CN112595847A - Shrimp immunofluorescence detection test strip and application - Google Patents
Shrimp immunofluorescence detection test strip and application Download PDFInfo
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- CN112595847A CN112595847A CN202011627620.XA CN202011627620A CN112595847A CN 112595847 A CN112595847 A CN 112595847A CN 202011627620 A CN202011627620 A CN 202011627620A CN 112595847 A CN112595847 A CN 112595847A
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Images
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
Abstract
The invention provides a shrimp immunofluorescence detection test strip and application, and relates to the technical field of detection test strips. The immunofluorescence detection test strip comprises a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially assembled and adhered to a PVC base plate; the combined pad is coated with a mixed antibody marked by fluorescent latex microspheres, and the cellulose nitrate membrane is sequentially coated with an antigen myosin antibody (T1 line), an anti-arginine kinase antibody (T2 line), an anti-hemocyanin antibody (T3 line), an anti-troponin/myosin light chain antibody (T4 line) and a rabbit anti-mouse IgG antibody (C line), wherein the T1 line, the T2 line, the T3 line and the T4 line are detection lines, and the C line is a quality control line. The method can accurately and quantitatively detect the contents of tropomyosin, arginine kinase, hemocyanin and troponin/myosin light chain in food, and has the advantages of simple and convenient operation, high accuracy and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of test strips, and particularly relates to a shrimp immunofluorescence test strip and application thereof.
Background
The safety problem of food allergens has attracted a wide attention, and their potential allergenicity is a new topic to be researched urgently. In the past 20 years, the incidence of allergy has risen dramatically, and food allergens have become one of the food safety hazards. Along with the development of economy and the improvement of the living standard of people, the attention and the attention of people to anaphylaxis are greatly improved. Crustaceans such as shrimps and crabs and products thereof are important in 8 major allergic foods proposed by food and agriculture organizations of the united nations, and the allergen thereof is mainly tropomyosin (Pen al) existing in muscle fibers. Food allergy endangers the health of allergic people to a great extent, and protecting the food safety of consumers is an important problem faced by food safety management departments and food production enterprises. However, the existing allergen detection method can only detect one allergen at a time, and has low sensitivity.
Disclosure of Invention
In view of the above, the invention aims to provide a shrimp immunofluorescence detection test strip and an application thereof, wherein the immunofluorescence detection test strip is simple and convenient to operate, high in accuracy and sensitivity, and capable of rapidly and quantitatively detecting shrimp allergens.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a shrimp immunofluorescence detection test strip, which comprises a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are connected from left to right and are fixed on a PVC base plate in sequence; the combination pad is coated with a mixed antibody marked by fluorescent latex microspheres; the mixed antibody comprises: anti-myosin antibodies, anti-arginine kinase antibodies, anti-hemocyanin antibodies, and anti-troponin/myosin light chain antibodies;
the nitrocellulose membrane comprises 4 parallel detection lines and 1 quality control line; antigen myosin antibody, anti-arginine kinase antibody, anti-hemocyanin antibody and anti-troponin/myosin light chain antibody are respectively coated on the detection line;
and the coating antibody on the detection line and the mixed antibody marked by the fluorescent latex microspheres are paired antibodies.
Preferably, the particle size of the fluorescent latex microsphere is 50-500 nm.
Preferably, the preparation method of the fluorescent latex microsphere comprises the steps of adsorbing fluorescently-labeled streptavidin by using the latex microsphere; the fluorescent label comprises fluorescein isothiocyanate, tetraethylrhodamine, tetramethyl rhodamine isothiocyanate or fluorescein CY 5.
Preferably, in the mixed antibody, the mass ratio of the antigen myosin antibody, the anti-arginine kinase antibody, the anti-hemocyanin antibody and the anti-troponin/myosin light chain antibody is 1:1:1: 1.
Preferably, in the anti-troponin/myosin light chain antibody, the mass ratio of the anti-troponin antibody to the anti-myosin light chain antibody is 0.8 to 1.2: 1.
Preferably, the amount of the mixed antibody coated on the bonding pad is 2-10 mu l/cm.
Preferably, the quality control line is coated with a rabbit anti-mouse IgG antibody, and the coating concentration is 0.5-5 mu l/cm.
The invention also provides application of the immunofluorescence detection test strip in detection of shrimp allergic components.
Preferably, the shrimp allergenic component comprises tropomyosin, arginine kinase, hemocyanin, troponin, and/or myosin light chain.
The invention provides a shrimp immunofluorescence detection test strip, which has a structure shown in figure 1, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper are sequentially assembled and adhered on a PVC (polyvinyl chloride) bottom plate. The combined pad is coated with an antigen myosin antibody marked by a fluorescent latex microsphere, an anti-arginine kinase antibody, an anti-hemocyanin antibody and a mixed antibody of an anti-troponin/myosin light chain antibody, and the nitrocellulose membrane is sequentially coated with the antigen myosin antibody (T1 line), the anti-arginine kinase antibody (T2 line), the anti-hemocyanin antibody (T3 line), the anti-troponin/myosin light chain antibody (T4 line) and a rabbit anti-mouse IgG antibody (C line), wherein the T1 line, the T2 line, the T3 line and the T4 line are detection lines, and the C line is a quality control line. The method can accurately and quantitatively detect the contents of tropomyosin, arginine kinase, hemocyanin and troponin/myosin light chain in food, and has the advantages of simple and convenient operation, high accuracy, high recovery rate (90-110 percent) and high sensitivity (less than or equal to 0.84 ng/ml).
Drawings
FIG. 1 is a structural diagram of an immunofluorescence detection test strip according to the present invention;
FIG. 2 is a standard curve of fluorescence immunochromatography assay.
Detailed Description
The invention provides a shrimp immunofluorescence detection test strip, which comprises a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are connected end to end from left to right and are sequentially fixed on a PVC (polyvinyl chloride) base plate; the combination pad is coated with a mixed antibody marked by fluorescent latex microspheres; the mixed antibody comprises: anti-myosin antibodies, anti-arginine kinase antibodies, anti-hemocyanin antibodies, and anti-troponin/myosin light chain antibodies;
the nitrocellulose membrane comprises 4 parallel detection lines and 1 quality control line; antigen myosin antibody, anti-arginine kinase antibody, anti-hemocyanin antibody and anti-troponin/myosin light chain antibody are respectively coated on the detection line; and the coating antibody on the detection line and the mixed antibody marked by the fluorescent latex microspheres are paired antibodies.
The structure of the immunofluorescence detection test strip is shown in figure 1, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper are sequentially assembled and adhered to a PVC base plate. The combined pad is coated with a mixed antibody marked by fluorescent latex microspheres, and the particle size of the fluorescent latex microspheres is preferably 50-500 nm. The preparation method of the fluorescent latex microsphere preferably comprises the steps of adsorbing fluorescence-labeled streptavidin by using the latex microsphere; the fluorescent label comprises fluorescein isothiocyanate, tetraethylrhodamine, tetramethyl rhodamine isothiocyanate or fluorescein CY 5.
The preparation method of the bonding pad preferably comprises the following steps:
(a) adsorbing and combining fluorescence-labeled streptavidin and latex microspheres to prepare fluorescent latex microspheres;
(b) combining biotin with the mixed antibody to prepare a biotinylated mixed antibody;
(c) mixing the fluorescent latex microspheres and the biotinylated mixed antibody to obtain a mixed antibody marked by the fluorescent latex microspheres;
(d) spraying the mixed antibody marked by the fluorescent latex microspheres on a bonding pad; there is no chronological order between steps (a) and (b).
In the present invention, the mass ratio of the fluorescence-labeled streptavidin and the latex microspheres in step (a) is preferably 1: 40; the volume ratio of the biotin to the mixed antibody in the step (b) is preferably 1: 4; the volume ratio of the fluorescent latex microspheres to the biotinylated mixed antibody in step (c) is preferably 10: 1. In the mixed antibody of the present invention, the mass ratio of the antigen myosin antibody, the anti-arginine kinase antibody, the anti-hemocyanin antibody, and the anti-troponin/myosin light chain antibody is preferably 1:1:1: 1. In the present invention, the source of each antibody in the mixed antibody is not particularly limited, and a conventional commercially available antibody is preferable. The anti-troponin/myosin light chain antibody according to the present invention preferably includes an anti-troponin antibody and an anti-myosin light chain antibody, and the mass ratio of the anti-troponin antibody to the anti-myosin light chain antibody is preferably 1: 1. The spraying amount of the mixed antibody marked by the fluorescent latex microspheres on the bonding pad in the step (d) is preferably 2-10 mu l/cm. In the invention, the mixed antibodies marked by the fluorescent latex microspheres are both secondary antibodies.
The nitrocellulose membrane comprises 4 parallel detection lines and 1 quality control line; antigen myosin antibody (T1 line), anti-arginine kinase antibody (T2 line), anti-hemocyanin antibody (T3 line) and anti-troponin/myosin light chain antibody (T4 line) are respectively coated on the detection lines; the quality control line is preferably coated with a rabbit anti-mouse IgG antibody (C line, coating concentration is 0.5-5 mu l/cm). The antibody coated on the detection line of the nitrocellulose membrane is equivalent to a primary antibody, and is respectively matched with the mixed antibody (secondary antibody).
The preparation method of the nitrocellulose membrane is not particularly limited, and preferably includes diluting the antigen myosin antibody, the anti-arginine kinase antibody, the anti-hemocyanin antibody, the anti-troponin/myosin light chain mixed antibody and the rabbit anti-mouse IgG antibody respectively with a coating buffer solution, and scratching the five diluted antibodies respectively in parallel on the nitrocellulose membrane, so that when the antibodies permeate the nitrocellulose membrane, detection regions respectively coated with the antigen myosin antibody, the anti-arginine kinase antibody, the anti-hemocyanin antibody, the anti-troponin/myosin light chain mixed antibody and quality control regions coated with the rabbit anti-mouse IgG antibody are respectively formed. When the detection area is scribed, the detection area is preferably dried by air blowing for 12 hours at 20 ℃ in a drying oven according to the diluted antibody concentration of 3mg/ml, the liquid feed amount of a peristaltic pump of 0.4ml/min and the scribing speed of 50m/20 min; when the quality control area is scribed, preferably according to the concentration of a diluted antibody of 5mg/ml, the liquid feed amount of a peristaltic pump of 0.4ml/min and the scribing speed of 50m/20min, scribing on the nitrocellulose membrane, wherein the line is parallel to the line of the detection area, and putting the nitrocellulose membrane into a drying oven for drying for 12h by blowing at 20 ℃. After the scribing, the invention preferably further comprises the steps of sealing the nitrocellulose membrane for 60min at 37 ℃ by using a sealing solution, taking out the nitrocellulose membrane, drying the nitrocellulose membrane for 2h at 37 ℃, and sealing the nitrocellulose membrane for later use.
In the invention, the sample pad, the combination pad, the nitrocellulose membrane and the absorbent filter paper are preferably assembled and adhered on the PVC base plate in sequence, and cut into the test paper strip shown in figure 1 by a slitter according to the requirement (4 mm).
The invention also provides application of the immunofluorescence detection test strip in detection of shrimp allergic components.
The shrimp allergenic component of the present invention preferably includes tropomyosin, arginine kinase, hemocyanin, troponin, and/or myosin light chain.
The invention also provides a method for detecting the shrimp allergy components by using the immunofluorescence detection test strip, which preferably comprises the following steps: and (3) dropwise adding 100-120 mu l of a sample to be detected on the sample pad, after 15min, putting the test strip in a special fluorescent signal reading instrument after the quality control area reaction is finished, reading the size of a fluorescent signal, and carrying out quantitative determination. After the sample to be detected is dripped on the sample pad, the sample is reacted and combined through the mixed antibody on the combination pad, and then the sample is reacted through the antigen myosin antibody, the anti-arginine kinase antibody, the anti-hemocyanin antibody and the anti-troponin/myosin light chain antibody in the detection area in sequence, and finally the reaction in the quality control area is finished.
The present invention provides a test strip for shrimp immunofluorescence detection and the application thereof, which are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of the conjugate pad
1) Preparation of fluorescent latex microspheres: diluting the latex microspheres with the particle size of 400nm to a final concentration of 30mg/ml and a volume of 6ml by using an adsorption buffer (50mM, pH5.8 citrate buffer) to prepare a latex microsphere suspension; adding red fluorescein rhodamine-labeled hyphomycetin into an adsorption buffer solution according to the volume ratio of 1: 50-500, wherein the final volume is 6 ml; adding the latex microsphere suspension into the adsorption buffer solution containing the red fluorescein rhodamine-labeled avidin to prepare a mixed solution; and (3) carrying out warm bath on the obtained mixed solution at room temperature for 1-2 h, continuously stirring, centrifuging, collecting precipitates, dissolving the precipitates by using a storage buffer solution (an adsorption buffer solution containing 0.06% BSA), and standing at 4 ℃ for storage for later use.
2) Preparation of biotinylated Mixed antibodies
An antigen myosin antibody (U.S. Indor, cat # PA-SHM); anti-arginine kinase antibody (Abcam, cat # ab225937, USA); anti-hemocyanin antibodies (Sibao organism, cat # H0892); the mixed antibody of the same amount of anti-troponin/myosin light chain (Hangzhou Mintai organism, product number: MT-0702 mixed according to the ratio of 1: 1) is diluted to 3ml by 0.2M pH4.7 sodium acetate buffer solution, and the mixed antibody is fully dialyzed by 0.2M pH4.7 sodium acetate buffer solution for interaction; dissolving 1ml of NHSB in 1ml of DMSO to obtain a NHSB solution; and adding 25 mu L HSB into the 3ml mixed antibody, stirring for 2-4 h, continuing stirring at room temperature for 10min, and dialyzing by using 20mM PBS (phosphate buffer solution) with pH3.9 to obtain the biotinylated mixed antibody.
3) Preparation of fluorescent latex microsphere labeled shrimp antibody
Mixing the fluorescent latex microspheres prepared in the step 1) and the biotinylated mixed antibody (according to the proportion of 10: 1) prepared in the step 2), reacting for 30min, centrifuging, dissolving the precipitate by using a storage buffer solution, and recovering to the original volume.
4) And spraying the fluorescent latex microsphere labeled shrimp mixed antibody on the bonding pad according to the dosage of 2-10 mul/cm.
2. Preparation of nitrocellulose membranes
1) Taking a nitrocellulose membrane for membrane treatment, and soaking in pH membrane treatment solution (TBS) for 5-10 min after marking;
2) assembling a sample application device, placing the soaked nitrocellulose membrane on a flat-laid mat, placing an antibody sample application plate, and reserving a place for adhering the marking paper.
3) Preparation of shrimp antibody detection zone: an antigen myosin antibody (Hangzhou Mintai biology, a product number is MO-0801), an anti-arginine kinase antibody (Hangzhou Mintai biology, a product number is MO-0802), an anti-hemocyanin antibody (Hangzhou Mintai biology, a product number is: MO-0803), anti-troponin/myosin light chain antibody (hangzhou mintai organism, cat #: MO-0804) according to the concentration of 3mg/ml, the liquid feed amount of a peristaltic pump of 0.4ml/min and the scribing speed of 50m/20min, and drying by air blast in a drying oven at 20 ℃ for 12 h.
4) Preparation of a quality control region: the rabbit anti-mouse IgG antibody is streaked on the nitrocellulose membrane according to the concentration of 8mg/ml, the liquid feed amount of a peristaltic pump of 0.4ml/min and the streaking speed of 50m/20min, the line is parallel to the line of the detection area, and the nitrocellulose membrane is put into a drying oven to be dried by air blasting for 12 hours at the temperature of 20 ℃.
5) Blocking the nitrocellulose membrane with blocking solution (100ml PBS and 0.5g BSA preparation) at 37 deg.C for 60min, taking out, oven drying at 37 deg.C for 2h, and sealing for use.
3. Test strip assembly
The sample pad, the bonding pad, the nitrocellulose membrane and the absorbent filter paper are assembled and adhered on a PVC base plate in sequence, and cut into the test paper strip shown in figure 1 by a slitter according to the requirement (4 mm).
4. Detection of antigen to be detected
And dropwise adding 100-120 mu l of a sample to be detected on a sample pad, reacting and combining the sample with an anti-shrimp mixed antibody on a combination pad, reacting the sample with an antigen myosin antibody, an anti-arginine kinase antibody, an anti-hemocyanin antibody and an anti-troponin/myosin light chain mixed antibody in a detection area in sequence, and finally finishing the reaction in a quality control area, putting the test strip into a special fluorescent signal reading instrument, reading the size of a fluorescent signal, and carrying out quantitative determination.
Linearity of dose-response curve: the concentrations of the calibration solutions prepared by the calibrators in the determination kit are respectively 100.00, 50.00, 17.50, 3.50 and 0.35IU/ml, the calibration solutions are fitted by using a double logarithm or other appropriate mathematical modes, and the fitting result of the model is required to meet the requirement that the internal precision (CV%) of analysis is less than or equal to 10.0 percent; the precision (CV%) between analyses should be less than or equal to 15.0%.
The results of the linear analysis of the dose-response curve show that each concentration and the tested value are in a smooth increasing curve within the range of 0.35-100 IU/ml, the lower limit of measurement (CV < the lowest concentration which can be detected by a 15% test system) is 0.35IU/ml, and the curve equation is shown in figure 2:
the foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A shrimp immunofluorescence test strip is characterized in that the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are connected from left to right and are fixed on a PVC base plate in sequence; the combination pad is coated with a mixed antibody marked by fluorescent latex microspheres; the mixed antibody comprises: anti-myosin antibodies, anti-arginine kinase antibodies, anti-hemocyanin antibodies, and anti-troponin/myosin light chain antibodies;
the nitrocellulose membrane comprises 4 detection lines and 1 quality control line which are arranged in parallel; antigen myosin antibody, anti-arginine kinase antibody, anti-hemocyanin antibody and anti-troponin/myosin light chain antibody are respectively coated on the detection line;
and the coating antibody on the detection line and the mixed antibody marked by the fluorescent latex microspheres are paired antibodies.
2. The immunofluorescence detection test strip according to claim 1, wherein the particle size of the fluorescent latex microsphere is 50-500 nm.
3. The immunofluorescence detection test strip according to claim 2, wherein the preparation method of the fluorescent latex microspheres comprises adsorbing fluorescently-labeled streptavidin by using the latex microspheres; the fluorescent label comprises fluorescein isothiocyanate, tetraethylrhodamine, tetramethyl rhodamine isothiocyanate or fluorescein CY 5.
4. The immunofluorescence detection test strip of claim 1, wherein, in the mixed antibody, the mass ratio of the antigen myosin antibody, the anti-arginine kinase antibody, the anti-hemocyanin antibody and the anti-troponin/myosin light chain antibody is 1:1:1: 1.
5. The immunofluorescence detection test strip according to claim 1 or 4, wherein, in the anti-troponin/myosin light chain antibody, the mass ratio of the anti-troponin antibody to the anti-myosin light chain antibody is 0.8-1.2: 1.
6. The immunofluorescence detection test strip according to claim 4, wherein, the concentration of the mixed antibody coated on the bonding pad is 2-10 μ l/cm.
7. The immunofluorescence detection test strip according to claim 1, wherein the quality control line is coated with rabbit anti-mouse IgG antibody at a coating concentration of 0.5-5 μ l/cm.
8. Use of the immunofluorescence detection test strip of any one of claims 1 to 7 in the detection of shrimp allergy components.
9. The use according to claim 8, wherein the shrimp allergenic component comprises tropomyosin, arginine kinase, hemocyanin, troponin, and/or myosin light chain.
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| AU2021101507A AU2021101507A4 (en) | 2020-12-31 | 2021-03-24 | Immunofluorescent assay test strip for shrimp and use thereof |
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| CN112924700A (en) * | 2021-01-26 | 2021-06-08 | 海南医学院 | Coating process of Keyhole Limpet Hemocyanin (KLH) and quantitative detection kit for mouse KLH specific IgG antibody |
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