CN1790025A - Test paper for lateral flow immunochromatographic semi-quantitative detection of ractopamine - Google Patents
Test paper for lateral flow immunochromatographic semi-quantitative detection of ractopamine Download PDFInfo
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- CN1790025A CN1790025A CN 200510126415 CN200510126415A CN1790025A CN 1790025 A CN1790025 A CN 1790025A CN 200510126415 CN200510126415 CN 200510126415 CN 200510126415 A CN200510126415 A CN 200510126415A CN 1790025 A CN1790025 A CN 1790025A
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a semi-quantitative detecting paper of Lac-dopamine effluent immune chromatography, which comprises the following parts on the test paper back: sample liquid absorption part, colloidal gold mark part, detecting reaction part and water-absorbing part, wherein the detecting reaction part contains 1-3 Lac-dopamine antibody as detecting line, which contains 1-3 anti-second animal protein lgG as reference line simultaneously; the amount of combined detecting line and reference line isn't more than one simultaneously; the amount of combined line is 2-4. The invention displays strong specificity to accomplish semi-quantitative detection at 4-40 deg.c temperature for 3 min, which is fit for unit or personnel to detect the Lac-dopamine medicine in the animal source food sample rapidly.
Description
Technical Field
The invention relates to a detection tool for beta-stimulant residues in animal food, in particular to ractopamine lateral flow immunochromatography semi-quantitative detection test paper and a preparation method and a use method thereof.
Background
Ractopamine is a beta-stimulant, commonly called clenbuterol, and the residue of the ractopamine in animal food can cause damage to human health, so that the ractopamine is definitely regulated and forbidden to be used as a nutrient distributor in China. However, due to the driving of economic interest, illicit abuse of ractopamine still occurs at times.
The detection of ractopamine residue currently mainly employs High Performance Liquid Chromatography (HPLC). However, when HPLC is used, the sample pretreatment is very complicated, the determination method is quite complex, the operation can be carried out by specially trained personnel, and the detection flux is very small. Under the condition that the animal breeding is dispersedly managed in China at present, uniform management is difficult, so that the quality of animal-derived food is difficult to be consistent, the quality of the product is monitored by only adopting a large instrument, the method is unrealistic, the instrument detection cost is high, and the popularization and the use are difficult. The ELISA method is another veterinary drug residue detection method which is researched more at present, and has the advantages of high sensitivity, large detection amount, low cost and the like, but the method also needs part of laboratory equipment such as an enzyme-linked immunosorbent assay (ELISA) instrument, and the analysis time is generally 1-4 hours, so that the method has certain limitation. Therefore, the corresponding ractopamine detection technology in China is not complete at present, and a simpler, quicker and more convenient detection tool is needed to be developed.
Disclosure of Invention
(I) technical problem to be solved
The invention aims to provide a ractopamine semi-quantitative detection test paper which is simple and convenient to use, high in sensitivity and low in cost, and a preparation method and a use method thereof.
(II) technical scheme
The invention provides ractopamine lateral flow immunochromatography semi-quantitative detection test paper, which is characterized in that a sample liquid absorption part, a colloidal gold labeling part, a detection reaction part and a water absorption part are sequentially adhered to a backing of a non-water-absorbing flexible material coated with non-setting adhesive.
The colloidal gold labeling part is a glass fiber or acetate fiber or nylon membrane, and the substance labeled on the colloidal gold labeling part is a mixture of a second species animal protein and a ractopamine antibody, or a mixture of the second species animal protein and an antigen for detecting ractopamine.
1-3 ractopamine detection antigens are coated on the detection reaction part to be used as detection lines, or 1-3 ractopamine antibodies are coated on the detection reaction part to be used as detection lines, and meanwhile, 1-3 IgG resisting second species animal protein is coated on the detection reaction part to be used as reference lines. The combination of the detection lines and the reference lines cannot be more than 1 at the same time, the number of the combination lines is 2-4, and the combination mode is five forms of 1 detection line +1 reference line, 1 detection line +2 reference lines, 1 detection line +3 reference lines, 2 detection lines +1 reference line and 3 detection lines +1 reference line. According to the accuracy requirement of semi-quantitative detection, the more the number of combined lines is, the more accurate the semi-quantitative is.
The antigen for detection is conjugate of ractopamine and carrier material, wherein the carrier material comprises protein, protein fragment, synthetic polypeptide, semisynthetic polypeptide, and polysaccharide, such as serum albumin, globulin, lipoprotein, polyamino acid, and dextran, and preferably carrier material comprises bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, and L-polylysine. The carrier material may be other synthetic or natural polymers having active groups, such as diphtheria toxin, tetanus toxin, yeast, nylon, dextrane, cellulose, etc., and may also be used to prepare the antigen for detection. The second animal protein is a protein of animal of non-antibody origin, for example, the antibody is of murine origin, and the second animal protein may be chicken, rabbit or other non-murine animal protein such as ovalbumin, rabbit serum albumin, etc.
The preparation method and the function of each part of the test paper are as follows:
1. backing: is a non-absorbent and tough material with one surface coated with non-setting adhesive, such as a PVC plate, and plays a role in fixing and supporting other components of the test paper.
2. Preparation of sample solution absorption part: soaking filter paper or glass fiber paper in PBS (pH7.0-8.4) for 2min, taking out, oven drying at 80 deg.C or drying in other manners to obtain sample solution absorption part, which can absorb sample solution during detection to facilitate upward movement of the sample solution.
3. Preparation of colloidal gold-labeled moiety: this portion functions to immobilize the colloidal gold-labeled antigen or antibody. The preparation steps comprise the preparation of colloidal gold sol, the labeling of ractopamine antibody or ractopamine antigen by colloidal gold, and the partial treatment of the labeling of colloidal gold. Wherein,
(1) preparing colloidal gold sol: mixing chloroauric acid (HAuCL)4) Preparing 1% mother solution with ultrapure water, taking 1mL of the mother solution, diluting to 100mL with ultrapure water to prepare 0.01% solution, heating to boiling, adding 1-5mL of 1% trisodium citrate aqueous solution, and continuously heating until transparent orange red appears, namely the colloidal gold sol.
(2) Colloidal gold labeled ractopamine antibody: dissolving and diluting ractopamine monoclonal antibody or polyclonal antibody and second species animal protein with PBS (0.01mol/L, pH7.0-7.5) to 2-4mg/mL, adding 1-3mL 2-4mg/mL ractopamine antibody and 1-1.5mL 2-4mg/mL second species animal protein per 100mL colloidal gold sol, shaking for 2min, and adding 0.2mol/L K2CO3Adjusting the pH value to 8.4, shaking for 5min, adding 11% PEG-100002mL, shaking for 5min, centrifuging for 15min at 15000r/min of 8000-.
(3) Colloidal gold labeled ractopamine antigen: detecting ractopamine antigen with a second species of animalDissolving and diluting the protein with PBS (0.01mol/L, pH7.0-8.4) to 2-4mg/mL, adding 1-3mL of 2-4mg/mL antigen for detection and 1-1.5mL of 2-4mg/mL second animal protein per 100mL colloidal gold sol, shaking for 2min, adding 0.2mol/L K2CO3Adjusting the pH value to 8.4, shaking for 5min, adding 11% PEG-100002mL, shaking for 5min, centrifuging for 15min at 15000r/min of 8000-.
(4) Treatment of a colloidal gold labeling part: pouring GAb or GAg into a tank, soaking glass fiber paper or filter paper for 1min, taking out, and drying at room temperature to obtain colloidal gold labeled part.
4. Preparation of a detection reaction part: soaking a nitrocellulose membrane or an acetate fiber membrane or a nylon membrane in 0.8% glutaraldehyde solution or 0.2% carbodiimide solution for 30min, taking out, drying at 37 ℃, coating 1-3 antigen threads or ractopamine antibody threads for detecting ractopamine with different concentrations on the upper side as detection lines, and coating 1-3 IgG threads resisting second species animal protein with different concentrations as reference lines. When the labeled objects of the colloidal gold labeled part are ractopamine antibody and second animal protein, the detection line is coated with ractopamine detection antigen; when the labeled objects of the colloidal gold labeled part are ractopamine detection antigen and second species animal protein, the detection line is coated with ractopamine antibody; the number of the detection lines and the reference lines cannot be more than 1 at the same time, and the number of the combined lines is 2-4. This is the detection reaction portion, which is primarily responsible for characterizing the reaction as a macroscopic color.
5. Preparation of a water absorption part: the glass fiber paper or filter paper or absorbent paper is dried at room temperature to obtain the water absorption part. This part mainly functions to absorb the excess sample solution that has moved up.
And sequentially adhering a sample liquid absorption part, a colloidal gold labeling part, a detection reaction part and a water absorption part on the backing to obtain the ractopamine lateral flow immunochromatography semi-quantitative detection test paper.
The detection principle of the test paper of the invention is slightly different because of different colloidal gold labeled objects.
When the labeled objects of the colloidal gold labeled part are ractopamine antibodies and second-species animal proteins, if the sample contains ractopamine, the sample solution is absorbed by the sample solution absorption part of the test paper and moves upwards to the colloidal gold labeled part through capillary action, the ractopamine in the sample solution reacts with the ractopamine antibodies labeled by the colloidal gold to form a conjugate, the conjugate continuously moves upwards to the detection line, and the detection antigen on the detection line cannot be combined with the ractopamine antibodies labeled by the colloidal gold after the ractopamine antibodies labeled by the colloidal gold are combined with the conjugate, so that the detection line is colorless; when no ractopamine exists in the sample, the ractopamine antibody marked by the colloidal gold reaches the detection line and is captured by the antigen for detection, and then the red color visible to naked eyes is formed, and the result is negative. Whether the sample contains ractopamine or not, when the colloidal gold-labeled second species animal protein moves upwards to reach the reference line, the colloidal gold-labeled second species animal protein can be captured by the anti-second species animal protein IgG coated on the reference line to form a macroscopic red color, namely the reference line.
When the labeled objects of the colloidal gold labeled part are ractopamine detection antigen and second animal protein, if the sample contains ractopamine, the sample solution is absorbed by the sample solution absorption part of the test paper and moves upwards through capillary action, the molecular weight of free ractopamine in the sample is small, the moving speed is high, the sample reaches the detection line first, and the sample is firstly combined with the ractopamine antibody coated on the detection line, so that the ractopamine antibody coated on the detection line only has one combination site, the combination capacity of the free ractopamine in the sample is stronger than that of the ractopamine detection antigen in a coupling state, the colloidal gold labeled detection antigen cannot be captured by the ractopamine antibody on the detection line any more, and the detection line is colorless, so that the detection line is positive; if no ractopamine exists in the sample, the ractopamine detection antigen marked by the colloidal gold reaches the detection line and then is captured by the ractopamine antibody coated on the detection line, and the color is visible red, which is negative. Whether the sample contains ractopamine or not, when the colloidal gold-labeled second species animal protein moves upwards to reach the reference line, the colloidal gold-labeled second species animal protein can be captured by the anti-second species animal protein IgG coated on the reference line to form a macroscopic red color, namely the reference line.
The test paper of the above two modes controls the color development depth of the detection line and the reference line during detection by adjusting the concentration of the coating substance of the detection line and the reference line during preparation of the test paper, and corresponds the color development depth or the number of the color development strips with the concentration of the standard substance, so that the purpose of semi-quantitative detection can be achieved.
(III) advantageous effects
The invention has the following positive effects:
1. the specificity is strong. The test paper has extremely high specificity, basically has no cross reaction with clenbuterol, salbutamol, diethylstilbestrol and the like, avoids the interference of other medicines on the detection, and meets the requirement of detecting the ractopamine compound medicine by the department of agriculture.
2. The sensitivity is high. The test paper has the lowest detection lower limit of 1ng/mL, and meets the requirement of rapid screening and detection.
3. Semi-quantitative detection can be realized. The test paper can not only carry out qualitative detection, but also achieve the purpose of semi-quantitative detection more importantly according to the comparison between the color of a detection line and the color of a reference line or the number of color generation strips of the detection line.
4. The operation is simple and convenient. The detection can be directly carried out on milk and animal urine, and the detection can be carried out after simple treatment of tissue samples such as meat and the like. The test paper does not need any professional training, does not need any instrument and equipment, can be operated by ordinary non-professional personnel, and can be read by naked eyes after being inserted into detection liquid. Therefore, the test paper has wide application range and can be used by health inspection supervision departments, animal breeding units and individual consumers.
5. High speed, high flux and stable color. The test paper is inserted into the sample solution, the result can be observed after 3min, one person can simultaneously and continuously detect, the detection flux is far higher than that of an HPLC method, the detection speed is higher than that of a ractopamine ELISA kit by more than 40-160 times, and the color can be permanently stored without changing color as fast as an ELISA substrate.
6. The detection temperature has wide suitable range. Can be used at 4-40 ℃, the result is normal, the operation is not needed at low temperature, heat preservation measures are not needed, and the product can be used indoors and outdoors.
7. The test paper has long shelf life. According to the result of the accelerated aging test, the quality guarantee period of the test paper under the dry condition can reach 2 years.
8. The cost is low. The detection cost of the test paper method is far lower than that of a ractopamine ELISA kit detection method, and the cost can be greatly reduced along with the large-scale production.
Drawings
FIG. 1 is a schematic structural diagram of the test paper of the present invention. Wherein 1 is a sample liquid absorption part, 2 is a colloidal gold labeling part, 3 is a detection reaction part, 4 is a detection line, 5 is a reference line, and 6 is a water absorption part.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
EXAMPLE 11 test strips for detection line +1 reference line
The detection line is coated with 1 ractopamine monoclonal antibody with the concentration of 0.1-30 mug/mL or ractopamine detection antigen, and the width is 1 mm; the reference line is coated with 0.01-30 mug/mL IgG 1 strip resisting the second species of animal protein, and the width is 1 mm; the interval between the two lines is 2-6 mm. When the color of the detection line is the same as that of the reference line, the sample is negative; when the detection line is lighter than the reference line, the sample is positive, the concentration is greater than A mug/kg, A is a set detection limit, and can be a certain concentration greater than or equal to 10 mug/kg. The reference line is always colored, and if the reference line is not colored, the test paper is invalid.
EXAMPLE 22 test strips for detection line +1 reference line
The 2 detection lines are coated with 0.01-30 mug/mL ractopamine monoclonal antibody or ractopamine detection antigen, and the concentration of the coating on the 2 nd detection line is less than that of the coating on the 1 st detection line reference line; the reference line is coated with 0.01-30 mug/mL of IgG for resisting the second animal protein, and the width of the reference line is 1 mm; the interval between each line is 2-6 mm. When the two detection lines show colors, the sample is negative; when the 1 st detection line of the detection lines is colored and the 2 nd detection line is not colored, the concentration of ractopamine in the sample is more than A [ mu ] g/kg and less than B [ mu ] g/kg; when neither test line is colored, the concentration of ractopamine in the sample is greater than B mug/kg. A. B is a predetermined detection limit, and may be a concentration of 10. mu.g/kg or more. The reference line is always colored, and if the reference line is not colored, the test paper is invalid.
EXAMPLE 33 test strips for detection line +1 reference line
The 3 detection lines are coated with 0.01-30 mug/mL ractopamine monoclonal antibody or ractopamine detection antigen, and the concentration of the coating on the three detection lines is decreased in sequence; the reference line is coated with IgG which is resistant to the second species of animal protein and has the concentration of 0.01-30 mug/mL; the interval between each line is 2-6 mm. When the 3 detection lines are all colored, the sample is negative; when the 1 st detection line and the 2 nd detection line of the detection lines are colored and the 3 rd detection line is not colored, the concentration of ractopamine in the sample is more than A [ mu ] g/kg and less than B [ mu ] g/kg; when the 1 st detection line is colored and the 2 nd and 3 rd detection lines are not colored, the concentration of ractopamine in the sample is more than B mu g/kg and less than C mu g/kg; when the 3 detection lines are not colored, the content of ractopamine in the sample bottle is more than C mug/kg; A. b, C is the detection limit set, and may be a concentration of 10. mu.g/kg or more. The reference line is always colored, and if the reference line is not colored, the test paper is invalid.
EXAMPLE 41 test strips for detection line +3 reference lines
The detection line is coated with 0.01-30 mug/mL ractopamine monoclonal antibody or ractopamine detection antigen, 3 reference lines are coated behind the detection line, the reference lines are coated with 0.01-30 mug/mL IgG resisting to the second species of animal protein, the coating concentration of the reference lines and the labeling concentration of the colloidal gold labeled animal protein of the 2 nd species are adjusted, so that the colors of the 3 reference lines are respectively the same as the color of the detection line when the ractopamine content in the standard sample is C, B, A mug/kg, and the coating concentration of the reference lines is determined accordingly; the interval between each line is 2-6 mm; when the color of the detection line is darker than that of the reference line 3, the sample is negative; when the color of the detection line is darker than that of the 3 rd reference line and lighter than that of the 2 nd detection line, the concentration of ractopamine in the sample is more than A [ mu ] g/kg and less than B [ mu ] g/kg; when the color of the detection line is darker than that of the 2 nd reference line and lighter than that of the 1 st reference line, the concentration of ractopamine in the sample is more than B [ mu ] g/kg and less than C [ mu ] g/kg; when the detection line is not colored, the concentration of ractopamine in the sample is more than C mug/kg.
EXAMPLE 51 test strips for detection line +2 reference lines
The detection line is coated with 0.01-30 mug/mL ractopamine monoclonal antibody or ractopamine detection antigen, 2 reference lines are coated behind the detection line, the reference lines are coated with 0.01-30 mug/mL IgG resisting to the second species of animal protein, the coating concentration of the reference lines and the labeling concentration of the colloidal gold labeled animal protein of the 2 nd species are adjusted, so that the color of the 2 reference lines is respectively the same as the color of the detection line when the ractopamine content in the standard sample is B, A mug/kg, and the coating concentration of the reference lines is determined accordingly; the interval between each line is 2-6 mm; when the color of the detection line is darker than that of the 2 nd reference line, the sample is negative; when the color of the detection line is darker than that of the 2 nd reference line and lighter than that of the 1 st detection line, the concentration of ractopamine in the sample is more than A [ mu ] g/kg and less than B [ mu ] g/kg; when the color of the detection line is darker than that of the 1 st reference line and lighter than that of the 1 st reference line, the concentration of the ractopamine in the sample is more than B mug/kg.
Example 6 Synthesis of ractopamine coupled antigen
The ractopamine coupling antigen is divided into an antigen for immunization and an antigen for detection, wherein the former is used for immunizing animals, and the latter is used for detecting and screening antibodies in antibody preparation and coating colloidal gold marks or detection lines in test paper preparation.
Preparation of antigen for immunization: 30mg of ractopamine hydrochloride and 10mg of succinic anhydride react in pyridine solution, the reaction solution is dried by nitrogen and then dissolved in dioxane, 25 mu L of tributylamine is added, stirring is carried out in ice bath for 15min, 13 mu L of isobutyl chloroformate is added, and stirring is carried out for 1 h. Adding BSA solution (100mg BSA in 0.11mol/L sodium borate solution, pH8.5), reacting at room temperature overnight, dialyzing the reaction solution in dialysis membrane bag for 3d, and packaging.
Antigen for detection: ovalbumin OVA is used for replacing bovine serum albumin BSA, and the rest steps are the same as the preparation of the antigen for immunization.
EXAMPLE 7 preparation of monoclonal antibody to ractopamine
Healthy 5-week-old female secondary Balb/c mice were immunized in 6 groups. In the first basal immunization, 0.1mL of ractopamine immunization antigen and an equal amount of complete Freund's adjuvant are fully and uniformly mixed and emulsified by a stirrer, and are injected into the abdominal cavity, wherein the injection amount is 0.2 mL/mouse. After four weeks, boosting is carried out, the adjuvant is replaced by incomplete Freund adjuvant, boosting is carried out once every two weeks, and the spleen is taken out and fused after the last immunization for 3 days.
The HAT culture solution, the IMDM incomplete culture solution, the IMDM complete culture solution and the 50% PEG-6000 solution are pre-warmed in a 37 ℃ water bath, and the other beaker containing water is pre-warmed in the 37 ℃ water bath. Mixing the prepared spleen cells, myeloma cells and spleen cells according to the ratio of 1: 5, washing the mixture with incomplete culture solution for 1 time in a 50mL plastic centrifugal tube, centrifuging the mixture at 1200r/min for 8min, removing supernatant, and sucking residual liquid by a dropper. The tube was placed in a 37 ℃ water bath, the pipette was inserted into the bottom of the tube, and preheated 1mL of 50% PEG-6000 was added to the tube while stirring for 90 seconds. After standing for 90s, 10mL of the complete culture broth preheated was added to the mixture in a 37 ℃ water bath for 60 s. The suspension was gently suspended once, centrifuged at 1000r/min for 6min, the supernatant was discarded, and the suspension was gently suspended with about 6mL of complete culture medium. According to the number of 96-well culture plates, HAT culture solution is supplemented to 76.8mL, the mixture is gently mixed, and the mixed suspension is inoculated into a 96-well plate with feeder cells, wherein each well is 0.1mL, and 8 plates are inoculated at a time. The plates were incubated in a 5% CO2 incubator at 37 ℃. At 7d after the fusion, the medium was changed 1 time with half the amount of HT medium, and after 13d, the complete medium of 20% NBS was used depending on the growth. After about 7d, hybridoma colonies appeared, which were large, round and clear. When colonies grow to the bottom of the wells 1/3, the supernatants are removed for detection of the corresponding specific antibodies by drawing a marker for clonal growth on the cover plates of the plates. Injecting the cells into the abdominal cavity of the mouse after the cells are completely cloned, taking ascites after a period of time when the ascites is sufficiently large, and purifying the ascites by protein A immunoaffinity chromatography to obtain the ractopamine monoclonal antibody.
EXAMPLE 8 treatment of sample liquid absorbing portion
Soaking filter paper or glass fiber paper in 0.1mol/LpH7.4 PBS for 2min, taking out, and oven drying at 80 deg.C or drying in other ways to obtain sample liquid absorbing part.
EXAMPLE 9 preparation and treatment of colloidal gold-labeled moieties
The preparation and treatment of the colloidal gold labeling part comprise the preparation of colloidal gold sol, the preparation of colloidal gold labeled ractopamine antibody and the treatment of the colloidal gold labeling part.
Preparing colloidal gold sol: mixing chloroauric acid (HAuCL)4) Preparing 1% mother solution by using ultrapure water, taking 1mL of the mother solution, using the ultrapure water to fix the volume to 100mL, preparing a 0.01% solution, heating to boiling, adding a certain amount of 1% trisodium citrate aqueous solution, and continuously heating until transparent orange red appears, namely the colloidal gold sol.
Preparing a colloidal gold labeled ractopamine monoclonal antibody: mixing ractopamine with waterDissolving and diluting the monoclonal antibody and the pig serum albumin to 3mg/mL by PBS (0.01mol/L, pH7.0-7.5), adding 1mL of ractopamine monoclonal antibody of 4mg/mL and 1mL of pig serum albumin of 2mg/mL into each 100mL of colloidal gold sol, shaking for 2min, and adding K of 0.2mol/L2CO3Adjusting the pH value to 8.4, shaking for 5min, adding 11% PEG-100002mL, shaking for 5min, centrifuging for 15min at 15000r/min, removing the supernatant, redissolving with PBS (0.01mol/L, pH7.4), centrifuging for 15min at 6000-13000r/min, removing the supernatant, diluting the precipitate 1000 times with PBS (0.01mol/L, pH7.5), and taking the product as a gold-labeled ractopamine monoclonal antibody.
Treatment of a colloidal gold labeling part: pouring the gold-labeled ractopamine monoclonal antibody into a tank, immersing glass fiber paper or filter paper for 1min, taking out, and drying at room temperature or freeze-drying in vacuum to obtain the colloidal gold-labeled part.
EXAMPLE 101 test strips +2 reference strips in combination test reaction partial treatment
Soaking the nitrocellulose membrane in 0.8% glutaraldehyde solution or 0.2% carbodiimide solution for 30min, taking out, drying at 37 ℃, spraying 1 piece of antigen line for detecting ractopamine on the nitrocellulose membrane by a spraying machine to serve as a detection line, wherein the concentration is 3 mug/mL; the IgG line coated with 2 goat anti-porcine serum albumin was used as a reference line, and the concentration near the lower end was 2. mu.g/mL, while the other was 1. mu.g/mL. The detection reaction part is formed by combining 1 detection line and 2 reference lines.
EXAMPLE 11 treatment of the Water-absorbing moiety and test strip Assembly
The water-absorbing paper is dried at room temperature to be used as a water-absorbing part.
And sequentially adhering a sample liquid absorption part, a colloidal gold labeling part, a detection reaction part and a water absorption part on the backing to obtain the ractopamine rapid semi-quantitative detection test paper in a combined form of 1 detection line and 2 reference lines.
Example 12 detection method
1. Animal urine samples: 2 drops of urine were directly added to the test portion of the test strip, and after 2min, the color was observed at about 100. mu.L. If the 2 detection lines all show colors, the sample is negative; if the 1 st detection line is colored and the 2 nd detection line is not colored, the ractopamine content in the sample is more than 2 ng/mL; if the 1 st detection line and the 2 nd detection line do not develop color, the content of ractopamine in the sample is more than 8 ng/mL. The reference line should develop color regardless of the ractopamine content of the sample, and if the reference line does not develop color, the test paper is invalid.
2. Animal liver, kidney, muscle, etc. tissue samples: a10 g tissue sample from which fat was removed was triturated, added to 30mL of PBS (0.01mol/L pH7.4), incubated at 80 ℃ for 30min, then placed in an ice bath for 10min, centrifuged, and the fat layer on the surface was carefully removed and the supernatant was used as a detection solution. The remainder of the procedure was performed with urine samples.
Claims (6)
1. A ractopamine lateral flow immunochromatography semi-quantitative detection test paper comprises a backing, a sample liquid absorption part, a colloidal gold labeling part, a detection reaction part and a water absorption part, wherein the sample liquid absorption part, the colloidal gold labeling part, the detection reaction part and the water absorption part are pasted on the backing, 1-3 ractopamine antibodies or 1-3 detection antigens are coated on the detection reaction part as detection lines, 1-3 IgG (immunoglobulin G) resisting second animal protein is coated on the detection reaction part as reference lines, the number of the detection lines and the reference lines cannot be more than 1 at the same time during combination, and the number of the combination lines is 2-4.
2. The strip of claim 1, wherein said test antigen is a conjugate of ractopamine with a carrier substance selected from the group consisting of proteins, protein fragments, synthetic polypeptides, semi-synthetic polypeptides and polysaccharides.
3. The strip of claim 2, wherein the carrier material is selected from the group consisting of bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, L-polylysine, diphtheria toxin, tetanus toxin, yeast, nylon, dextrane, and cellulose.
4. The strip of claim 1, wherein said second species of animal protein is a protein from a non-antibody source species of animal.
5. The test strip of any one of claims 1 to 4, wherein the detection line and the reference line are combined in 5 formats of 1 detection line and 1 reference line, 1 detection line and 2 reference lines, 1 detection line and 3 reference lines, 2 detection lines and 1 reference line, and 3 detection lines and 1 reference line.
6. The method of using a test strip according to any one of claims 1 to 4, wherein the semi-quantitative detection is achieved by adjusting the concentrations of the coating substances on the detection line and the reference line during the preparation of the test strip, controlling the color development depth of the detection line and the reference line during the detection, and correlating the color development depth or the number of developed color bands with the concentration of the standard substance.
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| CN 200510126415 CN1790025A (en) | 2005-12-08 | 2005-12-08 | Test paper for lateral flow immunochromatographic semi-quantitative detection of ractopamine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101539578A (en) * | 2009-02-17 | 2009-09-23 | 李红玉 | Colloidal gold test strip for testing melamine content |
| CN100570323C (en) * | 2006-08-08 | 2009-12-16 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of ractopamine column and its production and use |
| CN102507944A (en) * | 2011-11-09 | 2012-06-20 | 河北省科学院生物研究所 | Reagent card for semiquantitatively detecting salbutamol |
| CN102565384A (en) * | 2011-12-22 | 2012-07-11 | 正元盛邦(天津)生物科技有限公司 | Method for diagnosing ractopamine in semi-quantitative mode by employing double-indicatrix immunochromatography |
| CN103399154A (en) * | 2013-07-29 | 2013-11-20 | 武汉中博生物股份有限公司 | Test paper for colloidal gold immunochromatograohic assay of IgG (immunoglobulin G) antibody of dog anti-rabies virus and preparation method of test paper |
| CN105277665A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Test paper strip for rapidly detecting overstandard preservative benzoic acid |
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- 2005-12-08 CN CN 200510126415 patent/CN1790025A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100570323C (en) * | 2006-08-08 | 2009-12-16 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of ractopamine column and its production and use |
| CN101539578A (en) * | 2009-02-17 | 2009-09-23 | 李红玉 | Colloidal gold test strip for testing melamine content |
| CN102507944A (en) * | 2011-11-09 | 2012-06-20 | 河北省科学院生物研究所 | Reagent card for semiquantitatively detecting salbutamol |
| CN102565384A (en) * | 2011-12-22 | 2012-07-11 | 正元盛邦(天津)生物科技有限公司 | Method for diagnosing ractopamine in semi-quantitative mode by employing double-indicatrix immunochromatography |
| CN103399154A (en) * | 2013-07-29 | 2013-11-20 | 武汉中博生物股份有限公司 | Test paper for colloidal gold immunochromatograohic assay of IgG (immunoglobulin G) antibody of dog anti-rabies virus and preparation method of test paper |
| CN103399154B (en) * | 2013-07-29 | 2015-07-15 | 武汉中博生物股份有限公司 | Test paper for colloidal gold immunochromatograohic assay of IgG (immunoglobulin G) antibody of dog anti-rabies virus and preparation method of test paper |
| CN105277665A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Test paper strip for rapidly detecting overstandard preservative benzoic acid |
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