阅读说明：本技术 一种五氯酚钠半抗原、人工抗原及其制备方法与应用 (Sodium pentachlorophenate hapten, artificial antigen, and preparation methods and applications thereof ) 是由 李斌 黄家怡 苏振贤 梁志玲 石松 任季玉 于 2021-09-18 设计创作，主要内容包括：本发明公开了一种五氯酚钠半抗原、人工抗原及其制备方法与应用,该五氯酚钠半抗原结构如式I所示,五氯酚钠人工抗原的结构式如式V所示。本发明中的五氯酚钠半抗原的制备方法简单,一个半抗原分子中含有两个五氯苯氧基的特征结构,使得基于其制备得到的人工抗原在空间结构上更容易突显出小分子的结构特性,提高了免疫原性。而且,基于本发明中的五氯酚钠半抗原制备得到的人工抗原和抗体特异性好,能够准确的识别靶向物质。基于其制备得到的检测试纸条特异性高,灵敏度强,对五氯酚钠的实际检测灵敏度可达1μg/kg,具有较好的检测效果。(The invention discloses a sodium pentachlorophenate hapten, an artificial antigen, a preparation method and an application thereof. The preparation method of the sodium pentachlorophenate hapten is simple, and one hapten molecule contains two characteristic structures of pentachlorophenoxy, so that the artificial antigen prepared based on the hapten can more easily show the structural characteristics of small molecules on the spatial structure, and the immunogenicity is improved. Moreover, the artificial antigen and the antibody prepared based on the sodium pentachlorophenate hapten have good specificity and can accurately identify the target substance. The detection test strip prepared based on the sodium pentachlorophenate has high specificity and strong sensitivity, the actual detection sensitivity to the sodium pentachlorophenate can reach 1 mug/kg, and the detection effect is better.)

1. The sodium pentachlorophenate hapten is characterized by having a structure shown in a formula I:

2. the method of preparing sodium pentachlorophenate hapten as recited in claim 1, comprising the steps of:

(1) reacting the compound shown in the formula II and the compound shown in the formula III at room temperature to obtain a compound shown in a formula IV;

(2) adding an alkaline solution into the compound shown in the formula IV for hydrolysis to obtain the compound;

wherein the structural formula of the compound shown in the formula II is as follows:

the structural formula of the compound shown in the formula III is as follows:

the compound shown in the formula IV has a structural formula:

wherein, the step (1) preferably also contains a catalyst, a solvent and an acid-binding agent; (ii) a

The acid-binding agent preferably comprises at least one of sodium bicarbonate, sodium carbonate and potassium carbonate;

the solvent preferably includes at least one of N, N-dimethylformamide, acetonitrile and acetone;

the catalyst preferably comprises at least one of benzyltriethylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride and tetrabutylammonium iodide.

3. The sodium pentachlorophenate artificial antigen is characterized in that the sodium pentachlorophenate artificial antigen is obtained by coupling the sodium pentachlorophenate hapten as claimed in claim 1 with a carrier protein.

4. The sodium pentachlorophenate artificial antigen of claim 3, wherein the carrier protein includes at least one of bovine serum albumin, ovalbumin, human serum albumin, and hemocyanin.

5. The sodium pentachlorophenate artificial antigen of claim 3, wherein the structural formula of the sodium pentachlorophenate artificial antigen is shown as formula V,

wherein protein in formula V represents a carrier protein.

6. Use of sodium pentachlorophenate hapten as defined in claim 1 or sodium pentachlorophenate hapten as defined in claim 2 in any one of the following (1) to (2);

(1) preparing a sodium pentachlorophenate specific antibody;

(2) detecting sodium pentachlorophenate specific antibody.

7. The sodium pentachlorophenate specific antibody is characterized in that the sodium pentachlorophenate specific antibody is obtained by immunizing an animal with the sodium pentachlorophenate artificial antigen as the immunogen of any one of claims 3 to 5.

8. Use of the sodium pentachlorophenate-specific antibody of claim 7 in any one of the following (1) to (2);

(1) detecting sodium pentachlorophenate;

(2) preparing a sodium pentachlorophenate detection product;

wherein the product preferably comprises: at least one of a detection reagent, a detection kit, a detection test paper and a detection card.

9. A detection reagent or test strip comprising the sodium pentachlorophenate specific antibody of claim 7.

10. A test kit comprising the detection reagent or test strip of claim 9.

Technical Field

The invention relates to the field of biochemical engineering, and in particular relates to a sodium pentachlorophenate hapten, an artificial antigen, and preparation methods and applications thereof.

Background

Sodium pentachlorophenolate, Sodium pentachlorophenolate (PCP-Na), is a compound with the structural characteristics of pentachlorophenoxy, and is widely used as a fungicide, herbicide, insecticide and wood preservative. Sodium pentachlorophenate is chemically stable and not easy to degrade, and can enter the food chain through the action of biological enrichment. Sodium pentachlorophenate has teratogenic, carcinogenic and mutagenic effects, and after the sodium pentachlorophenate is taken into a human body for a long time, the sodium pentachlorophenate can cause damage to the immunologic function and the nerve function of the human body, so that the sodium pentachlorophenate has great harmfulness. In the world, relevant laws and regulations are established in most countries, regions and organizations, sodium pentachlorophenate is listed in animal medicines forbidden for food animals, is forbidden to be used in aquatic animal breeding, and cannot be detected in animal-derived foods.

In the related art, the detection method of pentachlorophenol or its sodium salt (sodium pentachlorophenol) in food is mainly gas chromatography, gas chromatography-electron capture method, gas chromatography-hydrogen flame method, etc. In the methods such as gas chromatography-mass spectrometry, gas chromatography and the like, equipment and instruments are expensive, the detection time is long, and professional operation is required, so that on-site detection and rapid temporary detection cannot be really realized, and great inconvenience is brought to daily detection work.

The key of immunoassay detection technology is the performance determination of antigen and antibody, and the key of antigen and antibody is hapten, so the structural design of hapten is important for obtaining the antigen and antibody with excellent performance. In the related technology, the artificial antigen directly prepared based on sodium pentachlorophenate has the defects of poor sensitivity and low cross reaction rate, and cannot meet the actual use requirement of the existing market. Therefore, the development of the sodium pentachlorophenate hapten or artificial antigen with high specificity has great significance for the rapid, high-sensitivity and low-cost detection method of sodium pentachlorophenate.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the hapten not only has an active group, but also completely retains the structural characteristics of sodium pentachlorophenate, and one hapten molecule contains two characteristic structures of pentachlorophenol, so that the prepared artificial antigen can more easily show the structural characteristics of small molecules on a spatial structure, and the immunogenicity is improved.

The invention provides a sodium pentachlorophenate hapten, which has a structure shown in a formula I:

the sodium pentachlorophenate hapten designed by the invention not only has an active group, but also completely retains the structural characteristics of pentachlorophenoxy, and one hapten molecule contains two characteristic structures of pentachlorophenoxy, so that the prepared artificial antigen can more easily show the structural characteristics of small molecules on a spatial structure, and the immunogenicity is improved. Based on the structure, the prepared specific antibody has extremely strong specificity.

In a second aspect of the present invention, there is provided a method for preparing sodium pentachlorophenate hapten as described in the first aspect of the present invention, comprising the steps of:

(1) reacting the compound shown in the formula II and the compound shown in the formula III at room temperature to obtain a compound shown in a formula IV;

(2) and (3) adding an alkaline solution into the compound shown in the formula IV for hydrolysis to obtain the compound.

Wherein the structural formula of the compound shown in the formula II is as follows:

the structural formula of the compound shown in the formula III is as follows:

the compound shown in the formula IV has a structural formula:

according to a second aspect of the present invention, in some embodiments of the present invention, the step (1) further comprises a catalyst, a solvent and an acid-binding agent.

In some preferred embodiments of the present invention, the acid scavenger comprises at least one of sodium bicarbonate, sodium carbonate, and potassium carbonate.

In some more preferred embodiments of the invention, the acid scavenger is potassium carbonate.

In some preferred embodiments of the present invention, the solvent comprises at least one of N, N-dimethylformamide, acetonitrile and acetone.

In some more preferred embodiments of the invention, the solvent is N, N-Dimethylformamide (DMF).

In some preferred embodiments of the present invention, the catalyst comprises at least one of benzyltriethylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride and tetrabutylammonium iodide.

In some more preferred embodiments of the invention, the catalyst is tetrabutylammonium iodide.

In some preferred embodiments of the present invention, the alkaline solution in step (2) comprises at least one of lithium hydroxide, sodium hydroxide and potassium hydroxide.

In some more preferred embodiments of the invention, the alkaline solution is lithium hydroxide.

In some more preferred embodiments of the present invention, the preparation method comprises the following specific steps:

mixing a compound with a structural formula shown as a formula II, DMF (dimethyl formamide) and a compound with a structural formula shown as a formula III, adding potassium carbonate and tetrabutylammonium iodide, and reacting for more than 24 hours at room temperature (23-27 ℃). Extraction with ethyl acetate, removal of the solvent (ethyl acetate), and purification by column chromatography gave the compound of formula IV. Dissolving the compound shown in the formula IV in methanol, adding a lithium hydroxide aqueous solution, and reacting for more than 8 hours at room temperature (23-27 ℃). And (4) extracting with ethyl acetate, adjusting the pH value of the water phase to 3-4, and separating out solid substances. And filtering to obtain the sodium pentachlorophenate hapten.

In the third aspect of the invention, a sodium pentachlorophenate artificial antigen is provided, and the sodium pentachlorophenate artificial antigen is obtained by coupling the sodium pentachlorophenate hapten and a carrier protein.

According to a third aspect of the invention, in some embodiments of the invention, the carrier protein comprises at least one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

Of course, other carrier proteins can be coupled by those skilled in the art according to the actual use requirement.

According to the third aspect of the invention, in some embodiments of the invention, the structural formula of the sodium pentachlorophenate artificial antigen is shown as formula V,

wherein protein in formula V represents a carrier protein.

In a fourth aspect of the present invention, there is provided a use of the sodium pentachlorophenate hapten of the first aspect of the present invention or the sodium pentachlorophenate hapten prepared by the preparation method of the second aspect of the present invention in any one of the following (1) to (2);

(1) preparing a sodium pentachlorophenate specific antibody;

(2) detecting sodium pentachlorophenate specific antibody.

According to a fourth aspect of the invention, in some embodiments of the invention, the sodium pentachlorophenate-specific antibody comprises a monoclonal antibody and a polyclonal antibody.

In some preferred embodiments of the invention, the sodium pentachlorophenol-specific antibody is a monoclonal antibody.

In the fifth aspect of the invention, a sodium pentachlorophenate specific antibody is provided, and the sodium pentachlorophenate specific antibody is obtained by immunizing an animal with the sodium pentachlorophenate artificial antigen as the immunogen in the third aspect of the invention.

According to a fifth aspect of the invention, in some embodiments of the invention, the animal comprises at least one of a rabbit, a sheep, a guinea pig, a mouse, a chicken, a goat and a horse.

According to a fifth aspect of the invention, in some embodiments of the invention, the sodium pentachlorophenate-specific antibody comprises a monoclonal antibody and a polyclonal antibody.

In some preferred embodiments of the invention, the sodium pentachlorophenol-specific antibody is a monoclonal antibody.

In a sixth aspect of the present invention, there is provided a use of the sodium pentachlorophenate-specific antibody according to the fifth aspect of the present invention in any one of the following items (1) to (2);

(1) detecting sodium pentachlorophenate;

(2) preparing sodium pentachlorophenate detection product.

According to a sixth aspect of the invention, in some embodiments of the invention, the sodium pentachlorophenate-specific antibody comprises a monoclonal antibody and a polyclonal antibody.

In some preferred embodiments of the invention, the sodium pentachlorophenol-specific antibody is a monoclonal antibody.

According to a sixth aspect of the invention, in some embodiments of the invention, the product comprises: at least one of a detection reagent, a detection kit, a detection test paper or a detection card.

In a seventh aspect of the present invention, a detection reagent or a detection test paper is provided, wherein the detection reagent or the detection test paper contains the sodium pentachlorophenate specific antibody according to the fifth aspect of the present invention.

According to a seventh aspect of the invention, in some embodiments of the invention, the sodium pentachlorophenate-specific antibody comprises a monoclonal antibody and a polyclonal antibody.

In some preferred embodiments of the invention, the sodium pentachlorophenol-specific antibody is a monoclonal antibody.

In an eighth aspect of the present invention, there is provided a test kit, wherein the test kit contains the detection reagent or test strip according to the seventh aspect of the present invention.

The invention has the beneficial effects that:

1. the sodium pentachlorophenol hapten is unique in design, not only has an active group, but also completely retains the structural characteristics of pentachlorophenol, and one hapten molecule contains the characteristic structures of two pentachlorophenols, so that the artificial antigen prepared based on the sodium pentachlorophenol hapten can more easily highlight the structural characteristics of small molecules on a spatial structure, and the immunogenicity is improved.

2. The artificial antigen and the antibody prepared based on the sodium pentachlorophenate hapten have good specificity, can accurately identify a target substance, have an IC50 value of 1.07 mu g/L, can be used for preparing a rapid detection test strip, a kit and other related detection equipment aiming at the sodium pentachlorophenate, and have extremely high application prospect.

3. The detection test strip prepared based on the artificial antigen and the specific antibody has high specificity and strong sensitivity, the actual detection sensitivity to sodium pentachlorophenate is at least 1 microgram/kg, and the detection test strip has better detection effect to sodium pentachlorophenate.

Drawings

FIG. 1 is a mass spectrum of sodium pentachlorophenate hapten in the example of the invention;

FIG. 2 is a reaction scheme of sodium pentachlorophenate hapten in an example of the present invention;

FIG. 3 is a comparison chart of the detection results of the sodium pentachlorophenate colloidal gold immunochromatographic test strip in the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Preparation of sodium pentachlorophenate hapten

Taking 0.5g (1.94mmol) of a compound shown in a structural formula II in a 100mL round-bottom flask, sequentially adding 10mL of N, N-Dimethylformamide (DMF) and 2.22g (7.76mmol) of a compound shown in a structural formula III, stirring until the compound is completely dissolved, sequentially adding 0.54g (3.88mmol) of potassium carbonate and 0.10g of N-tetrabutylammonium iodide, and reacting for more than 24 hours at room temperature (23-27 ℃). After the reaction was completed, 90mL of purified water was added, extraction was performed with ethyl acetate 2 times, the solvent (ethyl acetate) was removed under reduced pressure, and column chromatography was used to purify the product to obtain 0.83g of the compound represented by formula IV (mobile phase was an ethyl acetate/petroleum ether mixture, and the volume ratio of ethyl acetate/petroleum ether was 3: 4). 0.83g (1.32mmol) of the compound represented by the formula IV is dissolved in 5mL of methanol, 5mL of 4mol/L lithium hydroxide aqueous solution is added, and the reaction is carried out at room temperature (23-27 ℃) for more than 8 h. After the reaction is finished, 30mL of sodium chloride aqueous solution is added, 30mL of ethyl acetate is used for extraction for 2 times, the pH of the aqueous phase is adjusted to 3-4 by using 4M dilute hydrochloric acid, and solid matters are separated out. Filtering and drying to obtain the sodium pentachlorophenate hapten.

Wherein the structural formula of the compound shown in the formula II is as follows:

the structural formula of the compound shown in the formula III is as follows:

the compound shown in the formula IV has a structural formula:

in this example, 0.47g of sodium pentachlorophenate hapten was obtained.

The ESI-MS of sodium pentachlorophenate hapten obtained in this example was: 600.6[ M-1 ].

The mass spectrum is shown in figure 1.

The reaction scheme for sodium pentachlorophenate hapten is shown in figure 2.

Preparation of sodium pentachlorophenate artificial antigen

(1) Bovine serum albumin-sodium pentachlorophenate hapten is coupled to obtain an artificial antigen:

the sodium pentachlorophenate hapten (10mg) prepared in the above example was taken and dissolved sufficiently in 0.2mL of Dimethylformamide (DMF). Then, 5mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 5mg of N-hydroxysuccinimide (NHS) were added thereto and stirred at room temperature for 4 hours to allow complete reaction, thereby obtaining sodium pentachlorophenate hapten activated ester.

Then, under the stirring condition, the obtained sodium pentachlorophenol hapten activated ester is dropwise added into a bovine serum albumin solution (BSA, 35mg BSA +3.5mL of 0.01mol/L PBS buffer), and stirred at room temperature in a dark place for 16-24 h. Dialyzing with 0.01mol/L PBS at room temperature for 3 days, and changing the dialyzate 3 times a day to remove unreacted small molecular substances in the solution to obtain the sodium pentachlorophenate artificial antigen.

The obtained sodium pentachlorophenate artificial antigen can be stored at 4 ℃ for later use.

(2) Artificial antigen obtained by coupling hemocyanin-sodium pentachlorophenate hapten:

the method is the same as (1) and is characterized in that:

in this example, the addition of EDC and NHS was increased to 10 mg.

Hemocyanin (KLH) solution was prepared from 35mg KLH +3.5mL of 0.01mol/L PBS buffer.

Application of sodium pentachlorophenate artificial antigen

(1) The application of the sodium pentachlorophenate artificial antigen in the preparation of the sodium pentachlorophenate monoclonal antibody comprises the following steps:

the preparation method comprises the following steps:

taking the artificial antigen obtained by coupling the hemocyanin-sodium pentachlorophenate hapten in the embodiment as an immunogen, adding an equal volume of Freund's adjuvant, fully emulsifying, and then carrying out immune BALB/C mouse treatment. The immunogen dose of each BALB/C mouse is 50-100 mu g, and each immunization interval is 2 weeks. After 3 times of immunization, tail venous blood of the mice is collected, and the antibody titer in serum is detected. If the antibody titer does not meet the requirement, the immunization needs to be strengthened.

After the antibody titer is not increased any more, the subcutaneous booster immunization is carried out by using 100 mu g of the whole antigen (the artificial antigen obtained by coupling the hemocyanin-sodium pentachlorophenate hapten).

5 days after the booster immunization, the test mouse spleen cells were taken and subjected to cell fusion with SP20 cells (mouse myeloma cells). The fused cells are incubated and screened in HAT medium, and cultured in complete medium after 5 days of incubation.

And (3) detecting the obtained fusion cell supernatant by using an ELISA method, carrying out cloning culture on the fusion cells with strong positive detection results by using a limiting dilution method, and detecting by 3 times of cloning culture to obtain the fusion cells with positive results, namely the hybridoma of the pentachlorophenol sodium monoclonal antibody. After the hybridoma cells are subjected to amplification culture, the hybridoma cells are inoculated to the abdominal cavity of a mouse to generate ascites containing the pentachlorophenol sodium monoclonal antibody. And purifying the ascites by using an octanoic acid-ammonium sulfate precipitation method, and freeze-drying to obtain the sodium pentachlorophenate monoclonal antibody.

(2) Application of sodium pentachlorophenate artificial antigen in immunoassay:

in this embodiment, the ELISA method is used as an example to show the application of sodium pentachlorophenate artificial antigen in immunoassay, and the specific steps are as follows:

the artificial antigen obtained by coupling the bovine serum albumin-sodium pentachlorophenate hapten in the above example was diluted to 0.1. mu.g/mL with a carbonate buffer (pH 9.6) as a coating diluent. Then added to a polystyrene microplate in an amount of 100. mu.L/well and coated overnight at 4 ℃. The wells were drained, 1% BSA (phosphate buffer as a diluent) was added at 250. mu.L/well, blocked at 37 ℃ for 1h, and dried to obtain coated assay plates. Sodium pentachlorophenate standard solution (100. mu.L/well) and sodium pentachlorophenate monoclonal antibody (20. mu.L/well) with different concentrations are added into the coated detection plate in sequence, and the incubation is carried out for 0.5h at 37 ℃. The liquid in the holes was drained, 280. mu.L/hole of washing solution was added, washed 3 times, and patted dry. Add 100 u L/hole enzyme labeled second antibody, 37 degrees C reaction for 0.5 h. Washing for 3 times again, drying, adding 50 μ L/hole color developing solution, and reacting at 37 deg.C in dark for 15 min. The reaction was stopped by adding 50. mu.L/well of 2M sulfuric acid, and the OD value of each well was measured at a wavelength of 450 nm. The results are shown in Table 1.

The sodium pentachlorophenate monoclonal antibody in this example was diluted to 0.08. mu.g/mL with a phosphate buffer (pH 7.4) containing 0.05% sodium azide, and stored at 4 ℃ until use.

TABLE 1 OD values of sodium pentachlorophenate standard solutions of different concentrations

Concentration (μ g/L)	0	0.2	0.4	0.8	1.6	3.2	6.4
								OD value	2.255	1.845	1.766	1.285	0.862	0.497	0.207

The data in table 1 were fitted to a four parameter Logistic curve using ELISA Calc software to draw a standard curve.

It can be found that the linear equation of the sodium pentachlorophenate standard solution obtained is:

y＝(A-D)/[1+(x/C)^B]+ D; wherein r is²＝0.998，A＝2.26140，B＝1.21123，C＝1.07348，D＝-0.00919；

x represents the concentration of the substance in the sample;

y represents an OD value.

Found by calculation, the obtained IC₅₀The value is 1.07. mu.g/L, and the linear relationship is between 0.4 and 6.4. mu.g/L.

(3) The application of the sodium pentachlorophenate artificial antigen in preparing the colloidal gold immunochromatographic test strip for detecting sodium pentachlorophenate comprises the following steps:

the preparation method comprises the following steps:

a. preparing a reaction film:

the method comprises the steps of taking a nitrocellulose membrane (NC membrane) as a reaction membrane, regulating the concentration of an artificial antigen obtained by coupling the bovine serum albumin-sodium pentachlorophenate hapten in the embodiment to be 0.1-0.5 mg/mL by using a coating buffer solution, and regulating the concentration of mouse IgG to be 0.1-0.5 mg/mL by using the coating buffer solution. And respectively spraying the artificial antigen and the mouse IgG on the surfaces of a detection area (T line) and a control area (C line) corresponding to the reaction membrane according to the membrane liquid amount of 0.8-1.2 mu L/cm. The interval between the detection area and the control area is 2.5mm, and the detection area and the control area are placed in an oven for treatment for 12-16 h at the temperature of 45 ℃.

Wherein, the coating buffer solution is 0.01M PBS buffer solution (pH 7.6) containing 1 percent of sucrose and 0.05 percent of sodium azide by mass percentage.

b. Preparing colloidal gold micropores:

1g of chloroauric acid is taken, and pure water is added to be completely dissolved under the ultrasonic condition to be 100 mL. Then, 1mL of the solution with constant volume is taken, 100mL of pure water is added, 0.5mL of 0.06% sodium citrate solution is added after the solution is heated to boiling, and the heating is continued for 10 minutes. And cooling to room temperature. Adding pure water to restore to the original volume to obtain a nano gold solution, and storing the nano gold solution at normal temperature in the dark. Wherein, it should be noted that, in the above steps, all glassware needs to be soaked overnight with a mixed solution of potassium permanganate and sulfuric acid, and then washed and dried for use. Taking 1mL of nano-gold solution, and using 0.1mol/L K₂CO₃And 0.1mol/L HCl to adjust the pH of the solution. And adding 5 mu g of the sodium pentachlorophenate monoclonal antibody prepared in the above embodiment into nano gold solutions with different pH values, and reacting for 5 minutes at room temperature. The solution was observed for color change and the pH at which the solution color remained red was recorded. Then, 10. mu.L of 10% bovine serum albumin was added to the mixture solution which remained red in color for blocking, and the mixture was centrifuged at 12000rpm to discard the whole supernatant. Adding colloidal gold diluent (pure water solution containing 2% Tris, 5% bovine serum albumin, 0.05% thimerosal, and 5% sucrose) for redissolving, subpackaging at 20 μ l/hole in micropores, drying at 37 deg.C for 16 hr, and storing for use.

c. Assembling the colloidal gold immunochromatographic test strip:

the reaction membrane is superposed in the middle of the PVC plate back lining, and a sample pad (obtained by soaking the sample pad in a sample pad treatment solution for 5min and drying at 37 ℃ for 16h, wherein the sample pad treatment solution comprises 0.01M PB buffer solution containing 0.3% of Tween 20, 1% of sucrose, 0.5% of BSA and 0.05% of sodium azide by mass percent) and a water absorption pad are respectively superposed at two ends of the PVC plate back lining. The reaction membrane is respectively connected with the water absorption pad and the sample pad, the detection area is close to the sample pad, the control area is close to the water absorption pad, the colloidal gold immunochromatography test paper board for detecting sodium pentachlorophenate is obtained, and the test paper board is cut to obtain the test paper strip (the width is 3 mm).

The colloidal gold immunochromatographic test strip obtained in this example was used for detection, and the detection sensitivity was evaluated.

0.01M PBS buffer solution is used for preparing sodium pentachlorophenate standard solutions with different concentrations, then 100 mu L of sodium pentachlorophenate standard solutions with different concentrations are respectively added into the gold-labeled micropores prepared in the above examples, and the solution is repeatedly blown and beaten to be redissolved uniformly. Standing for 3min, transferring the solution in the gold-labeled micropores to the colloidal gold immunochromatographic test strip (in the sample adding hole) obtained in the embodiment, observing the result 5-8 min after sample addition, and reading the result inefficiently after sample addition for 8 min. Test replicates 3 groups.

The result interpretation adopts a naked eye interpretation method (as shown in figure 3):

the color development of the T line is stronger than that of the C line or has no obvious difference with that of the C line, the detection result is negative (-), and the detection sample does not contain sodium pentachlorophenate. The T line color development is obviously weaker than the C line color development or the T line color development is not realized, the detection result is positive (+), and the fact that the detection sample contains sodium pentachlorophenate is indicated. If the C line does not appear, the operation is incorrect or the test strip is failed, and the detection needs to be carried out again.

The results are shown in Table 2.

Table 2 test results of sodium pentachlorophenate standard substance detected by sodium pentachlorophenate colloidal gold immunochromatographic test strip

After repeated tests, the colloidal gold immunochromatographic test strip obtained in the embodiment has the detection sensitivity of at least 2 mug/L to sodium pentachlorophenate, and has higher sensitivity.

Meanwhile, the colloidal gold immunochromatographic test strip obtained in the above embodiment is used for stability detection.

The normal storage condition of the colloidal gold immunochromatographic test strip obtained in the above example is room temperature storage. However, in this example, in order to more truly show the stability of the test strip, an accelerated destructive test was performed on the test strip, and the colloidal gold immunochromatographic test strip obtained in the above example was continuously placed at room temperature and 37 ℃ (high temperature accelerated product aging) for 90 days, and comparative tests were performed on samples with different concentrations of sodium pentachlorophenate on days 0, 7, 14, 30, 45, 60, 75, and 90 (repeat 3 groups).

The results are shown in Table 3.

TABLE 3 detection results of the colloidal gold immunochromatographic test strip with different storage times

Where, + indicates a positive result and-indicates a negative result.

As can be seen from the above table, after the colloidal gold immunochromatographic test strip obtained in the above example is stored for 90 days under the sealed storage condition at room temperature and 37 ℃, the T/C color development conditions of the two test strips do not change significantly, and the detection results are consistent, which indicates that the colloidal gold immunochromatographic test strip obtained in the above example can be stably stored for 90 days at least under the extreme condition (37 ℃). The colloidal gold immunochromatographic test strip stored at normal temperature is stably stored at room temperature for more than one year, has no significant influence on the detection effect, and can completely meet the requirements of the market in the storage and transportation processes.

Actual detection effect of sodium pentachlorophenate colloidal gold immunochromatographic test strip

In order to further verify the detection effect of the sodium pentachlorophenate colloidal gold immunochromatographic test strip prepared in the above embodiment in the detection of actual samples, 10 different livestock and aquatic samples were selected as detection samples for evaluation.

The samples are mixed into the sodium pentachlorophenate standard solution in a standard adding mode, and the adding concentration gradient is 0, 0.5, 1, 2 and 4 mu g/kg.

The specific detection steps are as follows:

(1) sample pretreatment:

taking more than 100g of detection sample, crushing, randomly taking 2.0g of the detection sample, adding the crushed detection sample into a 10mL centrifuge tube, adding 2mL of ethyl acetate, fully shaking and uniformly mixing, and extracting for 2 min. Then centrifuged at 4000rpm for 3 min. Taking 1mL of centrifugal supernatant, concentrating and drying at 65 ℃, adding 300 mu L of 0.01M PBS for redissolving, and obtaining the solution to be detected.

(2) Sample detection:

taking 100 mu L of the solution to be detected, and detecting according to the using method of the sodium pentachlorophenate colloidal gold immunochromatographic test strip in the embodiment.

The results are shown in Table 4.

TABLE 4 test results of different test samples

Where, + indicates a positive result and-indicates a negative result.

As can be seen from Table 4, the sodium pentachlorophenate colloidal gold immunochromatographic test strip in the above embodiment has consistent detection results among 10 different detection samples, has good repeatability of the detection results, and is negative when the content of sodium pentachlorophenate in the detection samples is lower than 1 μ g/kg; above 1. mu.g/kg, all were positive. Therefore, it can be shown that the practical detection limit of the test strip for sodium pentachlorophenate colloidal gold immunochromatography in the above examples for sodium pentachlorophenate in a sample is 1 μ g/kg.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.