensively used as a buffer in enzymic reactions. Its
organic chemistry nomenclature is 2-amino-2-
thydroxymethyl)-1,3-propanediol. The pH of the ~ris is
7.5 + 0.05, and the buffer contains 0.01% sodium azide
(NaN3~. The sodium azide serves as a preservative and
inhibits catalase.
. . . . . . ..
W~92/11385 PCT/US91/09316
2~92~3~l7
1 Vial C contains the conjugate of anti-ciguatoxin
horseradish peroxidase. This reagent, along with the
buffer reagent of vial B, is preferably stored in
lyophilized form at 4C. The stability of buf~ers
stored in lyophilized fashion is eight months, and, once
reconstituted with 15 mls o~ distilled water, it is
stable for one month. The sta~ility of the conjugate
in lyophilized form is sight months and, once
reconstituted, is stable for one day.
Vial D contains a substrate whi~h, in this case,
is 4-chloro-1-naphthol. As with vial ~ and vial C, it
is preferred that this sample be stored in lyophilized
form and stored at 4C. It may be reconstituted with
15 mls of distilled water and 30 drops (1.5 ml) of 3%
hydrogen peroxide solution. The stability o~ vial D in
lyophilized form is eight months, and, once
reconstituted, one day. This substrate is a substance
acted upon and changed by the enzyme horseradish
peroxidase.
Vial E contains the distilled water used to
reconstitute the lyophilized reagents in the other
vials.
Vial F contains acetone as a precipitant for the
serum samples.
.. . . , . ~
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WO92/11385 PCT/US91/09316
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l Example l
Detection of Ciquatoxin
Ciguatoxin extracts were prepared by the method
described by Y. Hokama et al. (Toxicon, _15, 317-325
(1977)), which is incorporated herein by reference. The
ciguatoxin crude extracts were serial diluted in
methanol. Dilutions of the extract used were: 50, 25,
12.5, 6.25, 3.63, 1.76, 0.88, ().44, 0.22, and O.ll
mg/ml. The concentrations o~ the ciguatoxin samples
were assayed by binding the ciguatoxin to a bamboo stick
coated with correction fluid. The resultant toxin-
support complex was fixed in 0.3% hydrogen peroxide and
97.7% methanol, air dried and rinsed in 0.0~ M Tris HCl,
pH 7.5, to remove unbound toxin and to remove any
residual fixer. The ciguatoxin was then detected by
dipping the fixed toxin-support into a solution
containing anti-toxin conjugated to horse radish
peroxidase. Excess anti-toxin-enzyme conjugate was
removed by washing with 0.05 M Tris HCl, pH 7.5.
The horseradish peroxidase was assayed by dipping
the antitoxin-bound supports into a substrate solution
prepared by mixing 25 ml of 0.3% hydrogen peroxide in
0.05 ~ Tris HCl, pH 7.5, with lO mg of 4 chloro-l-
naphthol dissolved in 0.125 ml of absolute ethanol,
filtered through Whatman ~l filter paper to remove any
insoluble residue. The color change of the enzyme assay
reaction was quantitated by pipetting the reacted
substrate solutions into 96 well microtiter plates and
determining the optical density (O.D.~ of the samples
at 405 nanometers (nm) in a microplate spectrophoto-
meter. The results obtained are summarized in Table I.
'
WO9~/11385 2 a 9 ~ r~ PCT/US91/0931~
_g_
1 Table I
Concentration of O.D. at 405 nm O.D. at 405 nm
ciguatoxin (mq~ml) Readinq 1 Readina 2
0.11 0.084
0~22 0.09
0.44 0.093
0.88 0.136
1.76 0.094 0.074
3.63 0.085 0.078
6.25 0.095 0,09
12.5 0.107
25.0 0.12
50.0 0.113
Table 1 shows that the optical density at 405 nm
obtained in the reaction increases with increasing
concentrations of ciguatoxin but level off at higher
ciguatoxin concentrations.
Exam~le ?
Assay ~or Ciquatoxin Added to Normal Human Serum
Normal human serum samples were obtai~ed from
volunteers. Different concentrations of ciguatoxin were
added to the normal serum to mimic the serum o a person
with ciguatera poisoning.
Serial dilutions of ciguatoxin were made, with
ciguatoxin concentrations of 6.~5, 3.63, 1.76, 0.88,
0.44, 0.22 and 0.11 mg/ml being used. one ml of normal
serum was added to the ciguatoxin samples. The samples
were mixed and incubated at 37C for one hour. Serum
samples which contained no ciguatoxin were used as a
control. The samples were then assayed for ciguatoxin
as described in Example 1, except the serum~ciguatoxin
samples were pxecipitated with the addition of five ml
of acetone per ml of serum. The precipitate was
separated ~xom the supernatant by centrifugation at
1,500 x g for 10 min. The supernatant was recovered,
evaporated to dryness under a stream of air and the
residue was re~uspended in 1 ml of absolute methanol.
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WO 92~1 138~ r PCI`/US91/09316
,2,~Q,C,:j3~ ~ -10-
1 The resultant reaction mixtures were read on a
microtiter-plate spectrophotometer.
The results obtained were compared to the results
obtained with samples where no serum was added to the
ciguatoxin sample. The results are summarized in Tabl~
II.
Table II
O.D. at 405 nm
Concentration of O.D. at 405 nm 1 ml serum
Ciauatoxin (m~/ml) No serum acetone ppkn
0 - 0.077
0.11 0.084 0.088
0.22 0.09 O.OBl
0.44 0.093 0.091
0.8~ 0.104 0.099
1.76 o.09~ 0.114
3.63 0.085 0.108
6.25 0.095
The results presented in Table II indicate that
ciguatoxin can be detected by the above described method
after incubation with human serum and that the results
are comparable to those obtained in the a~sence of
serum.
In an addîtional series of experiments, serial
dilutions of ciguatoxin were made, with ciguatoxin
concentrations of 25-,-12.5, 6.25, 3.63, and 1.76 and one
ml of serum was added and incubated as described above.
Each of the samples was assayed ~or the added
cigua~oxin, as described above except the acatone
precipitation step was omitted. The results are
summarized in ~able III.
Tabl~ III
O.D. at 405 nm
Concentration of O.D. at 405 nm 1 ml serum
Ciquatoxin ~mq~ml) No serum no acetone pptn
1.76 0.074 0.112
3.63 0.078 0.131 .
6.2~ ~0.09 0.142
12.5 0.107 0.146
25.0 0.12 0.132
:
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WO92/11385 2 ~ 9 ~ P~/US9~/09316
The results show an increase in O.D. with an
increase ciguatoxin, however bac~ground values are
higher.
5Example 3
Assay for Ciauat:oxln
in the Serum of Afflicted Individuals
Sexum samples were obtained from patient from New
~ersey and from the Marshall and Majuro Islands. These
serum samples were from patients who exhibited clinical
symptoms of what was categorized as ciguatera poisoning.
The serum samples ~rom the patients exhibiting the
characteristics of ciguatera poisoning were analyzed as
described above, withoùt acetone precipitation.
Results ~rom the data from the patient and the
15 normal serum were compared. Normal human serum forms
a baseline at 0.106+0.009. All of the serum ~rom
patients diagnosed with ciguatera poisoning had higher-
than-normal optical density values having a range ~rom
0.130+0.023 ~o 0.237+0.0~3.
The average optical density o~ normal human serum
is 0.106+0.009, while the average optical density o~
patients with known ciguatera poisoning is 0~173+0~03~o
The results of these comparisons between normal and
patient sera is summarized--in Ta~le IV.
Table IV
PatientBefore Treatment After Treatment
O~ 187+0 ~ 0~30
2 0.143+0.0~1 0.199+0.056
3 0.157+0.020 0.161+0.025
4 0.237+0.053 0.288+0.04g
0.159+0.029 0.118+0.015
6 0.130+0.023 0.167l0.045
7 0.125+0.024
average 0.169+0.038 0.176+0.062
8 0 ~ 196+0.070 0.095+0.062
35total
average 0.173+0.036 0.095
WO92/11385 ~ ~ :9 ~ 12- PCT/USgl/09316
l Patient 8 was treated with plasmapheresis. All o her
patients were treated with mannitol.
Normal Serum
First Second Third Total
Number Run _ Run~un_ Average
l 0.1~7 0.0880.104 0.l00+0.0l0
2 0.107 0.1020.093 0.l0l+0.007
3 0.098 0.1~50.107 0.103+0.005
4 0.097 0.1210.096 0.105+0 014
0.124 0.1450.099 0.123~0 023
0.106~0.009
One patient's serum resulted in a reading of
0.l96+0.070. However, after a plasmapheresis treatment,
the reading was reduced to 0.095 -- a difference of
0.l0l. ~he result suggests that, b~ore the plas-
mapheresis treatment, the patient had a high concentra-
tion of ciguatoxin, and that the plasmapheresis treat-
ment decreased the ciguatoxin concentration to normal
levels, i.e., to levels of serum containing no
ciguatoxin.
Another group of patients had a combined total
average o~ 0.169. These patients were treated by
infusion with 250 ml of 20% mannitol within first 24
hours of exposure. After ~annitol in~usion, a treatment
for ciguatoxin poisoning, the optical density value of
the reaction mixtures increased to 0.176+0.062.
~ rom Table IV, it is evident that the optical
density value for most patients increased after the
mannitol treatment. It has been postulated that the
mannitol treatment merely displaces the ciguatoxin from
affected tissues and moves it into the bloodstream.
Therefore, ~ollowing mannitol treatment, it is expected
that the optical density reading would increase.
