AU2021103258A4 - Fluorescence immunochromatography assay (fica) test strip for fish, use thereof, and detection method using same - Google Patents
Fluorescence immunochromatography assay (fica) test strip for fish, use thereof, and detection method using same Download PDFInfo
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- 241000251468 Actinopterygii Species 0.000 title claims abstract description 29
- 238000003556 assay Methods 0.000 title claims abstract description 9
- 238000003317 immunochromatography Methods 0.000 title claims abstract description 5
- 238000001514 detection method Methods 0.000 title abstract description 11
- JFUIHGAGFMFNRD-UHFFFAOYSA-N fica Chemical compound FC1=CC=C2NC(C(=O)NCCS)=CC2=C1 JFUIHGAGFMFNRD-UHFFFAOYSA-N 0.000 title 1
- 239000004816 latex Substances 0.000 claims abstract description 44
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- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 28
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 28
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- 239000004615 ingredient Substances 0.000 claims description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 8
- 108010090804 Streptavidin Proteins 0.000 claims description 8
- 239000011248 coating agent Substances 0.000 claims description 7
- 238000000576 coating method Methods 0.000 claims description 7
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 claims description 5
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 claims description 5
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 5
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 5
- 229960002685 biotin Drugs 0.000 claims description 4
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
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- 206010020751 Hypersensitivity Diseases 0.000 description 6
- 102000001675 Parvalbumin Human genes 0.000 description 6
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000007815 allergy Effects 0.000 description 3
- 208000010668 atopic eczema Diseases 0.000 description 3
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Classifications
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N33/02—Food
- G01N33/12—Meat; fish
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N2333/4603—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from fish
Abstract
The present disclosure provides a fluorescence immunochromatography assay (FICA) test strip
for quantitatively detecting fish allergens, 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 pad 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-parvalbumin antibody (TI line), an anti-ALDH
antibody (T2 line), an anti-collagen antibody (T3 line), an anti-total-fish-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 control line. The test strip of the present disclosure can rapidly and quantitatively detect the
type and content of fish allergens, with simple operation, high accuracy, high recovery rate (90% to
110%), and high sensitivity (< 0.84 ng/ml).
Description
[01] The present disclosure belongs to the technical field of immunofluorescent assay, and specifically relates to a fluorescence immunochromatography assay (FICA) test strip for quantitatively detecting fish allergens, use thereof, and a detection method using the same.
[02] As a major animal protein, fish is widely present in various foods. Therefore, allergies caused by fish and fish products have gradually become ubiquitous in countries all over the world, and seriously affect the life quality of allergic patients. Allergic symptoms to fish generally start at the infant stage. Allergic reactions may occur after the ingestion, contact, or even inhalation of fish products. The clinical symptoms of allergy include: urticaria, angioedema, allergic dermatitis, asthma, etc., and allergy may even cause death in severe cases.
[03] The most effective way for preventing and treating food allergy is to avoid contact with related foods. In recent years, researchers at home and abroad have conducted in-depth studies on fish allergens that cause allergic reactions. Studies have shown that parvalbumin is the main allergen protein in fish, and 95% of fish allergic reactions are caused by parvalbumin; and parvalbumin is mainly expressed in the muscle tissue of fish and shows high resistance to the action of heat and enzymes. At present, there are commercial fish allergen detection reagents abroad, but the reagents are expensive and can only be used to detect limitedfish species.
[04] In view of this, the present disclosure is intended to provide an FICA test strip for quantitatively detecting fish allergens, use thereof, and a detection method using the same. The FICA test strip can rapidly detect fish allergenic ingredients in foods both qualitatively and quantitatively, with simple operation, high accuracy, and high sensitivity.
[05] To achieve the objective of the present disclosure, the present disclosure provides the following technical solutions.
[06] The present disclosure provides an FICA test strip for quantitatively detecting fish allergens, including a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad 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-parvalbumin antibody, an anti-acetaldehyde dehydrogenase (ALDH) antibody, an anti-collagen antibody, and an anti-total-fish-protein antibody;
[07] the NC membrane includes 4 test lines and 1 control line that are parallel; the test lines are coated with an anti-parvalbumin antibody, an anti-ALDH antibody, an anti-collagen antibody, and an anti-total-fish-protein antibody, respectively; the control line is coated with a rabbit anti-mouse IgG antibody; and
[08] the antibodies coated on the test lines are paired with the pooled antibodies labeled with fluorescent latex microspheres.
[09] Preferably, the fluorescent latex microspheres may have a particle size of 50 nm to 500 nm.
[10] 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;
[11] b. attaching biotin to the pooled antibodies to obtain biotinylated pooled antibodies;
[12] c. mixing the fluorescent latex microspheres with the biotinylated pooled antibodies to obtain pooled antibodies labeled with fluorescent latex microspheres; and
[13] d. spray-coating the pooled antibodies labeled with fluorescent latex microspheres on the conjugate pad;
[14] where the steps a and b can be conducted in any order.
[15] Preferably, a fluorescent marker used in the step a may include fluorescein isothiocyanate (FITC), Rhodamine B, tetramethyl rhodamine isothiocyanate (TRITC), or fluorescein CY5.
[16] Preferably, in the step d, the pooled antibodies labeled with fluorescent latex microspheres may be spray-coated on the conjugate pad at an amount of 2 L/cm to 10 L/cm.
[17] Preferably, in the pooled antibodies, the anti-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody may have a mass ratio of 1:1:1:1.
[18] Preferably, the 4 test lines may be coated with the anti-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody at a coating concentration of 0.5 L/cm to 5.0 [L/cm; and
[19] the 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.
[20] The present disclosure also provides use of the FICA test strip described above in the detection of fish allergenic ingredients in foods.
[21] Preferably, the fish allergenic ingredients may include parvalbumin, ALDH, collagen, and total fish protein.
[22] The present disclosure also provides a method for detecting fish 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 FICA test strip, reading a fluorescence signal on the test strip 15 min later, and determining a fish allergenic ingredient and a content thereof according to a standard curve.
y = 1069.49796 - 1069.52970 0.5o462
[23] The standard curve is 1.0321x 1070 , R2 = 0.95645,
[24] where x is an allergen concentration in IU/ml, and y is a fluorescence signal ratio.
[25] The present disclosure provides a FICA test strip for fish with a structure shown in FIG. 1, including a sample pad, a conjugate pad, an NC membrane, and an absorbent pad 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-parvalbumin antibody, an anti-ALDH antibody, an anti-collagen antibody, and an anti-total-fish-protein antibody; and the NC membrane is sequentially coated with an anti-parvalbumin antibody (TI line), an anti-ALDH antibody (T2 line), an anti-collagen antibody (T3 line), an anti-total-fish-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 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 fish allergens, with simple operation, high accuracy, high recovery rate (90% to 110%), and high sensitivity (< 0.84 ng/mL).
[26] FIG. 1 illustrates a structure of the FICA test strip provided by the present disclosure; and
[27] FIG. 2 illustrates a standard curve for FICA.
[28] The present disclosure provides an FICA test strip for quantitatively detecting fish allergens with a structure shown in FIG. 1, including a sample pad, a conjugate pad, an NC membrane, and an absorbent pad 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-parvalbumin antibody, an anti-ALDH antibody, an anti-collagen antibody, and an anti-total-fish-protein antibody;
[29] the NC membrane includes 4 test lines and 1 control line that are parallel; the test lines are coated with an anti-parvalbumin antibody, an anti-ALDH antibody, an anti-collagen antibody, and an anti-total-fish-protein antibody, respectively; the control line is coated with a rabbit anti-mouse IgG antibody; and
[30] the antibodies coated on the test lines are paired with the pooled antibodies labeled with fluorescent latex microspheres.
[31] 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;
[32] b. attaching biotin to the pooled antibodies to obtain biotinylated pooled antibodies;
[33] c. mixing the fluorescent latex microspheres with the biotinylated pooled antibodies to obtain pooled antibodies labeled with fluorescent latex microspheres; and
[34] 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.
[35] In the present disclosure, a mass ratio of the fluorescently-labeled SA to the latex microspheres in step a may preferably be 1:40; a volume ratio of the biotin to the pooled antibodies in step b may preferably be 1:4; and a volume ratio of the fluorescent latex microspheres to the biotinylated pooled antibodies in step c may preferably be 10:1. 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-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody may have a mass ratio preferably of 1:1:1:1. The present disclosure has no special limitations on sources of the anti-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-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.
[36] In the present disclosure, the NC membrane includes 4 test lines and 1 control line that are parallel; the test lines are coated with an anti-parvalbumin antibody (TI line), an anti-ALDH antibody (T2 line), an anti-collagen antibody (T3 line), and an anti-total-fish-protein antibody (T4 line), respectively; and the 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-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, anti-total-fish-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 the NC membrane, test lines respectively coated with the anti-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody and a 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-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody at a coating concentration of 0.5 L/cm to 5.0 [L/cm; and the control line may be coated with the rabbit anti-mouse IgG antibody at a coating concentration of 0.5 pL/cm to 5 L/cm. In the present disclosure, in view of different operations of the test and control lines during streaking, when streaking the test lines, the anti-parvalbumin antibody, the anti-ALDH antibody, the anti-collagen antibody, and the anti-total-fish-protein antibody may preferably be streaked successively at a concentration of 3 mg/mL (liquid output of peristaltic pump 0.4 mL/min, streaking speed 50 m/20 min) and blast-dried in a drying oven at 20°C for 12 h. When streaking the control line, the rabbit anti-mouse IgG antibody may preferably be streaked on the NC membrane at a concentration of 8 mg/mL (liquid output of peristaltic pump 0.4 mL/min, streaking speed 50 m/20 min); the line is parallel to the test lines and blast-dried in the drying oven at 20°C for 12 h.
[37] 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.
[38] The present disclosure provides use of the FICA test strip described above in the detection of fish allergenic ingredients in foods.
[39] In the present disclosure, the fish allergenic ingredients may preferably include parvalbumin, ALDH, collagen, and total fish protein, where parvalbumin, ALDH, and collagen are not involved in total fish protein.
[40] The present disclosure also provides a method for detecting fish 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 FICA test strip, reading a fluorescence signal on the test strip 15 min later, and determining a fish allergenic ingredient and a content thereof according to a standard curve. y = 1069.49796 - 1069.52970 .5o462
[41] The standard curve is 1.0321x 10 , R2 = 0.95645,
[42] where x is an allergen concentration in IU/ml, and y is a fluorescence signal ratio.
[43] 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-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody on the test zones, and finally reaches the control zone where the test is completed.
[44] The FICA test strip for quantitatively detecting fish allergens, 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.
[45] Unless otherwise specified, the reagents used in the present disclosure are all conventionally-purchased products.
[46] Example 1
[47] 1. Preparation of a conjugate pad
[48] 1) Preparation of fluorescent latex microspheres
[49] 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.
[50] 2) Preparation of biotinylated pooled anti-fish antibodies
[51] An anti-parvalbumin antibody (Hangzhou Mintai Biology, Item No.: M01204), an anti-ALDH antibody (Hangzhou Mintai Biology, Item No.: M01205), an anti-collagen antibody (Hangzhou Mintai Biology, Item No.: MO1206), and an anti-total-fish-protein antibody (Hangzhou Mintai Biology, Item No.: MO1207) were mixed at a mass ratio of 1:1:1:1 (pooled antibodies M1), and then the pooled anti-fish 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-fish 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.
[52] 3) Preparation of anti-fish antibodies labeled with fluorescent latex microspheres
[53] 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.
[54] 4) The pooled anti-fish antibodies labeled with fluorescent latex microspheres were spray-coated on the conjugate pad at an amount of 2 L/cm to 10 L/cm.
[55] 2. Preparation of an NC membrane
[56] 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.
[57] 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.
[58] 3) Preparation of anti-fish antibody test zones: The anti-parvalbumin antibody (Hangzhou Mintai Biology, Item No.: MO1204-05), anti-ALDH antibody (Hangzhou Mintai Biology, Item No.: M01205-06), anti-collagen antibody (Hangzhou Mintai Biology, Item No.: M01206-07), and anti-total-fish-protein antibody (Hangzhou Mintai Biology, Item No.: MO1207-08) were streaked successively at a concentration of 3 mg/mL (liquid output of peristaltic pump 0.4 mL/min, streaking speed 50 m/20 min) and blast-dried in a drying oven at 20°C for 12 h.
[59] 4) Preparation of a control zone: A rabbit anti-mouse IgG antibody (Hangzhou HuaAn Biotechnology Co. Ltd, Item No.: M080825) was streaked on the NC membrane at a concentration of 8 mg/mL (liquid output of peristaltic pump 0.4 mLmin, streaking speed 50 m/20 min); and the line was parallel to the test lines and blast-dried in the drying oven at 20°C for 12 h.
[60] 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.
[61] 6) Assembly of a test strip
[62] 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.
[63] 3. Detection of a to-be-tested antigen
[64] 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-fish antibodies on the conjugate pad, then underwent reaction sequentially with the anti-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody on the test zones, and finally reached the 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.
[65] 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%.
[66] 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 = 1069.49796 - 1069.52970 0.50462
[671 1 1.0321x 0 , R2 = 0.95645.
[68] 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. A fluorescence immunochromatography assay (FICA) test strip for quantitatively detecting fish allergens, including a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad 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 include: an anti-parvalbumin antibody, an anti-acetaldehyde dehydrogenase (ALDH) antibody, an anti-collagen antibody, and an anti-total-fish-protein antibody; the NC membrane includes 4 test lines and 1 control line that are parallel; the test lines are coated with an anti-parvalbumin antibody, an anti-ALDH antibody, an anti-collagen antibody, and an anti-total-fish-protein antibody, respectively; the 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. The FICA test strip according to claim 1, wherein the fluorescent latex microspheres have a particle size of 50 nm to 500 nm.
3. The FICA test strip according to claim 1, wherein a preparation method of the conjugate pad coated with the pooled antibodies labeled with fluorescent latex microspheres includes: 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; wherein a fluorescent marker used in the step a includes fluorescein isothiocyanate (FITC), Rhodamine B, tetramethyl rhodamine 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 L/cm; wherein in the pooled antibodies, the anti-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody have a mass ratio of 1:1:1:1.
4. The FICA test strip according to claim 1, wherein the 4 test lines are coated with the anti-parvalbumin antibody, anti-ALDH antibody, anti-collagen antibody, and anti-total-fish-protein antibody at a coating concentration of 0.5 L/cm to 5.0 [L/cm; and the control line is coated with the rabbit anti-mouse IgG antibody at a coating concentration of 0.5 L/cm to 5.0 [L/cm.
5. A method for detecting fish 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 FICA test strip according to any one of claims 1 to 4, reading a fluorescence signal on the test strip 15 min later, and determining a fish allergenic ingredient and a content thereof according to a standard curve; wherein y = 1069.49796 - 1069.52970 .5o462
the standard curve is 1.0321x 17 ,R2 = 0.95645, wherein x is an allergen concentration in IU/mL, and y is a fluorescence signal ratio.
－1/1－ Jun 2021
T2 T4 line line T1 T3 C line line line 2021103258
NC Absorbent Sample Conjugate membrane pad pad pad PVC backing card
FIG. 1 Fluorescence signal ratio
Control product concentration (IU/ML) FIG. 2
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| AU2021103258A AU2021103258A4 (en) | 2021-06-10 | 2021-06-10 | Fluorescence immunochromatography assay (fica) test strip for fish, use thereof, and detection method using same |
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| AU2021103258A4 true AU2021103258A4 (en) | 2021-07-29 |
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