Disclosure of Invention
The application aims to provide a protein A and gentamicin residual detection test strip which has high sensitivity, simple operation, low cost and short detection time.
The test strip for detecting protein A and gentamicin residues 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 protein A and gentamicin hapten-carrier protein conjugate and a quality control line (6) coated with goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a protein A monoclonal antibody-colloidal gold marker and a gentamicin monoclonal antibody-colloidal gold marker.
The gentamicin hapten-carrier protein conjugate is obtained by coupling a gentamicin hapten with carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine and human serum albumin.
The protein A and gentamicin monoclonal antibody are prepared by taking a protein A and gentamicin hapten-carrier protein conjugate as immunogens respectively, and are secreted by a protein A monoclonal antibody hybridoma cell strain and a gentamicin monoclonal antibody hybridoma cell strain respectively; the goat anti-mouse antibody is obtained by immunizing a goat with a mouse-derived antibody.
The sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the water absorbing pad (4) are sequentially adhered to the bottom plate (7), and the conjugate releasing pad 1/3-1/2 is covered below the sample absorbing 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 absorbing pad is water absorbing paper; the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.
Another object of the present application is to provide a method for preparing the above test strip, comprising the steps of:
1) Preparing a conjugate release pad sprayed with a protein A monoclonal antibody-colloidal gold marker and a gentamicin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction film with a detection line coated with protein A and gentamicin hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
Specifically, the method comprises the following steps:
1) Gentamicin hapten preparation: carrying out a series of chemical reactions on gentamicin, 4 '-difluoro-3, 3' -dinitrodiphenyl sulfone and other substances to obtain gentamicin hapten;
2) Coupling the gentamicin hapten with carrier protein to obtain a gentamicin hapten-carrier protein conjugate;
3) Immunizing a mouse by using a protein A and gentamicin hapten-carrier protein conjugate respectively, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a protein A monoclonal hybridoma cell strain and a gentamicin monoclonal hybridoma cell strain;
4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibody;
5) Preparing colloidal gold by the reaction of trisodium citrate and chloroauric acid;
6) Respectively adding the protein A monoclonal antibody and the gentamicin monoclonal antibody prepared in the step 3) into the colloidal gold prepared in the step 5) to respectively obtain a protein A monoclonal antibody-colloidal gold marker and a gentamicin monoclonal antibody-colloidal gold marker;
7) Spraying a protein A monoclonal antibody-colloidal gold marker and a gentamicin monoclonal antibody-colloidal gold marker on a conjugate release pad respectively, drying at 37 ℃ for 1h, taking out, and storing in a dry environment for later use;
8) Coating the protein A hapten-carrier protein conjugate and the gentamicin 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 phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction) with pH of 7.2 and 0.1mol/L for 2h, and drying at 37deg.C for 2h;
10 The sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are sequentially stuck on the bottom plate, the sample absorbing pad covers the conjugate releasing pad, finally, the sample absorbing pad is cut into small strips with the width of 3mm, a plastic box is added, and the sample absorbing pad can be stored for 12 months at the temperature of 4-30 ℃ after vacuum packaging.
The application also provides a method for detecting residues of protein A and gentamicin in a vaccine by using the test strip, which comprises the following steps:
(1) Sample pretreatment;
(2) Detecting by using a test strip;
(3) And analyzing the detection result.
The protein A and gentamicin rapid detection test strip adopts a highly specific antibody antigen reaction and competitive inhibition immunochromatography analysis technology, a protein A monoclonal antibody-colloidal gold label and a gentamicin monoclonal antibody-colloidal gold label are fixed on a conjugate release pad, and the protein A and gentamicin in a sample are respectively combined with the protein A monoclonal antibody-colloidal gold label and the gentamicin monoclonal antibody-colloidal gold label on the conjugate release pad in the flowing process to form a drug-antibody-colloidal gold label. And the medicine in the sample competes with the protein A and the gentamicin hapten-carrier protein conjugate on the reaction film detection line to combine with the protein A monoclonal antibody-colloidal gold marker and the gentamicin monoclonal antibody-colloidal gold marker, and whether the sample liquid to be detected contains protein A and gentamicin residues is judged according to the existence or the darkness of the red strip of the detection line.
During detection, a sample is dripped into a test strip hole after treatment, when the concentration of the protein A and the gentamicin in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker is respectively combined with the protein A and the gentamicin 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) respectively; if the concentration of protein A and gentamicin in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold-labeled substance is bound to all of the protein A and gentamicin, respectively, so that red bands do not appear at the T-line due to competition reaction without binding to the hapten-carrier protein conjugate of protein A and gentamicin.
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. The method for detecting the residues of the protein A and the gentamicin by using the test strip is simple, convenient, quick, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Detailed Description
The application is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the application only and are not intended to limit the scope of the application.
Example 1 preparation of test strips for protein A and gentamicin detection
The preparation method of the test strip mainly comprises the following steps:
1) Preparing a conjugate release pad sprayed with a protein A monoclonal antibody-colloidal gold marker and a gentamicin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction film with a detection line coated with protein A and gentamicin hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the PVC bottom plate which are prepared in the steps 1) and 2) into the test strip.
The following is a stepwise detailed description:
1. preparation of gentamicin hapten
Dissolving 1.39g of gentamicin in 80ml of pure water, dissolving 0.344g of 4,4 '-difluoro-3, 3' -dinitrodiphenyl sulfone in 30ml of methanol, adding into an aqueous solution of gentamicin, adding 1.38g of anhydrous potassium carbonate, stirring at room temperature for 3 hours, stopping the reaction, adding 300ml of ethyl acetate for extraction, standing, separating out water phase, concentrating and evaporating an organic phase, and recrystallizing with 20ml of absolute ethyl alcohol to obtain 0.34g of fluoronitrobenzene-gentamicin hapten product, wherein the yield is 43.9%.
2. Preparation of immunogens
Taking 29mg of fluoronitrobenzene-gentamicin hapten product, and adding 2ml of DMF for dissolution to obtain hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.05M PB buffer solution for dissolution to obtain solution B, dripping solution A into solution B, reacting at 4 ℃ for 12h, dialyzing and purifying with 0.02M PBS for 3 days, and changing the solution three times a day to obtain the gentamicin-BSA conjugate which is an immunogen, and preserving at-20 ℃ for later use.
3. Preparation of coating Material
Taking 16mg of fluoronitrobenzene-gentamicin hapten product, and adding 1ml of DMF for dissolution to obtain hapten solution A; the method comprises the steps of carrying out a first treatment on the surface of the Taking 50mg of egg serum albumin (OVA), adding 0.05M PB buffer solution for dissolution to obtain solution B, dripping solution A into solution B, reacting at 4 ℃ for 12h, dialyzing and purifying with 0.02M PBS for 3 days, and changing the solution three times a day to obtain the gentamicin-OVA conjugate, namely the coating antigen, and preserving at-20 ℃ for later use.
4. Preparation of protein A monoclonal antibody and gentamicin monoclonal antibody
(1) Immunization of animals
Gentamicin immunogen/protein A was injected into Balb/c mice at an immunizing dose of 150 μg/mouse to generate antisera.
(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 an indirect competition ELISA method, 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/ml were stored in liquid nitrogen for a long period of time. 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, so that 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 protein A and gentamicin 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 in a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5ml 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 original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, and has no sediment or floaters.
(2) Preparation of protein A monoclonal antibody-colloidal gold marker and gentamicin monoclonal antibody-colloidal gold marker
Under the magnetic stirring, the pH value of the colloidal gold is regulated to 7.0 by using 0.2mol/L potassium carbonate solution, 20-50 mug of standard is added into each milliliter of colloidal gold solution, protein A monoclonal antibody and gentamicin monoclonal antibody are respectively added into the colloidal gold solution, the stirring and mixing are continued for 30min, 10% BSA is added, the final concentration of the solution in the colloidal gold solution is 1% (volume fraction), and the solution is kept stand 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: 0.02 to 0.1 percent (mass fraction) of casein, 0.05 to 0.2 percent (mass fraction) of tween-80 and 0.02mol/L of phosphate buffer solution with pH of 7.2.
7. Preparation of conjugate release pads
The conjugate release pad was soaked in a phosphate buffer containing bovine serum albumin (the concentration of bovine serum albumin in the buffer is 0.5%), having a pH of 7.2 and 0.5mol/L, and was uniformly soaked for 1h and baked at 37℃for 3 h. And uniformly spraying the prepared protein A and gentamicin monoclonal antibody-colloidal gold marker on a conjugate release pad by using an isolow film spraying instrument, spraying 0.01ml of the protein A and gentamicin monoclonal antibody-colloidal gold marker on each 1cm of conjugate release pad, placing the conjugate release pad in a 37 ℃ environment (humidity is less than 20%) for 60min, taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (humidity is less than 20%) for storage for later use.
8. Preparation of reaction film
Coating the protein A and gentamicin 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.
The coating process comprises the following steps: diluting the protein A and gentamicin hapten-ovalbumin conjugate to 5-30mg/ml by using a phosphate buffer solution, and coating the protein A and gentamicin hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using an isolow spot membrane tester, wherein the coating amount is 0.1-10 mu l/cm; the goat anti-mouse antibody is diluted to 100-500 mu g/ml by 0.01mol/L phosphate buffer solution with pH7.4, and the goat anti-mouse antibody is coated on a quality control line (C line) on a nitrocellulose membrane by an isolow spot membrane tester, wherein the coating amount is 0.1-10 mu L/cm. And (5) drying the coated reaction film for 2 hours at 37 ℃ for standby.
9. Preparation of sample absorbent pad
The sample absorbing pad is placed in phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction), pH7.2 and 0.1mol/L for 2 hours, and is baked at 37 ℃ for 2 hours 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 1/3 area of the initial end of the conjugate release pad is covered by the sample absorption pad, the tail end of the conjugate release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane 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 and a quality control line, 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 strip; the detection line is positioned at one side close to the tail end of the conjugate release pad; the quality control line is positioned at one side far away from the tail end of the conjugate release pad; cutting the test paper strip into small strips with the width of 3mm by a machine, and placing the test paper strip in a special plastic card, wherein the test paper strip can be stored for 12 months at the temperature of 4-30 ℃.
Example 2 detection of protein A and gentamicin residues in samples
1. Detection is carried out by using a test strip
Sucking the sample solution to be detected by a suction pipe, vertically dripping 3 drops of the sample solution into a sample adding hole, starting 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;
invalidation: indicating that retesting is required.
Example 3 key technical parameters of test strips
1. Test paper strip detection limit
Limit of protein a detection: 10mg/kg;
gentamicin limit of detection: 100. Mu.g/kg.
2. False positive rate and false negative rate test
The false negative rate is 0; the false positive rate is less than 5%.
3. Specificity test
Gentamicin is approximately equal to 100%.
Streptomycin less than 1%;
less than 1% of dihydrostreptomycin;
neomycin is less than 1%.