CN113267622B - Test strip and method for detecting chlorohydroxypyridine - Google Patents
Test strip and method for detecting chlorohydroxypyridine Download PDFInfo
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Abstract
The invention discloses a test strip and a method for detecting chlorohydroxypyridine. 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 chlorohydroxypyridine hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody, and the micropore reagent is freeze-dried with a chlorohydroxypyridine monoclonal antibody-colloidal gold marker. The method for detecting chlorohydroxypyridine 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
Technical Field
The invention relates to a test strip and a method for detecting chlorohydroxy pyridine, in particular to a colloidal gold test strip for detecting chlorohydroxy pyridine, which is particularly suitable for detecting chlorohydroxy pyridine residues in animal-derived foods.
Background
Chloropicrin (Clopidol) belongs to pyridine compounds, and is widely applied to livestock and poultry farming because of the strong inhibition effect on coccidiosis. Research shows that the medicine is stopped during livestock and poultry cultivation, coccidiosis outbreak is often caused, and continuous or excessive medicine application can cause the residual of chloropicrin in animal bodies and can be accumulated and transferred along with food chains. Chlorohydroxypyridine is teratogenic and constitutes a great 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 prescribes the maximum residual limit of chlorohydroxypyridine in foods.
The methods for detecting chlorohydroxypyridine 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 chloropicrin residue in animal-derived foods is developed, a large number of samples can be screened and monitored on site, and detection work can be better met by food supervision departments in China and the like.
Disclosure of Invention
The invention aims to provide a colloidal gold test strip capable of detecting chloropicrin residues in animal-derived foods, and a detection method which is efficient, accurate, simple and convenient and suitable for on-site monitoring and screening of a large number of samples.
The test strip for detecting chlorohydroxypyridine provided by the invention comprises test paper and a microporous 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 chlorohydroxypyridine hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody; the microporous reagent is freeze-dried with chlorohydroxypyridine monoclonal antibody-colloidal gold marker, which is provided with a microporous plug.
The chlorohydroxy pyridine monoclonal antibody is prepared by taking chlorohydroxy pyridine hapten-carrier protein conjugate as an immunogen.
The chlorohydroxy pyridine hapten-carrier protein conjugate is obtained by coupling chlorohydroxy pyridine hapten with carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin, and the chlorohydroxy pyridine hapten is obtained by reacting chlorohydroxy pyridine with 4-maleimide phenylacetic acid hydrazide, 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 microporous reagent freeze-dried with a chlorohydroxypyridine monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with chlorohydroxypyridine hapten-carrier protein conjugate and a quality control line coated with 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 chlorohydroxypyridine monoclonal antibody-colloidal gold marker prepared in the steps 1) and 3) and test paper into a test strip.
Specifically, the method comprises the following steps:
1) Reacting chlorohydroxypyridine with 4-maleimide phenylacetic acid hydrazide to prepare chlorohydroxypyridine hapten;
2) Coupling chlorohydroxypyridine hapten with carrier protein to prepare chlorohydroxypyridine hapten-carrier protein conjugate;
3) Immunizing a mouse by using chlorohydroxy pyridine hapten-carrier protein conjugate, and obtaining a hybridoma cell strain secreting chlorohydroxy pyridine monoclonal antibody by fusing and screening spleen cells of the mouse and myeloma cells of the mouse;
4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibody;
5) Coating chlorohydroxypyridine hapten-carrier protein conjugate and 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 chlorohydroxypyridine monoclonal antibody into the prepared colloidal gold to obtain a chlorohydroxypyridine monoclonal antibody-colloidal gold marker;
8) Freeze-drying chlorohydroxypyridine monoclonal antibody-colloidal gold marker in a microporous reagent, and adding a microporous plug into the microporous 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 sample absorption pad, reaction film and water absorption pad on the bottom plate in sequence, cutting into strips with width of 3.95mm, placing into special plastic card case, and sealing with aluminum foil bag;
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 chlorohydroxypyridine residue in animal-derived foods by using the above test strip, comprising the steps of:
(1) Sample pretreatment;
(2) Detecting by using a test strip;
(3) And analyzing the detection result.
The chloropicrin rapid detection test strip adopts a highly specific antibody antigen reaction and immunochromatographic analysis technology, a chloropicrin monoclonal antibody-colloidal gold marker is freeze-dried in a microporous reagent, and chloropicrin in a sample fully reacts with the chloropicrin to form a medicine-antibody-colloidal gold marker; and then dripping into the clamping holes of the test paper strips, wherein the medicine in the sample competes with the chlorohydroxypyridine hapten-carrier protein conjugate on the reaction film detection line to combine with the chlorohydroxypyridine monoclonal antibody-colloidal gold marker in the flowing process, and judging whether the chlorohydroxypyridine residue is contained in the sample liquid to be detected 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 card hole is added after full reaction, when the concentration of chloropicrin in the sample is lower than the detection limit or is zero, a monoclonal antibody-colloidal gold marker is combined with a chloropicrin hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography 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 chlorohydrin in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold-labeled substance is bound to all of chlorohydrin, so that no red band or less coloration than the C-line appears at the T-line because of competition reaction without binding to the chlorohydrin 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, a chlorohydroxypyridine monoclonal antibody with high specificity is adopted, thereby 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 chlorohydroxypyridine by using the test strip is simple, convenient, quick, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
FIG. 1 is a diagram showing the synthesis of chlorohydroxypyridine 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 clopidogrel
The preparation method of the test strip mainly comprises the following steps:
1) Preparing a microporous reagent freeze-dried with a chlorohydroxypyridine monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with chlorohydroxypyridine hapten-carrier protein conjugate and a quality control line coated with 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 chlorohydroxypyridine monoclonal antibody-colloidal gold marker prepared in the steps 1) and 3) and test paper into a test strip.
The following is a stepwise detailed description:
1. synthesis of chlorohydroxypyridine hapten (synthetic route see figure 1)
Taking 1.92g of chlorohydroxypyridine, adding 40mL of 2mol/L sodium hydroxide aqueous solution for dissolution, adding 20mL of N, N-Dimethylformamide (DMF) solution containing 4.93g of 4-maleimide phenylacetic acid hydrazide, adding 1.9g of cuprous iodide, heating, and reacting at 100 ℃ for 4h; stopping the reaction, adding 100mL of water, adding 6mol/L of hydrochloric acid to neutralize until the pH value is 7, adding 200mL multiplied by 3 of 1, 2-dichloroethane, extracting for three times, combining organic phases, evaporating to dryness, and recrystallizing with 120mL of absolute ethyl alcohol to obtain 0.74g of maleimido chlorohydroxypyridine hapten product, namely the chlorohydroxypyridine hapten.
2. Preparation of immunogens
Taking 50mg of Bovine Serum Albumin (BSA), adding 6mL of CB buffer solution with the pH value of 0.1mol/L and 9.5 for dissolution, adding 1mL of DMF solution containing 11.6mg of ammonia-succinyl argon ammonia-3 (2-pyridine dithio) acid ester (SPDP), reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying by using 0.02mol/L PBS, and removing unreacted SPDP to obtain BSA solution introduced with dithiopyridyl, and marking as A solution; taking 11mg of tris (2-acyl ethyl) phosphate hydrochloride (TCEP), adding 2mL of PB buffer solution with the pH of 0.5mol/L and 7.0 for dissolution, dripping into the solution A, and reacting at the temperature of 4 ℃ for 30min to obtain thiolated BSA, and marking the thiolated BSA as solution B; taking 15mg of chlorohydroxy pyridine hapten, adding 1mL of DMF for dissolution, adding into solution B, reacting for 3 hours at room temperature, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution for 3 times a day, centrifuging and subpackaging to obtain the chlorohydroxy pyridine hapten-BSA conjugate, namely the immunogen.
3. Preparation of coating Material
Taking 50mg of Ovalbumin (OVA), adding 6mL of CB buffer solution with the pH of 0.1mol/L and 9.5 for dissolution, adding 1mL of DMF solution containing 9mg of SPDP, reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying by using 0.02mol/L PBS, and removing unreacted SPDP to obtain OVA solution introduced with dithiopyridyl, and marking as solution A; taking TCEP 11mg, adding 2mL of PB buffer solution with the pH of 0.5mol/L and 7.0 for dissolution, dripping into the solution A, and reacting at the temperature of 4 ℃ for 30min to obtain thiolated OVA, which is marked as solution B; taking 8.2mg of chlorohydroxy pyridine hapten, adding 1mL of DMF for dissolution, adding the solution into the solution B, reacting for 3 hours at room temperature, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution for 3 times each day, centrifuging and split charging to obtain the chlorohydroxy pyridine hapten-OVA conjugate, namely the coating antigen.
4. Preparation of chlorohydroxypyridine 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 chlorohydroxypyridine 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 chlorohydroxypyridine 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 chlorohydroxypyridine 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 7.2 of pH.
7. Preparation of microwell reagents
Adding 100 mu L of chloropicrin 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 freeze-dried chloropicrin monoclonal antibody-colloidal gold marker micropore reagent, 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 chlorohydroxypyridine 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: the chlorohydroxypyridine hapten-OVA conjugate was diluted to 1mg/mL with 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coated on a detection line (T line) on a nitrocellulose membrane with a Bio dot-based membrane-drawing instrument in an amount of 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; cutting test paper into strips with the width of 3.95mm by a machine, putting the strips into a specially-made plastic card shell, and sealing the strips by an aluminum foil bag.
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 clopidol in animal-derived foods
1. Sample pretreatment
(1) Fresh egg sample
Breaking fresh egg to 100mL beaker, and mixing (egg white and yolk are stirred uniformly); weighing (2.00+/-0.05) g of the uniformly mixed fresh egg sample into a 10mL polystyrene centrifuge tube, adding 0.5mL of NaCl solution, swirling for 2min by a vortex meter, adding 3mL of sample extractant, swirling for 2min by the vortex meter, and centrifuging for 5min at room temperature (20-25 ℃) of not less than 3000 r/min; transferring 2mL of the upper organic phase into a 10mL polystyrene centrifuge tube, and blow-drying under water bath nitrogen or air flow at 50-60 ℃; and adding 300 mu L of sample complex solution, and whirling for 30s by using a vortex meter to obtain sample liquid to be detected.
(2) Tissue sample
Homogenizing the tissue sample with a homogenizer; weighing (2.00+/-0.05) g of homogenized tissue sample into a 10mL polystyrene centrifuge tube, adding 5mL of sample extracting solution, swirling for 2min by a vortex meter, and centrifuging for 5min at room temperature (20-25 ℃) of not less than 3000 r/min; the upper layer is a sample liquid (for example, when the part of the sample is too much grease and the upper layer is a white emulsion, the middle part is taken to be a relatively clear part), 150 mu L of the sample liquid is sucked, 900 mu L of sample extracting solution is added, and the sample liquid is uniformly mixed by vortex to obtain the liquid to be measured (a 2mL polystyrene centrifuge tube is taken as a dilution container).
(3) Fresh milk sample
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 100 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 3min at room temperature (20-25 ℃), 100 mu L of the mixed solution is sucked and vertically dropped into the sample adding hole; and (5) starting timing the liquid flow, reacting for 5-8 min, and judging the result.
3. Analyzing the detection result
Negative (-). The color development of the T line is deeper than or consistent with that of the C line, which indicates that the chloropicrin concentration in the sample is lower than the detection limit, as shown in figures 4a and 4b.
Positive (+): the T line shows less color than the C line or the T line shows no color, which indicates that the concentration of chlorohydroxypyridine in the sample is equal to or higher than the detection limit, as shown in FIGS. 4C and 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
Taking blank eggs, duck eggs and quail eggs, and respectively adding chloropicrin to the blank eggs, the duck eggs and the quail eggs to obtain final concentrations of 7.5 mug/kg, 15 mug/kg and 30 mug/kg; taking blank chicken, pork and beef samples, and respectively adding chloropicrin to the blank chicken, pork and beef samples to obtain final concentrations of 1mg/kg, 2mg/kg and 4mg/kg; taking blank raw milk, pasteurized milk and UHT sterilized milk samples, and respectively adding chloropicrin to the blank raw milk, pasteurized milk and UHT sterilized milk samples with final concentrations of 5 mug/L, 10 mug/L and 20 mug/L; test strips were taken for testing and each sample was assayed in triplicate.
When the test strip is used for detecting animal-derived foods, when the chloropicrin-free and the addition concentration thereof in egg, duck egg and quail egg samples are 7.5 mug/kg, the chloropicrin-free and the addition concentration thereof in chicken, pork and beef samples are 1mg/kg, and the chloropicrin-free and the addition concentration thereof in raw milk, pasteurized milk and UHT sterilized milk samples are 5 mug/L, the test strip shows that the color development of T line is deeper than or consistent with that of C line and is negative; when the chloropicrin addition concentration in the egg, duck egg and quail egg samples is 15 mug/kg and 30 mug/kg, and the chloropicrin addition concentration in chicken, pork and beef samples is 2mg/kg and 4mg/kg, and the chloropicrin addition concentration in raw milk, pasteurized milk and UHT sterilized milk samples is 10 mug/L and 20 mug/L, the test strip shows that the T line color development is shallower than the C line color development or the T line color development is not positive, and the test strip has the following detection limit on the chloropicrin in animal-derived foods: fresh egg sample 15. Mu.g/kg, tissue sample 2mg/kg, fresh milk sample 10. Mu.g/L.
2. False positive rate and false negative rate test
Taking 20 parts of blank eggs, duck eggs, quail eggs, chicken, pork, beef, raw milk, pasteurized milk and UHT sterilized milk samples, respectively, detecting the blank eggs, the duck eggs, the quail eggs, chicken, pork, beef, raw milk, pasteurized milk and UHT sterilized milk samples by using test strips produced in 3 batches, and calculating the negative-positive rate of the blank eggs, the duck eggs, the quail eggs, the chicken, the pork, the beef, the raw milk, the pasteurized milk and the UHT sterilized milk samples.
The results show that: when 3 batches of test strips are used for detecting positive animal-derived foods, the results are positive, the positive coincidence rate is 100%, and the false negative rate is 0; when 20 negative animal-derived foods were tested, the results were all negative, and it was found that the negative coincidence rate was 100% and the false positive rate was 0. The test strip for detecting chloropicrin can be used for rapidly detecting chloropicrin residues in animal-derived foods.
3. Specificity test
When the test strip is used for detecting similar medicines of 10mg/kg of diclazuril, dinitrato amine, nicarbazin, sulfaquinoxaline, sulfadimidine, ding Yangkui ester, decoquinate, chloropropane hydrochloride, chlorbenzoguanamine hydrochloride and the like, the test strip shows that the color development of the T line is deeper than or consistent with that of the C line, and is negative, so that the test strip has no cross reaction on the medicines.
Claims (4)
1. The test strip for detecting chloropicrin 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 a chloropicrin 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 a chloropicrin monoclonal antibody-colloidal gold marker; the chlorohydroxy pyridine monoclonal antibody is prepared by taking chlorohydroxy pyridine hapten-carrier protein conjugate as immunogen; the chlorohydroxypyridine hapten-carrier protein conjugate is obtained by coupling chlorohydroxypyridine hapten with carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin; the synthesis method of the chlorohydroxypyridine hapten is characterized by comprising the following steps of: taking 1.92g of chlorohydroxypyridine, adding 40mL of 2mol/L sodium hydroxide aqueous solution for dissolution, adding 20mL of N, N-dimethylformamide solution containing 4.93g of 4-maleimide phenylacetic acid hydrazide, adding 1.9g of cuprous iodide, heating, and reacting at 100 ℃ for 4 hours; stopping the reaction, adding 100mL of water, adding 6mol/L of hydrochloric acid to neutralize until the pH value is 7, adding 200mL multiplied by 3 of 1, 2-dichloroethane, extracting for three times, combining organic phases, evaporating to dryness, and recrystallizing with 120mL of absolute ethyl alcohol to obtain chlorohydroxypyridine hapten, wherein the molecular structural formula is as follows:
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 microporous reagent freeze-dried with a chlorohydroxypyridine monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with chlorohydroxypyridine hapten-carrier protein conjugate and a quality control line coated with 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 chlorohydroxypyridine monoclonal antibody-colloidal gold marker prepared in the steps 1) and 3) and test paper into a test strip.
4. A method for detecting chlorohydroxypyridine residue in animal-derived food, 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.
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