TIME-RESOLVED IMMUNOQUANTITATION TEST STRIP FOR DETECTING TETRODOTOXIN IN SHELLFISH FOOD

Abstract

The present invention discloses a time-resolved immunoquantitation test strip for detecting tetrodotoxin (TTX) in shellfish food, which belongs to the technical field of rapid detection of time-resolved immunoassay. In the present invention, fluorescent microspheres are adopted to replace the traditional colloidal gold; the fluorescent microsphere is used for labeling a TTX antibody complex; by utilizing a competitive immunization method, the fluorescent microsphere, serving as a fluorescent probe, is used for immunochromatography; and by reading the fluorescence value of a detection line on a fluoroimmunoassay instrument, the TTX in shellfish samples can be analyzed quantitatively and rapidly. The time-resolved immunoquantitation test strip of the present invention can be used for detecting the content of the TTX in various types of shellfish food quantitatively and rapidly and is strong in specificity and high in sensitivity, wherein when the concentration of the TTX is 0.5-40 ng/mL, the logarithmic value of the concentration has a linear relationship with T/To, a linear equation is: Y=0.57365-0.2668LgX, R.sup.2=0.9940, and the limit of detection can reach 0.047 ng/mL.

Claims

1. A time-resolved immunochromatography quantitative test strip for detecting tetrodotoxin (TTX), comprising a sample pad, a nitrocellulose membrane (an NC membrane) and absorbent paper, wherein a detection line (a T line) and a quality control line (a C line) are arranged on the NC membrane; a TTX complete antigen (TTX-BSA) is coated on the detection line; and a biotin (biotin-BSA) is coated on the quality control line, wherein the dosage of the TTX complete antigen is 0.3-8 mg/mL, and the dosage of the biotin is 0.1-1 mg/mL.

2. The time-resolved immunochromatography quantitative test strip according to claim 1, wherein the detection concentration of the TTX is 0.1-1000 ng/mL.

3. The time-resolved immunochromatography quantitative test strip according to claim 1, wherein in a preparation method of the TTX complete antigen (TTX-BSA), the TTX complete antigen (TTX-BSA) is obtained by coupling a TTX standard substance and a carrier protein.

4. A method of detecting TTX in shellfishes by a time-resolved immunoquantitation test strip, comprising the following steps: (1) extraction of a to-be-detected substance: homogenizing shellfish meat, heating, cooling, centrifuging, and collecting supernate; and then, degreasing to obtain the to-be-detected substance; (2) uniformly mixing a Eu.sup.3+-fluorescent microsphere labeled TTX monoclonal antibody Eu-TTX-mAb, a Eu.sup.3+-fluorescent microsphere labeled streptavidin Eu-SA, the to-be-detected substance and buffer solution for hatching, so as to obtain to-be-detected solution; (3) adding the to-be-detected solution obtained in step (2) into the sample pad in the time-resolved immunochromatography quantitative test strip described in the present invention, and then determining the fluorescence intensity value at the detection line of a sample: a T value; and (4) substituting the obtained T value into a quantitative curve, so as to obtain the concentration of the TTX in the to-be-detected solution.

5. The method according to claim 4, wherein the volume ratio of the Eu.sup.3+-fluorescent microsphere labeled TTX monoclonal antibody Eu-TTX-mAb, the Eu.sup.3+-fluorescent microsphere labeled streptavidin Eu-SA, the to-be-detected substance and the buffer solution in step (2) is 2:10:45:35.

6. The method according to claim 4, wherein the hatching in step (2) is carried out at 20-30° C. for 10-20 min.

7. The method according to claim 4, wherein the chromatography in step (3) is carried out at 37° C. for 15-20 min.

8. The method according to claim 4, wherein the quantitative curve in step (4) is Y=0.57365-0.2668LgX, R.sup.2=0.9940, IC50=1.713 ng/mL, and the limit of detection is 0.047 ng/mL, wherein Y represents T/To, and X represents the concentration of the TTX.

9. An application of the time-resolved immunochromatography quantitative test strip of claim 1 in the field of food detection.

10. An application of the method of claim 4 in the field of food detection.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0044] FIG. 1 is a transmission electron micrograph of a tetrodotoxin (TTX) antibody coupling to a fluorescent microsphere, wherein A is an electron micrograph of the antibody coupling to the fluorescent microsphere at a low magnification; and B is an electron micrograph of the antibody coupling to the fluorescent microsphere at a high magnification.

[0045] FIG. 2 is a diagram showing ultraviolet verification on a TTX complete antigen.

[0046] FIG. 3 is a structural diagram of an immune test stripe for TTX.

[0047] FIG. 4 is a diagram showing a standard curve A and a quantitative curve B of detecting TTX with a labeling fluorescent microsphere immunochromatographic assay and a test strip diagram C under an ultraviolet lamp.

[0048] FIG. 5 is a diagram showing optimization on concentration of a complete antigen of a detection line (T line) with TTX immunochromatographic assay.

[0049] FIG. 6 is a diagram showing T line immunodynamic curves of TTX immunochromatographic assay, wherein with fluorescence intensity values and T/C as ordinates and reaction times as abscissas, an immunoreaction kinetic curve (A) is drawn; and (B) shows changes on fluorescence intensity value at different immunoreaction times.

[0050] FIG. 7 is a diagram showing a standard curve A and a quantitative curve B of detecting TTX in shellfish.

[0051] FIG. 8 is a diagram showing a TTX specificity test.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0052] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention, but not used for limiting the present invention. In the embodiments, used streptavidin-fluorescent microspheres are purchased from Suzhou VDO Biotech Co., Ltd. with a solid content of 1% and are used after being diluted by 500 folds by a self-made microsphere reconstituted solution, wherein the self-made microsphere reconstituted solution is 0.05 M Tris-HCl containing 5% sucrose, 1% bovine serum albumin and 1% Tween-20 and having a pH value of 7.0.

Embodiment 1 Preparation of Tetrodotoxin Complete Antigen (TTX-BSA)

[0053] 1. Preparation of tetrodotoxin complete antigen (TTX-BSA)

[0054] A TTX standard substance was coupled to bovine serum albumin (BSA) to obtain an artificial antigen

[0055] 0.5 mg of TTX was taken and dissolved into 1 mL of ultrapure water, and a mixture was stirred for clarification, denoted as a reaction liquid A; 4 mg of BSA was weighed and dissolved into 2.0 mL of PBS (pH 7.4) to obtain a protein solution; the reaction liquid A was slowly added to the protein solution dropwise for uniform mixing, denoted as a reaction liquid B; 37% formaldehyde solution was slowly added to the reaction liquid B dropwise until a final volume fraction was 1.5%, and uniform mixing was performed; then, a mixed solution was slowly shaken in a water bath at 30° C. for reacting for 72 h and was dialyzed for 2 d at 4° C. by using 0.01 M phosphate buffer solution (PBS) with changing dialysate for 3 times every day, so as to remove residual free TTX; and a TTX-BSA conjugate was obtained, i.e., the TTX complete antigen (TTX-BSA), and was preserved at −20° C.

[0056] 2. Identification of TTX antibody

[0057] The TTX standard substance, a carrier protein and the TTX complete antigen were subjected to ultraviolet (200-300 nm) scanning determination for comparing differences among the three.

[0058] A result is shown in FIG. 2. From FIG. 2: compared with spectrum shapes or peak values of the TTX standard substance and the carrier protein, a spectrum shape or a peak value of the TTX complete antigen have an obvious change, which shows that synthesis of the TTX complete antigen is successful.

Embodiment 2 Preparation of Fluorescent Probe for Eu.SUP.3+.-Fluorescent Microsphere Labeled TTX Monoclonal Antibody

[0059] Preparation of Relevant Solution:

[0060] Activation buffer: 0.05 M 2-(N-morpholino) ethanesulfonic acid (MES, C.sub.6H.sub.13NO.sub.4SH.sub.2O) solution with a pH value of 6.0;

[0061] coupling buffer: 0.01 M phosphate buffer solution (PBS) with a pH value of 7.0 (a solvent with free amine is avoided from being used);

[0062] activator: 2 mg/mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, C.sub.8H.sub.17N.sub.3HC.sub.1) solution and 2 mg/mL N-hydroxysuccinimide (NHS, C.sub.4H.sub.5NO.sub.3) solution; blocking buffer: 0.01 M phosphate buffer solution (PBS) containing 1% BSA and 0.05% Tween-20 and having a pH value of 6.0;

[0063] antibody basic protective liquid: 0.05 M Tris-HCl containing 5% trehalose, 0.1% PVPK-30, 0.5% BSA and 0.1% TritonX-100 and having a pH value of 7.0;

[0064] label washing liquid: 0.05 M Tris-HCl containing 0.1% Tween-20 and having a pH value of 7.0; and

[0065] microsphere reconstituted solution/reaction slow-release liquid: 0.05M Tris-HCl containing 5% sucrose, 1% bovine serum albumin and 1% Tween-20 and having a pH value of 7.0.

[0066] A specific preparation method for Eu-TTX-mAb comprises the following steps:

[0067] (1) 10 μL of fluorescent microsphere solution wrapping Eu′ therein and modified with a carboxyl functional group (purchased from Taizhou Bionano New Material Science Co., Ltd. and having a particle diameter of 300 nm) (having a solid content of 1%) placed at 4° C. was taken for ultrasonic dispersion, the activation buffer with a concentration of 900 μL was added for centrifugation for 5 min at 15000 rpm and 4° C.

[0068] (2) A supernate was discarded, 600 μL of activation buffer was added for ultrasonic resuspension, and centrifugal cleaning was repeated for 3 times.

[0069] (3) A supernate was discarded, 200 μL of activation buffer was added for ultrasonic resuspension, 5 μL of 2 mg/mL EDC solution and 5 μL of 2 mg/mL NHS solution were added as the activator, and oscillation was performed in a shaker in the dark for 30 min at a room temperature and 500 rpm.

[0070] (4) After activation was completed, centrifugation was performed; a supernate was discarded; and the PBS was added for cleaning for 3 times.

[0071] (5) A supernate was discarded, 400 μL of coupling buffer was added for ultrasonic resuspension, then 10 μg of TTX monoclonal antibody (references for a preparation method: ([1] Lei Cong. Preparation of Tetrodotoxin Specific Monoclonal Antibody [D]. Shanghai Ocean University, 2011)) was added, and oscillation labeling was performed for 3 h in the dark at the room temperature.

[0072] (6) After labeling was completed, 10% (v/v) blocking buffer and 100 μL of antibody basic protective liquid were added, and oscillation was performed for 40 min in the dark at the room temperature.

[0073] (7) After blocking, centrifugation was performed to discard a supernate, a resultant was cleaned for 3 times with 900 μL of label washing liquid.

[0074] (8) A supernate was discarded, 200 μL of fluorescent microsphere reconstituted solution was added, and a Eu.sup.3+-fluorescent microsphere labeled TTX monoclonal antibody Eu-TTX-mAb (a transmission electron micrograph shown as FIG. 1) was obtained and preserved at 4° C. for standby application.

[0075] From FIG. 1: the Eu-TTX-mAb is stable and controllable in surface functional group, good in experiment repetition, strong in detection specificity, capable of achieving rapid quantitative detection, high in sensitivity and small in error and thus provides great convenience and rapidness for immediate detection.

Embodiment 3 Preparation of TTX Immune Test Stripe Based on Labeling Fluorescent Microsphere

[0076] A method of preparing a time-resolved immunochromatography quantitative test strip for detecting TTX, comprising the following steps:

[0077] (1) A tetrodotoxin complete antigen (TTX-BSA) solution (0.4 mg/mL) and a biotin solution (0.3 mg/mL) were sprayed on a nitrocellulose membrane (NC membrane) with the spraying amount in each of 1 μL/cm (an instrument parameter, referring to that 1 μL of to corresponding solution was sprayed on the NC membrane once a nozzle moves by 1 cm every time) to serve as a detection line (T line) and a quality control line (C line). Widths of the T line and the C line depend on a diameter of a pipeline of a membrane spraying instrument and are 2 mm; a distance between the T line and the C line is about 5 mm; and the T line and the C line are dried for 3 h at 37° C. An XYZ3050 model three-dimensional dispensing platform from American BioDot Inc. is employed for spraying.

[0078] A sample pad, the nitrocellulose membrane and absorbent paper were adhered to a PVC basal plate to assemble a test strip. The key for assembly of the test strip is to guarantee consistent transitivity among various parts, wherein the T line is distant from the sample pad by about 5 cm; the C line is distant from the absorbent paper by about 5 cm; the sample pad overlaps on the nitrocellulose membrane with an overlap of about 3 mm; and similarly, the absorbent paper overlaps on the nitrocellulose membrane with an overlap of about 3 mm.

[0079] The plate subjected to adhesion was cut into the test strip with a width of about 4 mm by a strip cutter, the test strip was assembled by using a plastic base and a clamping housing to obtain an immunochromatography quantitative test strip, and the immunochromatography quantitative test strip was sealed and preserved at 4° C. for standby application.

[0080] A structural diagram of the immunochromatography quantitative test strip is shown as FIG. 3. The sample pad, the nitrocellulose membrane and the absorbent paper are sequentially provided on the basal plate from left to right.

Embodiment 4 Drawing of Standard Curve of Tetrodotoxin Immunochromatography Quantitative Test Strip

[0081] A drawing method of a standard curve comprises the following steps:

[0082] A TTX standard substance was taken and added to a phosphate buffer solution (pH 7.0,0.01M) to prepare standard solutions with concentrations of TTX of 0 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.5 ng/mL, 1 ng/mL, 4 ng/mL, 8 ng/mL, 20 ng/mL, 40 ng/mL, 100 ng/mL, 200 ng/mL, 800 ng/mL and 1000 ng/mL respectively; and the standard solutions were used for fluorescent test strip detection.

[0083] The Eu.sup.3+-fluorescent microsphere labeled TTX monoclonal antibody (Eu-TTX-mAb) prepared in embodiment 2 served as a fluorescent probe; 45 μL of standard solution, 2 μL of fluorescent probe (Eu-TTX-mAb solution: 0.05 μg/μL fluorescent microsphere labeled TTX monoclonal antibody, 0.1 M Tris-HCl solution containing 5% sucrose, 1% bovine serum albumin and 1% Tween-20 and having a pH value of 7.5), 10 μL of streptavidin-fluorescent microsphere and 35 μL of standard substance buffer (the standard substance buffer is 0.01 M PBS (pH 7.4) containing 5% methanol (v:v)) were uniformly mixed; and a mixture was hatched for 10 min at a room temperature to obtain 92 μL of to-be-detected solution.

[0084] Then, 92 μL of to-be-detected solution was slowly added to the sample pad of the immunochromatography quantitative test strip in embodiment 3 dropwise for chromatography for 20 min at 37° C.; a fluorescence value of the T line on the test strip was recorded by using an HG-98 immunoquantitation analyzer with 6 replicates tested for each concentration; and setting a fluorescence value of the T line of the standard solution with the concentration of 0 ng/mL as T.sub.0 and fluorescence values of the T lines of the standard solutions with other concentrations as T, the standard curve was drawn with a logarithmic value of the concentration of each TTX standard substance as an abscissa and T/T.sub.0×100 (%) as an ordinate, wherein a competitive inhibition rate was set as (1-T/T.sub.0)×100%.

[0085] From A in FIG. 4, with the increase of the concentration of the TTX, fluorescence on a band of the T line of the test strip would become lighter and lighter, so T/To would become smaller and smaller. As shown in FIG. 4, B is a change curve (quantitative curve) of T/To along with the concentration of TTX; when the concentration of the TTX is 0.5-40 ng/mL, the logarithmic value of the concentration of the TTX has a linear relationship with T/T0; a linear equation is: Y=0.57365-0.2668LgX, R.sup.2=0.9940, and IC50=1.713 ng/mL; and a limit of detection is 0.047 ng/mL.

Embodiment 5

[0086] A method of detecting tetrodotoxin in shellfishes by a time-resolved immunoquantitation test strip, comprising the following steps:

[0087] (1) extraction of a to-be-detected substance:

[0088] 5 g of shellfish meat was collected, 25 mL of 0.1% glacial acetic acid solution was added for homogenizing, and a water bath was performed at 100° C. for 10 min after homogenizing; standing was performed for cooling, centrifugation was performed at 10,000 r/min for 5 min, a supernate was collected, an appropriate amount of 0.1% glacial acetic acid solution was added to precipitates for uniform mixing, and a water bath was performed for 5 min; standing was performed for cooling, centrifugation was performed at 10,000 r/min for 5 min, a supernate was collected, and the above step was repeated for precipitates once; the three supernates were collected to a constant volume of 50 mL; then, 5 mL of chloroform was added for degreasing for 30 min, during which shaking was performed for uniform mixing 5 times, centrifugation was performed at 10,000 r/min for 3 min to separate an aqueous phase and chloroform, an aqueous phase was collected, degreasing was performed once again, and a pH value of the aqueous phase was adjusted to 7.0 with NaOH, so as to obtain the to-be-detected substance; and if not being detected directly, the to-be-detected substance might be preserved at 4° C. (without adjusting the pH value) (GB/T5009).

[0089] (2) 2 μL of Eu.sup.3+-fluorescent microsphere labeled TTX monoclonal antibody Eu-TTX-mAb, 10 μLof Eu.sup.3+-fluorescent microsphere labeled streptavidin Eu-SA, 45 μL of to-be-detected substance and 35 μL of buffer solution were uniformly mixed for hatching for 10 min at a room temperature, so as to obtain 92 μL of to-be-detected solution.

[0090] (3) The to-be-detected solution obtained in step (2) was added to the sample pad in the time-resolved immunochromatography quantitative test strip described in embodiment 3 for chromatography for 20 min at 37° C., and then a fluorescence intensity value (: a T value) at the detection line of the to-be-detected solution was recorded by the HG-98 immunoquantitation analyzer.

[0091] (4) The obtained T value was substituted into the quantitative curve in embodiment 4, so as to obtain the concentration of the TTX in the to-be-detected solution.

Embodiment 6 Condition Optimization

[0092] (1) Optimization of tetrodotoxin complete antigen

[0093] In order to improve the detection sensitivity of a time-resolved immunoquantitation test strip, the effect of different dilution concentrations of the TTX complete antigens on a competitive inhibition rate between a negative control group and a sample with the TTX content of 0.25 μg/L in the experimental process is researched.

[0094] The specific experimental exploration process is as follows:

[0095] the concentration of a TTX complete antigen (TTX-BSA) solution sprayed at a detection line T in embodiment 3 was adjusted; specifically, dilution folds of the TTX-BSA were 5, 10, 15, 20 and 25, concentrations were specifically 1.6 mg/mL, 0.8 mg/mL, 0.5 mg/mL, 0.4 mg/mL and 0.3 mg/mL, and other kept consistent to those in embodiment 3; and then the time-resolved immunoquantitation test strips for different concentrations of the TTX complete antigen were obtained.

[0096] The negative control group (5% methanol-PBS) and a positive test group (with the TTX concentration of 0.25 μg/L) were analyzed, and then the effect of different concentrations of the TTX-BSA on a detection method was evaluated.

[0097] From FIG. 5, with the increase in the concentration of the TTX-BSA at the T line, the fluorescence intensity between the negative control group (5% methanol-PBS) and a positive test group (with the TTX concentration of 0.25 μg/L) tends to be stable after being gradually increased; and when the dilution fold of the TTX-BSA is 20, and the concentration of the TTX complete antigen (TTX-BSA) solution is 0.4 mg/mL, the competitive inhibition rate (1-T/T.sub.0) between a negative sample and a positive sample reaches a maximal value of 0.3. Also, at this time, the detection line also has a comparatively higher fluorescence signal value of 343000 a.u. (the fluorescence intensity of the negative control: 499171 a.u.). Therefore, 20-fold dilution is a preferred dilution fold of the TTX-BSA complete antigen at the T line, and a concentration of 0.4 mg/mL is a preferred concentration of the TTX-BSA complete antigen at the T line.

[0098] (2) Optimization on quality control line of time-resolved immunoquantitation test strip

[0099] The biotin solution of the quality control line in embodiment 3 was adjusted to be a goat anti-mouse IgG solution; other kept consistent to those in embodiment 3; and the time-resolved immunoquantitation test strip was obtained.

[0100] The three samples confirmed to be negative by UPLC-MS/MS were labeled; the time-resolved immunoquantitation test strips with two kinds of quality control lines were selected for detecting positive samples with concentrations of 0.1 ng/mL, 0.5 ng/mL and 2 ng/mL (TTX) respectively; a mean concentration value, a standard deviation and a variable coefficient of the positive samples were calculated; and the accuracy and the precision of the time-resolved immunoquantitation test strip were evaluated.

TABLE-US-00001 TABLE 1 Experimental comparison on precision and stability of time-resolved immunoquantitation test strips with different quality control lines Meretrix Labeling meretrix Quality concen- Astarte linnaeus Clam control Target tration Mean Recovery CV Mean Recovery CV Mean Recovery CV line object (ng/mL) (ng/mL) rate (%) (%) (ng/mL) rate (%) (%) (ng/mL) rate (%) (%) biotin- TTX 0.1 0.096 100.52 3.66 0.101 99.90 1.90 0.104 99.51 3.27 BSA 0.5 0.460 101.48 4.70 0.492 100.29 2.90 0.544 98.49 3.13 2 2.266 97.08 3.21 1.848 101.86 0.25 2.143 98.39 3.39 Goat 0.1 0.116 115.96 1.02 0.149 148.74 5.96 0.129 129.35 1.88 anti- 0.5 0.578 115.63 7.06 0.552 110.34 11.97 0.753 150.53 18.26 mouse 2 1.917 95.84 14.63 2.135 106.73 78.65 1.949 97.46 24.33 IgG

[0101] Results are shown in Table 1. Determination results CV of the quality control line being biotin-BSA are all smaller than 4.70%, and the recovery rates are 97.08%-101.48%. However, the recovery rate of a traditional test strip with a goat anti-mouse IgG quality control line is large in fluctuation range and between 95.84% and 150.53%.

[0102] (3) Optimization on chromatography time

[0103] A chromatography time of step (3) in embodiment 5 was adjusted to be 0-20 min, and other kept consistent to those in embodiment 5.

[0104] With the fluorescence intensity values as ordinates and reaction times as abscissas, the immunoreaction kinetic curve was drawn, changes of the fluorescence intensities of the T line and the C line over time were observed, and the time that the fluorescence value of the T line became stable served as a preferred detection time.

[0105] FIG. 6 is a diagram showing T line immunodynamic curves of TTX immunochromatographic assay. From A in FIG. 6, the fluorescence intensities of the two lines both present a strengthening trend within 0-20 min over time. After 20 min, the fluorescence intensity values of the T line and the C line have no obvious changes and trend to be stable. From B in FIG. 6, the fluorescence intensity between the negative control group (5% methanol-PBA) and the positive control group (TTX=0.5 μg/L, 2 μg/L) trends to be stable at 20 min Therefore, it is relatively proper to select 20 min as the chromatography time.

Embodiment 7

[0106] 1. Precision test

[0107] 8 TTX time-resolved immunoquantitation test strips were selected from one batch for detecting fluorescence intensity values (To) at detection lines of a negative control group (5% methanol-PBA) and fluorescence intensity values (T) at T lines of a positive control group (TTX 0.25 μg/L), and intra-batch differences were analyzed.

[0108] 8 TTX time-resolved immunoquantitation test strips in different batches were used for detecting the fluorescence intensity values (To) at the detection lines of the negative control group (5% methanol-PBA) and the fluorescence intensity values (T) at the T lines of the positive control group (TTX 0.25 μg/L), and inter-batch differences were analyzed.

[0109] From Table 2, through analysis with a variable coefficient computational formula, intra-batch variable coefficients of T.sub.0, T and T/To (%) are 7.61%, 7.51% and 2.37% respectively, and inter-batch variable coefficients of T.sub.0, T and T/T.sub.0 (%) are 6.53%, 6.90% and 2.81% respectively, indicating that the time-resolved immunoquantitation test strips are small in intra-batch variable coefficients and inter-batch variable coefficients, high in precision and good in accuracy and basically meet the requirements of the quantitative detection test strip.

TABLE-US-00002 TABLE 2 Detection results of precision of time-resolved immunoquantitation test strip for TTX Intra- T/T.sub.0 Inter- T/T.sub.0 SD batch T.sub.0 T (%) batch T.sub.0 T (%) (%) 1 398312 318000 79.84% 1 465546 372218 79.95% 1.15% 2 428656 330281 77.05% 2 426906 321875 75.40% 1.12% 3 458968 346031 75.39% 3 390765 296437 75.86% 0.60% 4 465875 346187 74.31% 4 449250 341484 76.01% 0.58% 5 464765 365656 78.68% 5 449250 337875 75.21% 3.19% 6 455546 350687 76.98% 6 458125 348968 76.17% 0.57% 7 453734 350078 77.15% 7 470843 340468 72.31% 2.89% 8 377812 284812 75.38% 8 407343 314859 77.30% 1.43% Mean 437959 336467 76.85% Mean 439754 334273 76.03% SD 33324 25278 1.82% SD 28698 23057 2.14% CV (%) 7.61% 7.51% 2.37% CV (%) 6.53% 6.90% 2.81%

[0110] 2. Accuracy test

[0111] Sample Treatment:

[0112] With regard to a sample pre-treatment mode of shellfish products, 5 g of shellfish meat was collected, 25 mL of 0.1% glacial acetic acid was added for homogenizing, and a water bath was performed at 100° C. for 10 min after homogenizing; standing was performed for cooling, centrifugation was performed at 10,000 r/min to collect a supernate, an appropriate amount of 0.1% glacial acetic acid was added into precipitates for uniform mixing, and a water bath was performed for 5 min; standing was performed for cooling, centrifugation was performed at 10,000 r/min for 5 min, a supernate was collected, and the above step was repeated for precipitates once; the three supernates were collected to a constant volume of 50 mL; then, 5 mL of chloroform was added for degreasing for 30 min, during which shaking was performed for uniform mixing 5 times, centrifugation was performed at 10,000 r/min for 3 min to separate an aqueous phase and chloroform, an aqueous phase was collected, degreasing was performed once again, and a pH value of the aqueous phase was adjusted to 7.0 with NaOH, so as to obtain the to-be-detected substance; and if not being detected directly, the to-be-detected substance might be preserved at 4° C. (without adjusting the pH value) (GB/T5009).

[0113] In order to verify the accuracy and the sensitivity of the TTX test strip, labeled recovery tests are performed on several kinds of shellfish samples (astarte, clams and Meretrix meretrix linnaeus); addition concentrations of each sample are set to be there different labeling concentrations in a high group, a medium group and a low group; and for each group of concentration gradient, three parallel tests are set. With the labeled recovery rate as an accuracy evaluation index, a relative standard deviation (RSD %) of a detection result of a certain concentration of sample is repeatedly determined as a precision evaluation index. Computational formulas of the labeled recovery rate and the relative standard deviation are shown as following formula (1) and formula (2):

[00001]$\begin{array}{cc}\mathrm{Labeled}\mathrm{recover}\mathrm{rate}\left(\%\right)=\frac{\mathrm{Actually}\mathrm{detected}\mathrm{concentration}\mathrm{of}\mathrm{labeled}\mathrm{sample}}{\mathrm{Labeling}\mathrm{concentration}}⨯100& \left(1\right)\end{array}$$\begin{array}{cc}\mathrm{Variable}\mathrm{coefficient}\left(\mathrm{CV},\%\right)=\frac{\mathrm{Standard}\mathrm{deviation}\left(\mathrm{SD}\right)}{\mathrm{Mean}}& \left(2\right)\end{array}$

[0114] TTX labeled recovery tests were performed on the samples confirmed to be negative according to three concentration gradients of 0.1 μg/L, 0.5 μg/L and 2 μg/L respectively. The test for each concentration was repeated for 10 times, a mean of T/To was taken, and a variable coefficient was calculated.

[0115] Results are shown in Table 3, the determination results CV are all smaller than 4.70%, which shows that in a linearity range, the time-resolved immunoquantitation test strip has relatively good accuracy. The whole exploration method takes the PBS containing 0.05% Tween-20 as a sample diluent, so as to guarantee the performance of the method.

[0116] 5 g of each of the astarte, the clams and the Meretrix meretrix linnaeus was collected, 25 mL of 0.1% glacial acetic acid was added to each for homogenizing, and a water bath was performed at 100° C. for 10 min after homogenizing; standing was performed for cooling, centrifugation was performed at 10,000 r/min for 5 min, a supernate was collected, an appropriate amount of 0.1% glacial acetic acid was added into precipitates for uniform mixing, and a water bath was performed for 5 min; standing was performed for cooling, centrifugation was performed at 10,000 r/min for 5 min, a supernate was collected, and the above step was repeated for precipitates once; the three supernates were collected to a constant volume of 50 mL; then, 5 mL of chloroform was added for degreasing for 30 min, during which shaking was performed for uniform mixing 5 times, centrifugation was performed at 10,000 r/min for 3 min to separate an aqueous phase and chloroform, an aqueous phase was collected, degreasing was performed once again, and a pH value of the aqueous phase was adjusted to 7.0 with NaOH; and resultants were diluted by 15 folds (astarte), 5 folds (clam) and 5 folds (Meretrix meretrix linnaeus) respectively for immunochromatographic assay.

TABLE-US-00003 TABLE 3 Detection results of accuracy of time-resolved immunoquantitation test strip for TTX Labeling Immunochromatographic test strip concentration Tested Actual Recovery CV Sample (ng/mL) T/T.sub.0 (%) T/T.sub.0 (%) rate (%) (%) Astarte 0.1 0.840450 0.844863 100.53% 3.66% 0.5 0.653965 0.663652 101.48% 4.70% 2 0.493335 0.478888 97.07% 3.21% Meretrix 0.1 0.840450 0.839679 99.91% 1.90% meretrix 0.5 0.653965 0.655895 100.30% 2.90% linnaeus 2 0.493335 0.502486 101.85% 0.25% Clam 0.1 0.840450 0.836378 99.52% 3.27% 0.5 0.653965 0.644125 98.50% 3.13% 2 0.493335 0.485355 98.38% 3.39%

[0117] From Table 3, by using the time-resolved immunoquantitation test strip for detecting the labeled shellfish samples, it is finally obtained that, for the shellfishes, the labeled recovery rate is between 97.07% and 101.85%, and the variable coefficient is lower than 4.70%, indicating that the time-resolved immunoquantitation test strip can be used for field rapid screening and detection.

[0118] 3. Standard curve for shellfish foods

[0119] In order to further guarantee the accuracy of the method in embodiment 5, with samples confirmed to be negative by LC-MS as a substrate, the TTX standard substance was added to the shellfish meat to prepare shellfish samples containing 0 ng/mL, 0.1 ng/mL TTX, 1 ng/mL TTX, 2 ng/mL TTX, 4 ng/mL TTX, 8 ng/mL TTX, 20 ng/mL TTX, 40 ng/mL TTX, 80 ng/mL TTX and 100 ng/mL TTX respectively, and a shellfish meat standard curve which was added to the shellfish meat to prepare shellfishsuitable for field immediate detection was drawn.

[0120] Results are shown in FIG. 7, when a concentration range of the TTX is from 0.1 ng/mL to 100 ng/mL, a logarithmic value of the concentration has a linear relationship with T/To; a linear equation is: Y=−0.22LgX+0.690, and R.sup.2=0.9976; and a limit of detection is 0.141 ng/mL.

[0121] 4. Cross reaction test

[0122] Other commonly used shellfish toxins (OA, DA, BTX, ATX and MC-LR) were selected for evaluating the specificity of the time-resolved immunoquantitation test strip.

[0123] The negative samples (5% methanol-PBS) and positive samples with different concentrations (0 ng/mL, 0.5 ng/mL, 1.25 ng/mL, 2.5 ng/mL, 5 ng/mL, 10 ng/mL and 50 ng/mL) of each toxin were detected by using the time-resolved immunoquantitation test strips in embodiment 3, and the fluorescence intensity values of the T lines thereof were recorded.

[0124] From FIG. 8 and Table 4, cross reaction between the TTX and other shellfish toxins is relatively high, which shows that the time-resolved immunoquantitation test strip is stable in performance, meets the market demands and has a wide market prospect.

TABLE-US-00004 TABLE 4 Results of cross reaction test Shellfish FL.sub.T (a.u.) toxins 0.5 ng/mL 1.25 ng/mL 2.5 ng/mL 5 ng/mL 10 ng/mL 50 ng/mL TTX 501250 404713 304286 275250 234760 128011 OA 505619 505200 504166 503437 501617 501086 DA 503422 504729 501687 506770 503922 508625 BTX 498656 481594 485766 498521 506703 500083 MC-LR 452864 478692 475179 469265 465390 480375 ATX 339807 333160 334197 338692 333505 339010

[0125] Although the present invention has been disclosed as above in the preferred embodiments, they are not intended to limit the present invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is subject to the protection scope in claims.