AU2021101488A4 - Immunofluorescent assay test strip for egg, use thereof, and detection method using same - Google Patents

Abstract

Documn4-23/03/2021 ABSTRACT The present disclosure provides an immunofluorescent assay test strip for egg, use thereof, and a detection method using the same, and relates to the technical field of immunofluorescent assay. The test strip of the present disclosure includes 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. The conjugate pad is coated with pooled antibodies labeled with fluorescent latex microspheres, and the NC membrane is sequentially coated with an anti-ovalbumin (OVA) antibody (TI line), an anti-ovomucoid (OVM) antibody (T2 line), an anti-ovotransferrin (OVT) antibody (T3 line), an anti-total-egg-protein 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. The test strip of the present disclosure can rapidly and quantitatively detect the type and content of egg allergens, with simple operation, high accuracy, high recovery rate (90% to 110%), and high sensitivity (< 0.84 ng/mL). Document4-23/03/2021 1/2 Samp -e ConuaepdT pe Conjugatepad membrane Absorbent paper PVC backing card FIG. 1

Description

Document4-23/03/2021

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Samp -e ConuaepdT pe Conjugatepad membrane Absorbent paper PVC backing card

FIG. 1

Documn4-23/03/2021

IMMUNOFLUORESCENT ASSAY TEST STRIP FOR EGG, USE THEREOF, AND DETECTION METHOD USING SAME TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of immunofluorescent assay, and

specifically relates to an immunofluorescent assay test strip for egg, use thereof, and a

detection method using the same.

BACKGROUND

[0002] With the development of food industry, allergic diseases have become a global

problem. In recent years, the incidence of allergies caused by eating eggs has been on the

rise. According to epidemiological statistics abroad, about 35% of children with food

allergies are allergic to eggs, which is more common among children under 1 year old. At

present, there is no effective treatment for egg allergies at home and abroad. Patients who

are severely allergic to egg antigens should avoid eating foods with egg ingredients,

which is one of the most effective prevention methods. In recent years, the food labeling

law in the United States and European Union (EU) countries stipulates that eggs must be

labeled as the main allergenic ingredient, and these countries start to subject some

imported foods to egg allergen detection. Moreover, exported foods of food export

enterprises in China are often reported by the United States Food and Drug

Administration (USFDA) and rejected due to incorrect labeling of food allergens. As

people pay more and more attention to food safety, China is bound to establish an

allergen labeling system, which requires corresponding detection methods to provide

guarantee for implementing the system. Therefore, it is necessary to develop a rapid egg

detection method. Egg allergens remain strongly stable during processing. It has been

confirmed from references that egg allergens, after being subjected to glycosylation

treatment, 6 mol/L urea treatment, and 95°C heat treatment, cannot be completely

desensitized.

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[0003] Imported egg allergen enzyme-linked immunosorbent assay (ELISA) kits are often used for detecting egg allergens in foods. However, imported kits are expensive, so

it is important to develop a rapid, effective, and low-cost allergen detection kit.

SUMMARY

[0004] In view of this, the present disclosure is intended to provide an

immunofluorescent assay test strip for egg, use thereof, and a detection method using the

same. The immunofluorescent assay test strip can rapidly detect egg allergenic

ingredients in foods both qualitatively and quantitatively, with simple operation, high

accuracy, and high sensitivity.

[0005] To achieve the above objective, the present disclosure provides the following technical solutions.

[0006] The present disclosure provides an immunofluorescent assay test strip for egg, 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-ovalbumin

(OVA) antibody, an anti-ovomucoid (OVM) antibody, an anti-ovotransferrin (OVT)

antibody, and an anti-total-egg-protein antibody;

the NC membrane includes 4 test lines and 1 quality-control line that are parallel; the test lines are coated with an anti-OVA antibody, an anti-OVM antibody, an anti-OVT antibody, and an anti-total-egg-protein antibody, respectively; the quality-control line is coated with a rabbit anti-mouse IgG antibody; and the antibodies coated on the test lines are paired with the pooled antibodies labeled with fluorescent latex microspheres.

[0007] Preferably, the fluorescent latex microspheres may have a particle size of 50 nm

to 500 nm.

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[0008] Preferably, a preparation method of the conjugate pad coated with the pooled

antibodies labeled with fluorescent latex microspheres may include: a. allowing latex

microspheres to adsorb fluorescently-labeled streptavidin (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.

[0009] Preferably, a fluorescent marker used in the step a may include fluorescein

isothiocyanate (FITC), Rhodamine B, tetramethylrhodamine isothiocyanate (TRITC), or

fluorescein CY5.

[0010] Preferably, in the step d, the pooled antibodies labeled with fluorescent latex

microspheres are spray-coated on the conjugate pad at an amount of 2 L/cm to 10

pL/cm.

[0011] Preferably, in the pooled antibodies, the anti-OVA antibody, anti-OVM antibody,

anti-OVT antibody, and anti-total-egg-protein antibody may have a mass ratio of 1:1:1:1.

[0012] Preferably, the 4 test lines may be coated with the anti-OVA antibody, anti-OVM

antibody, anti-OVT antibody, and anti-total-egg-protein antibody at a coating

concentration of 0.5 L/cm to 5.0 [L/cm; and

the quality-control line may be coated with the rabbit anti-mouse IgG antibody at a coating concentration of 0.5 L/cm to 5 L/cm.

[0013] The present disclosure also provides use of the immunofluorescent assay test

strip described above in the detection of egg allergenic ingredients in foods.

[0014] Preferably, the egg allergenic ingredients may include OVA, OVM, OVT, and total egg

protein.

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[0015] The present disclosure also provides a method for detecting egg allergenic

ingredients in foods, including the following steps: adding 100 L to 120 L 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 an egg allergenic

ingredient and a content thereof according to a standard curve.

y = 3.15114 - 3.26 .05 2 The standard curve is 1+(x /21.63173°41, R 0.9997,

where, x is an allergen concentration in IU/mL, and y is a fluorescence signal ratio.

[0016] The present disclosure provides an immunofluorescent assay test strip for egg

with a structure shown in FIG. 1, 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. The conjugate pad is coated with pooled

antibodies labeled with fluorescent latex microspheres; the pooled antibodies include: an

anti-OVA antibody, an anti-OVM antibody, an anti-OVT antibody, and an

anti-total-egg-protein antibody; and the NC membrane is sequentially coated with an

anti-OVA antibody (Ti line), an anti-OVM antibody (T2 line), an anti-OVT antibody (T3

line), an anti-total-egg-protein 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 test

strip of the present disclosure can rapidly and quantitatively detect the type and content

of egg allergens, with simple operation, high accuracy, high recovery rate (90% to110%),

and high sensitivity (< 0.84 ng/ml).

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BRIEF DESCRIPTION OF DRAWINGS

[0017] FIG. 1 is a structural diagram of the immunofluorescent assay test strip

according to the present disclosure; and

[0018] FIG. 2 shows a standard curve for fluorescence immunochromatographic assay.

DETAILED DESCRIPTION

[0019] The present disclosure provides an immunofluorescent assay test strip for egg

with a structure shown in FIG. 1, 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-OVA antibody, an anti-OVM antibody, an anti-OVT antibody, and an

anti-total-egg-proteinantibody;

the NC membrane includes 4 test lines and 1 quality-control line that are parallel; the test lines are coated with an anti-OVA antibody, an anti-OVM antibody, an anti-OVT antibody, and an anti-total-egg-protein antibody, respectively; the quality-control line is coated with a rabbit anti-mouse IgG antibody; and the antibodies coated on the test lines are paired with the pooled antibodies labeled with fluorescent latex microspheres.

[0020] In the present disclosure, the conjugate pad is coated with pooled antibodies

labeled with fluorescent latex microspheres; and a preparation method of the conjugate

pad coated with the pooled antibodies labeled with fluorescent latex microspheres 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.

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[0021] 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

and the biotinylated pooled antibodies in step c may have a volume ratio preferably of

:1. In the present disclosure, the fluorescent latex microspheres may have a particle

size preferably of 50 nm to 500 nm. In the present disclosure, a fluorescent marker used

in the step a may preferably include FITC, Rhodamine B, TRITC, or fluorescein CY5. In

the present disclosure, in the pooled antibodies, the anti-OVA antibody, anti-OVM

antibody, anti-OVT antibody, and anti-total-egg-protein antibody may preferably have a

mass ratio of 1:1:1:1. The present disclosure has no special limitations on sources of the

anti-OVA antibody, anti-OVM antibody, anti-OVT antibody, and anti-total-egg-protein

antibody, and conventional commercially-available antibodies in the art 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.

[0022] 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-OVA antibody

(TI line), an anti-OVM antibody (T2 line), an anti-OVT antibody (T3 line), and an

anti-total-egg-protein antibody (T4 line), respectively; and the quality-control line is

coated with a rabbit anti-mouse IgG antibody (C line). The present disclosure has no

special limitations on a preparation method of the NC membrane, and the preparation

method may preferably include: diluting the anti-OVA antibody, anti-OVM antibody,

anti-OVT antibody, anti-total-egg-protein antibody, and rabbit anti-mouse IgG antibody

with a coating buffer, separately; and streaking obtained five diluted antibody solutions

on the NC membrane in parallel, separately, where, when the antibodies penetrate into

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the NC membrane, test lines respectively coated with the anti-OVA antibody, anti-OVM

antibody, anti-OVT antibody, and anti-total-egg-protein antibody and a quality-control

line coated with the rabbit anti-mouse IgG antibody are formed. In the present disclosure,

the 4 test lines may be coated with the anti-OVA antibody, anti-OVM antibody, anti-OVT

antibody, and anti-total-egg-protein antibody at a coating concentration of 0.5 L/cm to

5.0 [L/cm; and the quality-control line may be coated with the rabbit anti-mouse IgG

antibody at a coating concentration of 0.5 L/cm to 5 L/cm. In the present disclosure,

according to different operations of the test lines and quality-control line, the anti-OVA

antibody, anti-OVM antibody, anti-OVT antibody, and anti-total-egg-protein antibody

may preferably be streaked under the following conditions to form the test lines:

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 rabbit

anti-mouse IgG antibody may preferably be streaked under the following conditions to

form the quality-control line: concentration: 8 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.

[0023] 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 test strips shown in the present disclosure can be

produced in batches, which are suitable for clinical rapid diagnosis and on-site rapid

detection and easy to store.

[0024] The present disclosure provides use of the immunofluorescent assay test strip described

above in the detection of egg allergenic ingredients in foods.

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[0025] The egg allergenic ingredients in the present disclosure may preferably include OVA, OVM,

OVT, and total egg protein, where, the total egg protein does not involve OVA, OVM, and OVT.

[0026] The present disclosure also provides a method for detecting egg allergenic

ingredients in foods, including the following steps: adding 100 L to 120 L 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 an egg allergenic

ingredient and a content thereof according to a standard curve. The standard curve is

y~3 .lll y = 3.15114 4 - 3.12726 - 3.26 .05 1+ (x /21.63173om , R2 = 0.9997,

where, x is an allergen concentration in IU/mL, and y is a fluorescence signal ratio.

[0027] 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-OVA antibody, anti-OVM antibody, anti-OVT antibody,

and anti-total-egg-protein antibody on the test zones, and finally reaches the quality-control zone

where the test is completed.

[0028] The immunofluorescent assay test strip for egg, use thereof, and a detection

method using the same 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.

[0029] Unless otherwise specified, the reagents used in the present disclosure are all

conventionally-purchased products, where:

anti-OVA antibody: Indoor Biotechnologies (Item No.: MA-3G4, MA-7D8); anti-OVM antibody: Indoor Biotechnologies (Item No.: MA-5G11, MA-2B2); anti-OVT antibody: CusAb (Item No.: CSB-PA00390FORb); and anti-total-egg-protein antibody: Hangzhou Zhongou Biomedical Technology Co., Ltd. (Item No.: CE-GAL-01).

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Example 1 1. Preparation of a conjugate pad 1) Preparation of fluorescent latex microspheres

[0030] 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 an appropriate ratio, 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 in a storage buffer (adsorption buffer with 0.06% BSA), and stored

at 4°C for later use.

2) Preparation of biotinylated pooled anti-egg antibodies

[0031] An anti-OVA antibody, an anti-OVM antibody, an anti-OVT antibody, and an

anti-total-egg-protein antibody were mixed at a mass ratio of 1:1:1:1 (pooled antibodies

M1), and then the pooled anti-egg antibodies M1 were diluted to 3 mL with a 0.2 M

sodium acetate buffer with a pH of 4.7 and 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 anti-egg antibodies M1, and a resulting solution was stirred for 2 h to 4 h

and then stirred at room temperature for 10 min; and then a 20 mM PBS buffer with a pH

of 3.9 was used for dialysis to obtain the biotinylated pooled antibodies M1.

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3) Preparation of anti-egg antibodies labeled with fluorescent latex microspheres

[0032] The fluorescent latex microspheres prepared in step 1) were mixed with the

biotinylated pooled antibodies prepared in step 2) at a volume 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-egg 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-egg antibody test zones: An anti-OVA antibody, an anti-OVM antibody, an anti-OVT antibody, and an anti-total-egg-protein antibody 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 °C in a dry oven. 4) Preparation of a quality-control zone: A rabbit anti-mouse IgG antibody was streaked under the following conditions: concentration: 8 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 °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, dried at 37°C for 2 h, and packaged for later use. 6) 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 (shown in FIG. 1) with an appropriate width (4 mm) as required with a strip cutter.

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3. Detection of a to-be-tested antigen

[0033] 100 L to 120 L of a to-be-tested sample was added dropwise on the sample

pad, which underwent binding reaction with the pooled anti-egg antibodies on the

conjugate pad, then underwent reaction sequentially with the anti-OVA antibody,

anti-OVM antibody, anti-OVT antibody, and anti-total-egg-protein 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.

[0034] 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%.

[0035] 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~3 .lll 4- 3.12726 y = 3.15114 - 3.26 0 .05 1+ (x /21.63173 om , R2 = 0.9997,

where, x is an allergen concentration in IU/mL, and y is a fluorescence signal ratio.

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[0036] 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 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 (5)

1. Documen4-23/03/2021

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   What is claimed is: 1. An immunofluorescent assay test strip for egg, 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-ovalbumin (OVA) antibody, an anti-ovomucoid (OVM) antibody, an anti-ovotransferrin (OVT) antibody, and an anti-total-egg-protein antibody; the NC membrane comprises 4 test lines and 1 quality-control line that are parallel; the test lines are coated with an anti-OVA antibody, an anti-OVM antibody, an anti-OVT antibody, and an anti-total-egg-protein antibody, respectively; the quality-control line is coated with a rabbit anti-mouse IgG antibody; and the antibodies coated on the test lines are paired with the pooled antibodies labeled with fluorescent latex microspheres.
2. 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, the 4 test lines are coated with the anti-OVA antibody, anti-OVM antibody, anti-OVT antibody, and anti-total-egg-protein antibody at a coating concentration of 0.5 pL/cm to 5.0 L/cm; and the quality-control line is coated with the rabbit anti-mouse IgG antibody at a coating concentration of 0.5 L/cm to 5 L/cm.
3. 3. The immunofluorescent assay test strip according to claim 1, wherein, a preparation method of the conjugate pad coated with the pooled antibodies labeled with fluorescent latex microspheres comprises: a. allowing latex microspheres to adsorb fluorescently-labeled streptavidin (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; wherein, the steps a and b can be conducted in any order;

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   wherein, a fluorescent marker used in the step a comprises fluorescein isothiocyanate (FITC), Rhodamine B, tetramethylrhodamine isothiocyanate (TRITC), or fluorescein CY5; wherein, in the step d, the pooled antibodies labeled with fluorescent latex microspheres are spray-coated on the conjugate pad at an amount of 2 L/cm to 10

   pL/cm; wherein, in the pooled antibodies, the anti-OVA antibody, anti-OVM antibody, anti-OVT antibody, and anti-total-egg-protein antibody have a mass ratio of 1:1:1:1.
4. 4. Use of the immunofluorescent assay test strip according to any one of claims 1 to 3 in the detection of egg allergenic ingredients in foods; wherein, the egg allergenic ingredients comprise OVA, OVM, OVT, and total egg protein.
5. 5. A method for detecting egg allergenic ingredients in foods, comprising the following steps: adding 100 L to 120 L of a food solution dropwise on the sample pad of the immunofluorescent assay test strip according to any one of claims I to 3, reading a fluorescence signal on the test strip 15 min later, and determining an egg allergenic ingredient and a content thereof according to a standard curve; wherein, y~3 .lll 4 - 3.12726 y = 3.15114 - 3.26 .05

   the standard curve is 1+(x /21.63173om , R2 = 0.9997,

   wherein, x is an allergen concentration in IU/mL, and y is a fluorescence signal ratio.

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   T4 line T2 line T1 line

   T3 line

   C line 2021101488

   Sample Conjugate pad NC pad membrane Absorbent paper PVC backing card

   FIG. 1

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   Fluorescence signal ratio

   Quality-control product concentration (IU/ML)

   FIG. 2