CN209764883U - ELISA plate and ELISA kit for detecting okadaic acid toxin in shellfish - Google Patents

Abstract

The utility model relates to an ELIAS plate and ELISA kit for detecting okadaic acid toxin in shellfish. The ELISA plate comprises an outer frame support and a plurality of enzyme label strips; the enzyme label strip comprises a plurality of enzyme label holes, and a coating layer, a sealing layer and a okadaic acid toxin monoclonal antibody layer are sequentially stacked on the inner wall of each enzyme label hole. Through the special structural design, the sensitivity and the detection range of the rapid detection of the okadaic acid toxins in the shellfish are further improved, so that the field detection of the okadaic acid toxins in the shellfish has feasibility.

Description

ELISA plate and ELISA kit for detecting okadaic acid toxin in shellfish

Technical Field

The utility model belongs to the biological detection field, concretely relates to ELIAS plate and ELISA kit for detecting okadaic acid toxin in shellfish.

Background

In recent years, due to frequent outbreaks of red tides, shellfish toxins produced by red tide algae have increasingly serious influence on marine fishery. It is generally accepted that contamination of shellfish toxins in seafood products will likely pose an acute or chronic health threat to human health.

Okadaic Acid (OA) is a small molecule diarrheal shellfish toxin that is predominantly present in algal plants. OA is a fat-soluble substance and is stable to general cooking treatment. OA is a major component of cathartic shell fish poison (DSP), is a colorless crystal, is soluble in organic solvents such as methanol, ethanol, chloroform and diethyl ether, and is insoluble in water.

There are many conventional methods for determining OA concentration, including mouse bioassay, High Performance Liquid Chromatography (HPLC) techniques, Protein Phosphatase Inhibition (PPI) detection, etc. The above method has limited practical application due to lack of specificity, time-consuming, false positive caused by interference of other substances, and biological ethical problems. Currently, high performance liquid chromatography coupled with various other detectors, such as liquid chromatography tandem mass spectrometry, liquid chromatography-fluorescence detection, is an effective method for identifying and quantifying OA, but is time consuming due to sample preparation, and requires expensive equipment and skilled operators.

There is a need to develop rapid testing and field testing methods. Point Of Care Testing (POCT) is a new subdivision industry Of In Vitro Diagnosis (IVD), and is a new method for analyzing samples at the sampling site immediately, saving the complex processing procedure Of the samples in laboratory inspectors and obtaining the test results quickly. The current POCT technology comprises biosensors, biochips, immune test strips and the like.

Among them, Enzyme-Linked ImmunoSorbent Assay (ELISA) has the advantages of being rapid, easy to operate, sensitive and specific, is still used as a reference or confirmation method by related food regulatory agencies at present, and is widely applied to the aspects of meat, food, agricultural products and the like.

Disclosure of Invention

An object of the utility model is to prior art not enough, provide an ELIAS plate for detecting the okadaic acid toxin in shellfish, further improve the sensitivity and the detection range of okadaic acid toxin short-term test in shellfish for the witnessed inspections of aquatic products okadaic acid toxin in shellfish has the feasibility.

The utility model provides a technical scheme does:

An ELISA plate for detecting okadaic acid toxin in shellfish comprises an outer frame bracket and a plurality of enzyme label strips; the enzyme label strip comprises a plurality of enzyme label holes, and a coating layer, a sealing layer and a okadaic acid toxin monoclonal antibody layer are sequentially stacked on the inner wall of each enzyme label hole.

The utility model discloses in the enzyme mark strip is detachable installs on the frame support.

the coating layer of the utility model is a goat anti-mouse IgG layer. The method is different from the traditional ELISA plate in that the goat anti-mouse IgG coats the ELISA plate, and the competitive reaction is realized in a solution phase by using a mouse monoclonal antibody, OA-HRP and OA in a sample.

In the utility model, the sealing layer is a casein layer. Since non-specific adsorption affects the sensitivity of the ELISA method, non-specific adsorption is reduced by using a blocking solution.

The utility model also provides an ELISA kit for detecting okadaic acid toxin in shellfish, which comprises a box body, wherein the box body is divided into an upper layer and a lower layer, the bottom layer is used for placing an ELISA plate, and the top layer is used for placing a reagent bottle;

The ELISA plate comprises an outer frame support and a plurality of enzyme label strips; the enzyme label strip comprises a plurality of enzyme label holes, and a coating layer, a sealing layer and a okadaic acid toxin monoclonal antibody layer are sequentially stacked on the inner wall of each enzyme label hole.

The utility model discloses in the enzyme mark strip is detachable installs on the frame support.

The coating layer of the utility model is a goat anti-mouse IgG layer. A direct competition ELISA method is established by adopting a goat anti-mouse IgG plate-wrapping mode.

In the utility model, the sealing layer is a casein layer. Since non-specific adsorption affects the sensitivity of the ELISA method, non-specific adsorption is reduced by using a blocking solution.

The utility model discloses in the reagent bottle includes: a phosphate buffer solution bottle, a washing solution bottle, a termination solution bottle, an OA standard substance solution bottle, an OA-HRP enzyme-labeled antigen bottle, a TMB color developing solution A bottle and a TMB color developing solution B bottle.

Compared with the prior art, the beneficial effects of the utility model are embodied in:

The utility model discloses well range upon range of being equipped with coating layer, seal and field okadaic acid toxin monoclonal antibody layer in proper order on the inner wall in enzyme-labeled hole, be different from current ELISA with OA antigen complex cladding ELISA plate, this scheme cladding sheep anti mouse IgG, through this special structural design, further improve the sensitivity and the detection range of field okadaic acid toxin short-term test in the shellfish for the field test of aquatic products field okadaic acid toxin in the shellfish has the feasibility.

Drawings

FIG. 1 is a schematic diagram showing the structure of an ELISA kit according to the present embodiment;

FIG. 2 is a top view of a reagent bottle layer in an embodiment;

FIG. 3 is a top view of the microplate in the example;

FIG. 4 is a sectional view of an ELISA plate according to the embodiment;

FIG. 5 is an enlarged view of the enzyme-labeled well in the examples.

Wherein, 1, a box body; 2. a top layer; 3. a bottom layer; 4. an outer frame support; 5. enzyme label strips; 6. enzyme-labeled holes; 7. an OA standard solution bottle; 8. a phosphate buffer solution bottle; 9. a washing liquid bottle; 10. a termination liquid bottle; 11. OA-HRP enzyme labeled antigen bottle; 12. a bottle of TMB color development liquid A; 13. a TMB color development liquid B bottle; 14. a coating layer; 15. a sealing layer; 16. a okadaic acid toxin monoclonal antibody layer.

Detailed Description

The present invention will be further described with reference to the following specific examples.

As shown in FIG. 1, the ELISA kit for detecting okadaic acid toxin in shellfish comprises a case 1, which may be a paper box or a box made of other materials, and has a cover on the top. The box body 1 is divided into an upper layer and a lower layer, the bottom layer 3 is used for placing an enzyme label plate, and the top layer 2 is used for placing a reagent bottle.

As shown in fig. 2, the reagent bottles include a phosphate buffer solution bottle 8, a washing solution bottle 9, a stop solution bottle 10, a plurality of OA standard solution bottles 7, an OA-HRP enzyme-labeled antigen bottle 11, a TMB color developing solution a bottle 12, and a TMB color developing solution B bottle 13.

In the phosphate buffer solution bottle 8: 0.01M PBS, pH 7.4. The preparation method comprises the following steps: 8g NaCL, 0.24g KH₂PO₄，2.9g Na₂HPO₄·12H₂O, 0.2g of KCL, diluting to 1000mL, and adjusting the pH value to 7.4.

The washing liquid bottle 9 comprises: PBST, pH 7.4. The preparation method comprises the following steps: 1000mL of PBS buffer solution is added with 10% Tween-20 and mixed evenly, and the mixture is stored at 4 ℃.

The termination liquid bottle 10 comprises: 2 mol. L^-1h of (A) to (B)₂SO₄。

In the OA-HRP enzyme-labeled antigen bottle 11, OA-HRP, OA monoclonal antibody (2.0 mg. mL) prepared by activated ester method is used^-1) Purchased from Stannum-free Jing Biotech limited, and Horseradish peroxidase (HRP) purchased from Fei-Bo Mei Biotech limited responsible, obtained by diluting OA-HRP with PBST at 1: 8000.

The TMB color developing solution a bottle 12 and the TMB color developing solution B bottle 13 were 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) purchased from tokyo koshijiu biotechnology limited.

As shown in FIGS. 3 and 4, the microplate used in the present embodiment is a 48-well microplate, but it is needless to say that a microplate having other specifications, for example, 16-well or 96-well microplate, may be used.

The ELIAS plate includes frame support 4 and eight enzyme label strips 5, and enzyme label strip 5 includes six enzyme-labeled holes 6, and enzyme label strip 5 is the detachable installation on frame support 4.

As shown in FIG. 5, a coating layer 14, a sealing layer 15 and a okadaic acid toxin monoclonal antibody layer 16 are sequentially laminated on the inner wall of the enzyme-labeled well 6. The coating layer is a goat anti-mouse IgG layer, and the sealing layer is a casein layer.

As one embodiment, the microplate is prepared as follows:

(1) goat anti-mouse IgG was diluted to 5 μ g/mL using carbonate buffer (0.05M CBS, pH 9.6)^-1mu.L of each well was coated with an enzyme-labeled plate, the plate was coated overnight at 4 ℃, and washed 3 times with PBST (pH 7.4) and then blotted dry. Wherein the goat anti-mouse IgG (20.0 mg. mL)^-1) From Wuxi Jinglin Biotech Ltd

(2) The reaction wells were completely filled with 1% CS/PBS solution, blocked at 37 ℃ for 60min, washed 3 times, and blotted dry. Wherein, the casein CS is purchased from Shanghai leaf Biotech limited.

(3) The OA monoclonal antibody (okadaic acid toxin monoclonal antibody) was diluted at 1:16000 using PBST, 50. mu.L of OA monoclonal antibody was added to step (2), inoculated for 60min at 37 ℃, washed 3 times, and patted dry.

As one of the embodiments, the standard curve is plotted:

Mixing OA standard sample 1 mg/mL^-1(dissolved in DMSO), diluted to different concentrations (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10. mu.g.L) with PBS^-1). And respectively adding 100 mu L of OA standard substance and 50 mu L of OA-HRP into the enzyme label plate in sequence, inoculating for 60min at 37 ℃, washing the plate for 3 times, and patting to dry. Adding a substrate for color development: taking 50 mu L of TMB substrate A and B respectively, inoculating for 15min at 37 ℃. And (3) terminating the reaction: adding the inoculated enzyme label plate into stop solution to stop reaction, and stopping reaction at 50 mu L well^-1. OD at 450nm/650nm was measured using a microplate reader. A standard curve was plotted with the inhibition ratio as the ordinate and lg (OA) as the abscissa.

As one embodiment, the preparation of the sample to be tested: the samples to be tested are shell products such as clams, mussels, razor clams, white scallops and the like, and are collected in agricultural trade cities of Lingan city in Zhejiang province and Shenshan Shenmen markets. Soaking a sample to be detected, spitting sand, cleaning, removing shells, draining, and taking an edible part for homogenate. Weighing 4.0g homogenate sample, placing in 50mL centrifuge tube, adding 10mL methanol/water (8:2, V/V), mixing well for 10min with shaking table, 5000 r.min^-1Centrifuge for 10 min. The resulting solution was filtered through a 0.45 μm organic filter, and the filtrate was diluted 10 times with PBS buffer (0.01, pH 7.4).

The detection steps are as follows:

Adding 100 mul of sample to be detected and 50 mul of OA-HRP into the enzyme label plate in sequence, inoculating for 60min at 37 ℃, washing the plate for 3 times, and patting to dry. Adding a substrate for color development: taking 50 mu L of TMB substrate A and B respectively, inoculating for 15min at 37 ℃. And (3) terminating the reaction: adding the inoculated enzyme label plate into stop solution to stop reaction, and stopping reaction in 50 mu L of well^-1. OD at 450nm/650nm is measured by using a microplate reader, and calculation is carried out according to a standard curve, so as to complete field detection of okadaic acid toxin.

Claims (7)

1. an ELISA plate for detecting okadaic acid toxin in shellfish is characterized by comprising an outer frame bracket and a plurality of enzyme label strips; the enzyme-labeled strip comprises a plurality of enzyme-labeled holes, wherein a coating layer, a sealing layer and a okadaic acid toxin monoclonal antibody layer are sequentially stacked on the inner wall of each enzyme-labeled hole, and the coating layer is a goat anti-mouse IgG layer.

2. The elisa plate for detecting okadaic acid toxin in shellfish according to claim 1, wherein the enzyme label strip is detachably mounted on the outer frame support.

3. The microplate for detecting okadaic acid toxin in shellfish according to claim 1, wherein said blocking layer is a casein layer.

4. an ELISA kit for detecting okadaic acid toxin in shellfish comprises a kit body, and is characterized in that the kit body is divided into an upper layer and a lower layer, the bottom layer is used for placing an ELISA plate, and the top layer is used for placing a reagent bottle;

The ELISA plate comprises an outer frame support and a plurality of enzyme label strips; the enzyme-labeled strip comprises a plurality of enzyme-labeled holes, wherein a coating layer, a sealing layer and a okadaic acid toxin monoclonal antibody layer are sequentially stacked on the inner wall of each enzyme-labeled hole, and the coating layer is a goat anti-mouse IgG layer.

5. The ELISA kit for detecting okadaic acid toxin in shellfish of claim 4 wherein said enzyme label strip is removably mounted on the frame.

6. The ELISA kit for detection of okadaic acid toxin in a shellfish according to claim 4, wherein said blocking layer is a casein layer.

7. The ELISA kit for detecting okadaic acid toxin in a shellfish of claim 4, wherein said reagent bottle comprises: a phosphate buffer solution bottle, a washing solution bottle, a termination solution bottle, an OA standard substance solution bottle, an OA-HRP enzyme-labeled antigen bottle, a TMB color developing solution A bottle and a TMB color developing solution B bottle.