CN116008554B - Test strip and method for detecting veterinary drug oxfendazole - Google Patents

Abstract

The invention discloses a test strip and a method for detecting the veterinary drug oxfendazole. The test strip comprises test paper and a micropore reagent, wherein the test paper comprises a reaction membrane, a sample absorption pad, a water absorption pad and a bottom plate, the reaction membrane is provided with a detection line coated with an oxfendazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the micropore reagent is freeze-dried with an oxfendazole monoclonal antibody-colloidal gold marker. The method for detecting the oxfendazole by using the test strip is simple, convenient, quick, visual, accurate, convenient to carry, wide in application range, low in cost and easy to popularize and use.

Description

Test strip and method for detecting veterinary drug oxfendazole

Technical Field

The invention relates to a test strip and a method for detecting veterinary drug oxfendazole, in particular to a colloidal gold test strip for detecting veterinary drug oxfendazole, which is particularly suitable for detecting oxfendazole residues in liquid milk.

Background

Oxfendazole (OFZ) is a high-efficiency, broad-spectrum, low-toxicity benzimidazole carbamate anthelmintic, also known as sulfobendazole or sulfobendazole, which is an oxide on the sulfur atom of fenbendazole, and has the chemical name of 5-benzene-2-benzimidazole-carbamate. The first development was successful by new thoxas in the united states at the beginning of the 70 s, and was carried by the british veterinary pharmacopoeia (1985). Is widely used for expelling parasites of domestic animals and poultry. Studies have shown that continuous or excessive administration causes residual oxfendazole in animals and can accumulate and deliver with the food chain, posing a threat to human health. With increasing importance of food safety, the limit of drug residues in animal-derived foods is also increasingly strict. The national standard GB 31650 specifies that the maximum residual limit of the oxfendazole in the cow/sheep milk is 100 mug/kg.

The methods for detecting the oxfendazole reported at present are mainly the methods of gas chromatography, high performance liquid chromatography, gas chromatography mass spectrometry, liquid chromatography mass spectrometry and other instruments. The methods are operated under laboratory conditions, the pretreatment of the samples is tedious and time-consuming, expensive instruments and equipment are needed to be equipped, the detection cost is high, the time consumption is long, the operation is complex, the method has great limitation in the practical application process, and the method is difficult to meet the requirements of rapid detection of a large number of samples and on-site samples. Therefore, the colloidal gold test strip which is simple and rapid and is suitable for the oxfendazole residue in the liquid milk is developed, a large number of samples can be screened and monitored on site, and the detection work of food supervision departments in China and the like can be better met.

Disclosure of Invention

The invention aims to provide a colloidal gold test strip capable of detecting the residue of the veterinary drug oxfendazole in liquid milk, and provides a detection method which is efficient, accurate, simple and convenient and is suitable for on-site monitoring and screening of a large number of samples.

The test strip for detecting the oxfendazole comprises test paper and a micropore reagent; the test paper comprises a bottom plate, a sample absorption pad, a reaction membrane and a water absorption pad, which are sequentially connected, wherein the reaction membrane is provided with a detection line coated with an oxfendazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody; the microporous reagent is freeze-dried with an oxfendazole monoclonal antibody-colloidal gold marker, and the oxfendazole monoclonal antibody-colloidal gold marker is provided with a microporous plug.

The oxfendazole monoclonal antibody is prepared by taking oxfendazole hapten-carrier protein conjugate as an immunogen.

The oxfendazole hapten-carrier protein conjugate is obtained by coupling oxfendazole hapten with carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin, the oxfendazole hapten is obtained by taking oxfendazole EP impurity C as a starting raw material, carrying out condensation reaction with p-carboxybenzaldehyde and then reducing the mixture with sodium borohydride, and the molecular structural formula is as follows:

the bottom plate is a PVC bottom plate or other hard non-water-absorbing materials; the sample absorption pad is made of polyester fiber or glass fiber; the water absorption pad is water absorption filter paper; the reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane.

Another object of the present invention is to provide a method for preparing the above test strip, comprising the steps of:

1) Preparing a micropore reagent freeze-dried with an oxfendazole monoclonal antibody-colloidal gold marker;

2) Preparing a reaction membrane with a detection line coated with an oxfendazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;

3) Assembling the reaction membrane prepared in the step 2) with a sample absorption pad, a water absorption pad and a bottom plate to form test paper;

4) And (3) assembling the microporous reagent freeze-dried with the oxfendazole monoclonal antibody-colloidal gold marker prepared in the steps 1) and 3) and the test paper into a test strip.

Specifically, the method comprises the following steps:

1) The method comprises the steps of taking an oxfendazole EP impurity C as a starting material, carrying out condensation reaction with p-carboxybenzaldehyde, and then reducing the mixture by sodium borohydride to prepare the oxfendazole hapten;

2) Coupling the oxfendazole hapten with carrier protein to prepare an oxfendazole hapten-carrier protein conjugate;

3) Immunizing a mouse by using an oxfendazole hapten-carrier protein conjugate, and fusing and screening spleen cells of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting oxfendazole monoclonal antibodies;

4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibody;

5) Coating the oxfendazole hapten-carrier protein conjugate and the goat anti-mouse anti-antibody on a detection line (T) and a quality control line (C) of a reaction membrane respectively;

6) Preparing colloidal gold by the reaction of trisodium citrate and chloroauric acid;

7) Adding the prepared oxfendazole monoclonal antibody into the prepared colloidal gold to obtain an oxfendazole monoclonal antibody-colloidal gold marker;

8) Freeze-drying the oxfendazole monoclonal antibody-colloidal gold marker in a micropore reagent, and adding a micropore plug into the micropore reagent;

9) Soaking the sample absorption pad in 0.2mol/L phosphate buffer solution containing 1% bovine serum albumin and having a pH of 7.2 for 2 hours, and drying at 37 ℃ for 2 hours for later use;

10 Adhering a sample absorption pad, a reaction film and a water absorption pad on a bottom plate in sequence, and finally cutting into strips with the width of 3.95 mm;

11 Assembling the prepared microporous reagent and test paper into a test paper strip, and storing for 12 months at the temperature of 2-8 ℃.

Another object of the present invention is to provide a method for detecting residual oxfendazole in liquid milk by using the above test strip, which comprises the steps of:

(1) Sample pretreatment;

(2) Detecting by using a test strip;

(3) And analyzing the detection result.

The invention discloses an oxfendazole rapid detection test strip, which adopts a highly specific antibody antigen reaction and immunochromatography analysis technology, so that oxfendazole monoclonal antibody-colloidal gold marker is freeze-dried in a micropore reagent, and oxfendazole in a sample is fully reacted with the oxfendazole to form a drug-antibody-colloidal gold marker; and then a test strip is inserted, the drugs in the sample compete with the oxfendazole hapten-carrier protein conjugate on the reaction membrane detection line to combine with the oxfendazole monoclonal antibody-colloidal gold marker in the flowing process, and whether the oxfendazole residue is contained in the sample liquid to be detected is judged according to the red stripe depth of the detection line.

During detection, a sample is dripped into a microporous reagent after being treated, a test strip is inserted into micropores after full reaction, when the concentration of the oxfendazole in the sample is lower than the detection limit or is zero, a monoclonal antibody-colloidal gold marker is combined with the oxfendazole hapten-carrier protein conjugate fixed on a reaction membrane in the chromatographic process, a red strip appears on a detection line (T) and a quality control line (C) respectively, and the color development of the T line is deeper than that of the C line or is consistent with that of the C line; if the concentration of oxfendazole in the sample is at or above the limit of detection, the monoclonal antibody-colloidal gold label will bind to all of the oxfendazole, such that no red bands or less coloration than the C-line will occur at the T-line due to competition reactions without binding to the oxfendazole hapten-carrier protein conjugate. As shown in fig. 4.

Negative: and when the quality control line (C) shows red stripes, the detection line (T) also shows red stripes, and the color of the (T) line is close to or deeper than that of the (C), the judgment is negative.

Positive: and judging positive when the quality control line (C) shows red stripes and the detection line (T) does not develop color or the color of the detection line (T) is lighter than that of the detection line (C).

Invalidation: when the quality control line (C) does not display red stripes, whether the detection line (T) displays red stripes or not, the test strip is judged to be invalid.

The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for various units, simple storage and long shelf life. Wherein, the high-specificity oxfendazole monoclonal antibody is adopted, thus ensuring the reliability of the detection result; the gold-labeled antibody is freeze-dried in a microporous reagent, and in the detection process, the gold-labeled antibody can be fully contacted with the sample liquid to be detected for full reaction, so that the error is reduced, and the reaction sensitivity of the whole system is increased. The method for detecting the oxfendazole by using the test strip is simple, convenient, quick, visual and accurate, has wide application range, low cost and easy popularization and use.

Drawings

FIG. 1 is a diagram showing the synthesis of the oxibendazole hapten.

FIG. 2 is a schematic diagram of a cross-sectional structure of the test paper.

FIG. 3 is a microwell reagent plot.

Fig. 4 is a test strip detection result judgment chart.

Detailed Description

The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.

Example 1 preparation of test strip for detecting oxfendazole

The preparation method of the test strip mainly comprises the following steps:

1) Preparing a micropore reagent freeze-dried with an oxfendazole monoclonal antibody-colloidal gold marker;

2) Preparing a reaction membrane with a detection line coated with an oxfendazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;

3) Assembling the reaction membrane prepared in the step 2) with a sample absorption pad, a water absorption pad and a bottom plate to form test paper;

4) And (3) assembling the microporous reagent freeze-dried with the oxfendazole monoclonal antibody-colloidal gold marker prepared in the steps 1) and 3) and the test paper into a test strip.

The following is a stepwise detailed description:

1. synthesis of the Albendazole hapten (synthetic route see FIG. 1)

Dissolving 2.57g of oxfendazole EP impurity C in 200mL of methanol, adding 1.8g of p-carboxybenzaldehyde under stirring at room temperature, adding 2mL of pyridine, heating to react for 4h at 60 ℃, stopping the reaction, cooling to room temperature, adding 0.76g of sodium borohydride, stirring at room temperature for 2h, stopping the reaction, removing methanol and pyridine by rotary evaporation, adding 200mL of water, adding 1mol/L hydrochloric acid to adjust the pH value to 6, precipitating a large amount of insoluble solids, standing at room temperature for 2h, suction filtering, fully pulping the solids with 100mL of absolute ethyl alcohol, suction filtering, removing the ethyl alcohol, and repeating for one time to obtain 1.76g of white solid compound, namely oxfendazole hapten, wherein the yield is 45%.

2. Preparation of immunogens

Taking 14.6mg of oxfendazole hapten, adding 2mL of N, N-Dimethylformamide (DMF), dissolving and clarifying, adding 6.44mg of N-hydroxysuccinimide (NHS) and 10.75mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), fully dissolving and mixing uniformly, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 500mg of Bovine Serum Albumin (BSA), adding 4mL of CB buffer solution of 0.1mol/LpH 9.5.5 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting for 6 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, changing the solution 3 times a day, centrifuging and subpackaging to obtain the oxfendazole hapten-BSA conjugate, namely the immunogen.

3. Preparation of coating Material

Taking 17.39mg of oxfendazole hapten, adding 3mL of DMF for dissolving and clarifying, adding 10mg of NHS and 12.78mg of EDC, fully dissolving and mixing uniformly, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 100mg of Ovalbumin (OVA), adding 9mL of CB buffer solution of 0.1mol/LpH 9.5.5 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting for 6 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, changing the solution 3 times a day, centrifuging and subpackaging to obtain the oxfendazole hapten-OVA conjugate, namely the coating antigen.

4. Preparation of oxfendazole monoclonal antibody

(1) Immunization of animals

The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 150 mug/mouse, so that antisera are generated.

(2) Cell fusion and cloning

Spleen cells of an immunized Balb/c mouse are fused with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative proportion), cell supernatant is measured by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.

(3) Cell cryopreservation and resuscitation

The hybridoma cells were prepared into 1X 10 by using a frozen stock solution ⁶ Cell suspensions of individual/mL were stored in liquid nitrogen for long periods. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.

(4) Preparation and purification of monoclonal antibodies

Incremental culture method: the hybridoma cells are placed in a cell culture medium, cultured at 37 ℃, and the obtained culture solution is purified by an octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, and the monoclonal antibodies are preserved at-20 ℃.

The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium, wherein the final concentration of the calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of the sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium was 7.4.

5. Preparation of goat anti-mouse antibody

Sheep is used as immune animals, and a murine antibody is used as immunogen to immunize pathogen-free sheep, so that the goat anti-mouse antibody is obtained.

6. Preparation of oxfendazole monoclonal antibody-colloidal gold marker

(1) Preparation of colloidal gold

Diluting 1% chloroauric acid into 0.01% (mass fraction) with double distilled deionized water, placing 100mL into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 1.5mL 1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering to original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, has no sediment or floating matters, and has a wine red color when observed in sunlight.

(2) Preparation of oxfendazole monoclonal antibody-colloidal gold marker

Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.2 by using 0.2mol/L potassium carbonate solution, adding the oxfendazole monoclonal antibody into the colloidal gold solution according to the standard of adding 5-50 mug antibody into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30min; after 10min of standing, 10% BSA was added to give a final concentration of 1% in the colloidal gold solution, and the mixture was left for 10min. Centrifuging at 12000r/min and 4deg.C for 40min, discarding supernatant, washing the precipitate twice with redissolving buffer, re-suspending the precipitate with redissolving buffer with volume 1/10 of that of initial colloidal gold, and standing at 4deg.C for use.

Reconstitution buffer: 0.02mol/L phosphate buffer solution containing 0.1-0.5% of BSA, 2-4% of sucrose and pH 7.2.

7. Preparation of microwell reagents

Adding 100 mu L of the oxfendazole monoclonal antibody-colloidal gold marker into a micropore reagent micropore plate, putting into a freeze dryer, pre-freezing for 3 hours at the cold trap temperature of minus 50 ℃, and then drying for 15 hours in vacuum, thus obtaining the micropore reagent freeze-dried with the oxfendazole monoclonal antibody-colloidal gold marker, and sealing and preserving.

8. Preparation of sample absorbent pad

The sample pad was soaked in 0.2mol/L phosphate buffer containing 1% bovine serum albumin at pH 7.2 for 2h and dried at 37℃for 2 h.

9. Preparation of reaction film

The oxfendazole hapten-OVA conjugate is coated on a reaction membrane to form a detection line, and the goat anti-mouse anti-antibody is coated on the reaction membrane to form a quality control line.

The coating process comprises the following steps: diluting the oxfendazole hapten-OVA conjugate to 1mg/mL by using 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coating the oxfendazole hapten-OVA conjugate on a detection line (T line) on a nitrocellulose membrane by using a Bio dot-based membrane marker, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse antibody was diluted to 200. Mu.g/mL with 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coated on a quality control line (C line) on a nitrocellulose membrane in an amount of 1.0. Mu.L/cm using a Bio dot-based membrane cutter. And (5) drying the coated reaction film for 16 hours at 37 ℃ for standby.

10. Assembly of test strips

According to the section structure of the test paper shown in figure 2, a sample absorption pad (1), a reaction membrane (2) and a water absorption pad (3) are sequentially stuck on a PVC bottom plate (6); the tail end of the sample absorption pad is connected with the initial end of the reaction film, the tail end of the reaction film is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line (4) and a quality control line (5), and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test paper; the detection line is positioned at one side close to the tail end of the sample absorption pad; the quality control line is positioned at one side far away from the tail end of the sample absorption pad; the test paper was cut into small strips of 3.95mm width with a machine.

According to the microwell reagent diagram shown in FIG. 3, microwell plugs 8 are provided on the microwell reagent 7.

The test paper and the microporous reagent are assembled into a test paper strip, and the test paper strip is stored in an environment of 2-8 ℃ and has a valid period of 12 months.

Example 2 detection of oxfendazole in liquid milk

1. Sample pretreatment

The sample is recovered to room temperature and then is directly detected, and the sample to be detected needs to be uniform liquid and cannot be agglomerated, soured or precipitated.

2. Detection by test strips

Sucking 200 mu L of sample liquid to be detected into the micropores by using a micropipette, slowly sucking and fully mixing with the reagent in the micropores; after incubation for 5min at 35 ℃, the test strip is inserted into the microwells; the flow of the liquid was started to time, the reaction was continued for 5 minutes, and the result was judged.

3. Analyzing the detection result

Negative (-). The color development of the T line is darker than or consistent with the color development of the C line, indicating that the concentration of oxfendazole in the sample is below the detection limit, as shown in FIGS. 4a and 4b.

Positive (+): the T-line developed less than the C-line developed or the T-line developed no color, indicating that the oxfendazole concentration in the sample was at or above the limit of detection, as shown in fig. 4C, 4d.

Invalidation: the absence of line C indicates an incorrect procedure or that the test strip has failed due to deterioration, as shown in fig. 4e, 4f.

Example 3 sample detection example

1. Limit of detection test

Blank raw milk, pasteurized milk and UHT sterilized milk samples are taken, oxfendazole is added to the samples to obtain final concentrations of 5 mug/L, 10 mug/L and 20 mug/L respectively, test strips are taken for detection, and each sample is repeatedly measured three times.

When the test paper strip is used for detecting raw milk, pasteurized milk and UHT sterilized milk samples, when the oxfendazole is not contained in the raw milk, the addition concentration of the oxfendazole is 5 mug/L, the test paper strip shows that the color development of the T line is deeper than or consistent with that of the C line, and the test paper strip is negative; when the addition concentration of the oxfendazole is 10 mug/L and 20 mug/L, the test strip shows that the color development of the T line is lighter than that of the C line or the T line is not developed, and the test strip is positive, so that the detection limit of the test strip on the oxfendazole in the liquid milk is 10 mug/L.

2. False positive rate and false negative rate test

Taking blank raw milk, pasteurized milk and UHT sterilized milk samples, and adding 20 parts of positive raw milk, pasteurized milk and UHT sterilized milk samples with final concentration of 10 mug/L of oxfendazole, respectively detecting with test strips produced in 3 batches, and calculating the negative-positive rate.

The results show that: when test strips produced in 3 batches are used for detecting positive raw milk, pasteurized milk and UHT sterilized milk samples, the results are positive, the positive coincidence rate is 100%, and the false negative rate is 0; when blank raw milk, pasteurized milk and UHT sterilized milk samples were tested, the results were all negative, and the negative coincidence rate was found to be 100% and the false positive rate was found to be 0. The test strip for detecting the oxfendazole can be used for rapidly detecting the oxfendazole residue in liquid milk.

3. Specificity test

When the test strip is used for detecting 500 mug/kg of albendazole sulfoxide, toldazole, albendazole, fenbendazole, propoxyimidazole and other benzimidazole medicaments, the test strip shows that the T line color development is lighter than the C line color development or the T line is not developed, and the test strip is positive, and the cross reaction rate is only 2%, so that the test strip has small cross reaction and good specificity on the similar medicaments.

Claims (4)

1. The test strip for detecting the oxfendazole comprises a test strip and a micropore reagent, wherein the test strip comprises a reaction membrane, a sample absorption pad, a water absorption pad and a bottom plate, the reaction membrane is provided with a detection line coated with an oxfendazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the micropore reagent is freeze-dried with an oxfendazole monoclonal antibody-colloidal gold marker; the oxfendazole monoclonal antibody is prepared by taking oxfendazole hapten-carrier protein conjugate as immunogen; the oxfendazole hapten-carrier protein conjugate is obtained by coupling an oxfendazole hapten with carrier protein; the method is characterized in that the synthesis method of the oxfendazole hapten comprises the following steps: dissolving 2.57g of oxfendazole EP impurity C by adding 200mL of methanol, adding 1.8g of p-carboxybenzaldehyde under stirring at room temperature, adding 2mL of pyridine, heating to react for 4h at 60 ℃, stopping the reaction, cooling to room temperature, adding 0.76g of sodium borohydride, stirring at room temperature for 2h, stopping the reaction, removing the methanol and the pyridine by rotary evaporation, adding 200mL of water, adding 1mol/L hydrochloric acid to adjust the pH value to 6, precipitating a large amount of insoluble solids, standing at room temperature for 2h, suction filtering, fully pulping the solids by adding 100mL of absolute ethyl alcohol, suction filtering, removing the ethanol, repeating for one time to obtain 1.76g of white solid compound, namely the oxfendazole hapten, wherein the molecular structural formula is as follows:

；

the preparation method of the oxfendazole hapten-carrier protein conjugate immunogen comprises the following steps: taking 14.6mg of oxfendazole hapten, adding 2mL of N, N-dimethylformamide for dissolving and clarifying, adding 6.44mg of N-hydroxysuccinimide and 10.75mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, fully dissolving and uniformly mixing, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 500mg of bovine serum albumin, adding 4mL of 0.1mol/L CB buffer solution with pH of 9.5 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting for 6 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, changing the solution 3 times a day, centrifuging and subpackaging to obtain the oxfendazole hapten-bovine serum albumin conjugate which is an immunogen;

the preparation method of the oxfendazole hapten-carrier protein conjugate coating antigen comprises the following steps: taking 17.39mg of oxfendazole hapten, adding 3mL of DMF for dissolving and clarifying, adding 10mg of NHS and 12.78mg of EDC, fully dissolving and mixing uniformly, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 100mg of ovalbumin OVA, adding 9mL of 0.1mol/L CB buffer solution with pH of 9.5 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting for 6 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, changing the solution 3 times a day, centrifuging and subpackaging to obtain the oxfendazole hapten-OVA conjugate, namely the coating antigen.

2. The test strip of claim 1, wherein the test strip comprises a sample absorbing pad, a reaction membrane, and a water absorbing pad sequentially adhered to a bottom plate, and the microporous reagent has a microporous plug thereon.

3. A method of preparing the test strip of any one of claims 1-2, comprising the steps of:

1) Preparing a micropore reagent freeze-dried with an oxfendazole monoclonal antibody-colloidal gold marker;

2) Preparing a reaction membrane with a detection line coated with an oxfendazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;

3) Assembling the reaction membrane prepared in the step 2) with a sample absorption pad, a water absorption pad and a bottom plate to form test paper;

4) And (3) assembling the microporous reagent freeze-dried with the oxfendazole monoclonal antibody-colloidal gold marker prepared in the steps 1) and 3) and the test paper into a test strip.

4. A method for detecting the residual presence of oxfendazole in a liquid milk comprising the steps of:

1) Sample pretreatment;

2) Detecting with the test strip of any one of claims 1-2;

3) And analyzing the detection result.