EP0563320A4 - Detecting ciguatoxin in serum using stick enzyme immunoassay - Google Patents
Detecting ciguatoxin in serum using stick enzyme immunoassayInfo
- Publication number
- EP0563320A4 EP0563320A4 EP19920904300 EP92904300A EP0563320A4 EP 0563320 A4 EP0563320 A4 EP 0563320A4 EP 19920904300 EP19920904300 EP 19920904300 EP 92904300 A EP92904300 A EP 92904300A EP 0563320 A4 EP0563320 A4 EP 0563320A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- toxin
- enzyme
- recited
- kit
- support
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- 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
-
- 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
-
- 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
Definitions
- the present invention relates to an immunoassay for ciguatoxin in afflicted individuals.
- Ciguatera poisoning is a particular type of fish poisoning which results from the ingestion of certain types of contaminated fish. Intoxication is associated with the consumption of toxins produced by the dino- flagellate Gambierdiscus toxicus and is subsequently passed along the marine food chain to man. Ciguatoxins are polyether marine toxins. Approximately 27 different ciguatoxins are known, approximately 23 of which are toxic to. man.
- Humans are susceptible to ciguatera poisoning, both from eating toxic herbivores that ingest the dino- flagellates while feeding on red or brown algae, and from eating carnivores which have eaten the toxic herbivores.
- Ciguatera poisoning affects the digestive system (resulting in abdominal pain, diarrhea, vomiting, nausea) ; the cardiovascular system (resulting in bradycardia, hypotension, tachycar- dia) ; and the neurological system (resulting primarily in parasthesia and dysesthesia) .
- 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.
- diagnosis is often incorrect, and the causative agent of the symptoms is infrequently identified.
- 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 of 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 and to remove 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- enzyme complex with a substrate for the enzyme. The amount of product formed by the enzyme is then quantitated. 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.
- the detection of ciguatoxin in serum employs an immunoassay using antibody to ciguatoxin. Serum samples from individuals suspected of suffering from ciguatoxin are obtained.
- Acetone-insoluble material maybe 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 centrifugation at about 1500 x g for about 10 minutes. The acetone solution is decanted and evaporated to dryness under a stream of air. The residue left behind, after the acetone is evaporated, is redissolved in one ml of absolute methanol. 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.
- a support such as a bamboo stick
- 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 the samples, about 3 times, to adsorb any ciguatoxin which is present in the serum sample onto the adsorbent material.
- the support is removed, air-dried, and any ciguatoxin present is fixed onto the adsorbent material by dipping in 0.3% hydrogen peroxide and 99.7% methanol.
- the support 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 HC1, 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, MO, Cat. No. T1503) for about five seconds, to remove any excess or unbound ciguatoxin, and blotted with tissue to remove excess buffer.
- a buffer solution 50 mM Tris HC1, 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, MO, Cat. No. T1503
- 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 peroxidase present is also proportional to the amount of ciguatoxin present.
- the presence of horseradish peroxidase can then be detected by 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, MO, Cat. No.
- a convenient means for measuring multiple samples is 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 spectrophotometer.
- An optical density reading above about 0.1 at 405 nm, or which is above the optical density readings obtained with the serum of normal individuals, is considered to be a positive reaction indicating the presence of ciguatoxin in the serum samples.
- the method for detecting ciguatoxin is also appropriate for 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 contains a fixation reagent, and it is preferred 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- (hydroxymethyl)-l,3-propanediol. The pH of the Tris is 7.5 ⁇ 0.05, and the buffer contains 0.01% sodium azide (NaN 3 ) . The sodium azide serves as a preservative and inhibits catalase.
- 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 4 ⁇ C.
- the stability of buffers stored in lyophilized fashion is eight months, and, once reconstituted with 15 is of distilled water, it is stable for one month.
- the stability of the conjugate in lyophilized form is eight months and, once reconstituted, is stable for one day.
- Vial D contains a substrate which, in this case, is 4-chloro-l-naphthol.
- this sample be stored in lyophilized form and stored at 4*C. It may be reconstituted with 15 mis of distilled water and 30 drops (1.5 ml) of 3% hydrogen peroxide solution.
- 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.
- 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, 0.44, 0.22, and 0.11 mg/ml.
- the concentrations of 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 0.05 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 M Tris HCl, pH 7.5, with 10 g of 4-chloro-l- naphthol dissolved in 0.125 ml of absolute ethanol, filtered through Whatman #1 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. Table I
- 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 5 ciguatoxin concentrations.
- Example 2 Assay for Ciguatoxin Added to Normal Human Serum Normal human serum samples were obtained from Q volunteers. Different concentrations of ciguatoxin were added to the normal serum to mimic the serum of a person with ciguatera poisoning.
- the precipitate was separated from the supernatant by centrifugation at 1,500 x g for 10 min. The supernatant was recovered, 5 evaporated to dryness under a stream of air and the residue was resuspended in 1 ml of absolute methanol. The resultant reaction mixtures were read on a microtiter-plate spectrophotometer.
- the serum samples from the patients exhibiting the characteristics of ciguatera poisoning were analyzed as described above, without acetone precipitation.
- Results from the data from the patient and the normal serum were compared. Normal human serum forms a baseline at 0.106 ⁇ 0.009. All of the serum from patients diagnosed with ciguatera poisoning had higher- than-normal optical density values having a range from 0.130 ⁇ 0.023 to 0.237 ⁇ 0.053.
- Patient 8 was treated with plasmapheresis. All other patients were treated with mannitol.
- Another group of patients had a combined total average of 0.169. These patients were treated by infusion with 250 ml of 20% mannitol within first 24 5 hours of exposure. After mannitol infusion, a treatment for ciguatoxin poisoning, the optical density value of the reaction mixtures increased to 0.176 ⁇ 0.062.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US63216590A | 1990-12-21 | 1990-12-21 | |
US632165 | 2000-08-03 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0563320A1 EP0563320A1 (en) | 1993-10-06 |
EP0563320A4 true EP0563320A4 (en) | 1993-11-18 |
Family
ID=24534355
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19920904300 Withdrawn EP0563320A4 (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
Non-Patent Citations (1)
Title |
---|
See references of WO9211385A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1992011385A1 (en) | 1992-07-09 |
JPH06511311A (en) | 1994-12-15 |
AU9177991A (en) | 1992-07-22 |
CA2098847A1 (en) | 1992-06-22 |
EP0563320A1 (en) | 1993-10-06 |
BR9107179A (en) | 1994-02-08 |
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Legal Events
Date | Code | Title | Description |
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PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
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Effective date: 19930618 |
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A4 | Supplementary search report drawn up and despatched |
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Effective date: 19950329 |
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STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
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18D | Application deemed to be withdrawn |
Effective date: 19950809 |