CN110441518B - Test strip and method for detecting gibberellin - Google Patents

Abstract

The invention discloses a test strip and a method for detecting gibberellin. 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 gibberellin 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 gibberellin monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting gibberellin in a sample 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 food supervision departments in China on-site monitoring and detection.

Description

Test strip and method for detecting gibberellin

Technical Field

The invention relates to a test strip and a method for detecting gibberellin, in particular to a colloidal gold test strip for detecting gibberellin, which is particularly suitable for detecting gibberellin residues in bean sprouts.

Background

Gibberellin is a broad-spectrum plant growth regulator, plays a regulating role almost in each stage of plant growth and development, and particularly has a remarkable yield-increasing effect on vegetables, fruits and the like. Gibberellins are currently widely used as growth regulators for rootless bean sprouts. Excessive gibberellin intake by human bodies can interfere the normal endocrine system of the human bodies, and can accumulate in the human bodies after long-term eating, so that chronic poisoning of organs can be caused, canceration can be caused, and the physical and psychological health of consumers can be seriously harmed. The stipulations in the bulletin of the national food and drug administration, ministry of agriculture, national health and family planning committee of China about the prohibition of using gibberellin and other substances in the bean sprout production process (No. 11 in 2015) are as follows: the producers cannot use gibberellin, 4-chlorophenoxyacetic acid sodium, 6-benzyladenine etc. in the production of bean sprouts, and the bean sprouts producers cannot produce bean sprouts containing gibberellin, 4-chlorophenoxyacetic acid sodium, 6-benzyladenine etc.

The methods for detecting gibberellin reported at present mainly comprise liquid chromatography, liquid chromatography tandem mass spectrometry and other instrument methods. 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 rapid and is suitable for gibberellin residue in bean sprouts is developed, a large number of samples can be screened and monitored on site, and detection work of food supervision departments and the like in China can be better met.

Disclosure of Invention

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

The test strip for detecting gibberellin 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 gibberellin hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody; gibberellin monoclonal antibodies-colloidal gold markers are sprayed on the conjugate release pads.

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

The gibberellin hapten-carrier protein conjugate is obtained by coupling gibberellin hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin, the gibberellin hapten is obtained by oxidizing gibberellin and then reacting with butyric hydroxamic 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 1/3-1/2 of the conjugate release pad is covered under the sample absorption pad.

The bottom plate can be a PVC bottom plate or other hard non-water-absorbing materials; the sample absorption pad can be suction filter paper or oil filter paper; the conjugate release pad may be a glass wool or polyester material; the water absorption pad is absorbent paper; the reaction membrane can be 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 gibberellin monoclonal antibody-colloidal gold label;

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

3) And (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 bottom plate to form the test strip.

Specifically, the steps include:

1) Oxidizing gibberellin and then reacting with butyric acid hydroximic acid to prepare gibberellin hapten;

2) Coupling gibberellin hapten and carrier protein to prepare a gibberellin hapten-carrier protein conjugate;

3) Immunizing a mouse by using a gibberellin 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 a gibberellin monoclonal antibody;

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

5) Coating the gibberellin 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 reacting trisodium citrate with chloroauric acid;

7) Adding the prepared gibberellin monoclonal antibody into the prepared colloidal gold to obtain a gibberellin monoclonal antibody-colloidal gold marker;

8) Spraying gibberellin 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, 0.1mol/L phosphate buffer solution with pH of 7.2 for 2h, and drying at 37 deg.C for 2h;

10 A sample absorbent pad, a conjugate release pad, a reaction membrane, and a water absorbent pad are sequentially attached to the base plate, and the conjugate release pad has a 1/3 area covered with the sample absorbent pad from the starting end. Finally cutting into small strips with the width of 3mm, adding a plastic box, vacuum packaging, and storing for 12 months at the temperature of 4-30 ℃.

The invention also aims to provide a method for detecting gibberellin residues in bean sprouts 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 paper strip for rapidly detecting gibberellin adopts a highly specific antibody-antigen reaction and immunochromatographic analysis technology, a gibberellin monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and gibberellin in a sample is combined with the gibberellin monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form a drug-antibody-colloidal gold marker. Drugs in the sample compete with the gibberellin hapten-carrier protein conjugate on the reaction membrane detection line to bind with the gibberellin monoclonal antibody-colloidal gold marker, and whether the sample liquid to be detected contains gibberellin residues or not 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 card hole, when the concentration of gibberellin in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker is combined with a gibberellin hapten-carrier protein conjugate fixed on a reaction membrane 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 gibberellin in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold label will bind to all gibberellins, and thus no red band or less coloration will appear at the T-line due to the competitive reaction with the gibberellin 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 band, the test strip is judged to be invalid whether the detection line (T) shows a red band 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 gibberellin residues 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 gibberellin hapten synthesis.

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 only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1 preparation of test strip for detecting gibberellin

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

1) Preparing a conjugate release pad sprayed with a gibberellin monoclonal antibody-colloidal gold marker;

2) Preparing a reaction membrane with a detection line coated with a gibberellin 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 gibberellin haptens (see the attached figure 1 for synthetic route)

Adding 0.35g of gibberellin into 50mL0.24mg/mL ozone aqueous solution, fully stirring for 1h, adding the reaction solution into a solution containing 1.34g of zinc powder and 1mL of acetic acid, continuously stirring for 2h, stopping the reaction, filtering, removing the zinc powder, adding 120mL of ethyl acetate and 50mL of water into the filtrate, extracting, separating, washing an organic phase with water, concentrating and evaporating to dryness, and recrystallizing 65mL of dichloromethane/n-hexane (V/V, 1/5) to obtain 0.18g of gibberellin oxide, wherein the yield is 51.42%;

taking 0.18g of gibberellin oxide, adding 50mL of ethanol for dissolving, clarifying, adding 0.12g of butyric hydroxamate and 0.2mL of trichloroacetic acid, stirring for 3h at room temperature, stopping reaction, performing rotary evaporation, removing ethanol, adding 60mL of water, adding 80mL of dichloromethane, extracting, separating out a water phase, evaporating an organic phase to dryness, applying to a silica gel column, and performing elution separation by petroleum ether/ethyl acetate (V/V, 1/1) to obtain a butyric acid-gibberellin hapten product of 0.17g with the yield of 74%.

2. Preparation of immunogens

Taking 17mg of gibberellin hapten, adding 1mL of N, N-Dimethylformamide (DMF) for dissolving, adding 9.4mg of carbodiimide (EDC) and 7.3mg of N-hydroxysuccinimide (NHS), blowing, uniformly mixing, and reacting at room temperature for 4 hours to obtain a hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), and adding 0.05mol/L PB buffer solution for dissolving to obtain solution B; dripping the A solution into the B solution, reacting for 2h, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times per day to obtain gibberellin hapten-BSA conjugate, i.e. immunogen, subpackaging, and storing at-20 ℃.

3. Preparation of coating antigen

Dissolving 11mg of gibberellin hapten in 1mL of DMF (dimethyl formamide), adding 0.2mL of ethylenediamine, cooling to 0-5 ℃, dropwise adding 71 mu L of isobutyl chloroformate, and continuously reacting for 4 hours to obtain hapten activating solution A; taking 50mg of Ovalbumin (OVA), and adding 0.05mol/LPB buffer solution for dissolving to obtain solution B; dripping the A solution into the B solution, reacting for 2h, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times per day to obtain gibberellin hapten-OVA conjugate, namely coating antigen, subpackaging, and storing at-20 ℃.

4. Preparation of gibberellin monoclonal antibodies

(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 8:1 (quantitative ratio), measuring cell supernatant by adopting indirect competition ELISA, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain which stably secretes the monoclonal antibody.

(3) Cell cryopreservation and recovery

Making hybridoma cell into 1 × 10 with frozen stock solution ⁶ Cell 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: the hybridoma cells were cultured in a cell culture medium at 37 ℃ and the culture solution obtained was purified by the octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which were stored at-20 ℃.

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 gibberellin 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 2.5mL 1% trisodium citrate under continuous stirring at high temperature, stirring at constant speed, heating 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 gibberellin 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 gibberellin monoclonal antibody into the colloidal gold solution according to the standard that 20-50 mu g of antibody is added into each milliliter of colloidal gold solution, and continuously stirring and uniformly mixing for 30min; after standing for 10min, 10% BSA was added so that the final concentration in the colloidal gold solution became 1%, and the mixture was allowed to stand for 10min. 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 with volume 1/10 of the original volume of colloidal gold, and standing at 4 deg.C for use.

Redissolving buffer solution: 0.1 to 0.3 percent of BSA, 0.05 to 0.2 percent of Tween-80 and 0.02mol/L of phosphate buffer solution with pH7.2.

7. Preparation of conjugate Release pad

The conjugate release pad was soaked in 0.5% BSA in 0.2 pH 7.5 mol/L phosphate buffer, soaked uniformly for 1h, and baked at 37 ℃ for 3h for use. And uniformly spraying the prepared gibberellin monoclonal antibody-colloidal gold marker on a conjugate release pad by using an Isoflow film spraying instrument, spraying 0.01mL of the gibberellin monoclonal antibody-colloidal gold marker on every 1cm of the conjugate release pad, placing the conjugate release pad in an environment at 37 ℃ (the humidity is less than 20%) for 60min, taking out, and placing the conjugate release pad in a dry environment (the humidity is less than 20%) for storage for later use.

8. Preparation of sample absorbent pad

The sample absorption pad is placed in a phosphate buffer solution containing 0.5 percent of bovine serum albumin, with the pH value of 7.2 and 0.1mol/L to be soaked for 2 hours, and dried for 2 hours at the temperature of 37 ℃ for standby.

9. Preparation of the reaction film

Coating the gibberellin 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 gibberellin hapten-ovalbumin conjugate to 1mg/mL by using a phosphate buffer solution, and coating the gibberellin 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, pH7.4 phosphate buffer, and coated on a control line (C line) on a nitrocellulose membrane in an amount of 1.0. Mu.L/cm using an Isoflow point membrane machine. 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 in a 1/3 area from the initial end, the tail end of the binder release 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 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 paper 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 gibberellin in Bean sprouts

1. Pretreatment of samples

Homogenizing the bean sprout sample with a homogenizer (cut and ground to powder); weighing 2.0g +/-0.05 g of sample into a 10mL polystyrene centrifuge tube, adding 3mL phosphate buffer solution, and whirling for 1min by using a vortex mixer to mix uniformly; centrifuging at 4000r/min for 5min at room temperature (20-25 ℃), and taking supernatant as sample liquid to be detected.

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 8min, 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 gibberellin in the sample is lower than the detection limit, as shown in FIGS. 3a and 3b.

Positive (+): the T line is lighter or not developed than the C line, indicating that the gibberellin concentration in the sample is equal to or above the detection limit, as shown in FIGS. 3C and 3d.

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 3f.

Example 3 sample testing example

1. Limit of detection test

Taking blank bean sprout samples, adding gibberellin into the blank bean sprout samples to the final concentration of 50 mug/kg, 100 mug/kg and 200 mug/kg respectively, taking test strips for detection, and repeatedly measuring each sample for three times.

When the test paper strip is used for detecting bean sprout samples, when no gibberellin exists and the addition concentration of the gibberellin is 50 mug/kg, the test paper strip 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 strip is negative; when the gibberellin addition concentration is 100 mug/kg and 200 mug/kg, the test strip shows that the T line color development is lighter than the C line color development or the T line color is not developed and is positive, which shows that the test strip has the detection limit of 100 mug/kg on the gibberellin in the bean sprouts.

2. Test of false positive and false negative rates

Taking 20 parts of blank bean sprout samples and positive bean sprout samples added with gibberellin to the final concentration of 100 mug/kg respectively, detecting by using test strips produced in 3 batches respectively, and calculating the negative and positive rates. The results are shown in Table 1.

Table 1 bean sprout sample test results

The results show that: when 3 test strips produced in batches are used for detecting positive bean sprout samples, the results are all positive, the positive coincidence rate is 100 percent, and the false negative rate is 0; when 20 negative bean sprout samples are detected, the results are all negative, the negative coincidence rate is 100 percent, and the false positive rate is 0. The test strip for detecting gibberellin can be used for rapidly detecting gibberellin residues in bean sprout samples.

3. Specificity test

When the test strip is used for detecting 1000 mu g/kg of 4-chlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 6-benzyladenine, naphthylacetic acid, indoleacetic acid, indolebutyric acid, paclobutrazol, thidiazuron and other plant growth regulators, the test strip shows that the T line color development is deeper than or consistent with the C line color development and is negative, which indicates that the test strip has no cross reaction to the medicines.

Claims (5)

1. A test strip for detecting gibberellin 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 gibberellin hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and a gibberellin monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad; the gibberellin monoclonal antibody is prepared by taking a gibberellin hapten-carrier protein conjugate as an immunogen; the gibberellin hapten-carrier protein conjugate is obtained by coupling gibberellin hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine protein or human serum albumin, and the gibberellin hapten is synthesized by the following steps: adding 0.35g of gibberellin into 50mL of 0.24mg/mL ozone water solution, fully stirring for 1h, adding the reaction solution into a solution containing 1.34g of zinc powder and 1mL of acetic acid, continuously stirring for 2h, stopping the reaction, filtering, removing the zinc powder, adding 120mL of ethyl acetate and 50mL of water into the filtrate, extracting, separating, washing an organic phase with water, concentrating and evaporating to dryness, and recrystallizing by using 65mL of dichloromethane/n-hexane with a volume ratio of 1/5 to obtain 0.18g of gibberellin oxide; taking 0.18g of gibberellin oxide, adding 50mL of ethanol for dissolving and clarifying, adding 0.12g of butyric hydroxamic acid and 0.2mL of trichloroacetic acid, stirring for 3 hours at room temperature, stopping reaction, performing rotary evaporation, removing ethanol, adding 60mL of water, adding 80mL of dichloromethane, extracting, separating a water phase, evaporating an organic phase to dryness, applying to a silica gel column, and performing elution and separation by petroleum ether/ethyl acetate with a volume ratio of 1/1 to obtain 0.17g of gibberellin hapten, wherein the molecular structural formula of the gibberellin hapten is as follows:

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

3. The strip of any one of claims 1-2, wherein 1/3 to 1/2 of the conjugate release pad is covered under the sample absorbent 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 gibberellin monoclonal antibody-colloidal gold marker;

2) Preparing a reaction membrane with a detection line coated with a gibberellin 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 gibberellin residues in bean sprout samples, comprising the steps of:

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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