CN111505293B - Test strip for detecting variegated aspergillin and application thereof - Google Patents
Test strip for detecting variegated aspergillin and application thereof Download PDFInfo
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Abstract
The invention discloses a test strip for detecting variolothricin and application thereof. The test strip comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), wherein the reaction membrane is provided with a detection line (5) coated with a variegated aspergillin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a variegated aspergillin monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting the residual variolomycin in grains such as wheat, corn and the like and feeds by applying the test strip for the variolomycin. The test strip provided by the invention has the characteristics of simple operation, high sensitivity, high detection speed, low cost and the like, and is suitable for screening and on-site monitoring of a large number of samples.
Description
Technical Field
The invention relates to a test strip for detecting variolothricin and application thereof, in particular to a colloidal gold test strip for detecting the variolothricin, which is particularly suitable for detecting the remaining variolothricin in grains such as wheat, corn and the like and feeds.
Background
Variegated aspergillin (ST for short) can induce experimental animals to generate a plurality of cancers. High incidence of liver cancer in certain regions of south africa may be associated with ST. ST is associated with gastric cancer in humans. ST can also be converted to the more potent carcinogen aflatoxin. ST can cause some animal diseases. ST can be produced by metabolism of more than 10 fungi, and the yield on artificial culture medium reaches 10g/kg. Foods known to be contaminated with ST include: rice, wheat, corn, peanut, soybean, ham, cheese, coffee, etc.
Since the damages of ST are large and the distribution is wide, the effective and fast detection of ST in food and feed is very important to reduce the influence of ST on human and livestock. Compared with the traditional biological detection method, the thin layer chromatography, the high performance liquid chromatography and the like, the immunochemical detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, automatic operation and the like when the mycotoxin is detected. The prerequisite for immunochemical detection of ST is the requirement for antibodies against ST. The invention is a rapid detection method, has short time, simple operation and lower cost, and is suitable for detecting samples in large batch by a basic unit.
Disclosure of Invention
The invention aims to provide a test strip for detecting the residual of the sterigmatocystin, which has the advantages of high sensitivity, simple operation, low cost and short detection time.
The test strip for detecting the residual variegated aspergillin provided by the invention comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7); the reaction membrane is provided with a detection line (5) coated with a variegated aspergillin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a variegated aspergillin monoclonal antibody-colloidal gold marker.
The varicolored hapten-carrier protein conjugate is obtained by coupling a varicolored hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein and human serum albumin.
The variegated aspergillomycin monoclonal antibody is prepared by taking a variegated aspergillomycin hapten-carrier protein conjugate as an immunogen and is obtained by secreting a variegated aspergillomycin monoclonal antibody hybridoma cell strain; the goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody.
The sample absorption pad (1), the conjugate release pad (2), the reaction membrane (3) and the water absorption pad (4) are sequentially adhered to the bottom plate (7), 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-absorbent 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 variolothricin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a variegated aspergillin 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) Hapten preparation: enabling the variegated aspergillomycin and 3-maleimide hydrazine propionate and other substances to undergo a series of chemical reactions to obtain a variegated aspergillomycin hapten;
2) Coupling the variolothricin hapten with carrier protein to obtain a variolothricin hapten-carrier protein conjugate;
3) Immunizing a mouse by using a heterochromomycin hapten-carrier protein conjugate, and fusing and screening splenocytes and myeloma cells of the mouse to obtain a heterochromomycin monoclonal hybridoma cell strain;
4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) Preparing colloidal gold by reacting trisodium citrate with chloroauric acid;
6) Adding the variolox aspergillon monoclonal antibody prepared in the step 3) into the colloidal gold prepared in the step 5) to obtain a variolox aspergillon monoclonal antibody-colloidal gold marker;
7) Spraying the varicolomycin 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;
8) Coating the heterochromomycin hapten-carrier protein 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;
9) Soaking the sample absorption pad in 0.5% bovine serum albumin (volume fraction), pH7.2, 0.1mol/L phosphate buffer solution for 2h, and drying at 37 deg.C for 2h;
10 A sample absorbing pad, a conjugate releasing pad, a reaction film and a water absorbing pad are sequentially adhered on the bottom plate, the sample absorbing pad covers the conjugate releasing pad, and finally the sample absorbing pad is cut into small strips with the width of 3mm, a plastic box is added, the small strips are packaged in vacuum, and the small strips can be stored for 12 months at the temperature of 4-30 ℃.
The invention also aims to provide a method for detecting the residual variolomycin in grains and feeds 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 rapid detection test strip for the variegated triptycene adopts a highly specific antibody-antigen reaction and competitive inhibition immunochromatography analysis technology, the variegated triptycene monoclonal antibody-colloidal gold marker is fixed on the conjugate release pad, and the variegated triptycene in the sample is combined with the variegated triptycene 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 heterochromomycin hapten-carrier protein conjugate on the reaction membrane detection line compete to combine with the heterochromomycin monoclonal antibody-colloidal gold marker, and whether the liquid sample to be detected contains the residual heterochromomycin is judged according to the absence or the shade of the red strip of the detection line.
During detection, a sample is processed and then dripped into a test strip hole, when the concentration of the variegated aspergillin in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker is combined with a variegated aspergillin hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, and a red strip appears in a detection line (T) and a quality control line (C); if the concentration of the variegated aspergillin in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold labeled substance will bind to the variegated aspergillin entirely, and thus no red band will appear at the T-line because the competitive reaction will not bind to the variegated aspergillin hapten-carrier protein conjugate.
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 variolomycin 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 synthesis of a chromotremycin hapten.
FIG. 2 is a schematic cross-sectional view of a 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 Heterocromycin
The preparation method of the test strip mainly comprises the following steps:
1) Preparing a conjugate release pad sprayed with a variolothricin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a heteroauxin 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 PVC base plate to form the test strip.
The following steps are detailed:
1. preparation of variolothricin hapten
Taking 16mg of variegated triptycene, adding 20ml of methanol for dissolution, adding 18mg of 3-maleimide hydrazine propionate, fully adding the triptycene, adding 1.7g of sodium acetate, stirring for dissolution, heating, carrying out reflux reaction for 12 hours, stopping the reaction, carrying out rotary evaporation, removing the methanol, adding 50ml of water, adding 60ml of chloroform, fully shaking, standing, separating an aqueous phase, adding 70ml of water, shaking, standing, separating an aqueous phase, drying and evaporating an organic phase by anhydrous sodium sulfate, adding 3ml of diethyl ether, fully washing, removing a supernatant, carrying out solid precipitation, and carrying out vacuum drying to obtain 13mg of a maleimido propionic acid variegated triptycene hapten product, wherein the yield is 53%.
2. Preparation of immunogens
Taking 25mg of Bovine Serum Albumin (BSA), adding 0.1M PB8.0 for dissolving, adding 1.9mg of dithiothreitol, and reacting at room temperature for 12g to obtain solution A; dissolving 9mg of maleimido propionic acid sterigmatocystin hapten in 1ml of DMF to obtain solution B, slowly dripping the solution B into the solution A, continuously reacting for 9 hours at room temperature, stopping the reaction, dialyzing and purifying by 0.02M PBS for three days, and changing the solution 3 times a day to obtain a BSA-sterigmatocystin conjugate which is the immunogen, subpackaging and storing at-20 ℃ for later use.
3. Preparation of coating antigen
Dissolving Ovalbumin (OVA) 20mg in PB8.0 0 0.1M, adding dithiothreitol 0.94mg, and reacting at room temperature for 12g to obtain solution A; dissolving 4mg of maleimidopropylated variegated aspergillin hapten in 1ml of DMF to obtain solution B, slowly dripping the solution B into the solution A, continuously reacting for 9 hours at room temperature, stopping the reaction, dialyzing and purifying by 0.02M PBS for three days, and changing the solution 3 times a day to obtain an OVA-variegated aspergillin conjugate, namely the coating antigen, subpackaging, and storing at-20 ℃ for later use.
4. Preparation of variolothricin 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 supernatants by adopting an indirect competition ELISA method, 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
Making hybridoma cell into 1 × 10 with frozen stock solution 6 Cell suspension per ml, preserved for long periods 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 variolothricin 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 in 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 and heating at constant speed until the solution is bright red, cooling to room temperature, recovering to original volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, transparency and no precipitate or floating substances.
(2) Preparation of variolothricin monoclonal antibody-colloidal gold marker
Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.0 by using 0.2mol/L potassium carbonate solution, adding 20-50 mu g of variolomycin monoclonal antibody into the colloidal gold solution according to the standard of adding per milliliter of the colloidal gold solution, continuously stirring and uniformly mixing for 30min, adding 10% BSA to ensure that the final concentration of the BSA in the colloidal gold solution is 1% (volume fraction), and standing 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: 0.02mol/L phosphate buffer solution containing casein 0.02-0.1% (mass fraction), tween-80 0.05-0.2% (mass fraction) and pH7.2.
7. Preparation of conjugate Release pad
The conjugate release pad was soaked in 0.5mol/L phosphate buffer containing bovine serum albumin (bovine serum albumin concentration in buffer is 0.5%), pH7.2, and uniformly soaked for 1h, and baked at 37 ℃ for 3h for use. And uniformly spraying the prepared varicolomycin monoclonal antibody-colloidal gold marker on a conjugate release pad by using an Isoflow film spraying instrument, spraying 0.01ml of the varicolomycin 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 the conjugate release pad, and placing the conjugate release pad in a dry environment (the humidity is less than 20%) for storage.
8. Preparation of the reaction film
Coating the conjugate of the heteroaralysin hapten and the ovalbumin 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.
And (3) coating process: diluting the varicolomycin hapten-ovalbumin conjugate to 10mg/ml by using a phosphate buffer solution, and coating the varicolomycin 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 0.8 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 quality control line (C line) 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.
9. Preparation of sample absorbent pad
The sample absorption pad is soaked in phosphate buffer solution containing 0.5 percent of bovine serum albumin (volume fraction), pH7.2 and 0.1mol/L for 2h, and is baked for 2h at 37 ℃ for standby.
10. Assembly of test strips
Sequentially adhering a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad on a PVC bottom plate; the binder release pad is covered by the sample absorption pad in a 1/3 area from 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 and a quality control line, 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 of the end away from the conjugate release pad; the test paper strip is cut into small strips with the width of 3mm by a machine, and the small strips are arranged in a special plastic card and can be stored for 12 months at the temperature of 4-30 ℃.
EXAMPLE 2 detection of residual Heterochromycin in a sample
1. Detection with test strip
And (3) sucking the sample solution to be detected by a suction pipe, vertically and dropwise adding the sample solution to be detected into the sample adding hole, timing when the liquid flows, reacting for 5-10 min, and judging the result.
2. Analyzing the detection result
The results were read by a colloidal gold analyzer (abbreviated as "analyzer"):
negative (-): indicating that the concentration of the substance to be detected in the sample is lower than the detection limit;
positive (+): indicating that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit;
and (4) invalidation: indicating that retesting is required.
Example 3 sample testing example
1. Limit of detection test
Taking a blank wheat sample and a corn sample, respectively adding the variolothricin into the blank wheat sample and the corn sample until the final concentration is 0.5, 1 and 2 mug/kg, taking a test strip for detection, and repeatedly measuring each sample for three times.
When the test strip is used for detecting the wheat and corn samples, when the addition concentration of the variegated aspergillin is 0.5 mug/kg, the analyzer shows negative; when the addition concentration of the variolothricin is 1 and 2 mug/kg, the analyzer showed positive.
2. Test of false positive and false negative rates
Taking 20 parts of each wheat and corn positive sample with the known content of the variegated aspergillin of 1 mug/kg and 2 parts of each wheat and corn negative sample with the content of less than 0.5 mug/kg, detecting by using three batches of test strips, and calculating the negative and positive rates. The results are shown in tables 1 and 2.
TABLE 1 wheat test sample results
TABLE 2 corn test sample results
The results show that: when 3 test strips produced in batches are used for detecting positive wheat and corn samples, the results are all positive, and the coincidence rate of the positive samples is 100 percent, and the false negative rate is 0; when 20 negative wheat and corn 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 the sterigmatocystin can quickly detect the sterigmatocystin residue in wheat and corn.
3. Specificity test
The test paper strip of the variegated aspergillin is used for detecting 500 mug/kg of aflatoxin B1, aflatoxin M1, ochratoxin and other medicaments. The result shows that the quality control line and the detection line of the test strip are both colored and negative. The test paper strip has no cross reaction to 500 mu g/kg of aflatoxin B1, aflatoxin M1, ochratoxin and other medicaments.
Claims (4)
1. The test strip for detecting the variegated aspergillin comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), and is characterized in that the reaction membrane is provided with a detection line (5) coated with a variegated aspergillin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, the conjugate release pad (2) is sprayed with a variegated aspergillin monoclonal antibody-colloidal gold marker, the variegated aspergillin hapten-carrier protein conjugate is obtained by coupling a variegated aspergillin hapten and a carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein and human serum albumin, and the preparation method of the variegated aspergillin hapten is as follows:
taking 16mg of variegated aspergillin, adding 20ml of methanol for dissolving, adding 18mg of 3-maleimide hydrazine propionate, fully stirring, adding 1.7g of sodium acetate, stirring for dissolving, heating, carrying out reflux reaction for 12 hours, stopping the reaction, carrying out rotary evaporation, removing the methanol, adding 50ml of water, adding 60ml of chloroform, fully shaking, standing, separating a water phase, adding 70ml of water, shaking, standing, separating a water phase, drying an organic phase with anhydrous sodium sulfate, drying to dryness, adding 3ml of diethyl ether, fully washing, removing a supernatant, carrying out solid precipitation, and carrying out vacuum drying to obtain 13mg of a maleimido propionic acid variegated aspergillin hapten product with the yield of 53%.
2. The test strip of claim 1, wherein the variegated aspergillin monoclonal antibody is prepared by taking a variegated aspergillin hapten-carrier protein conjugate as an immunogen, and the goat anti-mouse anti-antibody is obtained by immunizing a goat with a mouse-derived antibody.
3. A method of making the test strip of any one of claims 1-2, comprising the steps of:
1) Preparing a conjugate release pad sprayed with a variolothricin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a variegated aspergillin 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.
4. A method for detecting the residual variolomycin in wheat, corn, other grains and feeds comprises the following steps:
1) Sample pretreatment;
2) Performing detection with the test strip of any one of claims 1-3;
3) And analyzing the detection result.
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