CN111735952A - Test strip and method for detecting hexachlorobenzene - Google Patents

Abstract

The invention discloses a test strip and a method for detecting hexachlorobenzene. The test strip comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a hexachlorobenzene hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a hexachlorobenzene monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting hexachlorobenzene in feed by applying the test strip. The test strip provided by the invention has the advantages of simple operation, high sensitivity, high detection speed, low cost, suitability for screening large-batch samples and the like, and can meet the requirements of the national government regulatory departments on the field monitoring and detection work.

Description

Test strip and method for detecting hexachlorobenzene

Technical Field

The invention relates to a test strip and a method for detecting hexachlorobenzene, in particular to a colloidal gold test strip for detecting hexachlorobenzene, which is particularly suitable for detecting residual hexachlorobenzene in feed.

Background

Hexachlorobenzene has been mainly used for preventing and treating wheat smut, seed and soil disinfection, also used as a solvent, and used as a manufacturing intermediate or additive in the production of synthetic rubber, polyvinyl chloride plastic, fireworks, munitions, wood preservatives and dyes, has the characteristics of difficult degradation, biological accumulation, high toxicity and the like, can reach the human body through ways of inhalation, ingestion, percutaneous absorption and the like, has influence on the liver and nervous system, causes organ function damage and skin damage, and is one of human carcinogens. In addition, hexachlorobenzene also has extremely high toxicity to aquatic organisms, and produces bioaccumulation through the food chain, ultimately affecting human health. The national standard GB 13078-2017 feed sanitation Standard stipulates the limit requirement of hexachlorobenzene in the feed, the grease is less than or equal to 0.2mg/kg, and other feed raw materials, additive premixed feed, concentrated feed, concentrate supplement and compound feed are less than or equal to 0.01 mg/kg. Therefore, the method has important practical significance for monitoring the hexachlorobenzene.

The methods for detecting hexachlorobenzene reported at present mainly comprise instrument methods such as gas chromatography, gas chromatography-mass spectrometry, liquid chromatography tandem mass spectrometry and the like. The methods are operated under laboratory conditions, the sample pretreatment is complicated and time-consuming, expensive instruments and equipment are required, 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 requirement for rapidly detecting a large number of samples and field samples is difficult to meet. Therefore, the colloidal gold test strip which is simple and quick and is suitable for detecting the hexachlorobenzene residue in the feed is developed, so that the on-site screening and monitoring of a large number of samples can be met, and the detection work of national government supervision departments and the like can be better met.

Disclosure of Invention

The invention aims to provide a colloidal gold test strip capable of detecting hexachlorobenzene residue in feed, and provides a detection method which is efficient, accurate, simple and convenient and is suitable for field monitoring and large-scale sample screening.

The test strip for detecting hexachlorobenzene provided by the invention comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the reaction membrane is provided with a detection line coated with a hexachlorobenzene hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody; the hexachlorobenzene monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad.

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

The hexachlorobenzene hapten-carrier protein conjugate is obtained by coupling hexachlorobenzene hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, the hexachlorobenzene hapten is obtained by condensation reaction of pentachlorobenzaldehyde and 3-hydrazinopropionic acid, and the molecular structural formula is as follows:

the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate, and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.

The bottom plate is a PVC bottom plate or other hard non-absorbent materials; the sample absorption pad is made of polyester fiber or glass fiber; the conjugate release pad is made of glass wool or polyester material; the water absorption pad is absorbent 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 test strip, which comprises the steps of:

1) preparing a conjugate release pad sprayed with a hexachlorobenzene monoclonal antibody-colloidal gold marker;

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

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

Specifically, the steps include:

1) carrying out condensation reaction on pentachlorobenzaldehyde and 3-hydrazinopropionic acid to prepare hexachlorobenzene hapten;

2) coupling hexachlorobenzene hapten with carrier protein to prepare a hexachlorobenzene hapten-carrier protein conjugate;

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

4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;

5) respectively coating the hexachlorobenzene 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;

6) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;

7) adding the prepared hexachlorobenzene monoclonal antibody into the prepared colloidal gold to obtain a hexachlorobenzene monoclonal antibody-colloidal gold marker;

8) spraying the hexachlorobenzene monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 1h, taking out, and storing in a dry environment for later use;

9) soaking the sample absorption pad in 0.5% bovine serum albumin-containing phosphate buffer solution with pH of 7.2 and 0.1mol/L for 1h, and drying at 37 deg.C for more than 4 h;

10) a sample absorbing pad, a conjugate releasing pad, a reaction membrane and a water absorbing pad are sequentially adhered on the bottom plate, and the conjugate releasing pad is covered by the sample absorbing pad from the 1/3 area at the starting end. And finally cutting into small strips with the width of 3mm, adding a plastic box, carrying out vacuum packaging, and storing for 12 months at the temperature of 4-30 ℃.

The invention also aims to provide a method for detecting the residual hexachlorobenzene in the feed by using the test strip, which comprises the following steps:

(1) pretreating a sample;

(2) detecting by using a test strip;

(3) and analyzing the detection result.

The test strip for rapidly detecting the hexachlorobenzene adopts a highly specific antibody-antigen reaction and immunochromatography analysis technology, a hexachlorobenzene monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and the hexachlorobenzene in a sample is combined with the hexachlorobenzene monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form a drug-antibody-colloidal gold marker. The drug in the sample and the hexachlorobenzene hapten-carrier protein conjugate on the reaction membrane detection line compete to combine with the hexachlorobenzene monoclonal antibody-colloidal gold marker, and whether the sample liquid to be detected contains hexachlorobenzene residue is judged according to the depth of a red strip of the detection line.

During detection, a sample is treated and then dropped into a test strip clamping hole, when the concentration of hexachlorobenzene in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker can be combined with a hexachlorobenzene hapten-carrier protein conjugate fixed on a reaction film in the chromatography process, a red strip is respectively formed on a detection line (T) and a quality control line (C), 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 hexachlorobenzene in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold label will bind to all of the hexachlorobenzene, and thus no red band will appear or the color will be lighter than that of the C-line at the T-line because the competitive reaction will not bind to the hexachlorobenzene hapten-carrier protein conjugate. As shown in fig. 3.

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

Positive: and when the quality control line (C) shows a red strip, the detection line (T) does not show color or the color of the line (T) is lighter than that of the line (C), the test line is judged to be positive.

And (4) invalidation: when the quality control line (C) does not show a red strip, the test strip is judged to be invalid whether the detection line (T) shows a red strip or not.

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 quality guarantee period. The method for detecting the residual hexachlorobenzene by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.

Drawings

FIG. 1 is a diagram of the synthesis of hexachlorobenzene hapten.

FIG. 2 is a schematic cross-sectional view of a test strip.

FIG. 3 is a diagram showing the test result of the test strip.

Detailed Description

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 preparation of test strip for detecting hexachlorobenzene

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

1) preparing a conjugate release pad sprayed with a hexachlorobenzene monoclonal antibody-colloidal gold marker;

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

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

The following steps are detailed:

1. synthesis of hexachlorobenzene hapten (the synthetic route is shown in figure 1)

Dissolving 2.78g of pentachlorobenzaldehyde in 60mL of methanol, and clarifying; dissolving 1.05g of 3-hydrazinopropionic acid in 3mL of water, and dropwise adding the solution into a methanol solution of pentachlorobenzaldehyde; adding 0.2mL of trifluoroacetic acid, stirring at room temperature for 3h, stopping reaction, performing rotary evaporation, removing methanol, adding 50mL of water and 50mL multiplied by 3 of ethyl acetate, extracting for three times, combining organic phases, performing rotary evaporation and evaporation, purifying by using a silica gel column, and performing elution separation by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 3:1 to obtain the hexachlorobenzene hapten.

2. Preparation of immunogens

Taking 14mg of hexachlorobenzene hapten, adding 1mL of N, N-Dimethylformamide (DMF) for dissolving, adding 9.7mg of N-hydroxysuccinimide (NHS) and 11mg of carbodiimide (EDC), and reacting for 3h at room temperature to obtain a hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), and adding 0.05mol/LCB buffer solution for dissolving to obtain solution B; dripping the A solution into the B solution, reacting at 4 deg.C for 12h, dialyzing and purifying with 0.02mol/LPBS for 3 days, changing solution 3 times per day to obtain hexachlorobenzene hapten-BSA conjugate as immunogen, packaging, and storing at-20 deg.C.

3. Preparation of coating antigen

Dissolving hexachlorobenzene hapten 6mg in 1mL of dimethyl sulfoxide (DMSO), adding NHS 5.2mg and EDC 5.5mg, and reacting at room temperature for 3h to obtain a hapten solution A; dissolving Ovalbumin (OVA)50mg in CB buffer solution of 0.05mol/L to obtain solution B; dripping the A solution into the B solution, reacting at 4 deg.C for 12h, dialyzing and purifying with 0.02mol/L PBS for 3 days, changing solution 3 times per day to obtain hexachlorobenzene hapten-OVA conjugate, packaging, and storing at-20 deg.C.

4. Preparation of hexachlorobenzene monoclonal antibody

(1) Animal immunization

Injecting the immunogen obtained in the step 2 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.

(2) Cell fusion and cloning

Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), measuring cell supernatant by adopting indirect competitive ELISA, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.

(3) Cell cryopreservation and recovery

Preparing hybridoma cell into 1 × 10 with frozen stock solution⁶Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.

(4) Preparation and purification of monoclonal antibodies

An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.

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

5. Preparation of goat anti-mouse anti-antibody

The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.

6. Preparation of hexachlorobenzene monoclonal antibody-colloidal gold marker

(1) Preparation of colloidal gold

Diluting 1% chloroauric acid to 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 of 1% trisodium citrate under continuous stirring at high temperature, stirring and heating at constant speed until the solution is bright red, cooling to room temperature, recovering the volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, is transparent and bright, has no sediment or floating objects, and is wine red when observed in sunlight.

(2) Preparation of hexachlorobenzene 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 hexachlorobenzene monoclonal antibody into the colloidal gold solution according to the standard that 5-50 mu g of antibody is added into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30 min; after standing for 10min, 10% BSA was added to make the final concentration of the solution in colloidal gold 1%, and the solution was allowed to stand for 10 min. Centrifuging at 12000r/min at 4 deg.C for 40min, discarding supernatant, washing the precipitate with redissolving buffer twice, resuspending the precipitate with redissolving buffer whose volume is 1/10 of the original volume of colloidal gold, and standing at 4 deg.C for use.

Redissolving buffer solution: 0.02mol/L phosphate buffer solution containing 0.1-0.5 percent of BSA, 2-4 percent of sucrose and pH7.2.

7. Preparation of conjugate Release pad

The conjugate release pad was soaked in 0.5% BSA, pH7.2, 0.5mol/L phosphate buffer, soaked for 1h, and baked at 37 deg.C for 3 h. And uniformly spraying the prepared hexachlorobenzene monoclonal antibody-colloidal gold marker on a conjugate release pad by using an Isoflow film spraying instrument, spraying 0.01mL of hexachlorobenzene monoclonal antibody-colloidal gold marker on every 1cm of conjugate release pad, placing in an environment at 37 ℃ (humidity is less than 20%) for 60min, taking out, and placing in a dry environment (humidity is less than 20%) for storage.

8. Preparation of sample absorbent pad

The sample absorption pad is soaked in 0.1mol/L phosphate buffer solution containing 0.5 percent of bovine serum albumin and having the pH value of 7.2 for 1h, and is dried for more than 4h at the temperature of 37 ℃ for later use.

9. Preparation of the reaction film

Coating the hexachlorobenzene hapten-ovalbumin conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line.

Coating process: diluting the hexachlorobenzene hapten-ovalbumin conjugate to 1mg/mL by using 0.01mol/L, pH 7.4.4 phosphate buffer, and coating the hexachlorobenzene hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using an Isoflow point membrane instrument, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01mol/L, pH 7.4.4 phosphate buffer and coated on a quality control line (line C) on a nitrocellulose membrane in an amount of 1.0. mu.L/cm using an Isoflow dot membrane apparatus. And (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.

10. Assembly of test strips

According to the section structure of the test strip shown in the attached figure 2, a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3) and a water absorption pad (4) are sequentially adhered to a PVC bottom plate (7); the binder release pad is covered by the sample absorption pad from the 1/3 area at the starting end, the tail end of the binder release pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC base plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC base plate; the reaction membrane is provided with a detection line (5) and a quality control line (6), and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are vertical to the long phase of the test strip; the detection line is located on the side near the end of the conjugate release pad; the quality control line is positioned on the side away from the end of the conjugate release pad; cutting the test strip into small strips with the width of 3mm by a machine, putting the small strips into a special plastic card, and storing the small strips in an environment with the temperature of 4-30 ℃ for 12 months.

Example 2 detection of hexachlorobenzene in feed

1. Sample pretreatment

Weighing (4.00 +/-0.05) g of crushed sample into a 15mL polystyrene centrifuge tube, adding 5mL of n-hexane, whirling for 3min by using a vortex instrument, and centrifuging for 5min at room temperature (20-25 ℃) at a speed of not less than 3000 r/min; transferring 4mL of the upper organic phase into a 10mL polystyrene centrifuge tube, and drying by blowing under a water bath nitrogen flow or an air flow at 50-60 ℃; add 300 u L0.02 mol/L PBS buffer solution to redissolve, vortex with vortex instrument 20s after examination.

2. Detection with test strips

Sucking 80 mu L of sample liquid to be detected by a micropipette and vertically dripping the sample liquid into the sample adding hole; the liquid flow was started, the reaction was carried out for 10min, and the results were judged.

3. Analyzing the results of the detection

Negative (-): the color development of the T line is darker than that of the C line or consistent with that of the C line, which indicates that the concentration of hexachlorobenzene in the sample is lower than the detection limit, as shown in FIGS. 3a and 3 b.

Positive (+): the color development of the T line is lighter than that of the C line or the T line is not developed, which indicates that the concentration of hexachlorobenzene in the sample is equal to or higher than the detection limit, as shown in FIGS. 3C and 3 d.

And (4) invalidation: the absence of line C indicates an incorrect procedure or the test strip has deteriorated and failed, as shown in FIGS. 3e and 3 f.

Example 3 sample testing example

1. Limit of detection test

Taking blank feed raw materials, premix, concentrate and batch samples, respectively adding hexachlorobenzene into the blank feed raw materials, the premix, the concentrate and the batch samples until the final concentrations are 5 mug/kg, 10 mug/kg and 20 mug/kg, taking test strips for detection, and repeatedly measuring each sample for three times.

When the test paper is used for detecting feed raw materials, premix compounds, concentrated materials and batch samples, when hexachlorobenzene is not contained and the addition concentration of hexachlorobenzene is 5 mug/kg, the test paper shows that the color development of a T line is darker than that of a C line or is consistent with that of the C line, and the test paper is negative; when the addition concentration of the hexachlorobenzene is 10 mug/kg and 20 mug/kg, the test strip shows that the color development of a T line is lighter than that of a C line or the color development of the T line is not positive, which shows that the test strip has the detection limit of 10 mug/kg for the hexachlorobenzene in the feed raw materials, the premix, the concentrate and the batch, and meets the limit requirement of GB 13078.

2. Test for false positive and false negative rates

Taking blank feed raw materials, premix, concentrate and batch samples, adding hexachlorobenzene to the positive feed raw materials with the final concentration of 10 mug/kg, the premix, the concentrate and the batch samples by 20 parts respectively, detecting by using test strips produced in 3 batches respectively, and calculating the positive and negative rates.

The results show that: when 3 batches of test strips are used for detecting positive samples, the results are all positive, the positive coincidence rate is 100 percent, and the false negative rate is 0; when negative samples are detected, the results are all negative, and the negative coincidence rate is 100 percent and the false positive rate is 0. The test strip for detecting hexachlorobenzene can be used for rapidly detecting the residual hexachlorobenzene in the feed.

3. Specificity test

When the test strip is used for detecting other organochlorine pesticides such as 10mg/kg quintozene, hexachloro cyclohexane, dichlorodiphenyl trichloroethane, heptachlor, chlordane, aldrin, dieldrin and the like, the test strip shows that the color development of a T line is deeper than or consistent with that of a C line, and the test strip is negative, so that the test strip has no cross reaction to the drugs.

Claims (5)

1. A test strip for detecting hexachlorobenzene comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a hexachlorobenzene hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a hexachlorobenzene monoclonal antibody-colloidal gold marker; the hexachlorobenzene monoclonal antibody is prepared by taking a hexachlorobenzene hapten-carrier protein conjugate as an immunogen; the hexachlorobenzene hapten-carrier protein conjugate is obtained by coupling hexachlorobenzene hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, and is characterized in that the hexachlorobenzene hapten is obtained by condensation reaction of pentachlorobenzaldehyde and 3-hydrazinopropionic acid, and the molecular structural formula is as follows:

2. the strip of claim 1, wherein the sample absorbing pad, the conjugate releasing pad, the reaction membrane, and the absorbent pad are sequentially attached to the base plate.

3. The strip of any one of claims 1-2, wherein the conjugate release pad 1/3-1/2 is covered under a sample absorbing pad.

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

1) preparing a conjugate release pad sprayed with a hexachlorobenzene monoclonal antibody-colloidal gold marker;

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

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

5. A method for detecting hexachlorobenzene residue in feed comprises the following steps:

1) pretreating a sample;

2) performing a test using the test strip of any one of claims 1-3;

3) and analyzing the detection result.

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