JPH06511311A - Detection of Ciguatoxin in Serum Using Fixed Enzyme Immunoassay - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] □ Detection of diguatoxin in serum using fixed enzyme immunoassay The present invention provides immunoassay analysis for diguatoxin in affected individuals. related.

Phantom 5th baseman Sigwatella poisoning is caused by the ingestion of certain types of contaminated fish. It's addictive. Poisoning is Uzuben hair FIAgiGambierdiscus to associated with the consumption of toxins produced by C. xicus and subsequently transported by the marine food chain. This is communicated to people. Diguatoxin is a polyether marine toxin. Approximately 27 cypress types There are approximately 23 different diguatoxins known to be present in humans. It's poisonous.

Humans are poisonous herbivores that ingested Uzuben hair FIAs while ingesting red or brown algae. from eating carnivores that have eaten poisonous herbivores. Can be affected by Guatela poisoning.

The onset of clinical symptoms of Sigwatella poisoning is between 10 minutes and 24 hours after consumption of poisonous fish. Occurs within Sigwatella poisoning can cause symptoms in the digestive system (abdominal pain, diarrhea, vomiting, nausea). cardiovascular system (causing bradycardia, hypotension, and tachycardia) and nervous system (primarily sensory Currently, the diagnosis of Shigwatella poisoning is , based on the clinical manifestations of the syndrome. Paresthesia and paresthesia are poisoning It is considered to be a clinical feature of the disease. However, symptoms vary and treatment It is easy for doctors to understand, and it is easy to confuse it with other familiar diseases, so it is difficult to diagnose. diagnosis is often inaccurate and the toxin causing the symptoms can be identified. is rare.

Currently, there is no anthropometric testing for ciguatera poisoning, and treatment of affected individuals is not practical. It is limited. Therefore, ciguatera poisoning can be quickly and easily eliminated in affected individuals. A means of accurate confirmation is required.

i” Ω look 1 The present invention relates to diguatoxin or related polyether marine in human serum. Relating to means for detecting the presence of sex toxins. The means to detect diguatoxin , comprising isolation of serum specimens. Serum acentone insoluble elements precipitated from serum specimens Serum can be removed and removed, or as an alternative, serum can be prepared without precipitation means. It may also be treated as essential. The diguatoxin present in the serum is Adheres and is fixed. The resulting toxin-support complex is fixed and in a buffer to remove any toxins and also remove any remaining fixative. Rinse.

The presence of diguatoxin allows it to interact with the enzyme to form a toxin-antitoxin-enzyme complex. Binding antitoxin (antibody against diguatoxin) to toxin-support complex It is detected by Excess antitoxin enzyme conjugate should be washed away with buffer. removed by. The enzyme of the toxin-antitoxin-enzyme complex is The elementary complex is evaluated and analyzed by reaction with a substrate for the enzyme. Then the enzyme Measure the amount of product produced by. The amount of product produced is directly It is proportional to the amount of diguatoxin present in the serum specimen.

Kits for performing serum evaluation assays are also described.

1 woven plate For detection of diguatoxin in serum, an iminoassay using a diguatoxin antibody is used. from individuals at risk of diguatoxin poisoning. Obtain a serum specimen.

By adding approximately 5 ml of acentone to each ml of isolated serum specimen. acetone-insoluble material can be precipitated from serum specimens. The precipitate is approx. Separate by centrifugal rotation at approximately 1500 x g for 10 minutes. Acetone solution is , transferred to another container and evaporated to dryness under a stream of air. After evaporation of acetone The remaining residue is redissolved with 1 part of anhydrous methanol. Then, the specimen Acetone precipitation is an alternative that can be evaluated and analyzed for diguatoxin. Diguatoxin is directly assayed (evaluation analysis) from serum specimens, omitting the sedimentation method. can also be obtained from subsequent enzymatic assay procedures, as described below, Any color change detected by comparison with a color chart is determined by acetone precipitation means. It appears more clearly when used, making the assay more sensitive. Ru. however.

The results obtained without the need for acetone precipitation are consistent with those obtained using a spectrophotometer. In cases where the precipitate has a value above background, the omission of the precipitation method is more commonly followed in clinical trials. This provides a simpler assay method that is easier to follow.

Dilute from serum specimens (acetone precipitated or non-acetone precipitated). To isolate the guatoxin, a support such as a bamboo stick is used to isolate the guatoxin. organic-based products such as those sold by Pentyl-Saib America, Inc. Covered with an absorbent such as solvent correction fluid. Absorbent film support present in serum specimen In order to absorb the diguatoxin into the absorbent material, it is inserted into the serum specimen and Stir about 3 times. Remove the support, air dry, add 0.3% hydrogen peroxide and 9% By soaking in 9.7% methanol, the diguatoxin present is determined in the absorbent material. wear. After fixing, the support is air dried for formation of the toxin-support complex. poison The element-support complex is used to remove excess or unimmobilized diguatoxin. For about 5 seconds, add buffer solution (50% TrislCl.pH 7,5; dilute MCI to desired concentration). 2-Amino-2-(hydroxymethyl)-1,3-propane, adjusted to pH Diol; Sigma Chemical Company (Kata), St. Louis, Minnesota Clean by rinsing with blot with a tissue to remove excess buffer.

To assay for the presence of ziguatoxin, the fixed support was coated with an anti-ziguatoxin. Syn-antibody - horseradish peroxidase Immerse in a solution of conjugate to form an antitoxin-immobilized support. Extra, also fixed After approximately 1 minute to remove any antitoxin antibodies, the fixed support was heated to 50+ aM T. Rinse twice with risCl, pi (7,5) and aspirate. Antibodies are monoclonal. Alternatively, it may be a polyclonal antibody, produced by conventional techniques. monoclonal antibody is desirable.

If ziguatoxin is fixed to the support, the antibody will fix to ziguatoxin. . The amount of antibody immobilized on the support is proportional to the amount of diguatoxin immobilized on the support. Ru. The antibody binds to horseradish peroxidase, so it is immobilized on diguatoxin. Any antibodies produced by horseradish peroxidase are The amount of peroxidase is also proportional to the amount of diguatoxin present. So, west The presence of horseradish peroxidase can be detected by enzymatic assay.

Horseradish peroxidase was prepared immediately before use on antitoxin-immobilized supports. Assayed by placing in substrate solution. The solution was 0.05M I- 0.3z Hydrogen Peroxide in Lis HC1, pH 7.5 (St. Louis, MN) Supplied by Sigma Chemical Company (Cat. No. 1 (1009)) 4) dissolved in 0.125 ml of absolute ethanol -Chloro-1-naphthol (Sigma Chemical Co., Ltd., St. Louis, Minnesota) 10m (as supplied by Catalog No. C8890) g and filtered with Whatman No. 1 filter paper to remove insoluble material. Prepared by being filtered. Reaction with horseradish peroxidase substrate Depending on the reaction, the concentration (density) of the six color changes that result in a color change in the reaction mixture is is directly proportional to the amount of horseradish peroxidase present in the initial serum specimen. directly proportional to the amount of diguatoxin present in it.

Alternatively, more quantitative results can be obtained by using a spectrophotometer. can be obtained. Reacted substrates are a convenient means of measuring multiple samples. Pipette the stock solution into a 96-well microtiter plate and The optical density of the specimen at 405 nanometers (0,0,) in a spectrophotometer There is a means to determine. an optical density measurement of greater than about 0.1 at 405 nm, or Exceeding the optical density measurements obtained in normal human serum, serum specimens This is considered to be a positive reaction indicating the presence of diguatoxin.

The means for detecting diguatoxin are also suitable for use in kit format.

The kit includes a support, six reagent vials, a chemical dropper, test tubes, a plotter and Consists of filter paper. The support is preferably made of bamboo, with one end coated with correction fluid. ing.

Vial A contains the fixing reagent, which is 99.7% methyl alcohol. Preferably, the bottle B consists of Tris and 0.3% hydrogen peroxide. Contains a buffering agent known as It is widely used as a buffer in enzymatic reactions. It is a weak basic compound used in Its organic chemistry standard name is 2-amino-2 -(Hydroxymethyl)-1,3-propanediol. The pH of Tris is 7.5±0.05, and the buffer is 0.011 sodium azide (NaN3) Contains. Sodium azide acts as a preservative and inhibits catalase .

Vial C contains a combination of anti-diguatoxin horseradish peroxidase. There's something in there. This reagent is lyophilized together with the buffer reagent in vial B at 4 degrees Celsius. The safety of buffers stored in a freeze-dried state is The quality is 8 months, and it is stable for 1 month when reduced with 15 liters of distilled water.

7a The stability of the combined product in the dry state is 8 months, and when reduced, it is stable for 1 month. The vial contains the substrate, in this case 4-chloro-1-naphthol. It's in.

Small bottle B and small bottle I! Similar to LC, this specimen was lyophilized at 4 degrees Celsius. It is desirable to store 15% of distilled water and 30% of hydrogen peroxide solution. It can be reduced by dipping (1.5+sl). The stability of the small bottle in the freeze-dried state is 8. It is a month, and when reduced to one day. This substrate is produced by the enzyme horseradish reacts and changes with oxidase.

Vial E contains the distillate used to reduce the lyophilized reagents in the other vials. It contains water.

The small 11th floor contains acetone, a precipitant for serum specimens.

Takumi 1 Shin Shin Diguatoxin extract was prepared by Y. Hokama et al. (1977)) (such means are incorporated herein by reference). (included in the book). The diguatoxin crude extract was diluted with methanol. Next diluted. The dilution of the extract used is 50.25.12.5.6.2 5.3.63.1.7f3.0.8B, 0.44.0.22 and 0.11peng/ It was ugly. The concentration of the ziguatoxin specimen is determined by coating the ziguatoxin with correction fluid. The cells were assayed by attaching to bamboo sticks. The resulting toxin - The support composite was fixed with 0.3% hydrogen peroxide and 97.1% methanol. , air dry and remove unfixed toxins and residual fixative. Rinse with 5M Tris HCl, pH 7.5, where the settled toxin-bearing The body was treated with a solution containing an antitoxin combined with horseradish (wasabi radish) peroxidase. Diguatoxin was detected by immersion in the liquid. Extra antitoxin-enzyme conjugate was removed by washing with 0.05 M Tris I'[C1, pH 7.5. removed.

Horseradish peroxidase was added to 0.05M Tris HC1, pH 7.5. ．． 4. Dissolve 25s+1 of Iris Peroxide Water Slip in 0.125ml of absolute ethanol. - Mix with 101 g of chloro-1-naphthol to remove insoluble residues. Substrate produced by filtering with Whatman Number 1 filter paper for The analysis was performed by immersing the antitoxin-immobilized support in the solution. enzyme assay The color change of the reaction indicates that the reacted substrate solution is in a 96-well microtiter plate. Pipette into a 405 nanometer sample in a microplate spectrophotometer. ) was measured by determining the optical density of the specimen at (0,D, ). Obtained The results are summarized in Table 1.

议1 3.63 0.0B5 0.078 8.25 0.095 0.09 Table 1 shows that the optical density at 405 nm obtained in the reaction is increases with increasing concentration, but decreases at higher concentrations of diguatoxin. This indicates a dangerous situation.

Pan-λ ゛　Hi　　Sh　Shin　・Se Routine human serum specimens were obtained from volunteers. Serum of people with ziguatoxin poisoning Next, different concentrations of diguatoxin were added to Tsutomu's serum.

8.25.3.63.1. Flo, 0,88.0.44.0.22 and 0.11 layers Using diguatoxin at a concentration of g/111, serial dilutions of diguatoxy 1 ml of normal serum in which 10% of the serum was produced was added to the diguatoxin preparation. The specimen Mixed and allowed to stand at 37 degrees Celsius for 1 hour.

Serum specimens without diguatoxin were used as control specimens. There specimens were assayed for diguatoxin as described in Example 1, but only For serum/diguatoxin preparations, add 77 tons of 5 μl per 1 μl of serum. It was precipitated by this. The precipitate was centrifuged at 1,500 x g for 10 minutes. was separated from the supernatant by The supernatant liquid is recovered and dried under air flow. The residue is resuspended in 1+al of anhydrous methanol. result The resulting reaction mixture was measured on a microtiter plate spectrophotometer. Ta.

The results obtained were compared to those obtained with the diguatoxin specimen without the addition of serum. Compare with the results obtained. The results are summarized in Table 1.

deep red Diguatoxin parallel action! Ω row of 4 animals 0.11 0.0B4 0.088 0.22 0.09 0.081 0.44 0.093 0.091 0.8B 0.104 0.099 1.78 0.094 0.114 3, F13 0.0B5 0.108 B, 25 0.095 The results presented in Table I show that diguatoxin was that the results can be detected by the above-mentioned means after the This shows that the results can be compared with the actual results.

In an additional series of experiments, 25.12.5.6.25.3. 63 and 1. Serial dilutions of Diguatoxin, with concentrations of Diguatoxin of Flo. was produced, 1 liter of serum was added and allowed to stand. Each specimen was washed with acetone. Regarding the diguatoxin added, the above method was used except that the precipitation means was omitted. Assayed. The results are summarized in Table ■.

Table 1 The 0.0° result for ziguatoxin 405n11 is due to the increase in ziguatoxin. Although it shows an increase in 0,D,, the background value is high.

Legend”- Shi-ni 1 Shi-to-shin's-7 Serum specimens were collected from patients from New Chassis and from the Marshall and Majuro Islands. Obtained from. These serum specimens indicate a clinical condition classified as ciguatera intoxication. It was obtained from a patient who showed symptoms.

Serum specimens from patients showing properties of ciguatera intoxication were tested without acetone precipitation. , analyzed using the method described above.

Results of data from patient and normal serum were compared.1 Normal human serum is 0° A baseline is formed at 106±0.009. The disease was exaggerated as ciguatera poisoning. All sera from individuals ranged from 0.130±0.023 to 0.237±0.053. It had a higher than normal optical density value in the range of .

The average optical density of normal human serum is 0.108±0.009, while The average optical density of patients with known Ciguatera poisoning was 0.173 ± 0.036. The results of such a comparison of 0 normal and patient serum are summarized in Table ■.

deep red lji LIJ! I Anus-1-1 10,187±o, oa.

2 0.143±0.021 0.19Q±0.0563 0.157±0.0 20 0.161±0.0254 0.237±0.053 0.2138±0 ．． 0495 0.159±0.029 0.11B±0.0156 0.130 ±0.023 0°167±0.0457 0.125±0.024 Average 0.169±0.038 0.176±0.082B O, 19B±0. 070 0.095±0.062 Total average 0.173±0.036 0.09 5 cases 1! t8 had been treated with plasmapheresis. others All patients had been treated with mannitol.

Pass! ■Blood fake 1 U! ! ! No.11 No.11 Ryofuo Hitoshi 1 0.107 0.08B 0.1 04 0.100±0.0102 0.107 0.102 0.093 0. 101±0.0073 0.09B 0.105 0.107 0.103±o , oos4 0.097 0.121 0.098 0.105±0.0141 As a result, the measured value was 0.198±0.070 for the serum of 2 patients. death However, after the plasma export procedure, the measured value decreased to 0.095, with a deviation of 0.095. It was 101. The results show that before the plasmapheresis procedure, the patient , had high concentrations of diguatoxin, and by plasmapheresis treatment. , the concentration of ziguatoxin is at normal levels (i.e., contains ziguatoxin (not serum levels).

Another group of patients had a mixed total mean value of 0.169. these The patient experienced exudation of 250 ml of 203 mannitol within the first 24 hours of exudation. was treated.

Light of the reacted mixture after leaching of mannitol, treatment of diguatoxin poisoning The density had increased to 0.176±0.062.

From Table■, it can be seen that the optical density values of most patients increased after mannitol treatment. it is obvious. Mannitol treatment simply removes diguatoxin from the affected group. It has been hypothesized that it is only displaced from the tissues and transferred to the bloodstream. Therefore, mannit It is expected that optical density measurements will increase following treatment.

Translation of written amendment submitted Article 184-7-1 of the Patent Law) June 18, 1993

Claims (1)

[Claims] 1. To determine the presence of ciguatoxin or related polyether marine toxins in human serum. A method comprising the following steps for detection: isolating the serum specimen; a step of attaching the toxin contained in the precipitated serum specimen to a support; toxin-support complex; fixing the toxin to the support using a fixing solution to form a compound; The toxin- rinsing the support complex in a buffer; forming a toxin-antitoxin-enzyme complex; immobilizing the antitoxin-enzyme conjugate to the toxin-support complex; Unfixed antivenom by washing the toxin-antitoxin-enzyme complex with a buffer. removing the toxin-antitoxin-enzyme complex; and using the toxin-antitoxin-enzyme complex as a substrate for the enzyme. assaying the enzyme of the toxin-antitoxin-enzyme complex by reacting; Step of measuring the amount of product formed by the enzyme. 2.Furthermore, adding acetone to the serum specimen; mixing the acetone serum specimen; separating the precipitated material; The process of transferring the acetone fraction to another container and under air flow to form a residue. Drying the acetone and redissolving the residue with anhydrous methanol. , the serum specimen is further processed by precipitation of an acetone-insoluble material, The method according to claim 1. 3. The support according to claim 1, wherein the support comprises a hard member coated with a toxin-absorbing material. Method. 4. The method of claim 3, wherein the rigid member comprises a bamboo stick. 5. The method of claim 3, wherein the toxin absorbing material comprises liquid paper. 6 The antitoxin-enzyme conjugate antitoxin is a monoclonal compound directed against ciguatoxin. 2. The method of claim 1, comprising an anti-inflammatory antibody. 7 Fixer is 99.7% (v/v) methanol and 0.03% (v/v) peroxide 2. The method of claim 1, comprising hydrogen. 8. The buffer according to claim 1, wherein the buffer comprises 0.05M Tris-HCl having a pH of 7.5. Method. 9. Claim 1, wherein the enzyme of the antitoxin-enzyme conjugate comprises horseradish peroxidase. Method described. 10. Claim 1, wherein the substrate contains hydrogen peroxide and 4-chloro-1-naphthol. Method described. 11 The means for measuring the amount of product in an enzyme assay is determined by the color of the reaction mixture after the enzyme reaction. 2. A method as claimed in claim 1, comprising comparing the color chart with a color chart. 12 The means for measuring the amount of the product of the enzyme assay is 405 nm in a spectrophotometer. 2. The method of claim 1, comprising measuring the optical density of the reaction mixture at . 13. Ciguatoxin or related polyester in human serum with the following composition: Kit for detecting the presence of marine toxins. A precipitation solution for precipitating high molecular weight molecules from serum specimens; a support for fixing the toxin; a fixing solution for fixing the toxin on the support; a fixed support; a buffer for washing the carrier; an antitoxin-enzyme conjugate for immobilizing the toxin; Substrate for assaying enzymes of antitoxin-enzyme conjugates; in enzyme assays A means for measuring the amount of product formed by an enzyme. 14. Claim 13, wherein the support comprises a hard member coated with a toxin-absorbing material. Kit included. 15. The kit according to claim 14, wherein the rigid member comprises a bamboo stick. 16. The kit according to claim 13, wherein the toxin absorbing material comprises liquid paper. t. 17 Fixer is 99.7% (v/v) methanol and 0.03% (v/v) peracid 14. The kit according to claim 13, comprising hydrogen chloride. 18. The kit according to claim 13, wherein the precipitation solution contains acetone. 19 Claim wherein the buffer comprises 0.05M Tris-HCl, pH 7.5±0.05 The kit according to paragraph 13. 20 Claim wherein the buffer further comprises 0.01% (w/v) sodium azide The kit according to paragraph 13. 21 Antitoxin-enzyme conjugate antitoxin is a monoclonal antibody directed against the toxin 14. The kit of claim 13, comprising: 22 Claim No. 2, wherein the enzyme of the antitoxin-enzyme conjugate consists of horseradish peroxidase. The kit according to item 13. 23 Claim No. 2, wherein the substrate comprises hydrogen peroxidase and 4-chloro-1-naphthol The kit according to item 13. 24 The means for measuring the amount of product in an enzyme assay is determined by the color of the reaction mixture after the enzyme reaction. 14. The kit of claim 13, wherein the kit is for comparing the color chart with a color chart. 25 The means for measuring the amount of the product of the enzyme assay is 405 nm in a spectrophotometer. 14. The means according to claim 13, for measuring the optical density of a reaction mixture at .