CN116478273A - Antigen epitope peptide for constructing alpha-lactalbumin based on IgE linear epitope and method for detecting alpha-lactalbumin with low detection limit - Google Patents
Antigen epitope peptide for constructing alpha-lactalbumin based on IgE linear epitope and method for detecting alpha-lactalbumin with low detection limit Download PDFInfo
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- 102000004407 Lactalbumin Human genes 0.000 title claims abstract description 58
- 108090000942 Lactalbumin Proteins 0.000 title claims abstract description 58
- 235000021241 α-lactalbumin Nutrition 0.000 title claims abstract description 58
- 238000001514 detection method Methods 0.000 title claims abstract description 49
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 28
- 239000000427 antigen Substances 0.000 title claims abstract description 26
- 102000036639 antigens Human genes 0.000 title claims abstract description 26
- 108091007433 antigens Proteins 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 26
- 235000013305 food Nutrition 0.000 claims abstract description 17
- 241000699670 Mus sp. Species 0.000 claims abstract description 4
- 108060003552 hemocyanin Proteins 0.000 claims abstract description 4
- 230000000903 blocking effect Effects 0.000 claims description 21
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 4
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 2
- 229960001230 asparagine Drugs 0.000 claims description 2
- 235000009582 asparagine Nutrition 0.000 claims description 2
- 235000018417 cysteine Nutrition 0.000 claims description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 2
- 230000003053 immunization Effects 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 150000001413 amino acids Chemical group 0.000 claims 2
- 238000004458 analytical method Methods 0.000 abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 4
- 235000013365 dairy product Nutrition 0.000 abstract description 2
- 238000002965 ELISA Methods 0.000 description 12
- 235000013336 milk Nutrition 0.000 description 9
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- 210000004080 milk Anatomy 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 235000020247 cow milk Nutrition 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000013566 allergen Substances 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000000172 allergic effect Effects 0.000 description 3
- 208000010668 atopic eczema Diseases 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 208000004262 Food Hypersensitivity Diseases 0.000 description 2
- 206010016946 Food allergy Diseases 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 102000008192 Lactoglobulins Human genes 0.000 description 2
- 108010060630 Lactoglobulins Proteins 0.000 description 2
- 208000009793 Milk Hypersensitivity Diseases 0.000 description 2
- 102000014171 Milk Proteins Human genes 0.000 description 2
- 108010011756 Milk Proteins Proteins 0.000 description 2
- 201000010859 Milk allergy Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 235000020932 food allergy Nutrition 0.000 description 2
- 235000013350 formula milk Nutrition 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000014612 sandwich biscuits Nutrition 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 235000011149 sulphuric acid Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 201000005356 cow milk allergy Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- 238000003891 environmental analysis Methods 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 239000013568 food allergen Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000000774 hypoallergenic effect Effects 0.000 description 1
- 235000020215 hypoallergenic milk formula Nutrition 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention realizes quantitative detection and analysis of alpha-lactalbumin in food matrixes by adopting an sELISA method, builds AA 64-79 epitope peptide of the alpha-lactalbumin based on IgE linear epitope, wherein the amino acid sequence of the epitope peptide is NDSTEYGLFQINNKIW, couples the epitope peptide with hemocyanin to obtain complete antigen, immunizes mice by taking the complete antigen as antigen to prepare monoclonal antibody, takes the monoclonal antibody as capture antibody, and takes biotinylated alpha-lactalbumin polyclonal antibody as detection antibody, has extremely high specificity on peptide segments containing active epitopes in the food matrixes, and can accurately detect alpha-lactalbumin and sensitized residues thereof in dairy products of any food matrixes.
Description
Technical Field
The invention belongs to the field of quantitative detection methods of alpha-lactalbumin, and particularly relates to a method for detecting alpha-lactalbumin with low detection limit.
Background
Cow milk is a high-quality nutrition supplementary food commonly used by people and is one of common allergic foods, but cow milk allergy is confirmed to be one of the most common allergic of early-stage food allergy of children, and accounts for about one fifth of food allergy of all children, and is widely paid attention to all countries in the world. In China, the prevalence of cow's milk allergy in infants is as high as 2.69%, and even serious infants with cow's milk protein as low as 0.6mg can cause allergic reactions. Cow's milk is rich in protein content, contains about 30-35 g of protein per liter of cow's milk, contains more than 25 different proteins, and currently, main allergens in cow's milk are commonly considered to be casein, beta-lactoglobulin and alpha-lactalbumin, and whey is the most commonly used food raw material in the food industry, wherein beta-lactoglobulin and alpha-lactalbumin account for 15% of total milk protein, and are the most important allergens.
Food allergen detection is generally classified into three main categories according to principles: detection methods based on molecular biology, chromatography methods and detection methods based on immunology. Molecular biology-based detection methods are achieved by amplifying DNA fragments specific to allergic foods, which require high stability and high automation due to the long-term integrity of DNA under heat treatment and high pressure, but cannot detect allergen proteins, can only target the characteristic DNA fragments expressed by them, and have no quantitative relationship between DNA content and protein content, and are only suitable for detecting samples with high nucleic acid content, such as peanuts, nuts, soybeans, fish, crustaceans, and the like. The chromatography method has the advantages of high sensitivity, good accuracy, low detection limit, strong specificity and the like, but the instrument and the equipment are complex, special training personnel are needed, and the wide application of the method in the food industry can be limited due to high price. In the immunological detection method, an enzyme-linked immunosorbent assay (ELISA) is taken as the detection method with the widest application range, is one of the most effective detection methods for identifying biomolecules, has the advantages of strong specificity, high sensitivity, capability of realizing accurate quantitative detection of a large number of samples and the like, and has been widely applied to clinical diagnosis, food routine analysis, environmental analysis and laboratory research. Wherein it can be classified into an indirect competition type ELISA and a sandwich type ELISA according to the detection type of ELISA, wherein the indirect competition type ELISA is more suitable for detecting molecules or small molecules. False positives often occur due to cross-allergic reactions caused by the presence of similarly acting epitope allergens after the alpha-lactalbumin has been broken down into small fragments by hydrolysis. The sandwich ELISA is more suitable for small molecule allergens in food proteins, and the specificity of the system is ensured because the enzyme-labeled secondary antibodies can recognize the detection antibody and simultaneously are difficult to recognize the capture antibody. The key technical problem to be solved by the enzyme-linked immunosorbent assay (ELISA) is to find an epitope peptide corresponding to alpha-lactalbumin, so that the detection of the content of the alpha-lactalbumin in a food matrix with high sensitivity, high precision and low detection limit is realized.
Disclosure of Invention
The invention aims to provide a method for detecting alpha-lactalbumin with low detection limit, which solves the technical problems of higher detection limit and lower sensitivity in the existing method for detecting the content of the alpha-lactalbumin in a food matrix.
In order to solve the technical problems, the invention discloses an antigen epitope peptide for constructing alpha-lactalbumin based on IgE linear epitopes, and the amino acid sequence of the antigen epitope peptide is NDSTEYGLFQINNKIW.
The invention discloses a method for detecting alpha-lactalbumin with low detection limit, which comprises the following steps,
s1, synthesizing an antigen epitope peptide for constructing alpha-lactalbumin based on an IgE linear epitope, wherein the amino acid sequence of the antigen epitope peptide is NDSTEYGLFQINNKIW;
s2, coupling the epitope peptide and hemocyanin to obtain a complete antigen;
s3, immunizing a mouse by taking the complete antigen as an antigen, and preparing a monoclonal antibody;
s4, detecting the content of the alpha-lactalbumin in the food matrix by using the monoclonal antibody as a capture antibody and the biotinylated alpha-lactalbumin polyclonal antibody as a detection antibody through an sELISA method.
Preferably, the antigen epitope peptide is coupled with asparagine and cysteine to obtain a blocking peptide, the blocking peptide is used for screening mice and cell strains, and the amino acid sequence of the blocking peptide is N-NDSTEYGLFQINNKIW-C.
Preferably, the monoclonal antibody is used for screening mice and cell lines by using the blocking peptide.
Preferably, the working concentration of the monoclonal antibody as a capture antibody during detection in S4 by the sELISA method is 2 μg/mL.
Preferably, the working concentration of the biotinylated alpha-lactalbumin polyclonal antibody as a detection antibody in the detection process of S4 by using the sELISA method is 1 mug/mL.
Compared with the prior art, the invention has the beneficial effects that:
the method disclosed by the invention can be used for realizing the quantitative detection and analysis of alpha-lactalbumin in food matrixes by adopting an sELISA method, constructing an AA 64-79 epitope peptide of the alpha-lactalbumin based on IgE linear epitopes, coupling the antigen epitope peptide with hemocyanin to obtain a complete antigen, preparing a monoclonal antibody by taking the complete antigen as an antigen immune mouse, taking the monoclonal antibody as a capture antibody, and taking a biotinylated alpha-lactalbumin polyclonal antibody as a detection antibody, has extremely high specificity on peptide segments containing active epitopes in the food matrixes, and can accurately detect the alpha-lactalbumin and sensitization residues thereof in dairy products of any food matrixes.
Drawings
FIG. 1 is a diagram showing the mass spectrum of an epitope immunopeptides of the present invention for constructing alpha-lactalbumin based on linear epitopes of IgE.
FIG. 2 is an HPLC chromatogram of an epitope immunopeptides of the present invention for constructing alpha-lactalbumin based on linear epitopes of IgE.
FIG. 3 is a standard curve of low detection limit amount detection of alpha-lactalbumin based on IgE linear epitope construction of epitope immunopeptides of alpha-lactalbumin in the examples of the present invention.
Detailed Description
The present invention will be further described with reference to the drawings and detailed description, wherein the described embodiments are only some, but not all, of the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Referring to fig. 1 to 3, the content of alpha-lactalbumin in infant hydrolyzed hypoallergenic formula is quantitatively detected.
1. Sample pretreatment:
the 7 hydrolyzed hypoallergenic milk infant formulas of Table 1 were dissolved in PBS and diluted with binding protein to a protein concentration of 5mg/mL for use.
Table 1 hydrolyzed formula milk powder
2. Sample detection
(1) Coating: the alpha-lactalbumin monoclonal antibody was diluted to 2. Mu.g/mL with coating solution, 100. Mu.L per well, and coated overnight at 4 ℃.
(2) Washing: washed 4 times with PBST (containing 0.1% Tween-20) for 3min each time, and then dried.
(3) And (3) blocking: the wells were incubated with PBS and 4% gelatin (0.1% Tween-20) in PBST blocking solution at 37℃for 0.5h with 100. Mu.L per well. After the end of blocking, the mixture was washed with PBST for 4 times, 3min each time, and then was dried.
(4) Adding an antigen: the cow milk alpha-lactalbumin standard/sample is diluted to 2 mug/mL with blocking liquid in triplicate, 100 mu L of blocking liquid is taken as blank control per hole, incubated for 1h at 37 ℃, washed for 4 times with PBST after incubation, and buckled for 3min each time.
(5) Adding a detection antibody: after washing, the biotinylated α -lactalbumin polyclonal antibody was diluted to 1 μg/mL with blocking solution, 100 μl per well, and incubated for 1h at 37deg.C.
(5) HRP-labeled streptavidin: after washing, HRP-labeled streptavidin was diluted 60-fold with blocking solution, 100. Mu.L per well was added and incubated for 1h at 37 ℃.
(6) Color development: after washing, 100 mu L of TMB color development liquid is added into each hole, and the reaction is carried out for 20min at 37 ℃ in a dark place;
(7) Terminating the reaction: the color reaction was terminated by adding 50. Mu.L of 2mol/L H2SO4 per well.
(8) OD value: and detecting the light absorption value of the ELISA plate at 450nm by using an ELISA reader, wherein the average value of the multiple detection is the final detection value.
3. Analysis
And calculating the content of alpha-lactalbumin and sensitized residues in the sample according to a linear regression equation and a sample absorbance value.
4. Results
Referring to fig. 3, the detection standard curve equation is y=2.732 x-2.909, r 2 = 0.9951, sensitivity was 1.122ng/mL. A. B milk powder is not able to detect alpha-lactalbumin due to its high hydrolyzability; the content of alpha-lactalbumin in the C milk powder is 815.85 +/-10.819 mg/kg,the content of alpha-lactalbumin in the D milk powder is 2140.891 +/-37.91 mg/kg, the content of alpha-lactalbumin in the E milk powder is 1888.95 +/-81.90 mg/kg, the content of alpha-lactalbumin in the F milk powder is 12249.90 +/-883.59 mg/kg, and the content of alpha-lactalbumin in the G milk powder is 6747.12 +/-143.42 mg/kg.
Example 2
Referring to fig. 1 to 3, the content of alpha-lactalbumin in the milk sandwich biscuit is quantitatively detected.
1. Sample pretreatment
The sample was first ground to a powder, 2g of the powder was added to 40mL of the extract, stirred overnight at 4℃in a chromatography cabinet, centrifuged (4 ℃,10,000g,15 min) and the supernatant was taken. Finally, the supernatant was filtered through a 0.45 μm membrane.
2. Sample detection
(1) Coating: the alpha-lactalbumin monoclonal antibody was diluted to 2. Mu.g/mL with coating solution, 100. Mu.L per well, and coated overnight at 4 ℃.
(2) Washing: washed 4 times with PBST (containing 0.1% Tween-20) for 3min each time, and then dried.
(3) And (3) blocking: the wells were incubated with PBS and 4% gelatin (0.1% Tween-20) in PBST blocking solution at 37℃for 0.5h with 100. Mu.L per well. After the end of blocking, the mixture was washed with PBST for 4 times, 3min each time, and then was dried.
(4) Adding an antigen: the cow milk alpha-lactalbumin standard/sample is diluted to 2 mug/mL with blocking liquid in triplicate, 100 mu L of blocking liquid is taken as blank control per hole, incubated for 1h at 37 ℃, washed for 4 times with PBST after incubation, and buckled for 3min each time.
(5) Adding a detection antibody: after washing, the biotinylated α -lactalbumin polyclonal antibody was diluted to 1 μg/mL with blocking solution, 100 μl per well, and incubated for 1h at 37deg.C.
(5) HRP-labeled streptavidin: after washing, HRP-labeled streptavidin was diluted 60-fold with blocking solution, 100. Mu.L per well was added and incubated for 1h at 37 ℃.
(6) Color development: after washing, 100 mu L of TMB color development liquid is added into each hole, and the reaction is carried out for 20min at 37 ℃ in a dark place;
(7) Terminating the reaction: the color reaction was terminated by adding 50. Mu.L of 2mol/L H2SO4 per well.
(8) OD value: and detecting the light absorption value of the ELISA plate at 450nm by using an ELISA reader, wherein the average value of the multiple detection is the final detection value.
3. Analysis
And calculating the content of alpha-lactalbumin and sensitized residues in the sample according to a linear regression equation and a sample absorbance value.
4. Results
Referring to fig. 3, the detection standard curve equation is y=2.732 x-2.909, r 2 = 0.9951, sensitivity was 1.122ng/mL. The content of alpha-lactalbumin in the milk sandwich biscuit is 159.89 +/-10.691 mg/kg.
Claims (6)
1. An antigen epitope peptide for constructing alpha-lactalbumin based on IgE linear epitope is characterized in that the amino acid sequence of the antigen epitope peptide is NDSTEYGLFQINNKIW.
2. A method for detecting alpha-lactalbumin with low detection limit amount is characterized by comprising the following steps,
s1, synthesizing an antigen epitope peptide for constructing alpha-lactalbumin based on IgE linear epitopes;
s2, coupling the epitope peptide and hemocyanin to obtain a complete antigen;
s3, immunizing a mouse by taking the complete antigen as an antigen, and preparing a monoclonal antibody;
s4, detecting the content of the alpha-lactalbumin in the food matrix by using the monoclonal antibody as a capture antibody and the biotinylated alpha-lactalbumin polyclonal antibody as a detection antibody through an sELISA method.
3. The method for detecting alpha-lactalbumin with low detection limit as claimed in claim 2, wherein the antigen epitope peptide is a blocking peptide obtained by coupling asparagine and cysteine, and the amino acid sequence of the blocking peptide is N-NDSTEYGLFQINNKIW-C.
4. The method for detecting alpha-lactalbumin with low detection limit as claimed in claim 3, wherein the monoclonal antibody is used for screening of mice and cell strains by using the blocking peptide.
5. The method for detecting alpha-lactalbumin with low detection limit as claimed in claim 2, wherein the working concentration of the monoclonal antibody as a capture antibody in the detection process of S4 by the sELISA method is 2 μg/mL.
6. The method for detecting alpha-lactalbumin with low detection limit as claimed in claim 2, wherein the working concentration of the biotinylated alpha-lactalbumin polyclonal antibody as a detection antibody in the detection process of S4 by the sELISA method is 1 μg/mL.
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| CN202310276532.7A CN116478273A (en) | 2023-03-21 | 2023-03-21 | Antigen epitope peptide for constructing alpha-lactalbumin based on IgE linear epitope and method for detecting alpha-lactalbumin with low detection limit |
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| CN202310276532.7A CN116478273A (en) | 2023-03-21 | 2023-03-21 | Antigen epitope peptide for constructing alpha-lactalbumin based on IgE linear epitope and method for detecting alpha-lactalbumin with low detection limit |
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