CN112526142A - Egg immunofluorescence detection test strip and application and detection method thereof - Google Patents
Egg immunofluorescence detection test strip and application and detection method thereof Download PDFInfo
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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
- G01N33/68—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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- 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
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- 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
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Abstract
The invention provides an egg immunofluorescence detection test strip and application and a detection method thereof, and relates to the technical field of immunofluorescence detection. The test strip comprises a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are connected end to end from left to right and are sequentially fixed on a PVC base plate; the combination pad is coated with a mixed antibody marked by fluorescent latex microspheres; an anti-ovalbumin antibody (T1 line), an anti-ovomucoid antibody (T2 line), an anti-ovotransferrin antibody (T3 line), an anti-egg total antibody (T4 line) and a rabbit anti-mouse IgG antibody (C line) are coated on a nitrocellulose membrane (NC membrane), 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 test strip can rapidly and quantitatively detect the type and the content of the egg allergen, is simple and convenient to operate and high in accuracy, and has the recovery rate of 90-110% and high sensitivity (less than or equal to 0.84 ng/ml).
Description
Technical Field
The invention belongs to the technical field of immunofluorescence detection, and particularly relates to an egg immunofluorescence detection test strip and an application and a detection method thereof.
Background
With the development of the food industry, allergic diseases have become a global problem. In recent years, the incidence of allergic diseases caused by eating eggs is on the rise. According to foreign epidemiological statistics, about 35% of food-allergic children are allergic to eggs, with infants before the age of 1 year being more common. At present, no effective treatment method for egg food allergy diseases at home and abroad exists, and the prevention of eating egg component-containing food for patients with severe egg antigen allergy is one of the most effective prevention methods at present. In recent years, in the food labeling law of the united states and the european union, it is required to mark eggs as main allergen components and to start egg allergen detection on some imported foods, and food export enterprises in China are often notified by the FDA in the united states and suffer from returns due to incorrect labeling of food allergens. With the increasing importance of people on food safety, China also has to establish a set of allergen identification system, and a corresponding detection method is needed to provide guarantee for the implementation of the system. Therefore, it is necessary to develop a rapid detection method for eggs. The egg allergen has strong stability in the processing process, and the egg allergen can not be completely desensitized after being respectively treated by glycosylation, 6mol/L urea and heating at 95 ℃ as proved by documents.
The imported egg allergen enzyme-linked immunosorbent assay kit is mostly used as a detection target of egg allergens in food. But the imported kit is expensive, so that the development of the rapid, effective and low-cost allergen detection kit has important significance.
Disclosure of Invention
In view of the above, the invention aims to provide an egg immunofluorescence detection test strip, an application and a detection method thereof, which can be used for rapidly, qualitatively and quantitatively detecting egg allergen components in food, and are simple and convenient to operate, high in accuracy and high in sensitivity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an egg 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 in an end-to-end manner 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-ovalbumin antibody, anti-ovomucoid antibody, anti-ovotransferrin antibody and anti-egg total antibody;
the nitrocellulose membrane comprises 4 parallel detection lines and 1 quality control line; the detection line is respectively coated with an anti-ovalbumin antibody, an anti-ovomucoid antibody, an anti-ovotransferrin antibody and an anti-egg total antibody; the quality control line is coated with a rabbit anti-mouse IgG antibody;
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 method for preparing the conjugate pad coated with the mixed antibody labeled by the fluorescent latex microspheres comprises the following steps: a. adsorbing and combining fluorescence-labeled streptavidin and latex microspheres to obtain fluorescent latex microspheres;
b. combining biotin with the mixed antibody to obtain 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;
the steps a and b do not have a chronological relationship.
Preferably, the fluorescent label in step a comprises fluorescein isothiocyanate, tetraethylrhodamine, tetramethyl rhodamine isothiocyanate or fluorescein CY 5.
Preferably, in the step d, the mixed antibody marked by the fluorescent latex microspheres is sprayed on the bonding pad according to the dosage of 2-10 μ l/cm.
Preferably, the mass ratio of the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-chicken total antibody in the mixed antibody is 1:1:1: 1.
preferably, the coating concentration of the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody coated on the 4 detection lines is 0.5-5.0 mu l/cm;
the coating concentration of the rabbit anti-mouse IgG antibody coated on the quality control line is 0.5-5 mul/cm.
The invention also provides application of the immunofluorescence detection test strip in detection of the allergen components of the eggs in food.
Preferably, the peanut allergen component comprises ovalbumin, ovomucoid, ovotransferrin and total egg protein.
The invention also provides a method for detecting the allergen components of the eggs in the food, which comprises the following steps: dripping 100-120 mu l of food solution on a sample pad of the immunofluorescence detection test strip, reading a fluorescence signal of the detection test strip after 15min, and determining the components and the content of the peanut allergen according to a standard curve;
the standard curve is
R2＝0.9997；
Wherein x is the allergen concentration, IU/ml, and y is the ratio of fluorescence signals.
The invention provides an egg immunofluorescence detection test strip, which has a structure shown in figure 1 and 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 in an end-to-end manner 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-ovalbumin antibody, anti-ovomucoid antibody, anti-ovotransferrin antibody and anti-egg total antibody; an anti-ovalbumin antibody (T1 line), an anti-ovomucoid antibody (T2 line), an anti-ovotransferrin antibody (T3 line), an anti-egg total antibody (T4 line) and a rabbit anti-mouse IgG antibody (C line) are coated on a nitrocellulose membrane (NC membrane), 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 mixed antibody and the antibody coated on the NC membrane are respectively paired, the mixed antibody is similar to a secondary antibody, and the antibody coated on the NC membrane is similar to a primary antibody. The test strip can rapidly and quantitatively detect the type and the content of the egg allergen, is simple and convenient to operate and high in accuracy, and has the recovery rate of 90-110% 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 an egg immunofluorescence detection test strip, the structure of which is shown in figure 1, wherein 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 in an end-to-end manner 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-ovalbumin antibody, anti-ovomucoid antibody, anti-ovotransferrin antibody and anti-egg total antibody;
the nitrocellulose membrane comprises 4 parallel detection lines and 1 quality control line; the detection line is respectively coated with an anti-ovalbumin antibody, an anti-ovomucoid antibody, an anti-ovotransferrin antibody and an anti-egg total antibody; the quality control line is coated with a rabbit anti-mouse IgG antibody;
and the coating antibody on the detection line and the mixed antibody marked by the fluorescent latex microspheres are paired antibodies.
The preparation method of the combined pad coated with the mixed antibody marked by the fluorescent latex microspheres preferably comprises the following steps: a. adsorbing and combining fluorescence-labeled streptavidin and latex microspheres to obtain fluorescent latex microspheres;
b. combining biotin with the mixed antibody to obtain 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; the steps a and b do not have a chronological relationship.
The mass ratio of the fluorescence-labeled streptavidin to the latex microspheres in the 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. The particle size of the fluorescent latex microsphere is preferably 50-500 nm. The fluorescent label in step a of the present invention preferably comprises fluorescein isothiocyanate, tetraethylrhodamine, tetramethyl rhodamine isothiocyanate or fluorescein CY 5. The mass ratio of the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody of the mixed antibody is preferably 1:1:1: 1. the sources of the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-chicken total antibody are not particularly limited in the present invention, and are preferably those conventionally available in the art. 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 (NC membrane) comprises 4 parallel detection lines and 1 quality control line; the detection lines are respectively coated with an anti-ovalbumin antibody (T1 line), an anti-ovomucoid antibody (T2 line), an anti-ovotransferrin antibody (T3 line) and an anti-egg total antibody (T4 line); the quality control line is coated with a rabbit anti-mouse IgG antibody (C line). The method for producing the NC film of the present invention is not particularly limited, and preferably includes: respectively diluting an anti-ovalbumin antibody, an anti-ovomucoid antibody, an anti-ovotransferrin antibody, an anti-egg total antibody and a rabbit anti-mouse IgG antibody by using a coating buffer solution, respectively scribing the five diluted antibodies on a nitrocellulose membrane in parallel, and respectively forming a detection line for coating the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody and a quality control line for coating the rabbit anti-mouse IgG antibody after the antibodies permeate the nitrocellulose membrane. The coating concentrations of the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody coated on the 4 detection lines are all 0.5-5.0 mu l/cm; the coating concentration of the rabbit anti-mouse IgG antibody coated on the quality control line is 0.5-5 mul/cm. According to the invention, when the marking is carried out, the operation aiming at the detection limit and the quality control line is different, and when the marking is carried out on the detection limit, the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody are preferably subjected to forced air drying for 12 hours in a drying oven at 20 ℃ according to the concentration of 3mg/ml, the liquid feed amount of a peristaltic pump of 0.4ml/min and the marking speed of 50m/20min respectively and sequentially; when the quality control line is scribed, preferably, the rabbit anti-mouse IgG antibody is scribed on the nitrocellulose membrane at a scribing speed of 50m/20min according to the concentration of 8mg/ml, the liquid feed amount of a peristaltic pump of 0.4ml/min, and the line is parallel to the line of the detection line, and the nitrocellulose membrane is put into a drying oven to be dried by air blow for 12h at 20 ℃.
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 test strip disclosed by the invention can be produced in batch, is suitable for clinical rapid diagnosis and field rapid detection, and is easy to store.
The invention provides application of the immunofluorescence detection test strip in detection of the allergen components of eggs in food.
The egg allergen component according to the present invention preferably comprises ovalbumin, ovomucoid, ovotransferrin and egg total protein, wherein ovalbumin, ovomucoid and ovotransferrin are not involved in egg total protein.
The invention also provides a method for detecting the allergen components of the eggs in the food, which comprises the following steps: dripping 100-120 mu l of food solution on the sample pad of the immunofluorescence test strip, reading the fluorescence signal of the test strip after 15min, and obtaining the standard curveMeasuring the components and content of peanut allergen; the standard curve is
R2＝0.9997；
Wherein x is the allergen concentration, IU/ml, and y is the ratio of fluorescence signals.
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 anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody which sequentially pass through the detection area react, and finally the reaction in the quality control area is finished.
The present invention provides an egg immunofluorescence test strip and the application and detection method 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.
The reagents used in the invention are all conventional purchased products unless otherwise specified, wherein:
anti-ovalbumin antibodies: IndorBiotechnologies (cat # MA-3G4, MA-7D 8);
anti-ovomucoid antibodies: IndorBiotechnologies (cat # MA-5G11, MA-2B 2);
anti-ovotransferrin antibody: CusAb (cat # CSB-PA00390F0 Rb);
anti-egg total antibodies: hangzhou faithful Europe biomedical science and technology park, Inc. (Cat: CE-GAL-01).
Example 1
1. Preparation of the conjugate pad
1) Preparation of fluorescent latex microspheres
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 proper red fluorescein rhodamine-labeled hyphomycetin into the adsorption buffer solution, 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 anti-egg Mixed antibody
Mixing an anti-ovalbumin antibody, an anti-ovomucoid antibody, an anti-ovotransferrin antibody and an anti-egg total antibody according to the mass ratio of 1:1:1:1 (mixed antibody M1), diluting to 3ml by using 0.2M sodium acetate buffer solution with pH4.7, and fully dialyzing the egg antibody M1 by using 0.2M sodium acetate buffer solution with pH4.7 for interaction; dissolving 1ml of NHSB in 1ml of DMSO to obtain a NHSB solution; adding 25 mu l of NHSB into the 3ml of egg mixed antibody M1, stirring for 2-4 h, continuing stirring for 10min at room temperature, and dialyzing by using 20mM PBS buffer solution with pH3.9 to obtain the biotinylation mixed antibody M1.
3) Preparation of fluorescent latex microsphere labeled egg antibody
Mixing the fluorescent latex microspheres prepared in the step 1) and the biotinylated mixed antibody prepared in the step 2) according to the volume ratio of 10:1, 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 egg 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) Preparing an egg antibody detection area: sequentially carrying out air drying on the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody in a drying oven at 20 ℃ for 12h 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/20 min.
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) Sealing the nitrocellulose membrane with the sealing solution at 37 deg.C for 60min, taking out, oven drying at 37 deg.C for 2h, and sealing.
6) Test strip assembly
The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent filter paper were sequentially assembled and adhered to a PVC base plate, and cut into test strips of appropriate width (4mm) as required by a slitter (shown in fig. 1).
3. 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 with the egg-resistant mixed antibody on the combination pad, reacting with the egg-resistant mixed antibody, the egg-resistant mucin antibody, the egg-resistant transferrin antibody and the egg-resistant total antibody which sequentially pass through the detection area, and finally finishing the reaction in the quality control area, putting the test strip in 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:
wherein x is the allergen concentration, IU/ml, and y is the ratio of fluorescence signals.
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 (10)
1. An egg immunofluorescence detection 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 in an end-to-end manner and are sequentially fixed on a PVC base plate; the combination pad is coated with a mixed antibody marked by fluorescent latex microspheres; the mixed antibody comprises: anti-ovalbumin antibody, anti-ovomucoid antibody, anti-ovotransferrin antibody and anti-egg total antibody;
the nitrocellulose membrane comprises 4 parallel detection lines and 1 quality control line; the detection line is respectively coated with an anti-ovalbumin antibody, an anti-ovomucoid antibody, an anti-ovotransferrin antibody and an anti-egg total antibody; the quality control line is coated with a rabbit anti-mouse IgG antibody;
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 1, wherein, the preparation method of the binding pad coated with the mixed antibody marked by the fluorescent latex microspheres comprises the following steps: a. adsorbing and combining fluorescence-labeled streptavidin and latex microspheres to obtain fluorescent latex microspheres;
b. combining biotin with the mixed antibody to obtain 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;
the steps a and b do not have a chronological relationship.
4. The immunofluorescence detection test strip of claim 3, wherein, in step a, the fluorescence label comprises fluorescein isothiocyanate, tetraethylrhodamine, tetramethyl rhodamine isothiocyanate or fluorescein CY 5.
5. The immunofluorescence detection test strip according to claim 3, wherein, in the spraying of step d, the mixed antibody marked by the fluorescent latex microspheres is sprayed on the bonding pad according to the dosage of 2-10 μ l/cm.
6. The immunofluorescence detection test strip according to claim 1 or 3, wherein, the mass ratio of the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody of the mixed antibody is 1:1:1: 1.
7. the immunofluorescence detection test strip according to claim 1, wherein the coating concentration of the anti-ovalbumin antibody, the anti-ovomucoid antibody, the anti-ovotransferrin antibody and the anti-egg total antibody coated on 4 detection lines is 0.5-5.0 μ l/cm;
the coating concentration of the rabbit anti-mouse IgG antibody coated on the quality control line is 0.5-5 mul/cm.
8. Use of the immunofluorescence detection test strip of any one of claims 1 to 7 in detection of an egg allergen component in a food.
9. The use of claim 8, wherein the peanut allergen component comprises ovalbumin, ovomucoid, ovotransferrin, and total egg protein.
10. A method for detecting allergen components in eggs in food is characterized by comprising the following steps: dripping 100-120 mu l of food solution on a sample pad of the immunofluorescence test strip of any one of claims 1-7, reading a fluorescence signal of the test strip after 15min, and determining the components and the content of peanut allergen according to a standard curve;
the standard curve is
R2＝0.9997；
Wherein x is the allergen concentration, IU/ml, and y is the ratio of fluorescence signals.
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| AU2021101488A AU2021101488A4 (en) | 2020-11-25 | 2021-03-23 | Immunofluorescent assay test strip for egg, use thereof, and detection method using same |
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| CN112526124A (en) * | 2020-11-25 | 2021-03-19 | 杭州福德敏生物技术有限公司 | Peanut immunofluorescence detection test strip and application and detection method thereof |
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