AU2021101387A4

AU2021101387A4 - Immunofluorescent assay test strip for wheat and use thereof

Info

Publication number: AU2021101387A4
Application number: AU2021101387A
Authority: AU
Inventors: Xin Li; Xuanyi MENG; Ping Tong; Yong Wu; Juanli YUAN
Original assignee: Hangzhou Fudemin Biotechnology Co Ltd
Current assignee: Hangzhou Fudemin Biotechnology Co Ltd
Priority date: 2020-12-31
Filing date: 2021-03-17
Publication date: 2021-05-13
Anticipated expiration: 2029-03-17
Also published as: CN112630423A

Abstract

Documn7-16/03/2021 ABSTRACT The present disclosure provides an immunofluorescent assay test strip for wheat and use thereof, and relates to the technical field of test strips. The immunofluorescent assay test strip of the present disclosure includes a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent paper that are sequentially assembled and adhered on a PVC backing card. The conjugate pad is coated with pooled antibodies labeled with fluorescent latex microspheres, and the NC membrane is sequentially coated with an anti-Tri a 12 antibody (TI line), an anti-Tri a 14 antibody (T2 line), an anti-Tri a 19 antibody (T3 line), an anti-Tri a 26 antibody (T4 line), and a rabbit anti-mouse IgG antibody (C line). The T, T2, T3 and T4 lines are test lines, and the C line is a quality-control line. The present disclosure can accurately and quantitatively detect the contents of Tri a 12, Tri a 14, Tri a 19, and Tri a 26 in foods, with simple operation, high accuracy, and high sensitivity. Doument7-16/03/2021 Milli Sample Conjugate pad NC Absorbent paper pad membrane PVC backing card FIG. 1 3.0 y=7.19789-+ 7.22725 1+(X/263.23601 R2 =0.96043 2.5 0 2.0 1.5 tjI 1.0 0.5 0.0 .353.5 17.5 50 100 Quality-control product concentration (IU/ML) FIG. 2

Description

Doument7-16/03/2021

Milli

Sample Conjugate pad NC Absorbent paper pad membrane PVC backing card

FIG. 1

3.0 y=7.19789-+ 7.22725 1+(X/263.23601 2 R =0.96043

2.5

2.0 0

1.5

1.0 tjI

0.5

0.0 .353.5 17.5 50 100 Quality-control product concentration (IU/ML)

FIG. 2

Documn7-16/03/2021

IMMUNOFLUORESCENT ASSAY TEST STRIP FOR WHEAT AND USE THEREOF TECHNICAL FIELD The present disclosure belongs to the technical field of test strips, and specifically relates to an immunofluorescent assay test strip for wheat and use thereof. BACKGROUND Food allergy refers to an IgE-mediated immune response of the human body to allergens in food, which leads to an allergic response in the digestive system or the whole body. As an incidence of food allergy continues to rise globally, food allergy has become one of the food safety issues that people are increasingly concerned about in the 21st century. Food allergy specifically has the epidemiological characteristics of regional distribution, population distribution, time distribution, and type distribution. The European Union (EU) launched the EuroPrevall project in 2005, which is implemented by the collaboration of 56 laboratories and research institutes from China, India, Ghana, and 19 European countries to provide research foundation and reference data for the risk assessment and management of food allergens. In this project, Wong et al. selected 16,866 pupils in urban and rural areas of Beijing for questionnaires and skin prick tests, and an incidence of IgE-mediated food allergy was estimated to be 4% among pupils in urban areas and was estimated to be 2% among pupils in rural areas, where, food allergens involved wheat. The first international-level nationwide epidemiological survey of allergies in the general population in Chinese history was officially launched in Beijing on October 18, 2009. According to the characteristics of food allergies in China, the Ministry of Health of the People's Republic of China issued the recommended national standard "Allergenic Ingredients in Prepackaged Foods" (GB/T 23779-2009) in 2009, and gluten-containing grains and products thereof rank first among recommended identified allergens listed in the national standard. Wheat and related products thereof are widely used in people's common diets, which are not only a main food source, but also one of the important food protein sources for people. Wheat allergy can affect the health of internal organs, respiratory tract, and skin, causing exercise-induced allergies, asthma, rhinitis, etc. Wheat allergy is more commonly manifested as a delayed allergic reaction, with a high prevalence and missed diagnosis rate and many complications, which seriously affects the lives of allergic patients. Therefore,

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wheat allergy is a food safety issue that cannot be ignored. Therefore, it is of vital importance to develop a detection method that can detect wheat allergens and contents thereof in foods and to provide related packaging labels, which can avoid many risks of wheat allergy for allergy sufferers. SUMMARY In view of this, the present disclosure is intended to provide an immunofluorescent assay test strip for wheat and use thereof. The immunofluorescent assay test strip can rapidly and quantitatively detect wheat allergens, with simple operation, high accuracy, and high sensitivity. To achieve the above objective, the present disclosure provides the following technical solutions. The present disclosure provides an immunofluorescent assay test strip for wheat, including a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent paper that are connected end-to-end and sequentially fixed on a PVC backing card from left to right, where, the conjugate pad is coated with pooled antibodies labeled with fluorescent latex microspheres; the pooled antibodies include: an anti-Tri a 12 antibody, an anti-Tri a 14 antibody, an anti-Tri a 19 antibody, and an anti-Tri a 26 antibody; the NC membrane includes 4 test lines and 1 quality-control line that are parallel; the test lines are coated with an anti-Tri a 12 antibody, an anti-Tri a 14 antibody, an anti-Tri a 19 antibody, and an anti-Tri a 26 antibody, respectively; the quality-control line is coated with a rabbit anti-mouse IgG antibody; and the coated antibodies on the test lines are paired with the pooled antibodies labeled with fluorescent latex microspheres. Preferably, the fluorescent latex microspheres may have a particle size of 50 nm to 500 nm. Preferably, a preparation method of the fluorescent latex microspheres may include allowing latex microspheres to adsorb fluorescently-labeled streptavidin (SA); and a fluorescent marker may include fluorescein isothiocyanate (FITC), Rhodamine B, tetramethylrhodamine isothiocyanate (TRITC), or fluorescein CY5. Preferably, in the pooled antibodies, the anti-Tri a 12 antibody, anti-Tri a 14 antibody, anti-Tri a 19 antibody, and anti-Tri a 26 antibody may have a mass ratio of 1:1:1:1. Preferably, an anti-Tri a 12 antibody, an anti-Tri a 14 antibody, an anti-Tri a 19 antibody, and an anti-Tri a 26 antibody may be coated on the 4 test lines at a coating concentration of 0.5 l/cm to 5.0 l/cm.

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Preferably, the quality-control line may be coated with a rabbit anti-mouse IgG antibody at a coating concentration of 0.5 l/cm to 5 l/cm. The present disclosure also provides use of the immunofluorescent assay test strip described above in the detection of wheat allergenic ingredients. Preferably, the wheat allergenic ingredients may include Tri a 12, Tri a 14, Tri a 19, and Tri a 26. The present disclosure also provides a method for detecting wheat allergenic ingredients in foods, including the following steps: adding 100 1 to 120 1 of a food solution dropwise on the sample pad of the immunofluorescent assay test strip, reading a fluorescence signal on the test strip 15 min later, and determining a wheat allergenic ingredient and a content thereof according to a standard curve.

y = 7.19789 - 7275).14 The standard curve is 1 + (x /263.23601)° R2 = 0.96043,

where, x is an allergen concentration in IU/ml, and y is a fluorescence signal ratio. The present disclosure provides an immunofluorescent assay test strip for wheat with a structure shown in FIG. 1, where, a sample pad, a conjugate pad, an NC membrane, and an absorbent paper are sequentially assembled and adhered on a PVC backing card. The conjugate pad is coated with pooled antibodies of anti-Tri a 12 antibody, anti-Tri a 14 antibody, anti-Tri a 19 antibody, and anti-Tri a 26 antibody that are labeled with fluorescent latex microspheres, and the NC membrane is sequentially coated with an anti-Tri a 12 antibody (TI line), an anti-Tri a 14 antibody (T2 line), an anti-Tri a 19 antibody (T3 line), an anti-Tri a 26 antibody (T4 line), and a rabbit anti-mouse IgG antibody (C line). The TI, T2, T3 and T4 lines are test lines, and the C line is a quality-control line. In the present disclosure, the pooled antibodies are paired with the antibodies coated on the NC membrane, respectively. The pooled antibodies serve as secondary antibodies, and the antibodies coated on the NC membrane serve as primary antibodies. The present disclosure can accurately and quantitatively detect the contents of Tri a 12, Tri a 14, Tri a 19, and Tri a 26 in foods, with simple operation, high accuracy, high recovery rate (90% to110%), and high sensitivity (< 0.84 ng/ml). BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a structural diagram of the immunofluorescent assay test strip according to the present disclosure; and

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FIG. 2 shows a standard curve for fluorescence immunochromatographic assay.

DETAILED DESCRIPTION The present disclosure provides an immunofluorescent assay test strip for wheat, including a sample pad, a conjugate pad, an NC membrane, and an absorbent paper that are connected end-to-end and sequentially fixed on a PVC backing card from left to right, where, the conjugate pad is coated with pooled antibodies labeled with fluorescent latex microspheres; the pooled antibodies include: an anti-Tri a 12 antibody, an anti-Tri a 14 antibody, an anti-Tri a 19 antibody, and an anti-Tri a 26 antibody; the NC membrane includes 4 test lines and 1 quality-control line that are parallel; the test lines are coated with an anti-Tri a 12 antibody, an anti-Tri a 14 antibody, an anti-Tri a 19 antibody, and an anti-Tri a 26 antibody, respectively; and the coated antibodies on the test lines are paired with the pooled antibodies labeled with fluorescent latex microspheres. The immunofluorescent assay test strip of the present disclosure has a structure shown in FIG. 1, where, a sample pad, a conjugate pad, an NC membrane, and an absorbent paper are sequentially assembled and adhered on a PVC backing card. The conjugate pad of the present disclosure is coated with pooled antibodies labeled with fluorescent latex microspheres, and the fluorescent latex microspheres may have a particle size preferably of nm to 500 nm. In the present disclosure, a preparation method of the fluorescent latex microspheres may preferably include allowing latex microspheres to adsorb fluorescently-labeled SA; and a fluorescent marker may include FITC, Rhodamine B, TRITC, or fluorescein CY5. In the present disclosure, a preparation method of the conjugate pad may preferably include: (a) allowing latex microspheres to adsorb fluorescently-labeled SA to obtain fluorescent latex microspheres; (b) attaching biotin to the pooled antibodies to obtain biotinylated pooled antibodies; (c) mixing the fluorescent latex microspheres with the biotinylated pooled antibodies to obtain pooled antibodies labeled with fluorescent latex microspheres; and (d) spray-coating the pooled antibodies labeled with fluorescent latex microspheres on the conjugate pad; where, the steps (a) and (b) can be conducted in any order. In the present disclosure, the fluorescently-labeled SA and the latex microspheres in step (a) may have a mass ratio preferably of 1:40; the biotin and the pooled antibodies in step (b) may have a volume ratio preferably of1:4; and the fluorescent latex microspheres

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and the biotinylated pooled antibodies in step (c) may have a volume ratio preferably of :1. In the pooled antibodies of the present disclosure, the anti-Tri a 12 antibody, anti-Tri a 14 antibody, anti-Tri a 19 antibody, and anti-Tri a 26 antibody may have a mass ratio preferably of 1:1:1:1. The present disclosure has no special limitations on a source of each antibody in the pooled antibodies, and a conventional commercially-available antibody may preferably be used. In the step (d) of the present disclosure, the pooled antibodies labeled with fluorescent latex microspheres may be spray-coated on the conjugate pad at an amount preferably of 2 l/cm to 10 l/cm. In the present disclosure, the pooled antibodies labeled with fluorescent latex microspheres are secondary antibodies. In the present disclosure, the NC membrane includes 4 test lines and 1 quality-control line that are parallel; the test lines are coated with an anti-Tri a 12 antibody (TI line), an anti-Tri a 14 antibody (T2 line), an anti-Tri a 19 antibody (T3 line), and an anti-Tri a 26 antibody (T4 line), respectively; and the quality-control line may preferably be coated with a rabbit anti-mouse IgG antibody (C line, at a coating concentration of 0.5 l/cm to 5 1I/cm). In the present disclosure, the antibodies coated on the test lines on the NC membrane serve as primary antibodies, which are paired with the above pooled antibodies (secondary antibodies), respectively. The present disclosure has no special limitations on a preparation method of the NC membrane, and the preparation method may preferably include: diluting the pooled antibodies of anti-Tri a 12 antibody, anti-Tri a 14 antibody, anti-Tri a 19 antibody, and anti-Tri a 26 antibody and the rabbit anti-mouse IgG antibody with a coating buffer; and streaking obtained five diluted antibody solutions on the NC membrane in parallel, separately, where, when the antibodies penetrate into the NC membrane, test zones respectively coated with the anti-Tri a 12 antibody, anti-Tri a 14 antibody, anti-Tri a 19 antibody, and anti-Tri a 26 antibody and a quality-control zone coated with the rabbit anti-mouse IgG antibody are formed. In the present disclosure, the test zones may preferably be streaked under the following conditions: diluted antibody concentration: 3 mg/ml, delivery volume of a peristaltic pump: 0.4 ml/min, streaking speed: 50 m/20 min, and fan-drying for 12 h at 20°C in a dry oven; and the quality-control zone may preferably be streaked under the following conditions: diluted antibody concentration: 5 mg/ml, delivery volume of a peristaltic pump: 0.4 ml/min, streaking speed: 50 m/20 min, and fan-drying for 12 h at 20°C in a dry oven. The test lines and the quality-control line are streaked on the NC membrane in parallel. After the streaking, the present disclosure may

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preferably further include blocking the NC membrane at 37°C for 60 min with a blocking solution, drying the membrane at 37°C for 2 h, and packaging for later use. In the present disclosure, the sample pad, conjugate pad, NC membrane, and absorbent filter paper may preferably be assembled and adhered on the PVC backing card sequentially, and a resulting product may be cut into test strips as shown in FIG. 1 as required (4 mm) with a strip cutter. The present disclosure also provides use of the immunofluorescent assay test strip described above in the detection of wheat allergenic ingredients. In the present disclosure, the wheat allergenic ingredients may preferably include Tri a 12, Tri a 14, Tri a 19, and Tri a 26. The present disclosure also provides a method for detecting wheat allergenic ingredients using the above immunofluorescent assay test strip, preferably including: adding 100 1 to 120 1 of a to-be-tested sample dropwise on the sample pad, and after reaction in the quality-control zone is completed 15 min later, placing the test strip in a special fluorescence signal reader for reading a fluorescence signal to achieve quantitative determination. In the present disclosure, after the to-be-tested sample is added dropwise on the sample pad, the sample undergoes binding reaction with the pooled antibodies on the conjugate pad, then undergoes reaction sequentially with the anti-Tri a 12 antibody, anti-Tri a 14 antibody, anti-Tri a 19 antibody, and anti-Tri a 26 antibody on the test zones, and finally reaches the quality-control zone where the test is completed. The immunofluorescent assay test strip for wheat and use thereof provided in the present disclosure will be described in detail below with reference to examples, but these examples cannot be understood as limiting the claimed scope of the present disclosure. Example 1 1. Preparation of a conjugate pad 1) Preparation of fluorescent latex microspheres: Latex microspheres with a particle size of 400 nm were diluted with an adsorption buffer (50 mM citrate buffer with a pH of 5.8) to obtain a latex microsphere suspension with a final concentration of 30 mg/ml and a volume of 6 ml; red rhodamine-labeled SA was added to an adsorption buffer at a volume ratio of 1:(50-500), with a final volume of 6 ml; the above latex microsphere suspension was added to the above adsorption buffer with red rhodamine-labeled SA to obtain a mixed solution; the resulting mixed solution was warmed at room temperature for 1 h to 2 h under constant stirring and then centrifuged; and a resulting precipitate was collected, dissolved

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in a storage buffer (adsorption buffer with 0.06% BSA), and stored at 4°C for later use. 2) Preparation of biotinylated pooled antibodies The pooled antibodies of anti-Tri a 12 antibody (Abcam, America; Item No.: ab96676), anti-Tri a 14 antibody (Abcam, America; Item No.: ab53538), anti-Tri a 19 antibody (Seebio Biotech; Item No.: A1052), and anti-Tri a 26 antibody (Exalpha, America; Item No.: AWG) were diluted to 3 ml with a 0.2 M sodium acetate buffer with a pH of 4.7, and the pooled antibodies were fully dialyzed alternately using a 0.2 M sodium acetate buffer with a pH of 4.7; 1 ml of NHSB was dissolved in 1 ml of DMSO to obtain an NHSB solution; 25 L of the NHSB solution was added to 3 ml of the above pooled antibodies, and a resulting solution was stirred for 2 h to 4 h and then stirred at room temperature for min; and then a 20 mM PBS buffer with a pH of 3.9 was used for dialysis to obtain the biotinylated pooled antibodies. 3) Preparation of anti-wheat antibodies labeled with fluorescent latex microspheres The fluorescent latex microspheres prepared in step 1) were mixed with the biotinylated pooled antibodies prepared in step 2) at a ratio of 10:1; after reaction was conducted for 30 min, a resulting reaction solution was centrifuged; and a resulting precipitate was dissolved in a storage buffer and diluted to the original volume. 4) The pooled anti-wheat antibodies labeled with fluorescent latex microspheres were spray-coated on the conjugate pad at an amount of 2 l/cm to 10 l/cm. 2. Preparation of an NC membrane 1) Membrane treatment: A piece of NC membrane was taken, marked, and soaked in a pH membrane treatment solution (TBS) for 5 min to 10 min. 2) Assembly of a spotting device: The soaked NC membrane was placed on a flat mat, and an antibody spotting board was placed, leaving a place for attaching marker paper. 3) Preparation of anti-wheat antibody test zones: The anti-Tri a 12 antibody (Hangzhou Mintai Biology, Item No.: MO-0806), anti-Tri a 14 antibody (Hangzhou Mintai Biology, Item No.: MO-0807), anti-Tri a 19 antibody (Hangzhou Mintai Biology, Item No.: MO-0808), and anti-Tri a 26 antibody (Hangzhou Mintai Biology, Item No.: MO-0809) were sequentially streaked under the following conditions: concentration: 3 mg/ml, delivery volume of a peristaltic pump: 0.4 ml/min, streaking speed: 50 m/20 min, and fan-drying for 12 h at 20°C in a dry oven. 4) Preparation of a quality-control zone: The rabbit anti-mouse IgG antibody was streaked under the following conditions: concentration: 8 mg/ml, delivery volume of a

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peristaltic pump: 0.4 ml/min, streaking speed: 50 m/20 min, and fan-drying for 12 h at °C in a dry oven. The test lines and the quality-control line were streaked on the NC membrane in parallel. 5) The NC membrane was blocked at 37°C for 60 min with a blocking solution (prepared from 100 ml of PBS and 0.5 g of BSA), dried at 37°C for 2 h, and packaged for later use. 3. Assembly of a test strip The sample pad, conjugate pad, NC membrane, and absorbent filter paper were sequentially assembled and adhered on a PVC backing card, and a resulting product was cut into test strips as shown in FIG. 1 as required (4 mm) with a strip cutter. 4. Detection of a to-be-tested antigen 100 1 to 120 1 of a to-be-tested sample was added dropwise on the sample pad, which underwent binding reaction with the pooled anti-wheat antibodies on the conjugate pad, then underwent reaction sequentially with the anti-Tri a 12 antibody, anti-Tri a 14 antibody, anti-Tri a 19 antibody, and anti-Tri a 26 antibody on the test zones, and finally reached the quality-control zone where the test was completed. Then the test strip was placed in a special fluorescence signal reader for reading a fluorescence signal to achieve quantitative determination. Linearity of a dosage-response curve: Calibration solution series prepared from calibrators in a kit were detected, with concentrations of 100.00 IU/ml, 50.00 IU/ml, 17.50 IU/ml, 3.50 IU/ml, and 0.35 IU/ml, respectively; and the double-log model or another appropriate mathematical model was used for fitting. A model fitting result should conform to an intra-assay precision (CV%) < 10.0% and an inter-assay precision (CV%) < 15.0%. Linearity analysis results of the dosage-response curve show that, within a range of 0.35 IU/ml to 100 IU/ml, a relationship between concentrations and test values presents a smooth increasing curve, with a measurement lower limit (CV < 15%, the lowest concentration that the test system can detect) of 0.35 IU/ml. A curve equation is shown in FIG. 2:

y = 7 .19 7 8 9 - 7.22725 1+ (x /263.23601)° , R2 = 0.96043.

The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several

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improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Claims

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What is claimed is: 1. An immunofluorescent assay test strip for wheat, comprising a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent paper that are connected end-to-end and sequentially fixed on a PVC backing card from left to right, wherein, the conjugate pad is coated with pooled antibodies labeled with fluorescent latex microspheres; the pooled antibodies comprise: an anti-Tri a 12 antibody, an anti-Tri a 14 antibody, an anti-Tri a 19 antibody, and an anti-Tri a 26 antibody; the NC membrane comprises 4 test lines and 1 quality-control line that are parallel; the test lines are coated with an anti-Tri a 12 antibody, an anti-Tri a 14 antibody, an anti-Tri a 19 antibody, and an anti-Tri a 26 antibody, respectively; and the coated antibodies on the test lines are paired with the pooled antibodies labeled with fluorescent latex microspheres.
2. The immunofluorescent assay test strip according to claim 1, wherein, the fluorescent latex microspheres have a particle size of 50 nm to 500 nm; wherein, a preparation method of the fluorescent latex microspheres comprises allowing latex microspheres to adsorb fluorescently-labeled streptavidin (SA); and a fluorescent marker comprises fluorescein isothiocyanate (FITC), Rhodamine B, tetramethylrhodamine isothiocyanate (TRITC), or fluorescein CY5.
3. The immunofluorescent assay test strip according to claim 1, wherein, in the pooled antibodies, the anti-Tri a 12 antibody, anti-Tri a 14 antibody, anti-Tri a 19 antibody, and anti-Tri a 26 antibody have a mass ratio of 1:1:1:1; wherein, the pooled antibodies are coated on the conjugate pad at a concentration of 2 pl/cm to 10 l/cm.
4. The immunofluorescent assay test strip according to claim 1, wherein, the quality-control line is coated with a rabbit anti-mouse IgG antibody at a coating concentration of 0.5 l/cm to 5 l/cm.
5. Use of the immunofluorescent assay test strip according to any one of claims 1 to 4 in the detection of wheat allergenic ingredients; wherein, the wheat allergenic ingredients comprise Tri a 12, Tri a 14, Tri a 19, and Tri a 26.

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T4 line T2 line T1 line

T3 line

C line 2021101387

Sample Conjugate pad NC Absorbent paper pad membrane PVC backing card

FIG. 1 Fluorescence signal ratio

Quality-control product concentration (IU/ML)

FIG. 2