CA2098847A1 - Detecting ciguatoxin in serum using stick enzyme immunoassay - Google Patents
Detecting ciguatoxin in serum using stick enzyme immunoassayInfo
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- CA2098847A1 CA2098847A1 CA002098847A CA2098847A CA2098847A1 CA 2098847 A1 CA2098847 A1 CA 2098847A1 CA 002098847 A CA002098847 A CA 002098847A CA 2098847 A CA2098847 A CA 2098847A CA 2098847 A1 CA2098847 A1 CA 2098847A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
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Abstract
The present invention relates to a method for detecting the presence of ciguatoxin or related polyether marine toxin in human serum. The steps for detecting the ciguatoxin comprise isolating a serum sample, attaching and fixing the ciguatoxin to a support, and rinsing the resultant toxin-support complex in a buffer to remove unbound toxin and to remove any residual fixer. The presence of ciguatoxin is detected by binding anti-toxin, an antibody against the ciguatoxin conjugated to an enzyme, to the toxin-support complex to form a toxin-antitoxin-enzyme complex. The enzyme of the toxin-anti-toxin-enzyme complex is assayed, and the amount of product formed by the enzyme is then quantitated. A kit for conducting the assay is also described.
Description
WO92/ll385 2 ~ n ~3 ~ 1~ 7 PCT/US91/09316 ~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 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.
...... .
3~ :
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 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 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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~ 8-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 5 ~ ~ :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.
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 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.
...... .
3~ :
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 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 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.
.. . . , . ~
.: . . .
, . . : . . ,. ,, ~ . , , ~ . :
~ 8-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.
~, ' . ~ , " ., ' . ' . ~, :;: ' ' ' . ., ' . , ;. '. ' . . . :
', ~
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 :
.. . . ~ ... . ,. . - . - . -, .,, - , . , , ;, , , ~
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 5 ~ ~ :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.
Claims (25)
1. A method for detecting the presence of ciguatoxin or related polyether marine toxin in human serum comprising the steps of:
isolating a serum sample;
attaching any toxin contained in the precipitated serum sample to a support;
fixing the toxin to the support with a fixer to form a toxin-support complex;
rinsing the toxin-support complex in a buffer to remove unbound toxin and to remove any residual fixer;
binding an anti-toxin-enzyme conjugate to the toxin-support complex to form a toxin-antitoxin-enzyme complex;
removing any unbound anti-toxin-enzyme conjugate by washing the toxin-antitoxin-enzyme complex with a buffer;
assaying the enzyme of the toxin-anti-toxin-enzyme complex by reacting the toxin-anti-toxin-enzyme complex with a substrate for the enzyme; and quantitating the amount of product formed by the enzyme.
isolating a serum sample;
attaching any toxin contained in the precipitated serum sample to a support;
fixing the toxin to the support with a fixer to form a toxin-support complex;
rinsing the toxin-support complex in a buffer to remove unbound toxin and to remove any residual fixer;
binding an anti-toxin-enzyme conjugate to the toxin-support complex to form a toxin-antitoxin-enzyme complex;
removing any unbound anti-toxin-enzyme conjugate by washing the toxin-antitoxin-enzyme complex with a buffer;
assaying the enzyme of the toxin-anti-toxin-enzyme complex by reacting the toxin-anti-toxin-enzyme complex with a substrate for the enzyme; and quantitating the amount of product formed by the enzyme.
2. The method as recited in claim 1, wherein the serum sample is further processed by precipitation of acetone insoluble material comprising the additional steps of:
adding acetone to the serum sample;
mixing the acetone serum sample;
separating precipitated material;
decanting the acetone fraction;
drying the acetone under a stream of air to form a residue; and redissolving the residue in absolute methanol.
adding acetone to the serum sample;
mixing the acetone serum sample;
separating precipitated material;
decanting the acetone fraction;
drying the acetone under a stream of air to form a residue; and redissolving the residue in absolute methanol.
3. The method as recited in claim 1, wherein the support comprises a solid member coated with a toxin-adsorbing material.
4. The method as recited in claim 3, wherein the solid member comprises a bamboo stick.
5. The method as recited in claim 3, wherein the toxin adsorbing material comprises LIQUID PAPER.
6. The method as recited in claim 1, wherein the anti-toxin of the anti-toxin-enzyme conjugate comprises a monoclonal antibody directed against the ciguatoxin.
7. The method as recited in claim 1, wherein the fixer comprises 99.7% (v/v) methanol and 0.03% (v/v) hydrogen peroxide.
8. The method as recited in claim 1, wherein the buffer comprises 0.05 M Tris HCl at a pH of 7.5.
9. The method as recited in claim 1, wherein the enzyme of the anti-toxin-enzyme conjugate comprises horseradish peroxidase.
10. The method as recited in claim 1, wherein the substrate comprises hydrogen peroxide and 4-chloro-1-naphthol.
11. The method as recited in claim l, wherein the means for quantitating the product of the enzyme assay comprises comparing the color of the reaction mixture 9 after the enzyme reaction, to a color chart.
12. The method as recited in claim 1, wherein the means for quantitating the product of the enzyme assay comprises reading the optical density of the reaction mixtures at 405 nm in a spectrophotometer.
13. A kit for detecting the presence of ciguatoxin or related polyether marine toxin in human serum comprising:
a precipitant to precipitate high-molecular-weight molecules from serum samples;
a support for binding toxin from the serum sample;
a fixer to fix the toxin to the support;
a buffer to wash the fixed support;
an anti-toxin-enzyme conjugate for binding to the toxin;
a substrate for assaying the enzyme of the anti-toxin-enzyme conjugate; and means for quantitating the amount of product formed by the enzyme in the enzyme assay.
a precipitant to precipitate high-molecular-weight molecules from serum samples;
a support for binding toxin from the serum sample;
a fixer to fix the toxin to the support;
a buffer to wash the fixed support;
an anti-toxin-enzyme conjugate for binding to the toxin;
a substrate for assaying the enzyme of the anti-toxin-enzyme conjugate; and means for quantitating the amount of product formed by the enzyme in the enzyme assay.
14. The kit as recited in claim 13, wherein the support comprises a solid member coated with a toxin-adsorbing material.
15. The kit as recited in claim 14, wherein the solid member comprises a bamboo stick.
16. The kit as recited in claim 13, wherein the toxin adsorbing material comprises LIQUID PAPER.
17. The kit as recited in claim 13, wherein the fixer comprises 99.7% (v/v) methanol and 0.03% (v/v) hydrogen peroxide.
18. The kit as recited in claim 13, wherein the precipitant comprises acetone.
19. The kit as recited in claim 13, wherein the buffer comprises 0.05 M Tris HCl, pH 7.5?0.05.
20. The kit as recited in claim 13, wherein the buffer further comprises 0.01% (w/v) sodium azide.
21. The kit as recited in claim 13, wherein the anti-toxin of the anti-toxin-enzyme conjugate comprises a monoclonal antibody directed against the toxin.
22. The kit as recited in claim 13, wherein the enzyme of the anti-toxin-enzyme conjugate comprises horseradish peroxidase.
23. The kit as recited in claim 13, wherein the substrate comprises hydrogen peroxidase and 4-chloro-1-naphthol.
24. The kit as recited in claim 13, wherein the means for quantitating the product of the enzyme assay comprises comparing the color of the reaction mixture, after the enzyme reaction, to a color chart.
25. The kit as recited in claim 13, wherein the means for quantitating the product of the enzyme assay comprises reading the optical density of the reaction mixtures at 405 nm in a spectrophotometer.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63216590A | 1990-12-21 | 1990-12-21 | |
| US632,165 | 1990-12-21 | ||
| PCT/US1991/009316 WO1992011385A1 (en) | 1990-12-21 | 1991-12-06 | Detecting ciguatoxin in serum using stick enzyme immunoassay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2098847A1 true CA2098847A1 (en) | 1992-06-22 |
Family
ID=24534355
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002098847A Abandoned CA2098847A1 (en) | 1990-12-21 | 1991-12-06 | Detecting ciguatoxin in serum using stick enzyme immunoassay |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0563320A4 (en) |
| JP (1) | JPH06511311A (en) |
| AU (1) | AU9177991A (en) |
| BR (1) | BR9107179A (en) |
| CA (1) | CA2098847A1 (en) |
| WO (1) | WO1992011385A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5286498A (en) * | 1992-05-01 | 1994-02-15 | Hawaii Chemtect Incorporated | Rapid extraction of ciguatoxin from contaminated tissues |
| CN101963614A (en) * | 2010-09-03 | 2011-02-02 | 青岛科技大学 | The capillary electrophoresis electrochemical enzyme-linked immuno assay detects the method for ciguatoxin |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4816392A (en) * | 1984-10-02 | 1989-03-28 | Research Corporation Of The University Of Hawaii | Rapid stick test for detection of ciguatoxin and other polyether toxins from tissues |
-
1991
- 1991-12-06 CA CA002098847A patent/CA2098847A1/en not_active Abandoned
- 1991-12-06 AU AU91779/91A patent/AU9177991A/en not_active Abandoned
- 1991-12-06 BR BR919107179A patent/BR9107179A/en unknown
- 1991-12-06 WO PCT/US1991/009316 patent/WO1992011385A1/en not_active Application Discontinuation
- 1991-12-06 EP EP19920904300 patent/EP0563320A4/en not_active Withdrawn
- 1991-12-06 JP JP4504310A patent/JPH06511311A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| WO1992011385A1 (en) | 1992-07-09 |
| JPH06511311A (en) | 1994-12-15 |
| EP0563320A4 (en) | 1993-11-18 |
| AU9177991A (en) | 1992-07-22 |
| EP0563320A1 (en) | 1993-10-06 |
| BR9107179A (en) | 1994-02-08 |
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