CN110317255A - αs1The monoclonal antibody and milk allergen detection method that the epitope of casein is prepared - Google Patents

αs1The monoclonal antibody and milk allergen detection method that the epitope of casein is prepared Download PDF

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CN110317255A
CN110317255A CN201910653868.4A CN201910653868A CN110317255A CN 110317255 A CN110317255 A CN 110317255A CN 201910653868 A CN201910653868 A CN 201910653868A CN 110317255 A CN110317255 A CN 110317255A
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casein
antibody
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monoclonal antibody
epitope
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丛艳君
吕晓哲
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Beijing Technology and Business University
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
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    • G01—MEASURING; TESTING
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    • 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
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4731—Casein

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Abstract

The invention discloses the α that amino acid sequence is SEEIVPNSVEQKHIQKEDVPSERs1The epitope of casein, and its effective monoclonal antibody is prepared using the epitope, it is further provided using the monoclonal antibody as primary antibody, α is detected by indirect competitive ELISAs1The method of casein or its hydrolysate anaphylactogen.Utilize α of the inventions1The monoclonal antibody of the epitope preparation of casein, detects α by indirect competitive ELISA methods1Casein is antigen, and detection limit is less than 10.49ng/mL, and its detection method accuracy, repeatability are preferably, can be effectively used for detection milk allergen, have application value.

Description

αs1Monoclonal antibody and the milk allergen detection that the epitope of casein is prepared Method
Technical field
The present invention relates to biotechnologys and field of food, in particular to αs1The epitope of casein, and its system Standby obtained monoclonal antibody, and utilize the method for monoclonal antibody detection milk allergen.
Background technique
Cow's milk is a kind of protein product full of nutrition, while being also eight common big allergin (eggs, fish, first One of shell class, dairy produce, peanut, soybean, nut, wheat), sensitization has seriously affected the health (Mills of infant E N C,Valovirta E,Madsen C,et al.Information provision for allergic Consumers-where are we going with food allergen labelling? [J] .Allergy, 2004,59 (12):1262-1268).Cow's milk protein rich content, every liter of cow's milk contain about 30-35 grams protein, containing having more than 25 kinds of differences Protein, for theoretical any protein therein have potential sensitization (Martorell-Aragon é s A, Echeverría-Zudaire L,Alonso-Lebrero E,et al.Position document:IgE-mediated cow's milk allergy[J].Allergologia et Immunopathologia,2015,43(5):507-526).But Casein (casein, CN), beta lactoglobulin (beta-lactoglobulin, β-lg) and α-lactalbumin are generally believed at present (alpha-lactalbumin, α-la) is main milk allergen (Galan-Malo P, L ó pez M, Ortiz J, et al.Detection of egg and milk residues on working surfaces by ELISA and lateral flow immunoassay tests[J].Food Control,2017,74:45-53).Lactalbumin accounts for cow's milk Albumen about 20%, wherein discovery alpha lactalbumin (Bosd4), beta lactoglobulin (Bosd5), immunoglobulin (Bosd7), ox blood The lactoferrin (Bosd lactoferrin) of pure albumen (BSA, Bosd6) and trace is anaphylactogen.And α-lactalbumin and Beta lactoglobulin is the most important anaphylactogen of whey portion, account for total milk protein 5% and 10% (Hochwallner H, Schulmeister U,Swoboda I,et al.Cow’s milk allergy:From allergens to new forms of diagnosis,therapy and prevention[J].Methods,2014,66(1):22-33；Docena G H, Fernandez R,Chirdo F G,et al.Identification of casein as the major allergenic and antigenic protein of cow's milk[J].Allergy,1996,51(6):412-416).Casein is about The 80% of cow's milk total protein is accounted for, (Wang Meng is opened and quick-boiled, and is gone into business it has been reported that about 65% cow's milk sensitization person is to casein allergy It is small beautiful, wait Typical allergic original and its allergy characteristic [J] Chinese food and nutrition, 2008 (11): 62-64 in food).Casein By αs1Casein (32%), αs2Casein (10%), (10%) four kind of protein group of beta-casein (28%) and κ-casein At wherein αs1Casein is the most important anaphylactogen of casein fraction, occupies 30% or more of cow's milk total protein (Schulmeister U,Hochwallner H,Swoboda I,et al.Cloning,Expression,and Mapping of Allergenic Determinants ofαs1-Casein,a Major Cow's Milk Allergen[J].The Journal of Immunology,2009,182(11):7019-7029)。αs1Casein is a single-stranded phosphorylated protein Matter, by 199 Amino acid profiles, proline content is higher, without disulfide bond, fairly simple (the Kumosinski T of tertiary structure F,Brown E M,Farrell H M.Three-Dimensional Molecular Modeling of Bovine Caseins:αs1-Casein[J].Journal of Dairy Science,1991,74(9):2889-2895).Completely αs1Casein or its biggish IgE fragment reaction are (Schulmeister U, Hochwallner the reason of causing allergic reaction H,Swoboda I,et al.Cloning,Expression,and Mapping of Allergenic Determinants ofαs1-Casein,a Major Cow's Milk Allergen[J].The Journal of Immunology,2009,182 (11):7019-7029)。
Allergic reaction caused by milk allergen is a kind of hypersensitivity mediated by IgE and non-IgE is mediated, can cause skin The reaction of skin, intestines and stomach, respiratory system results even in inflammation reaction or shock (Alessandro F, Jan when serious B,Holger S,et al.World Allergy Organization(WAO)Diagnosis and Rationale for Action against Cow’s Milk Allergy(DRACMA)Guidelines[J].Pediatric Allergy and Immunology,2010,21:1-125).Milk allergy be infancy and childhood most common phagopyrism (Sicherer S H,Sampson H A.Food allergy:Epidemiology,pathogenesis,diagnosis,and treatment[J].Journal of Allergy and Clinical Immunology,2014,133(2):291-307)。 At present both at home and abroad to Milk allergy disease without thoroughly effective radical cure method, prevent unique effective means of Milk allergy from being Avoid eating food (Han Xuelan lactalbumin enzyme hydrolysis process and hydrolysate Study on functional properties containing milk and milk products [D] University Of Science and Technology Of Tianjin, 2017).But so, the energy of upgrowth and development of children and amino acid are supplied to a certain extent It will reduce, influence normal development and health (Luyt D, Ball H, Makwana N, the et al.BSACI of allergy children guideline for the diagnosis and management of cow's milk allergy[J].Clinical& Experimental Allergy,2014,44(5):642-672).Cow's milk protein is frequently as a kind of food processing auxiliary material simultaneously, Many aspects in food production, such as ice cream, biscuit etc. are applied, these remaining ingredients may cause sensitivity response (Villa C,Costa J,Oliveira M B P P,et al.Bovine Milk Allergens:A Comprehensive Review[J].Comprehensive Reviews in Food Science and Food Safety,2018,17(1): 137-164).Therefore for maintenance food safety and protection consumer health, the technique that research reduces the content of anaphylactogen in cow's milk And establish fast and accurately milk allergen external detection method, it appears very urgent.
There is allergen protein in lot of documents report hydrolysis cow's milk that can reduce its antigenicity at present, but all because of protease There is its specific hydrolytic sites, the antigenic reduction of hydrolysate is not directly proportional to the increase of degree of hydrolysis.Even if hydrolysate water Xie Du is very big, molecular weight very little, but as long as epitope is not destroyed, still has anaphylaxis.And with the increasing of hydrolysis degree A large amount of free amino acid will certainly be generated, will lead to the raising of internal osmotic pressure after consumption by infants, and then generate sense of discomfort.Cause This, will prepare good milk protein hydrolysate, have to control degree of hydrolysis, improve hydrolysate Small Peptides mass fraction, drop Low or control free amino acid ratio.Although appropriate lactoalbumin hydrolysate matter hydrolysate is applied to the life of babies ' formula milk powder It produces, but since protease hydrolytic anaphylactogen effect epitope lacks specificity, so that milk protein hydrolysate still has sensitization Property, can have in order to avoid the problems such as protein processing and the change of Physiological Properties, needing to research and develop caused by blindness hydrolysis The method of antigen residual quantity after effect detection lactoalbumin hydrolysate matter.
And with the development and application of new technology, the detection of anaphylactogen is towards highly sensitive, simplicity is quick, the high direction of accuracy Development, but there are still some problems during practice.When using round pcr, relevant gene piece is obtained in food Section has certain difficulty, and technical requirements are high, instrument and equipment costly, in application aspect by certain constraint (van Hengel A J.Food allergen detection methods and the challenge to protect food- allergic consumers[J].Analytical and Bioanalytical Chemistry,2007,389(1):111- 118).Equally, capillary electrophoresis technique and instrumental method are not suitable for quickly detecting due to equipment valuableness and complicated for operation In.Both at home and abroad to using reversed-phased high performace liquid chromatographic analysis detection cow's milk protein, there are also research (El-Manzalawy Y,Honavar V.Recent advances in B-cell epitope prediction methods[J].Immunome research,2010,6(2):S2；[71]Le Houérou C,Bennetau-Pelissero C,Lamothe V,et al.Syntheses of Novel Hapten–Protein Conjugates for Production of Highly Specific Antibodies to Formononetin,Daidzein and Genistein[J].Tetrahedron, 2000,56 (2): 295-301), accuracy rate is higher, but because major defect is at high cost.ELISA method sensitivity height, high specificity, Quickly, expense is low, thus is widely used in Allergic skin test, examines especially suitable for anaphylactogen a small amount of in food It surveys.But this method also has its limitation, for there are the anaphylactogens of Cross-reactivity, i.e., for there are similar action epitopes Anaphylactogen between, often there is false positive in ELISA method, therefore prepares antibody with high specificity to allergy in ELISA method detection cow's milk It is former particularly significant.Simultaneously in applied immunology method context of detection, the quality and reaction pattern of antibody are also to influence testing result Key factor (Ni little Qin, Lai Weihua bovine casein detection method progress [J] Food Science, 2014,35 (03): 290-294).It when using such method, needs to obtain the antibody of high quality, choose suitable reaction pattern and effectively extracted Quick original method.In terms of pesticide and medicine, antibody is prepared as haptens about synthesis polypeptide, (Cao Z, Zhang has been reported W,Ning X,et al.Development of Monoclonal Antibodies Recognizing Linear Epitope:Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton,Maize,and Tobacco[J].Journal of Agricultural and Food Chemistry,2017,65(46):10115-10122；Feng G,Boyle M J,Cross N,et al.Human Immunization With a Polymorphic Malaria Vaccine Candidate Induced Antibodies to Conserved Epitopes That Promote Functional Antibodies to Multiple Parasite Strains [J] .The Journal of Infectious Diseases, 2018,218 (1)), but in field of food, antibody Preparation mostly be with holoprotein as antigen, the antibody specificity that will result in preparation in this way is not strong, and accuracy is not high.
To sum up, although cow's milk protein hydrolysate has been applied to infant formula exploitation, hydrolytic sites are still had Specificity is not strong, and anaphylactogen residual quantity is higher, the heavier problem of hydrolysate bitter taste.αs1Casein is the main composition of casein Component is anaphylactogen important in cow's milk, occupies 30% or more of cow's milk total protein, has anaphylactoid infant to take in it After can seriously affect physical and mental health, micro intake will make Milk allergy patient generate strong allergic reaction.Therefore it establishes The protease of allergen sensitisation, optimization hydrolysis work is effectively reduced in the detection method of anaphylactogen residual quantity accurately and fast, screening The research of skill is very urgent.
Summary of the invention
In view of the drawbacks of the prior art and current demand, the present invention is directed to seek to establish anaphylactogen residual accurately and fast The detection method of amount technically includes the selection of epitope, the preparation of monoclonal antibody and the foundation of detection method.
The present inventor's early period identifies identification α by vitro tests1Casein IgE effect epitope has aa141~155, aa21 ~35, aa91~105, aa186~200, aa26~40 (Yanjun Cong, et al.Identification of the critical amino acid residues of immunoglobulin E and immunoglobulin G epitopes onα(s1)-casein by alanine scanning analysis[J].Journal of Dairy Science,2013,96(11):6870-6876).(Ruiter, B.et al.Characterization of T such as Ruiter cell epitopes inαs1-casein in cow’s milk allergic,atopic and non-atopic Children.Clin.Exp.Allergy 36:303-310) using 32 overlapping peptide analysis T cells identification epitopes, wherein peptide chain 43~66,73~96,91~114,127~180 can be identified by the T cell of 3 patients.But in view of effect The sensitization intensity (discrimination of aa91~105 is 100%) and conservative of epitope (avoid its sequence from having to other albumen similar Property), the present inventor by it has been reported that main function epitope aa73~96, aa91~114 and aa91~105 are combined, And consider the characteristic that epitope is distributed on anaphylactogen conformation surface, choose αs1SEEIVPNSVEQKHIQKEDVPSER in casein (aa83~105) are used as haptens.Using the hydrophily of the Protean program forecasting sequence of DNAStar software it is equal > 0, indicate this A sequence has relatively high hydrophilicity；With the surface accessibility overwhelming majority > 1 in time series, indicate easily to occur to fold simultaneously It is exposed to outside；Antigenic index is equal > 0, then it represents that this sequence formed a possibility that epitope it is very high (El-Manzalawy Y, Honavar V.Recent advances in B-cell epitope prediction methods[J].Immunome research,2010,6(2):S2.).Therefore present invention selection synthesis SEEIVPNSVEQKHIQKEDVPSER (aa83~105) As haptens, show it with extraordinary effect through follow-up study.
Therefore, first aspect of the present invention is to provide αs1The epitope of casein, amino acid sequence are SEEIVPNSVEQKHIQKEDVPSER or its both ends are based on αs1Casein and increase 1-5 amino acid (i.e. increased ammonia in both ends Base acid and αs1Corresponding sequence is identical in casein), it can more specifically increase by 1,2,3,4 and 5 amino Acid.It can use the epitope and prepare effective monoclonal antibody.
The second aspect of the present invention is to provide one kind by above-mentioned αs1The epitope and bovine serum albumin(BSA) of casein (BSA) it is coupled obtained comlete antigen.
Third aspect of the present invention provides the preparation method of above-mentioned comlete antigen, wherein being synthesized first by solid-phase synthesis Above-mentioned αs1Casein acts on epitope, and is coupled bovine serum albumin(BSA) (BSA) and prepares comlete antigen.It is further preferred that institute Stating coupling is realized by glutaraldehyde method.
The 4th aspect of the present invention provides a kind of for above-mentioned αs1The antibody of the epitope of casein, especially Dan Ke Grand antibody.More preferably by by the αs1Epitope-BSA the comlete antigen of casein is immunized mouse and is prepared Monoclonal antibody.When experiment is shown using the monoclonal antibody as first antibody, the spirit of the indirect competitive ELISA method of foundation Sensitivity, accuracy and repeatability, and opposite αs1Casein holoprotein be immunized Balb/c mouse preparation monoclonal antibody and Speech, the indirect competitive ELISA method detection limit that monoclonal antibody of the invention is established is lower, i.e. sensitivity is higher.Meanwhile it preparing Comlete antigen monoclonal antibody and αs1Casein holoprotein monoclonal antibody can and αs1In casein and skimmed milk αs1Specific immune response occurs for casein, and cross reaction does not occur with soybean protein isolate.
The 5th aspect of the present invention provides a kind of detection αs1The method of casein, wherein being using aforementioned present invention αs1The monoclonal antibody of the epitope of casein detects α by indirect competitive ELISA as primary antibodys1Casein.Its In, the operating method of indirect competitive ELISA is referred to conventional method progress.
Further, the present invention provides a kind of method for detecting milk allergen, wherein being detection pair with cow's milk to be measured As using the α of aforementioned present inventions1The monoclonal antibody of the epitope of casein passes through indirect competitive ELISA as primary antibody To detect α in cow's milks1Casein or its protease hydrolysate.α can be learnt by the detections1The protease hydrolysate of casein is residual The amount of remaining anaphylactogen, to determine the quality of cow's milk or whether properly prepare milk powder, especially infant formula.
The present invention further provides the methods for preparing low sensitization milk protein hydrolysate comprising with hydrolase to lactoprotein into Row hydrolysis, passes through the α with aforementioned present inventions1The monoclonal antibody of the epitope of casein passes through indirect competition as primary antibody ELISA detects αs1The remaining anaphylactogen of casein hydrolysate.Based on detection may determine that aforementioned hydrolysising condition whether be suitable for, To determine optimum hydrolysising condition, it also may determine that whether milk protein hydrolysate properly prepares milk powder, especially infant formula.More Specific step includes: with protease, and lactoprotein is hydrolyzed in preferably papain, obtains enzyme hydrolyzate, is freeze-dried Freeze-dried powder is obtained, antigenic dilution is added and is diluted to suitable concentration, according to indirect competitive ELISA method program, using in the present invention The monoclonal antibody for the comlete antigen stated detects the antigen residual quantity of the protease hydrolytic liquid of lactoprotein as primary antibody.
Utilize α of the inventions1The epitope of casein, the monoclonal antibody of preparation are better than αs1Casein holoprotein Make the monoclonal antibody of antigen acquisition.α is detected by indirect competitive ELISA methods1Casein is antigen, α of the inventions1- Its detection limit of monoclonal antibody of the epitope preparation of casein is less than 10.49ng/mL, and αs1Casein holoprotein When monoclonal antibody is as primary antibody, detection is limited to 60.50ng/mL, the former detection limit has apparent advantage.In addition, α of the inventions1- The most suitable working concentration of the monoclonal antibody of the epitope preparation of casein is 10000 times of dilution, and αs1Casein holoprotein The most suitable working concentration of monoclonal antibody be then 5000 times of dilution, while with the accuracy and again of its indirect competitive ELISA method established Renaturation is also more excellent.Simultaneously by verifying, method accuracy, the repeatability of monoclonal antibody suggestion of the invention are preferable, can It is effectively used for detection milk allergen, there is application value.
Detailed description of the invention
The HPLC map of Fig. 1 synthesis polypeptide.
The MS map of Fig. 2 synthesis polypeptide.
The ultraviolet scanning spectrum figure of Fig. 3 synthesis polypeptide, BSA and comlete antigen.
Wherein, curve 1 indicates synthesis polypeptide ultraviolet spectra, and maximum absorption band is at 196nm.Curve 2 indicates BSA ultraviolet light Spectrum, maximum absorption band is at 278nm.Curve 3 indicates comlete antigen ultraviolet spectra, has absorption peak at 196nm and 278nm.
The SDS-PAGE electrophoresis of Fig. 4 soybean protein isolate is composed
Wherein, swimming lane 1 is Marker, and swimming lane 2 is α s1- casein, and swimming lane 3 is skimmed milk, and swimming lane 4 is soybean separation Albumen.
Pvdf membrane amino black colored graph after Fig. 5 transfer.
The western blot figure of Fig. 6 comlete antigen monoclonal antibody
Wherein, 1 α s1- casein is indicated, 2 indicate skimmed milk, and 3 indicate soybean protein isolate.
The western blot figure of Fig. 7 α s1- casein monoclonal antibody
Wherein, 1 α s1- casein is indicated, 2 indicate skimmed milk, and 3 indicate soybean protein isolate.
The western blot figure (negative control) of Fig. 8 negative serum
Wherein, 1 α s1- casein is indicated, 2 indicate skimmed milk, and 3 indicate soybean protein isolate.
The indirect competitive ELISA suppression curve of Fig. 9 comlete antigen monoclonal antibody.
Figure 10 αs1The indirect competitive ELISA suppression curve of casein monoclonal antibody.
Figure 11 αs1The reversed-phase high performance liquid chromatography figure of casein mark product.
The standard curve of Figure 12 rp-hplc determination α s1- casein standard items.
The hydrolyzate antigen residual quantity of Figure 13 comlete antigen monoclonal antibody measurement.
Figure 14 αs1The hydrolyzate antigen residual quantity of casein monoclonal antibody measurement.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments, and will further illustrate this in conjunction with the embodiments Advantageous effect of the invention.
Embodiment 1: the comlete antigen preparation of epitope
1, the synthesis of haptens, that is, polypeptide and identification
Based on analysis and research, α is chosens1SEEIVPNSVEQKHIQKEDVPSER (aa83~105) in casein makees For haptens.Using the Fmoc solid phase method of peptide synthesis, C- end amino acid is connected on Wang resin, using conventional Fmoc method into Row is gradually condensed.After synthesis, sequence is cut down from solid phase carrier with strong acid, is purified through HPLC, Mass Spectrometric Identification is cold It is lyophilized dry rear spare.The wherein references such as concrete operation method step: Cong Yanjun, Ren Fazheng, Yun Zhanyou αs1Casein IgE antigen Identification [J] Food Science of determinant, 2010,31 (17): 232-235.
It is analyzed using high performance liquid chromatography and mass spectrum.Chromatographic column: C18 column (4.6mm × 150mm, 5 μm), mobile phase: A be 0.1% trifluoroacetic acid aqueous solution of volume fraction, B be 80% acetonitrile and 20% water, and add volume fraction be 0.09% three Fluoroacetic acid.Using by volume fraction gradient elution program: Mobile phase B increases in 0~20min, shared volume fraction from 15% 45%.Flow velocity: 1.0mL/min, Detection wavelength: 220nm；Column temperature: 20 DEG C.
Mass Spectrometry Conditions: ESI ion source is used；Atomisation pressure: 15psi；Dry temperature degree: 350 DEG C；Flow velocity: 5L/min；It sweeps Retouch mass range: m/z 500~2200.
The polypeptide after synthesis is purified by high performance liquid chromatography, purity can reach 92.664%, as shown in Figure 1. Simultaneously with the relative molecular mass of mass spectrometric determination polypeptide (site is linked in order to increase peptide C end, has synthesized one the C-terminal more Lysine, therefore the polypeptide molecular weight of mass spectroscopy will add the relative molecular mass of a lysine).Fig. 2 indicates synthesis The MS map of sequence SEEIVPNSVEQKHIQKEDVPSER, the relative molecular mass of polypeptide are 2781.1, show to have synthesized correct Polypeptide.
2, the preparation and identification of comlete antigen
The synthesis polypeptide purified and bovine serum albumin(BSA) (BSA) are coupled using glutaraldehyde method, obtain comlete antigen (behaviour Make method reference: Immunoenzyme techniques [J] the China laboratory medicine magazine in Guo Jiyan immunological test, 2005,28 (2): 221-224；Teng Haiying, Yu Yuyan, Lu Ling are waited in glutaraldehyde method synthesis huperzine comlete antigen and its identification Fujian [J] Medical pharmaceutical university journal, 2012,22 (06): 41-44.).
The synthesis polypeptide for taking 15mg to purify is dissolved in 6mL0.01mol/L phosphate buffer solution, and BSA25.5mg is added 1.5mL0.2% glutaraldehyde is added in stirring under ice bath, and 1.9mL1mol/L glycine is added after 60min, continues to stir 60min, system It is spare that the polypeptide-BSA compound obtained prepares freeze-dried powder after isolating and purifying by Sephadex G200.
The coupling ratio of comlete antigen is measured using uv scan method.Synthesis polypeptide, BSA are complete after the two coupling Holoantigen can generate apparent absorption peak in ultra-violet (UV) band.Synthesis polypeptide, BSA and comlete antigen are made into a certain concentration with PBS Solution after, spectral scan is carried out under 190~1100nm all-wave length respectively.Measured respectively by colorimetric method synthesis polypeptide (A), BSA (B) and comlete antigen (C) respectively at A and B maximum absorption band wavelength m, n absorption value (Absorbence, A), i.e. AAm、 AAn、ABm、ABn、ACm、ACn.According to Lambert-Beer's law, according to formula A=kcl, (A indicates absorbance, and c indicates extinction material Concentration, l indicates absorption light path, that is, absorber thickness of sample, and k is molar absorption coefficient) calculate A substance and B substance and exist respectively A, the molecular extinction value at B substance absorption peak wavelength, i.e. kAm、kAn、kBm、kBn.A substance and B object are calculated by formula (1) The molecular ratio (i.e. the coupling ratio of synthesis polypeptide and BSA) of matter:
Molecular ratio=(ACm·kBn- ACn·kBm)/(ACn·kAm- ACm·kAn) (1)
Synthesis polypeptide, BSA and comlete antigen are subjected to spectral scan under 190~1100nm all-wave length respectively, as a result such as Shown in Fig. 3, carrier protein BSA has maximum absorption band at 278nm, and synthesis polypeptide has maximum absorption band at 196nm.Coupling BSA polypeptide has the characteristic absorption peak of synthesis polypeptide and BSA at 190~1100nm and has corresponding spectrum superposition situation. Therefore synthesis polypeptide and BSA are tentatively coupled success.
Measure synthesis polypeptide, BSA and the comlete antigen ultraviolet absorptivity at synthesis polypeptide and BSA maximum absorption band respectively Value, as shown in table 1.The molecular ratio i.e. coupling ratio that synthesis polypeptide and BSA are calculated according to formula (1) is 6.31.
1 synthesis polypeptide of table, BSA, comlete antigen ultraviolet absorptivity
Embodiment 2: the preparation and identification of monoclonal antibody
The present embodiment αs1Balb/c mouse is immunized in the comlete antigen of the sensitization epitope of casein, prepares monoclonal Antibody, while preparing αs1The monoclonal antibody of casein holoprotein is as control.The effect of monoclonal antibody is detected by indirect elisa method Valence, Western blot identify the specificity of antibody.
1, the preparation of monoclonal antibody
The comlete antigen of SEEIVPNSVEQKHIQKEDVPSER coupling BSA prepared by embodiment one, it is small to be immunized Balb/c Mouse takes subcutaneous multi-point injection method, and mouse docking takes blood examination to survey serum titer since being exempted from third, and potency is not up to standard, continues Immune, booster immunization after potency is up to standard carries out cell fusion after booster immunization 3 days.Booster immunization uses tail vein injection method.
After four times immune, the casein mouse of booster immunization is taken, dislocates and puts to death after eye socket blood sampling.Eye blood is placed at room temperature for 2h, and 4 DEG C overnight, next day centrifuging and taking supernatant detection.Booster immunization mouse spleen is taken, connective tissue is removed, prepares splenocyte suspension, cell It is stand-by after counting.The good myeloma cell of cell state is taken to count.By splenocyte: myeloma cell 5-10:1 is mixed, 1500r/min is centrifuged 4min, discards culture supernatants after centrifugation, the cell mixing of centrifugation bottom of the tube is struck and is scattered, at 40 DEG C Water-bath 1min in beaker, backward cell mixing in be slowly added into 1mL37 DEG C preheating PEG (molecular weight 1500), in 40 seconds plus It is complete, water-bath 30 seconds again are added after PEG.1640 basic culture solutions are added later, it is desirable that 1640 basis training of 3mL is added in 1min Nutrient solution, finally plus 50mL, 800r/min are centrifuged 4min.Supernatant is abandoned after centrifugation, and 1640 basic culture solutions are added and wash again once, i.e., 800r/min is centrifuged 4min.HAT culture solution is slowly added to required volume after discarding supernatant, and cell is resuspended, gently mixes it It is even, it is added in preprepared feeder cells plate, the 100 every holes μ L.Tissue culture plate is finally cleaned disinfection with alcohol swab, is put In 37 DEG C, 5%CO2In incubator, syncretizing effect is observed after 5 days.
5th day observation cell fusion effect after the screening fusion of positive hybridoma cell, is observed simultaneously under inverted microscope The cell heap number in each hole of tissue culture plate is recorded, cell changes after liquid until cell heap size is about cell culture plate well 1/10 when can draw cell conditioned medium, with ELISA method detection cell conditioned medium antibody titer.
The positive hybridoma cell that filters out simultaneously repeatedly is subcloned.Atoleine sensitization Balb/c mouse, 6-8 weeks, Ascites can be prepared after 7-10 days.Hybridoma is collected, takes ten thousand cell infusion of 100-150 in mouse abdominal cavity, can be aged after a week Mouse state is inactive and the abdominal cavity enlargement of mouse.Injection cell uses asepsis injector to acquire ascites in mouse abdominal cavity after a week, It is primary every acquisition in one to two days, it is put to death after acquisition 3 times.Freezen protective is dispensed after the ascites detection potency of acquisition and inhibition.Most Obtain measuring the monoclonal antibody for the one plant of good comlete antigen of effect that meets the requirements afterwards.One plant is prepared by identical method simultaneously αs1The monoclonal antibody of casein is as control.
Using mouse Ig G1、Ig G2、Ig G2a, Ig E ELISA kit identification, operated according to operation instructions, reflect The hypotype of fixed above-mentioned two plants of monoclonal antibodies is IgG1。
2, the measurement of antibody titer
The potency for the monoclonal antibody that above-mentioned preparation is mentioned is measured using indirect enzyme-linked immunosorbent (ELISA) method.
It is measured by square matrix method, antigen coat concentration is 5 μ g/mL, and monoclonal antibody successively presses 1:5000,1:10000,1: The dilution of 20000,1:40000,1:80000,1:160000,1:320000,1:640000 multiple.With the normal mouse not being immunized Ascites as negative control.
(1) antigen coat: the antigen that coating buffer was diluted is added in ELISA Plate with 100 holes μ l/, was placed at 4 DEG C Night.
(2) it washs: in the liquid that second day outwells in ELISA Plate, with PBST board-washing 3 times of every 200 μ l of hole, drying.
(3) it closes: confining liquid is added with 100 holes μ l/ in ELISA Plate and is closed, stands 1h at 37 DEG C.
(4) add primary antibody: the monoclonal antibody of different dilutions being added in (1) ELISA Plate with the amount in 100 holes μ l/ respectively, In 37 DEG C of incubation 2h, board-washing.
(5) add ELIAS secondary antibody: the sheep anti-mouse igg of the horseradish peroxidase label after dilution is added with the amount in 100 holes μ l/ Into ELISA Plate, 37 DEG C of standing 1h, board-washing.
(6) develop the color: the substrate application liquid of fresh configuration, 100 holes μ l/ are added in ELISA Plate, and room temperature dark place is reacted about 20min。
(7) it terminates reaction: the sulfuric acid of 2mol/L being added with 50 holes μ l/, color is by Lan Bianhuang.
(8) absorbance value in each hole in ELISA Plate colorimetric: is measured at Single wavelength 450nm with microplate reader.
The preparation of reagent needed for indirect ELISA:
(1) phosphate buffer of the 0.02mol/L of PBS:pH7.4.
(2) PBST: the PBS containing 0.05% Tween-20.
(3) confining liquid: the PBST containing 1%BSA.
(4) antigenic dilution: the carbonate solution of the 50mmol/L of pH9.6.
(5) substrate application liquid: by the phosphate buffer of the pH6.0 of 10mL 0.1M, 100 μ l TMB application liquid are (by 60mg TMB is dissolved in 10mL dimethyl sulfoxide and forms), the 30% hydrogen peroxide Fresh of 15 μ l forms.
With the potency of indirect elisa method measurement comlete antigen monoclonal antibody, the results are shown in Table 2.P indicates the OD value of antibody, N table Show the OD value for the mouse ascites not being immunized.When detection using value>2.1 P/N and P > 0.2 determine testing result as the positive, P/N value< 2.1 or P < 0.2 determines that testing result is feminine gender.It the results are shown in Table 2, when antibody extension rate is more than or equal to 320000, P/N value Greater than 2, but when dilution is 640000, the OD value < 0.2 of antibody, therefore can reach by monoclonal antibody potency prepared by comlete antigen 320000, it can be used for subsequent research.
The OD value of 2 comlete antigen antibody titer of table
Above-mentioned indirect elisa method measures αs1The potency of casein monoclonal antibody, P indicate the OD value of antibody, and N expression is not immunized Mouse ascites OD value.When detection using value>2.1 P/N and P > 0.2 determine testing result as the positive, value<2.1 P/N or P < 0.2 determines that testing result is feminine gender.The results are shown in Table 3, antibody extension rate be more than or equal to 320000 when, P/N value be greater than 2, but When dilution is 640000, the OD value < 0.2 of antibody, therefore by αs1Casein antibody titer can reach 320000, can be used for Subsequent research.
3 α of tables1The OD value of casein antibody titer
3, Western blot identifies antibody specificity
(1)SDS-PAGE
Sample: the preparation of skimmed milk.4 DEG C of cow's milk, 4000r/min is centrifuged 30min, makes fat floating, removes floating Fat.Repeatedly, skimmed milk is obtained, and dilutes suitable multiple.With the α of the suitable concentration of configurations1Casein, soybean Protein isolate solution is to be measured together as sample protein.
The separation gel of 12.5% concentration and the concentration glue of 4.5% concentration are prepared, glue connection degree is 3.6%.Sample treatment liquid with Sample protein 1:1 mixing, is added bromophenol blue indicator, mixes, the loading after water-bath boils 5min or so, dries in the air to room temperature, often A swimming lane applied sample amount is 20 μ L.Pre-dyed albumen Marker is added in first swimming lane.
Vertical electrophoresis, constant current 20mA.Migrate in bromophenol blue indicator to gel lower end about 1cm or so, stop electrophoresis, closes Power supply.Lower clamp plate is unloaded from electrophoretic apparatus, takes out gel.30min is dyed with Coomassie brilliant G-250 dyeing liquor after taking-up, so It is repeatedly decolourized with destainer afterwards, until protein band is clear in gel, takes out gel, analyze its opposite point with gel imaging system Protonatomic mass.
(2) electrotransfer
(1) size that PVDF membrane (pvdf membrane) is cut out into PAGE gel to separation gel, in pvdf membrane Not only sliding surface on make sample-adding with pencil and comb the label that each tooth position is set, 3~5s is impregnated in anhydrous methanol, then turns slow in electricity 10min is impregnated in fliud flushing.The filter paper that seven Zhang great little are about 6 × 8cm is cut out, seven filter paper, electrotransfer sponge, electrophoresis are finished SDS-PAGE separation gel is soaked in electricity together and turns 10min in buffer.
(2) clip of electrotransfer is opened and is laid on clean smooth experimental bench, make black grid below and kept Level spreads processed sponge on black grid face, is rolled with smooth glass bar from a side to the other side, drives bubble out of. Tile three filter paper impregnated on foam-rubber cushion, equally drives bubble out of.Gel is carefully placed on filter paper.Then by PVDF Film is carefully placed in gel face.Then tile on film three layers of filter paper impregnated again, finally impregnated at another piece of laying Sponge puts down white grid, clamps " gel sandwich " and is fitted into electrophoresis tank, fills electrotransfer buffer.Each layer when operation Placement will be careful, and constantly catches up with bubble, prevents aeration result.I.e. transferring film stacks sequence are as follows: gel splint (white Grid) → sponge → tri- layer filter paper → pvdf membrane → gel → tri- layer filter paper → sponge → gel splint (black grid).
(3) entire electrophoresis tank is placed in ice bath, pvdf membrane opens power supply and start electricity close to anode, gel close to cathode Swimming transferring film, constant current 200mA, electrotransfer 2h (pay attention to cooling).
(4) it is unloaded " gel sandwich " after transferring, takes out pvdf membrane and cut off in duplicate from centre, portion amino black 10B dyeing liquor dyes 5min, and transferring effect and protein band position are observed in decoloration to the band that can be seen clearly on film, another For immunoblotting.
(3) immunoblotting
1) pvdf membrane after transfer is used into dH in sizeable plate2O washes 5min, then with TBST at 37 DEG C, drift It washes 4 times, each 15min discards liquid.
2) it is put into the plate added with confining liquid (10~20mL), at 37 DEG C, carries out 1h and be quenched, close pvdf membrane, so After use dH2O washes 5min.
3) by the monoclonal antibody of pvdf membrane and suitable concentration in 4 DEG C overnight, then with dH2O wash 5min, wash 3 times with TBST, every time 10min。
4) HRP of the pvdf membrane and suitable concentration sheep anti mouse secondary antibody marked is washed in 37 DEG C of incubation 2h, then with dH2O 5min, TBST are washed 3 times, each 10min.
5) chromogenic reaction: the pvdf membrane after rinsing is immersed in the substrate solution of Fresh, colour developing (37 DEG C, 35~ 40min), clear to protein band colour developing, i.e., available high purity water rinsing terminates reaction.
6) pvdf membrane is washed down with high purity water, is placed in double-layer filter paper and air-dries preservation.
αs1The SDS-PAGE electrophoresis of casein, skimmed milk, soybean protein isolate is composed as shown in figure 4, swimming lane 1 is Marker, swimming lane 2 are αs1Casein, swimming lane 3 are skimmed milk, and respectively lactotransferrin, serum are white from top to bottom for band Albumen, immunoglobulin, alpha-casein, beta-casein, κ-casein, beta lactoglobulin and α-lactalbumin.Swimming lane 4 is soybean Albumen, band from top to bottom be respectively α ' subunit, α subunit, β subunit, A3,11S acidity chain, 38kDa, 11S alkalinity chain, 14.4kDa。
The protein band on gel is transferred on pvdf membrane by electricity transfer, it is clear then to be observed with amino black dyeing Map, and albumen is fully transferred on film, such as Fig. 5.
To the monoclonal antibody and α of comlete antigens1The specificity of the monoclonal antibody of casein is identified.Pass through Immunoblot experiment is identified, as a result sees Fig. 6 and Fig. 7, the monoclonal antibody and α of comlete antigens1Casein monoclonal antibody With αs1α in casein and skimmed milks1Specific immune response has occurred in casein, and does not have with soybean protein isolate It reacts.Meanwhile in order to exclude false positive reaction, western blot test is carried out with mouse negative serum, as a result such as Fig. 8 It is shown, negative mice serum and αs1Casein, skimmed milk, soybean protein isolate do not react.The result shows that: brand-new Standby comlete antigen and αs1The monoclonal antibody of casein can be with αs1α in casein and skimmed milks1Casein Specific immune response occurs, therefore, freshly prepd antibody can be used for subsequent establishing indirect competitive ELISA method.
Embodiment 3: indirect competitive ELISA method measures hydrolyzate antigen residual quantity
1, single enzymolysis αs1The method of casein
By αs1Casein is configured to the aqueous solution of 3% (w/v), and protease optimum temperature, pH give item according to producer Part, such as table 4.Additive amount is respectively 1500,2000,2500,3000,3500U/g protein, molten with the HCl or NaOH of 1mol/L Liquid adjusts the pH of reaction system to protease optimum pH, hydrolyzes 3 hours.Maintain temperature and the pH of solution permanent in hydrolytic process Fixed, hydrolysis carries out in thermostatic control oscillator vibration, and hydrolyzing terminates 85 DEG C of water-bath enzyme deactivation 10min immediately, cold in cooling postposition refrigerator Freeze use to be analyzed.
The various protease hydrolytic α of table 4s1The optimum condition of casein
2, indirect competitive ELISA method program
(1) antigen coat: the antigen that coating buffer was diluted is added in ELISA Plate with 100 holes μ l/, was placed at 4 DEG C Night.
(2) antigen or sample to be tested and the monoclonal antibody diluted of 1:1 antigen and antibody response: are added in reaction tube.With Antigen or sample to be tested is not added as uncontested reaction system, 4 DEG C of refrigerator overnights.
(3) it washs: in the liquid that second day outwells in ELISA Plate, with PBST board-washing 3 times, each 5min of every 200 μ l of hole, Drying.
(4) it closes: confining liquid is added with 100 holes μ l/ in ELISA Plate, stands 1h at 37 DEG C.
(5) add primary antibody: antigen in step (2) and antibody mixture are added to the ELISA Plate of step (1) with the hole 100ul/ In, in 37 DEG C of incubation 2h, board-washing 4 times, each 5min, dry.
(6) add ELIAS secondary antibody: the sheep anti-mouse igg of the horseradish peroxidase label after dilution is added with the amount in 100 holes μ l/ Into ELISA Plate, 37 DEG C of placement 1h, board-washing.
(7) develop the color: the substrate application liquid of fresh configuration, 100 holes μ l/ are added in ELISA Plate, and room temperature dark place is reacted about 20min。
(8) it terminates reaction: adding the sulfuric acid of 2mol/L, 50 holes μ l/, color is by Lan Bianhuang.
(9) colorimetric: with the light absorption value in each hole in microplate reader dual wavelength measurement ELISA Plate.
3, the determination of the most suitable working concentration of ELIAS secondary antibody
Envelope antigen is diluted to 5 μ g/ml, 100 holes μ l/ are added in ELISA Plate, 4 DEG C overnight, and next day is added with containing 1% The HRP- sheep anti-mouse igg of the PBST gradient dilution of BSA, extension rate are followed successively by 500,1000,2000,4000,8000,16000, One dilution of each column, is separately added into the reacting hole of ELISA Plate.Remaining step is the same as above-mentioned indirect competitive ELISA method journey Sequence.Measure the OD value of each dilution, corresponding concentration when selection is closest to 1.0, so that it is determined that the most suitable work of ELIAS secondary antibody is dense Degree.
It the results are shown in Table 5 and table 6.Using OD value closest to 1 as the most suitable working concentration of ELIAS secondary antibody criterion.Therefore, When primary antibody is comlete antigen monoclonal antibody, the most suitable extension rate of ELIAS secondary antibody is 1000.When primary antibody is αs1When casein monoclonal antibody, The most suitable extension rate of ELIAS secondary antibody is 2000.
The most suitable working concentration for the ELIAS secondary antibody that table 5 is reacted with comlete antigen monoclonal antibody
The most suitable working concentration for the ELIAS secondary antibody that table 6 is reacted with α s1- casein monoclonal antibody
4, the determination of envelope antigen and monoclonal antibody working concentration
Using square matrix titration measuring, steps are as follows:
(1) envelope antigen does gradient dilution, and concentration is 1.25 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml and 20 respectively μ g/ml, one dilution of every row, 100 holes μ l/, 4 DEG C stand overnight, and next day, which seals up, closes fluid-tight and close.
(2) monoclonal antibody does gradient dilution, extension rate is respectively 2500,5000,10000,20000,40000, 80000,160000,320000, one dilution of each column, 100 holes μ l/.Antibody is not added in last column, as blank control.37 DEG C incubate 2h after board-washing.
Remaining step selects OD value 1.0 or so with above-mentioned indirect competitive ELISA method program, the OD value in more each hole Corresponding condition is best antigen and monoclonal antibody working concentration.
On the basis of the working concentration of most suitable ELIAS secondary antibody, with αs1Casein is as antigen, respectively with comlete antigen And αs1The corresponding monoclonal antibody of casein is primary antibody, by indirect competitive ELISA method measure the monoclonal antibodies of different dilutions with not With the reaction OD value of the antigen of concentration, it the results are shown in Table 4.7 and 4.8.
As shown in Table 7, being continuously increased with the monoclonal antibody dilution of comlete antigen, OD value decreases.With OD value is closest to 1.0 as the standard for determining the most suitable working concentration of antigen-antibody, then the most suitable peridium concentration of antigen is 2.5 μ g/mL, The most suitable working concentration of the monoclonal antibody of comlete antigen is 10000.
The determination of the monoclonal antibody working concentration of 7 antigen coat concentration of table and comlete antigen
As shown in Table 8, with αs1Casein monoclonal antibody dilution is continuously increased, and OD value decreases.With OD Being worth closest to 1.0 as the standard for determining the most suitable working concentration of antigen-antibody, then the most suitable peridium concentration of antigen is 2.5 μ g/mL, αs1The most suitable working concentration of the monoclonal antibody of casein is 5000.
8 antigen coat concentration of table and αs1The determination of the monoclonal antibody working concentration of casein
5, the foundation of indirect competitive ELISA suppression curve
On the basis of optimizing envelope antigen and the most suitable working concentration of monoclonal antibody, ELIAS secondary antibody most suitable working concentration, According to indirect competitive ELISA program, respectively with comlete antigen monoclonal antibody and αs1Casein monoclonal antibody is primary antibody, with αs1Casein protein The common logarithm for measuring concentration is abscissa, and Competitive assays rate is ordinate, establishes indirect competitive ELISA standard curve.It is wherein competing It strives inhibiting rate to calculate by OD value, formula such as (2).
Competitive assays rate (%)=B/B0 (2)
B is the α of each respective concentrations1OD value when casein Competitive assays, B0For no αs1When casein Competitive assays OD value.
Detection limit: 10 holes of random selection carry out the detection of zero standard product indirect competitive ELISA, calculate D450The average value of value (D0) and standard deviation (SD), LOD is calculated according to formula (3).
LOD=(D0-2SD)/D0× 100% (3)
Monitoring lower-cut (LOD) (the concrete operations ginseng that corresponding antigenic quality concentration is the method is calculated on standard curve According to: Zhu Yepei, Wang Wei, Lv Qing Qin, Qian Hang, Xu Xinglian, the macro zoo-anaphylactogen bovine serum albumin(BSA) indirect competitive ELISA of period-luminosity Foundation [J] the Agricultural University Of Nanjing journal of detection method, 2016,39 (02): 305-311.).
On the basis of optimizing antigen and the most suitable working concentration of monoclonal antibody, ELIAS secondary antibody most suitable working concentration, establish Indirect competitive enzyme-linked immunosorbent (ELISA) suppression curve.By αs1Casein standard items press 1280ng/mL, 640ng/mL, 320ng/ ML, 160ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 0ng/mL gradient dilution, with αs1Casein quality is dense The common logarithm of degree is abscissa, using Competitive assays rate as ordinate, draws standard curve.As the Dan Ke that primary antibody is comlete antigen When grand antibody, antigen concentration 640ng/mL, 320ng/mL, 160ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL It when with 0ng/mL, establishes standard curve and preferable linear relationship is presented, as shown in figure 9, equation of linear regression is y=-9.22x+ 100.78(R2=0.9806), detection is limited to 10.49ng/mL.When primary antibody is αs1When the monoclonal antibody of casein, antigen is dense Degree be 1280ng/mL, 640ng/mL, 320ng/mL, 160ng/mL, 80ng/mL, 40ng/mL when, establish standard curve present compared with Good linear relationship, as shown in Figure 10, equation of linear regression are y=-23.44x+135.77 (R2=0.9697), detection is limited to 60.50ng/mL。
6, the accuracy and repeatability of method
With batch in error and batch between error evaluate the accuracy and repeatability of the method for building up of the competition inhibition curve.With In batch error and batch between error evaluate the accuracy and repeatability of indirect competitive ELISA method.
When using the monoclonal antibody of comlete antigen as primary antibody, by αs1Casein designs 5 dilution gradient concentration, and each concentration makees 3 Secondary parallel, 3 repetitions, ELISA test carries out under conditions of agents useful for same, reaction condition are identical with detection device, counts Standard deviation is calculated, batch interior error is indicated with the coefficient of variation between its hole, is shown in Table 9, the coefficient of variation is in 1.159%~4.406% range.
It is repeated 3 times in different time with different ELISA Plates.Standard deviation and the coefficient of variation are calculated, with its interassay coefficient of variation Indicate error between criticizing.It the results are shown in Table 10, the coefficient of variation is in 0.826%~4.362% range.
The withinrun precision of 9 comlete antigen monoclonal antibody of table is examined
The betweenrun precision of 10 comlete antigen monoclonal antibody of table is examined
With αs1When the monoclonal antibody of casein is as primary antibody, by αs1Casein designs 5 dilution gradient concentration, each concentration Make 3 parallel, 3 repetitions, ELISA test carries out under conditions of agents useful for same, reaction condition are identical with detection device, Standard deviation is calculated, and batch interior error is indicated with the coefficient of variation between its hole, is shown in Table 11, the coefficient of variation is in 0.828%~5.191% model In enclosing.
It is repeated 3 times in different time with different ELISA Plates.Standard deviation and the coefficient of variation are calculated, with its interassay coefficient of variation Indicate error between criticizing.It the results are shown in Table 12, the coefficient of variation is in 0.099%~4.849% range.
11 α of tables1The withinrun precision of casein monoclonal antibody is examined
12 α of tables1The betweenrun precision of casein monoclonal antibody is examined
The above results, with the monoclonal antibody and α of comlete antigens1When the monoclonal antibody of casein is respectively as primary antibody, illustration method is accurate Property it is good, and repeatability is good, but comparatively, the former accuracy and repeatability is more preferably.
7, the accuracy of reversed-phased high performace liquid chromatographic verifying indirect competitive ELISA method
In order to further evaluate the accuracy of detection method, also using reversed-phased high performace liquid chromatographic respectively to 6 kinds of albumen Enzyme hydrolysis αs1Casein hydrolysate anaphylactogen residual quantity is measured, and is carried out with the testing result of indirect competitive ELISA method Compare
The preparation of standard items stock solution
αs1The preparation of casein standard items stock solution.Prepare the stock solution of 2mg/mL: the α of accurate weighing 20mgs1Junket egg White mark product, constant volume.- 20 DEG C save backup.
Chromatographic condition
Chromatographic column: Agilent Zorbax 300SB-C8 chromatographic column (4.6 × 150mm, 3.5 μm).Mobile phase A is 0.1% TFA/ aqueous solution, the TFA/ACN solution that Mobile phase B is 0.1%, mobile phase is spare after filtering, deaerating.Using linear ladder Degree elution, the initial concentration of Mobile phase B are to rise to 35% in 33%, 5min, rise to 37% by 35% in 5~9min, 40% is risen to by 37% in 9~18min, in 18~22min, Mobile phase B concentration rises to 41% by 40%, then protects 5.5min is held, in 27.5~28min, concentration continues to rise to 43%, and in 28~36min, concentration is risen to by 43% 45%, near 33% in 1min, and keep to 8min.Flow velocity is 0.5m L/min, Detection wavelength 214nm, column temperature 45 DEG C, 10 μ L of sample volume.
The α as measured by reversed-phased high performace liquid chromatographics1Casein mark product map is as shown in figure 11.It is found that αs1Casein Retention time be 20.28min.
The drafting of standard curve
αs1The drafting of casein standard curve.By αs1Casein stock solution is diluted to 0.05,0.1,0.2,0.4,0.8, 1、2mg/mL。
According to above-mentioned chromatographic condition respectively to αs1Casein mark product are detected, to mark quality concentration as abscissa, respectively The corresponding peak area of mass concentration is ordinate, draws standard curve.
According to chromatographic condition, the α of each mass concentration is measureds1Casein mark product, it is as shown in table 13 to obtain its peak area value.
The α of 13 reversed-phased high performace liquid chromatographic quantitative detection different quality concentration of tables1Casein standard items
According to αs1Each mass concentration of casein standard items and its corresponding peak area, with αs1The mass concentration of casein For abscissa, corresponding peak area is ordinate, draws αs1The standard curve of casein is shown in Figure 12, y=9193.1x- 165.92, R2=0.9997.
Rp-hplc determination αs1The antigen residual quantity of casein hydrolysate
Measure 6 kinds of α respectively by reversed-phased high performace liquid chromatographics1Antigen residual quantity (the protease addition of casein hydrolysate Amount is the hydrolysate of 1500U/g), calculate hydrolysate αs1The peak area of casein, and it is fixed according to the standard curve that Figure 12 is established Measure out α in each samples1The content of casein, is shown in Table 14.Each sample does 3 times in parallel, and 3 repetitions take mean value.
α in 14 reversed-phased high performace liquid chromatographic of table and indirect competitive ELISA method measurement hydrolysates1Casein residual quantity (mg/mL)
Protease Protease M Neutral proteinase Compound protease Pepsin Alkali protease Papain
Liquid phase 1.119±0.098 0.912±0.079 0.421±0.053 0.421±0.035 0.109±0.011 0.062±0.006
Comlete antigen monoclonal antibody 1.281±0.125 0.887±0.082 0.447±0.065 0.410±0.045 0.113±0.012 0.058±0.008
Evaluate the accuracy of indirect competitive ELISA method
Using the method for paired-sample t test, α in the method detection hydrolysate to verify foundations1Casein residual quantity The accuracy of (table 14), i.e., the antigen residual quantity for the indirect competitive ELISA method measurement established with monoclonal antibody prepared by comlete antigen Value carry out t inspection with the value of liquid phase measurement respectively, p value is as shown in Table 15.
α in 15 hydrolysate of tables1The t of casein residual quantity examines p value
Protease Protease M Neutral proteinase Compound protease Pepsin Alkali protease Papain
Comlete antigen monoclonal antibody 0.301 -0.205 -0.310 0.132 0.213 0.331
By data in table 15 it is found that p value is greater than 0.05, i.e. reversed-phased high performace liquid chromatographic method and indirect competitive ELISA Method does not have difference to the measured result of same sample.Therefore, the α of foundations1Casein indirect competitive ELISA side The measurement result of method is more accurate, can be used as the theoretical foundation of anaphylactogen kit research and development.
8, hydrolyzate antigen residual quantity is measured
By αs1Antigenic dilution (the carbonate of the 50mmol/L of pH9.6 is added in the different enzymolysis liquid freeze-dried powders of casein Solution) be diluted to suitable concentration, i.e. antigen concentration within the scope of the indirect competitive ELISA suppression curve of foundation and detects It limits in range.According to indirect competitive ELISA method program, using αs1Casein monoclonal antibody and comlete antigen monoclonal antibody are two different anti- Body detects the antigen residual quantity of the hydrolyzate of variety classes enzyme and different enzyme additive amounts as primary antibody.
By unhydrolysed αs1Casein solution is diluted to suitable concentration, i.e. indirect competitive ELISA of the antigen concentration in foundation Within the scope of suppression curve and within the scope of detection limit.According to indirect competitive ELISA method program, using αs1Casein monoclonal antibody and It is as shown in table 16 to detect antigenic content as primary antibody for the two different antibody of comlete antigen monoclonal antibody.
The non-hydrolyzing alpha of table 16s1The antigenic content of casein solution
When comlete antigen monoclonal antibody is primary antibody, six kinds of αs1In casein enzymolysis liquid antigen residual quantity with additive amount increase And the case where reducing, is as shown in Figure 13.The enzymolysis liquid antigen residual quantity of Protease M is maximum, in identical protease additive amount When, residual quantity is significantly higher than other enzymes (p < 0.05), between 1.281-0.364mg/mL.Protease M enzymolysis liquid antigen is residual Allowance substantially reduces (p < 0.05) with the increase of enzyme additive amount.
For neutral proteinase in identical protease additive amount, antigen residual quantity is significantly higher than compound protein in hydrolyzate (p < 0.05) of enzyme, pepsin, alkali protease and papain.The antigen residual quantity of neutral protein enzymatic hydrolysis liquid exists Between 0.887-0.189mg/mL.Increase residual quantity with enzyme concentration in 1500-2000U/g to substantially reduce, in 2500-3500U/g When, the reduction of antigen residual quantity tends towards stability.
For compound protease when enzyme additive amount is 1500U/g, antigen residual quantity is significantly higher than basic protein in hydrolyzate Enzyme and papain this 2 kinds of enzymolysis liquids.But when enzyme concentration is in 2000-3500U/g, antigen residual quantity is significantly higher than stomach cardia This 3 kinds of enzymolysis liquids (p < 0.05) of enzyme, alkali protease and papain.Compound protein digests liquid and increases residual quantity with enzyme concentration Significant (p < 0.05) is reduced, between 0.447-0.088mg/mL.
Antigen residual quantity is greater than alkali protease and pawpaw egg in identical protease additive amount in stomach cardia enzymolysis liquid White enzyme, the significant difference (p < 0.05) when additive amount is 1500U/g, but in 2000-3500U/g, difference is not significant, and with adding The increase residual quantity of enzyme amount reduces not significant.Stomach cardia enzymolysis liquid antigen residual quantity is between 0.410-0.020mg/mL.
The antigen residual quantity of hydrolysis by novo liquid is greater than papain in identical enzyme concentration, but difference is not significant (p>0.05).Basic protein digests liquid and increases residual quantity reduction with enzyme concentration, between 0.113-0.013mg/mL.
The antigen residual quantity that Papain digests liquid is minimum compared to other enzymes in identical enzyme concentration, increases with enzyme concentration Add and reduce, between 0.058-0.010mg/mL.Papain hydrolysis liquid antigen reduced rate (does not hydrolyze antigen residual Amount-measurement hydrolysate residual quantity)/antigen residual quantity is not hydrolyzed, when enzyme additive amount is 1500U/g-3500U/g, antigen drop Low rate is respectively 99.80%, 99.89%, 99.91%, 99.96%, 99.97%.
Work as αs1When the monoclonal antibody of casein is primary antibody, 6 kinds of αs1In casein enzymolysis liquid antigen residual quantity with additive amount increasing The case where adding and reducing, is as shown in Figure 14.The enzymolysis liquid of Protease M resists in hydrolyzate when enzyme additive amount is 1500U/g Former residual quantity is significantly higher than other 5 kinds of enzymolysis liquids, and antigen residual quantity is maximum.But when enzyme concentration is in 2000-3500U/g, antigen Residual quantity is greater than compound protease neutral proteinase and papain this 3 kinds of enzymes significantly less than pepsin, alkali protease Solve liquid.Protease M enzymolysis liquid antigen residual quantity is between 8.280-0.934mg/mL, when enzyme additive amount is 1500-2500U/ Increase when g with enzyme concentration and substantially reduce (p < 0.05), in 2500-3500U/g, residual quantity reduces not significant.
For pepsin in identical protease additive amount, antigen residual quantity is significantly higher than basic protein in hydrolyzate (p < 0.05) of enzyme, compound protease, neutral proteinase and papain.Stomach cardia enzymolysis liquid increases residual quantity with enzyme concentration Significant (p < 0.05) is reduced, between 6.603-1.310mg/mL.
For alkali protease in identical protease additive amount, antigen residual quantity is significantly higher than compound protein in hydrolyzate (p < 0.05) of enzyme, neutral proteinase and papain.Basic protein digests liquid and increases residual quantity reduction significantly with enzyme concentration (p < 0.05), between 4.077-1.195mg/mL.
Compound protein digests liquid antigen residual quantity and is greater than neutral proteinase and pawpaw egg in identical protease additive amount White enzyme.Compound protein digests liquid and increases residual quantity reduction significantly (p < 0.05) with enzyme concentration, between 1.925-0.616mg/mL.
Neutral protein digests liquid antigen residual quantity noticeably greater than papain in identical protease additive amount.It is neutral Protein enzymatic hydrolyzate increases residual quantity with enzyme concentration and reduces significant (p < 0.05), between 1.729-0.310mg/mL.
Papain digests the antigen residual quantity of liquid in identical enzyme concentration significantly less than other enzymes (p < 0.05), with Enzyme concentration increases and reduces, between 0.420-0.172mg/mL.Papain hydrolysis liquid antigen reduced rate, i.e., (do not hydrolyze Antigen residual quantity-measurement hydrolysate residual quantity)/antigen residual quantity is not hydrolyzed, is 1500U/g-3500U/g in enzyme additive amount When, antigen reduced rate is respectively 98.60%, 99.08%, 99.21%, 99.30%, 99.43%.
The monoclonal antibody and α of comlete antigens1The reduction effectiveness ranking of antigen residual quantity in each enzymolysis liquid that casein monoclonal antibody is surveyed Also different.When comlete antigen monoclonal antibody be primary antibody, 6 kinds of protein enzymatic hydrolyzates in identical protease additive amount, antigen residual quantity from It arrives greatly small for Protease M > neutral proteinase > compound protease > pepsin > alkali protease > papain.Work as αs1- When the monoclonal antibody of casein is primary antibody, when enzyme additive amount is 1500U/g, Protease M enzymolysis liquid resists 6 kinds of protein enzymatic hydrolyzates Former residual quantity is significantly higher than other 5 kinds of enzymolysis liquids.But when enzyme concentration is in 2000-3500U/g, 6 kinds of protein enzymatic hydrolyzate antigens are residual Allowance is pepsin > alkali protease > Protease M > compound protease > neutral proteinase > Papain from big to small Enzyme.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Beijing Technology and Business University
<120>monoclonal antibody and milk allergen detection method that the epitope of α s1- casein is prepared
<160> 1
<170> Patent-In 3.3
<210> 1
<211> 23
<212> PRT
<213>artificial sequence
<220>
<223>artificial sequence description: the epitope of α s1- casein
<400> 1
SEEIVPNSVE QKHIQKEDVP SER 23

Claims (10)

1. a kind of αs1The epitope of casein, amino acid sequence SEEIVPNSVEQKHIQKEDVPSER or its both ends Based on αs1Casamino acid sequence and increase 1-5 amino acid.
2. one kind is by α described in claim 1s1The epitope of casein obtains complete with bovine serum albumin(BSA) (BSA) coupling Holoantigen.
3. comlete antigen as claimed in claim 2 is to realize to be coupled by glutaraldehyde method.
4. the preparation method of comlete antigen as described in claim 1, which is characterized in that synthesize institute by solid-phase synthesis first State αs1Casein acts on epitope, and is coupled bovine serum albumin(BSA) (BSA) and prepares comlete antigen, it is preferable that the coupling is It is realized by glutaraldehyde method.
5. a kind of antibody obtained with epitope described in claim 1, especially monoclonal antibody.
6. antibody as claimed in claim 5, which is characterized in that it is by by the αs1Epitope-the BSA of casein Mouse is immunized and the monoclonal antibody that is prepared in comlete antigen.
7. a kind of method for detecting milk allergen, wherein using cow's milk to be measured as test object, using such as claim 5 or with 6 The antibody detects α by indirect competitive ELISA as primary antibodys1Casein or its protease hydrolysate.
8. a kind of detection αs1The method of casein, wherein being to be led to using the antibody such as claim 5 or as described in 6 as primary antibody Indirect competitive ELISA is crossed to detect αs1Casein or its protease hydrolysate.
9. a kind of method for preparing low sensitization milk protein hydrolysate comprising be hydrolyzed with hydrolase to lactoprotein, pass through use Antibody as described in such as claim 5 or with 6 detects α by indirect competitive ELISA as primary antibodys1Casein hydrolysate it is residual Remaining anaphylactogen, and judge aforementioned hydrolysising condition whether be suitable for；Preferential specific steps include: with protease, preferably Papain Lactoprotein is hydrolyzed in enzyme, obtains enzyme hydrolyzate, and freeze-drying prepares freeze-dried powder, and addition antigenic dilution is diluted to suitable dense Degree, according to indirect competitive ELISA method program, using the monoclonal antibody of the above-mentioned comlete antigen of the present invention as primary antibody, detection The antigen residual quantity of the protease hydrolytic liquid of lactoprotein.
10. detecting the kit of milk allergen comprising the antibody as described in such as claim 5 or with 6.
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