DETECTING CIGUATOXIN IN SERUM USING STICK ENZYME IMMUNOASSAY

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: "DETECTING CIGUATOXIN IN SERUM USING STICK ENZYME BMUNOASSAY" Field of the Invention The present invention relates to an immunoassay for ciguatoxin in afflicted individuals. Background of the Invention 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. 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 (resulting in bradycardia, hypotension, tachycar- 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 agent of the symptoms is infrequently identified. Currently, there is no human diagnostic test for ciguatera poisoning, and treatment of afflicted in¬ dividuals is 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 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. Detailed Description 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. The color change, detected by comparison to a color chart, obtained 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 results 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 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. 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. To assay for the presence 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 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. H1009) in 0.05 M Tris HCl, pH 7.5, with 10 g of 4- chloro-1-naphthol (such as that supplied by Sigma Chemical Co. St. Louis, MO, Cat. No. C8890) dissolved in 0.125 ml of absolute ethanol filtered through Whatman #1 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 amount of horseradish 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 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 β 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. As with vial B and vial C, it is preferred that 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. The stability of 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. Example 1 Detection of Ciguatoxin 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 0 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. Serial dilutions of ciguatoxin were made, with ciguatoxin concentrations of 6.25, 3.63, 1.76, 0.88, 5 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 37°C for one hour. Serum sample ' s which contained no ciguatoxin were used as a control. The samples were then assayed for ciguatoxin 0 as described in Example 1, except the serum/ciguatoxin samples were precipitated with the addition of five ml of acetone per ml of serum. 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 results obtained were compared to the results obtained with samples where no serum was added to the ciguatoxin sample. The results are summarized in Table II. 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 absence of serum. In an additional 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 for the added ciguatoxin, as described above except the acetone precipitation step was omitted. The results are summarized in Table III. Table III Concentration of Ciguatoxin (mg/ml) 1.76 3.63 6.25 12.5 25.0 The results show an increase in O.D. with an increase ciguatoxin, however background values are higher. Example 3 Assay for Ciguatoxin in the Serum of Afflicted Individuals Serum samples were obtained from patient from New Jersey 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 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. The average optical density of normal human serum is 0.106+0.009, while the average optical density of patients with known ciguatera poisoning is 0.173±0.036. The results of these comparisons between normal and patient sera is summarized in Table IV. Table IV Patient 8 was treated with plasmapheresis. All other patients were treated with mannitol. Normal Serum 0 One patient's serum resulted in a reading of 0.196±0.070. However, after a plasmapheresis treatment, 5 the reading was reduced to 0.095 — a difference of 0.101. The result suggests that, before 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 Q levels, i.e., to levels of serum containing no ciguatoxin. 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. From 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, following mannitol treatment, it is expected that the optical density reading would increase.

Claims: WHAT IS CLAIMED IS : 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. 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. 3. The method as recited in claim 1, wherein the support comprises a solid member coated with a toxin- adsorbing material. . 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-l- naphtliol. 11. The method as recited in claim 1, 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. 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. 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-l- 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. AMENDED CLAIMS [received by the International Bureau on21 April 1992 (21.04.92); original claims 1,15,23-25 amended; remaining claims unchanged (4 pages)] 1. A method for detecting the presence of ciguatoxin or related polyether marine toxin in serum comprising the steps of: isolating a serum sample; precipitating acetone-insoluble material from the 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 precipita¬ tion 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. 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-molecu- lar-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 correction fluid. 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 com¬ prises 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 peroxide and 4-chloro-l- naphthol. 24. The kit as recited in claim 13, wherein the means for quantitating the product of the enzyme assay comprises a color chart. 25. The kit as recited in claim 13, wherein the means for quantitating the product of the enzyme assay comprises a spectrophotometer.

Inventors: HOKAMA YOSHITSUGI, HOKAMA, YOSHITSUGI
Applicants: HAWAII CHEMTECT INT, HAWAII CHEMTECT INTERNATIONAL
Application year: 1991
Main classification: C12Q1
IPC codes: C07K16, C12Q1, G01N33