CN110317255B - αs1Monoclonal antibody prepared from casein epitope and method for detecting cow milk allergen - Google Patents

Abstract

The invention discloses α with an amino acid sequence of SEEIVPNSVEQKHIQKEDVPSER_s1-an epitope of casein and its use for the preparation of an effective monoclonal antibody, further providing the use of said monoclonal antibody as a primary antibody for the detection of α by indirect competition with the E L ISA_s1Method of using the α of the present invention_s1Preparation of antigenic epitopes of CaseinMonoclonal antibody, detection α by indirect competitive E L ISA method_s1Casein is an antigen, the detection limit of the casein is lower than 10.49ng/m L, the detection method has high accuracy and repeatability, can be effectively used for detecting cow milk allergen, and has popularization and application values.

Description

αs1Monoclonal antibody prepared from casein epitope and bovine lactogenesisAllergen detection method

Technical Field

The invention relates to the field of biotechnology and food, in particular to α_s1Casein epitope, monoclonal antibody prepared by the method, and method for detecting cow milk allergen by using the monoclonal antibody.

Background

Cow's milk is a nutritious protein product and is also one of the eight common allergenic substances (eggs, fish, crustaceans, dairy products, peanuts, soybeans, nuts, wheat), and its allergenicity has a serious impact on the health of infants (MillsE N C, Valovirta E, Madsen C, et al]Allergy, 2004,59(12):1262-]Allergologia et Immunopathologia,2015,43(5):507-]Food Control,2017,74:45-53 whey protein makes up about 20% of milk protein, wherein α -whey protein (Bosd4), β -lactoglobulin (Bosd5), immunoglobulins (Bosd7), bovine serum albumin (BSA, Bosd6) and traces of lactoferrin (Bosd lactoferrin) were found to be allergens whereas α -lactalbumin and β -lactoglobulin are the most important allergens of the whey fraction, accounting for 5% and 10% of total milk protein (Hochwalner H, Schulmeister U, Swoboda I, et al].Methods,2014,66(1):22-33；Docena G H,Fernandez R,Chirdo F G,et al.Identification of casein as the major allergenicand antigenic protein of cow's milk[J]Allergy,1996,51(6): 412-. Casein accounts for about 80% of the total protein of cow's milk, and it has been reported in the literature that about 65% of all cow's milk sensitizers are allergic to casein (momon, quick-boiling, gaxiali, etc.. common allergens in food and their allergenic properties [ J]Chinese food and nutrition, 2008(11):62-64) Casein is prepared from α_s1Casein (32%), α_s2Casein (10%), β -casein (28%) and kappa-casein (10%) of which α_s1Casein is the most important allergen in the casein fraction and accounts for more than 30% of the total protein in cow's milk (Schulmeister U, Hochwallner H, Swoboda I, et al cloning, Expression, and making of Allergenic Determinants of α_s1-Casein,a Major Cow's Milk Allergen[J].TheJournal of Immunology, 2009,182(11):7019-7029)。α_s1Casein is a single-chain phosphorylated protein consisting of 199 amino acids, has a high proline content, no disulfide bonds, and a relatively simple tertiary structure (Kumosinskit F, Brown E M, Farrell H M.Three-Dimensional Molecular Modeling of BovineCasein: α)_s1-Casein[J]Journal of Dairy Science,1991,74(9):2889-_s1Casein or its larger IgE-reactive fragments are responsible for the allergic reaction (Schulmeister U, HochwallnerH, Swoboda I, et al. cloning, Expression, and Mapping of Allergic scavenging of α_s1-Casein,a Major Cow's Milk Allergen[J].The Journal of Immunology,2009,182(11):7019-7029)。

Cow Milk allergen caused allergic reaction is an IgE-mediated and non-IgE-mediated hypersensitivity reaction which may cause reactions of skin, gastrointestinal tract, respiratory system, and even cause systemic anaphylaxis or shock in severe cases (Alessandro F, JanB, Holger S, et al. World Allergy Organization (WAO) Diagnosis and ratio for active ingredient Cow Milk Cow Milk' S Milk Allergy (DRACMA) Guidelines [ J ]. Pediatric Allergy is the most common Food Allergy in infancy and childhood (Sicher S H, Sampson H. Food biscuit: epidemic, Cow Milk Allergy, Cow Milk, diet, Cow Milk, Cow Milk, Cow Milk, Cow.

There are a lot of reports on the reduction of antigenicity of allergenic proteins in hydrolyzed milk, but because proteases all have specific hydrolysis sites, the reduction of antigenicity of hydrolysates is not directly proportional to the increase of degree of hydrolysis. Even if the hydrolysate has a large degree of hydrolysis and a small molecular weight, it is still allergic as long as the epitope is not destroyed. And a large amount of free amino acid is inevitably produced along with the increase of the hydrolysis degree, so that the osmotic pressure in the body of the infant is increased after the infant eats the feed, and further discomfort is caused. Therefore, in order to prepare high-quality milk protein hydrolysate, the hydrolysis degree must be controlled well, the mass fraction of small peptides in the hydrolysate is increased, and the proportion of free amino acids is reduced or controlled. Although moderately hydrolyzed milk protein hydrolysates have been used in the production of infant milk formula, due to the lack of specificity of the protease hydrolysis allergen function epitope, milk protein hydrolysates are still allergenic, and in order to avoid problems of protein processing and changes in physiological functional properties due to blind hydrolysis, it is desirable to develop methods that can effectively detect the residual amount of antigen after hydrolysis of milk proteins.

The detection of allergens is developed to The high sensitivity, convenience, rapidness and high accuracy With The development of new technology, but some problems still exist in The practical application process, when PCR technology is used, related gene fragments are obtained in food, technical requirements are high, instruments and equipment are expensive, certain constraints are imposed on The application (non-Protein A J. food allergen detection methods and The passage to detect food-allergen Conjugates [ J ]. Analytical and biological Chemistry,2007,389(1): 111) 118. similarly, capillary electrophoresis technology and instrument analysis methods are not suitable for rapid detection of cow milk Protein by using reverse phase high efficiency liquid chromatography analysis and have some researches (El-Protein, Horvar V.in, B-Protein B-19) for preparing allergen specific Protein, and The preparation method is also applicable to The detection of cow milk Protein by using biological antigen specificity Protein (Protein) 1, Protein J. 12. A. 12. A. 7. A. The preparation of allergen specific Protein is also relates to detect bovine allergen specific Protein, a Protein.

To sum up, although cow's milk protein hydrolysate has been applied to the development of infant formula powder, there are still problems of poor hydrolysis site specificity, high allergen residue, and heavy bitter taste of hydrolysate α_s1Casein is a main component of casein, is an important allergen in cow milk, occupies more than 30% of the total protein of cow milk, and can seriously affect physical and mental health after being ingested by infants with allergic reaction, and strong allergic reaction can be generated by cow milk allergic patients after being ingested in a trace amount. Therefore, the research of establishing an accurate and rapid detection method for allergen residue, screening the protease for effectively reducing the allergen allergenicity and optimizing the hydrolysis process is urgent.

Disclosure of Invention

Aiming at the defects and the practical requirements of the prior art, the invention aims to seek to establish a precise and rapid detection method for allergen residue, which technically comprises the selection of an epitope, the preparation of a monoclonal antibody and the establishment of the detection method.

The inventor previously identified α through in vitro test identification_s1The IgE-acting epitopes of casein include aa 141-155, aa 21-35, aa 91-105, aa 186-200, aa 26-40 (Yanjun Cong, et al identification of the diagnostic amino acids residues of immunoglobulin E and immunoglobulin Gepitopes on α (s1) -casein by albumin screening analysis [ J1 ]]Journal of Dairy science,2013,96(11):6870-6876 Ruiter et al (Ruiter, B.et al. Characterisation of Tcell epitopes in α s1-casein in cow's milk a)Legend, atopic and non-atopicschildren. Clin. exp. allergy 36: 303- & 310) adopts 32 overlapping peptides to analyze T cell recognition epitopes, wherein the 43-66, 73-96, 91-114 and 127-180 peptide chains can be recognized by T cells of 3 patients, but considering the sensitization strength (the recognition rate of aa 91-105 is 100%) and the conservation (avoiding the similarity of the sequences with other proteins), the inventor combines the reported main effect epitopes aa 73-96, 91-114 and aa 91-105, and considering the characteristic of the conformational distribution of the epitopes on the surface of the allergen, selects α_s1SEEIVPNSVEQKHIQKEDVPSER (aa 83-105) in casein as hapten. Protean program using DNAStar software predicts hydrophilicity of sequence>0, indicating that the sequence has relatively high hydrophilic property; surface accessibility in simultaneous sequence is for the most part>1, indicating that folding is easily occurred while being exposed to the outside; antigen index number average>0, indicating that this sequence has a high probability of forming an epitope (El-Manzalaw Y, Honavar V.Recent advances in B-cell epitope prediction methods [ J ]]Immunomesearch, 2010,6(2): S2.). Therefore, the invention selects and synthesizes SEEIVPNSVEQKHIQKEDVPSER (aa 83-105) as hapten, and subsequent researches show that the hapten has very good effect.

Accordingly, a first aspect of the present invention is to provide α_s1An epitope of casein having the amino acid sequence SEEIVPNSVEQKHIQKEDVPSER, or both ends based on α_s1Casein to 1-5 amino acids (i.e. both ends of the added amino acid with α)_s1The corresponding sequences in the casein are identical), more particularly 1, 2, 3, 4 and 5 amino acids may be added. The epitope can be used to prepare effective monoclonal antibodies.

The second aspect of the present invention is to provide a method of the α_s1Complete antigens obtained by coupling the antigenic epitopes of casein with Bovine Serum Albumin (BSA).

The third aspect of the present invention provides the above complete antigen preparation method, wherein α above is first synthesized by solid phase synthesis method_s1Casein acts on epitopes and is coupled to Bovine Serum Albumin (BSA) to prepare complete antigens. Further preferably, said pairThe coupling is realized by a glutaraldehyde method.

A fourth aspect of the present invention provides a method for addressing α above_s1Antibodies, in particular monoclonal antibodies, to epitopes of casein more preferably by combining said α_s1Experiments show that when the monoclonal antibody is used as a primary antibody, the sensitivity, the accuracy and the repeatability of the established indirect competition E L ISA method are high, and the method is relative to α_s1For preparing monoclonal antibody by immunizing Balb/c mouse with casein holoprotein, the indirect competitive E L ISA method established by the monoclonal antibody has lower detection limit, namely higher sensitivity, and simultaneously, the prepared complete antigen monoclonal antibody and α_s1The casein whole protein monoclonal antibody can be combined with α_s1α in casein and skim milk_s1Casein is specifically immunoreactive, not cross-reacting with soy protein isolate.

A fifth aspect of the invention provides a test α_s1Method for casein, wherein α of the invention described above is used_s1Monoclonal antibodies against epitopes of casein as primary antibodies, detected α by indirect competition with the E L ISA_s1The method of operation of indirect competition for the E L ISA can be carried out according to conventional methods.

Furthermore, the present invention provides a method for detecting cow's milk allergens, wherein the cow's milk to be detected is used as the detection object, and α of the present invention is used_s1Monoclonal antibodies to epitopes of casein as primary antibodies, detection of α in cow's milk by indirect competition with E L ISA_s1α is known from the test_s1-the amount of allergen remaining from enzymatic hydrolysate of casein in order to determine the quality of cow's milk or whether it is suitable for preparing milk powder, especially infant formula.

The present invention further provides a process for producing hypoallergenic milk protein hydrolysate, which comprises hydrolyzing milk protein with a hydrolase by using α of the present invention described above_s1Monoclonal antibodies to epitopes of casein as primary antibodies, by indirect competition with the E L ISATo detect α_s1The more specific steps include hydrolyzing milk protein with protease, preferably papain, to obtain an enzyme hydrolysate, freeze-drying to obtain a lyophilized powder, adding an antigen diluent to dilute to a suitable concentration, and detecting the antigen residue of the protease hydrolysate of milk protein by using the monoclonal antibody of the complete antigen as a primary antibody according to the procedure of indirect competitive E L ISA method.

α utilizing the present invention_s1Casein epitope, monoclonal antibody prepared, superior to α_s1Monoclonal antibody obtained by using casein holoprotein as antigen α is detected by indirect competition E L ISA method_s1Casein as antigen, α of the invention_s1Monoclonal antibodies prepared with epitopes of casein having detection limits lower than 10.49ng/m L and α_s1When monoclonal antibody of casein holoprotein is used as primary antibody, the detection limit is 60.50ng/m L, and the detection limit of the primary antibody has obvious advantages_s1The optimal working concentration of monoclonal antibodies prepared from epitopes of casein is 10000-fold dilution, whereas α_s1The optimal working concentration of the monoclonal antibody of the casein holoprotein is diluted by 5000 times, and the accuracy and the repeatability of the indirect competition E L ISA method established by the method are excellent.

Drawings

FIG. 1 is an HP L C map of a synthetic polypeptide.

FIG. 2 MS spectra of synthetic polypeptides.

FIG. 3 UV scanning spectra of synthetic polypeptides, BSA and complete antigen.

Wherein, the curve 1 shows the ultraviolet spectrum of the synthesized polypeptide, and the maximum absorption peak is at 196 nm. Curve 2 shows the BSA UV spectrum with a maximum absorption at 278 nm. Curve 3 shows the complete antigen UV spectrum with absorption peaks at both 196nm and 278 nm.

FIG. 4 SDS-PAGE electrophoresis of soy protein isolate

Wherein, lane 1 is Marker, lane 2 is α s1-casein, lane 3 is skim milk, and lane 4 is soy protein isolate.

FIG. 5 is an amino black dye image of a PVDF film after transfer.

FIG. 6 immunoblotting of complete antigen mAbs

Wherein 1 represents α s1-casein, 2 represents skim milk, and 3 represents soy protein isolate.

FIG. 7 immunoblot of 7 α s1-Casein mAb

Wherein 1 represents α s1-casein, 2 represents skim milk, and 3 represents soy protein isolate.

FIG. 8 immunoblot of negative sera (negative control)

Wherein 1 represents α s1-casein, 2 represents skim milk, and 3 represents soy protein isolate.

FIG. 9 Indirect competitive E L ISA inhibition curve for the complete antigen mab.

FIG. 10 α_s1Indirect competition for casein mab E L ISA inhibition curve.

FIG. 11 α_s1-reversed phase high performance liquid chromatogram of casein standard.

FIG. 12 is a standard curve of α s1-casein standard measured by reversed phase high performance liquid chromatography.

FIG. 13 residual amount of hydrolysate antigen determined by complete antigen monoclonal antibody.

FIG. 14 α_s1-residual quantity of hydrolysate antigen determined by casein monoclonal antibody.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to the following embodiments with reference to the attached drawings, and advantageous effects of the present invention will be further described with reference to the embodiments.

Example 1: complete antigen preparation of antigenic epitopes

1. Synthesis and identification of haptens, i.e. polypeptides

Selection α based on analysis and study_s1SEEIVPNSVEQKHIQKEDVPSER (aa 83-105) in casein is used as hapten, an Fmoc solid phase peptide synthesis method is adopted, C-terminal amino acid is connected to Wang resin, the conventional Fmoc method is adopted for gradual condensation, after the synthesis is finished, a sequence is cut from a solid phase carrier by strong acid, and the product is purified by HP L C, identified by mass spectrum and frozen and dried for later use, wherein the specific operation method comprises the steps of referring to Junyan, administration, cloud fighter, α_s1Recognition of the Casein IgE epitope [ J]Food science, 2010,31(17): 232-.

The method comprises the steps of adopting high performance liquid chromatography and mass spectrometry to analyze, adopting a C18 column (4.6mm × 150mm, 5 mu m) as a chromatographic column, adopting a trifluoroacetic acid aqueous solution with the volume fraction of 0.1% as a mobile phase A, adopting acetonitrile with the volume fraction of 80% and water with the volume fraction of 20% as a B, adding trifluoroacetic acid with the volume fraction of 0.09%, adopting a gradient elution program according to the volume fraction, namely, adopting the mobile phase B with the volume fraction increased from 15% to 45%, adopting the flow rate of 1.0m L/min and the detection wavelength of 220nm, and adopting the column temperature of 20 ℃.

The mass spectrum conditions comprise ESI ion source adopted, spraying pressure of 15psi, drying gas temperature of 350 ℃, flow rate of 5L/min and scanning mass range of 500-2200 m/z.

The synthesized polypeptide is purified by high performance liquid chromatography, and the purity can reach 92.664%, as shown in figure 1. Meanwhile, the relative molecular mass of the polypeptide is determined by mass spectrometry (in order to increase the linking site at the C end of the polypeptide, one more lysine is synthesized at the C end, so the relative molecular mass of one lysine is added to the molecular mass of the polypeptide determined by mass spectrometry). FIG. 2 shows an MS map of the synthesized sequence SEEIVPNSVEQKHIQKEDVPSER, with the relative molecular mass of the polypeptide being 2781.1, indicating that the correct polypeptide was synthesized.

2. Preparation and characterization of complete antigens

The purified synthetic polypeptide is coupled with Bovine Serum Albumin (BSA) by a glutaraldehyde method to obtain a complete antigen (the operation method refers to Guobai Swallow, an enzyme immune technology in immunological test [ J ]. China test medical journal, 2005,28(2): 221-224; Tenghai, Yuyan, Luling, and the like; the glutaraldehyde method is used for synthesizing the huperzine A complete antigen and identifying the huperzine A complete antigen [ J ]. Fujian Chinese medicine university journal, 2012,22(06): 41-44.).

Dissolving 15mg of purified synthetic polypeptide in 6m L0.01.01 mol/L phosphoric acid buffer solution, adding BSA25.5mg, stirring under ice bath, adding 1.5m L0.2.2% glutaraldehyde, adding 1.9m L1 mol/L glycine after 60min, continuing stirring for 60min, and separating and purifying the prepared polypeptide-BSA compound by Sephadex G200 to prepare freeze-dried powder for later use.

The coupling ratio of the complete antigen was determined by UV spectrometry. Synthesizing polypeptide, BSA, and the complete antigen after coupling the polypeptide and the BSA can generate obvious absorption peaks in an ultraviolet region. And preparing the synthetic polypeptide, BSA and complete antigen into a solution with a certain concentration by using PBS, and respectively performing spectrum scanning at the full wavelength of 190-1100 nm. Colorimetric determination of the absorbance values of m, n (absorbance, A), i.e., AA, of the synthesized polypeptide (A), BSA (B) and the complete antigen (C) at the maximum absorption peak wavelengths of A and B, respectively_m、AA_n、AB_m、AB_n、AC_m、AC_n. Calculating molar extinction values of the substance A and the substance B at the absorption peak wavelength of A, B substance respectively, namely kA, according to the formula A ═ kcl (A represents absorbance, c represents the concentration of the light absorbing substance, l represents the absorption optical path of the sample, namely the thickness of the absorption layer, and k is the molar absorption coefficient) according to Lambert-beer law_m、kA_n、 kB_m、kB_n. Calculating the molar ratio of the A substance to the B substance (i.e. the coupling ratio of the synthetic polypeptide to BSA) according to the formula (1):

molar ratio (AC)_m·kB_n－AC_n·kB_m)/(AC_n·kA_m－AC_m·kA_n) (1)

The synthetic polypeptide, BSA and complete antigen are respectively subjected to spectrum scanning at the full wavelength of 190-1100 nm, and the result is shown in figure 3, wherein the carrier protein BSA has a maximum absorption peak at 278nm, and the synthetic polypeptide has a maximum absorption peak at 196 nm. The coupled BSA polypeptide has characteristic absorption peaks of synthetic polypeptide and BSA at 190-1100 nm and has corresponding spectrum superposition conditions. Therefore, the primary coupling between the synthetic polypeptide and BSA was successful.

The UV absorbance values of the synthetic polypeptide, BSA and complete antigen at the maximum absorbance peaks of the synthetic polypeptide and BSA, respectively, were determined as shown in Table 1. The molar ratio of synthetic polypeptide to BSA, i.e.the coupling ratio, was calculated according to formula (1) to be 6.31.

TABLE 1 ultraviolet absorbance of synthetic Polypeptides, BSA, complete antigens

Example 2: preparation and characterization of monoclonal antibodies

α for this embodiment_s1Complete antigen immunization of Casein sensitization epitope to Balb/c mice, preparation of monoclonal antibody, Simultaneous preparation of α_s1The titer of the monoclonal antibody is detected by an indirect E L ISA method, and the specificity of the antibody is identified by an immunoblotting method.

1. Preparation of monoclonal antibodies

The SEEIVPNSVEQKHIQKEDVPSER-coupled BSA complete antigen prepared in the first example is used for immunizing Balb/c mice, a subcutaneous multi-point injection method is adopted, the third non-immunized mouse is subjected to tail breaking and blood sampling to detect the serum titer, the immunization is continued if the titer does not reach the standard, the immunization is strengthened after the titer reaches the standard, and the cell fusion is carried out after the immunization is strengthened for 3 days. The tail vein injection method is adopted for strengthening the immunity.

After four times of immunization, taking a casein mouse for strengthening the immunity, collecting blood from an orbit, dislocating and killing the mouse, standing the blood at room temperature for 2h, standing overnight at 4 ℃, centrifuging the next day to take supernatant for detection, taking the spleen of the mouse for strengthening the immunity, removing connective tissue, preparing spleen cell suspension, counting cells for later use, taking myeloma cells with good cell state, mixing the spleen cells and the myeloma cells 5-10:1, centrifuging for 4min at 1500r/min, discarding supernatant culture solution after centrifuging, knocking out mixed cells at the bottom of a centrifuge tube, bathing in water for 1min at 40 ℃ in a beaker, slowly adding PEG (molecular weight 1500) preheated at 1m L ℃ into the mixed cells, adding the PEG within 40 s, bathing in water for 30 s after adding the PEG, then adding 1640 basic culture solution, requiring that 3m L1640 culture solution is added within 1min, finally adding 50m L, centrifuging for 4min at 800r/min, discarding supernatant 1640, and adding the basic 1640Centrifuging the culture solution for 4min at 800r/min, removing supernatant, slowly adding HAT culture solution to desired volume, resuspending cells, mixing, adding into prepared feeder cell plate (100 μ L per well), wiping cell culture plate with alcohol cotton, sterilizing, standing at 37 deg.C and 5% CO₂In the incubator, the fusion effect was observed after 5 days.

And (3) observing the cell fusion effect 5 days after the screening and fusion of the positive hybridoma cells, observing and recording the cell stack number in each hole of the cell culture plate under an inverted microscope, sucking cell supernatant when the cell stack size is about 1/10 of the hole of the cell culture plate after the cell liquid is changed, and detecting the antibody titer of the cell supernatant by using an E L ISA method.

Screening positive hybridoma cell, making several times of subcloning, making liquid paraffin sensitized Balb/c mouse, 6-8 weeks, after 7-10 days preparing ascites, collecting hybridoma cell, injecting 100-150 ten thousand cells into mouse abdominal cavity, after one week it can be seen that mouse state is inactive and mouse abdominal cavity is swollen, after one week injecting cell, using sterile injector to collect ascites into mouse abdominal cavity, collecting every two days, killing after 3 times of collecting ascites, testing titer and inhibiting, packaging and freezing and storing so as to obtain a monoclonal antibody with complete antigen, at the same time preparing α according to same method_s1Monoclonal antibodies to casein as control.

Using mouse Ig G₁、Ig G₂、Ig G_2aAnd Ig E L ISA kit identification, and the operation according to the instruction book identifies that the two monoclonal antibodies have IgG subtype₁。

2. Determination of the potency of monoclonal antibodies

The titer of the monoclonal antibody prepared above is determined by indirect enzyme-linked immunosorbent assay (E L ISA).

The antigen coating concentration is 5 mug/m L determined by a matrix method, monoclonal antibodies are diluted by 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000 and 1:640000 times in sequence, and ascites of normal mice which are not immunized is used as a negative control.

(1) Antigen coating: the antigen diluted in the coating solution was added to the microplate at 100. mu.l/well, and left overnight at 4 ℃.

(2) Washing: the next day, the liquid in the microplate was decanted, and the plate was washed 3 times with 200. mu.l per well of PBST and spun-dried.

(3) And (3) sealing: blocking by adding 100. mu.l of blocking solution to the microplate at a well, and allowing the mixture to stand at 37 ℃ for 1 hour.

(4) Adding a primary antibody: the monoclonal antibodies of different dilutions were added to the microplate in an amount of 100. mu.l/well (1), incubated at 37 ℃ for 2h, and the plate was washed.

(5) Adding an enzyme-labeled secondary antibody: adding the diluted horseradish peroxidase-labeled goat anti-mouse IgG into an enzyme label plate in the amount of 100 mu l/hole, standing for 1h at 37 ℃, and washing the plate.

(6) Color development: and adding the substrate application solution which is prepared freshly into the ELISA plate at a concentration of 100 mu l/hole, and reacting for about 20min at normal temperature in the dark.

(7) The reaction was stopped by adding 2 mol/L of sulfuric acid at 50. mu.l/well and changing the color from blue to yellow.

(8) Color comparison: and (3) measuring the absorbance value of each hole in the ELISA plate at the single wavelength of 450nm by using an ELISA reader.

Preparation of reagents required for indirect E L ISA:

(1) PBS 0.02 mol/L phosphate buffer, pH7.4.

(2) PBST: PBS containing 0.05% Tween-20.

(3) Sealing liquid: PBST with 1% BSA.

(4) Antigen diluent 50 mmol/L carbonate solution with pH 9.6.

(5) The substrate application solution was prepared from 10M L0.1.1M phosphate buffer pH6.0, 100. mu.l TMB application solution (prepared by dissolving 60mg TMB in 10M L dimethyl sulfoxide), and 15. mu.l 30% hydrogen peroxide.

The indirect E L ISA method is used to determine the complete antigen monoclonal antibody titer, the results are shown in Table 2, P represents the antibody OD value, N represents the OD value of the mouse ascites without immunity, the detection results are judged to be positive by P/N value >2.1 and P > 0.2, the detection results are judged to be negative by P/N value <2.1 or P < 0.2, the results are shown in Table 2, when the antibody dilution multiple is 320000 or more, the P/N value is greater than 2, but when the dilution rate is 640000, the antibody OD value is < 0.2, therefore, the complete antigen prepared monoclonal antibody titer can reach 320000, and can be used for subsequent research.

TABLE 2 OD value of full antigen monoclonal antibody titer

α of the indirect E L ISA method described above_s1The titer of the casein mab, P the OD of the antibody and N the OD of the ascites in the non-immunized mouse. The P/N value is used for detection>2.1 and P > 0.2, the detection result is judged to be positive, and the P/N value<2.1 or P < 0.2, the result was negative, and the result is shown in Table 3, where the P/N value was greater than 2 at a dilution of 320000 or more, but the OD value of the antibody was < 0.2 at a dilution of 640000, and thus was determined to be α_s1The casein antibody titer can reach 320000, and the casein antibody can be used for subsequent research.

TABLE 3 α_s1OD value of Casein monoclonal antibody titer

3. Identification of antibody specificity by immunoblotting

(1)SDS-PAGE

Preparing skim milk, centrifuging at 4 deg.C for 30min at 4000r/min to float fat, removing floating fat, repeating for several times to obtain skim milk, diluting with appropriate dilution, and mixing with α_s1Casein, soy protein isolate solution together as sample protein, to be tested.

Preparing 12.5% separation gel and 4.5% concentration gel, the degree of crosslinking is 3.6%, mixing the sample treatment solution with the sample protein at a ratio of 1:1, adding bromophenol blue indicator, mixing, boiling in a water bath for about 5min, airing to room temperature, and then loading, wherein the loading amount of each lane is 20 mu L.

Vertical electrophoresis, constant current 20 mA. And stopping electrophoresis when the bromophenol blue indicator migrates to about 1cm below the gel, and turning off the power supply. The splint was removed from the electrophoresis apparatus and the gel was removed. Taking out, dyeing with Coomassie brilliant blue G-250 staining solution for 30min, decolorizing with decolorizing solution for several times until protein bands in gel are clear, taking out gel, and analyzing relative molecular mass with gel imaging system.

(2) Electrotransfer

(1) Cutting a polyvinylidene fluoride membrane (PVDF membrane) to the size of separation gel in SDS-PAGE gel, marking the positions of teeth of a sample-adding comb on the non-smooth surface of the PVDF membrane by using a pencil, soaking in absolute methanol for 3-5 s, then soaking in an electrotransformation buffer solution for 10min, cutting seven pieces of filter paper with the size of about 6 × 8cm, and soaking seven pieces of filter paper, electrotransformation sponge and SDS-PAGE separation gel after electrophoresis in the electrotransformation buffer solution for 10 min.

(2) The clamp for electric transfer is opened and laid on a clean and flat experiment table, the black grid is kept horizontal below, the treated sponge is laid on the surface of the black grid, and a smooth glass rod is rolled from one side to the other side to expel air bubbles. Three soaked filter papers are laid on the sponge cushion to expel air bubbles. The gel was carefully placed on the filter paper. The PVDF membrane was then carefully placed on the gel side. Then spreading three layers of soaked filter paper on the membrane, finally spreading another piece of soaked sponge, putting down a white grid, clamping the 'gel sandwich', putting into an electrophoresis tank, and filling with an electrotransfer buffer solution. During operation, each layer needs to be carefully placed, and air bubbles are continuously removed, so that the air bubbles are prevented from influencing the result. Namely, the film transfer and stacking sequence is as follows: gel splint (white grid) → sponge → three layers of filter paper → PVDF film → gel → three layers of filter paper → sponge → gel splint (black grid).

(3) And (3) placing the whole electrophoresis tank in an ice bath, enabling the PVDF film to be close to the anode, enabling the gel to be close to the cathode, starting a power supply to start electrophoresis and film transfer, keeping constant current at 200mA, and performing electrotransfer for 2h (paying attention to cooling).

(4) After transfer, the "gel sandwich" was removed, the PVDF membrane was cut out from the middle in duplicate, one portion was stained with amino black 10B staining solution for 5min, and the band on the membrane was decolorized until it was visible, and the transfer effect and the position of the protein band were observed, and the other portion was used for immunoblotting.

(3) Immunoblotting

1) Transferring the PVDF membrane into a plate with proper size and using dH₂O washing for 5min, rinsing with TBST at 37 deg.C for 15min for 4 times, and discarding the liquid.

2) Placing into a dish with sealing liquid (10-20 m L), quenching for 1h at 37 ℃, sealing the PVDF membrane, and then using dH₂And washing for 5min by using an O washing machine.

3) PVDF membrane and appropriate concentration of monoclonal antibody at 4 degrees C overnight, and then dH2O washing 5min, TBST washing 3 times, each time 10 min.

4) PVDF membrane and appropriate concentration of HRP labeled goat anti mouse secondary antibody at 37 degrees C were incubated for 2h, then dH2O washing 5min, TBST washing 3 times, each time 10 min.

5) And (3) color development reaction: immersing the rinsed PVDF membrane in a freshly prepared substrate solution, developing (37 ℃, 35-40 min), rinsing with high-purity water when the protein band develops clearly, and stopping reaction.

6) The PVDF membrane is washed clean by high-purity water and placed in double-layer filter paper for air drying preservation.

α_s1SDS-PAGE of casein, skim milk, soy protein isolate is shown in FIG. 4, Marker in lane 1 and α in lane 2_s1Casein, lane 3 skim milk, lactoferrin, serum albumin, immunoglobulin, α -casein, β -casein, kappa-casein, β -lactoglobulin, and α -lactalbumin, respectively, from top to bottom, lane 4 soy protein, and lanes α' subunit, α subunit, β subunit, A3, 11S acidic chain, 38kDa, 11S basic chain, and 14.4kDa, respectively, from top to bottom.

The protein band on the gel was transferred to the PVDF membrane by electrical transfer, and then a clear pattern was observed by amino black staining, and the protein was completely transferred to the membrane, as shown in fig. 5.

Monoclonal antibodies to the complete antigen and α_s1-caseinThe specificity of the monoclonal antibody against the white was identified by immunoblotting, the results are shown in FIGS. 6 and 7, and the monoclonal antibody against the complete antigen and α_s1Monoclonal antibodies to casein α_s1α in casein and skim milk_s1The immunoblotting test was carried out using mouse-negative serum to eliminate false-positive reaction, and the results are shown in FIG. 8, in which the mouse-negative serum was mixed with α_s1Casein, skim milk, and soy protein isolate were not reacted the results show that the newly prepared complete antigen was α_s1Monoclonal antibodies to casein can be reacted with α_s1α in casein and skim milk_s1Casein is specifically immunoreactive, so the newly prepared antibody can be used for the subsequent establishment of indirect competition E L ISA methods.

Example 3 determination of residual quantity of antigens in hydrolysate by Indirect competitive E L ISA method

1. Single enzyme hydrolysis α_s1Method for producing casein

α will be mixed_s1Casein is prepared into 3% (w/v) aqueous solution, the optimal action temperature and pH of protease are determined according to the conditions given by manufacturers, as shown in Table 4, the addition amounts are 1500, 2000, 2500, 3000 and 3500U/g protein respectively, 1 mol/L HCl or NaOH solution is used for adjusting the pH of the reaction system to the proper pH value of the protease, the hydrolysis is carried out for 3 hours, the temperature and the pH of the solution are kept constant in the hydrolysis process, the hydrolysis is carried out in a constant-temperature water bath oscillator, the hydrolysis is carried out immediately at 85 ℃ in a water bath for inactivating the enzyme for 10min, and the solution is placed in a refrigerator for freezing to be analyzed after cooling.

TABLE 4 hydrolysis of various proteases α_s1Optimum conditions for casein

2. Indirect competition E L ISA method program

(1) Antigen coating, the antigen diluted by the coating solution is added into an enzyme label plate at a rate of 100 mu l/hole, and is placed at 4 ℃ overnight.

(2) And (3) reacting the antigen and the antibody, namely adding 1:1 of antigen or a sample to be detected and the diluted monoclonal antibody into the reaction tube. The non-antigen or the sample to be tested is used as a non-competitive reaction system and is kept in a refrigerator at 4 ℃ overnight.

(3) And (3) washing, namely pouring the liquid in the enzyme label plate on the next day, washing the plate for 3 times by using PBST (Poly-p-phenylene-succinate) with the volume of 200 mu l per hole, and drying by spinning for 5min each time.

(4) Blocking, adding blocking liquid into the ELISA plate at a rate of 100 μ l/hole, and standing at 37 deg.C for 1 h.

(5) Adding a primary antibody: and (3) adding the mixture of the antigen and the antibody in the step (2) into the enzyme label plate in the step (1) at the rate of 100 ul/hole, incubating for 2h at 37 ℃, washing the plate for 4 times, each time for 5min, and drying by spinning.

(6) Adding an enzyme-labeled secondary antibody: the diluted goat anti-mouse IgG labeled with horseradish peroxidase was added to an ELISA plate in an amount of 100. mu.l/well, and the plate was washed after standing at 37 ℃ for 1 hour.

(7) Color development: and adding the substrate application solution which is prepared freshly into the ELISA plate at a concentration of 100 mu l/hole, and reacting for about 20min at normal temperature in the dark.

(8) The reaction was stopped by adding 2 mol/L of sulfuric acid, 50. mu.l/well and changing the color from blue to yellow.

(9) Color comparison: and (4) measuring the light absorption value of each hole in the ELISA plate by using a double-wavelength ELISA reader.

3. Determination of optimum working concentration of enzyme-labeled secondary antibody

Diluting the coating antigen to 5 mu g/ml, adding 100 mu l/hole into an enzyme label plate, keeping the temperature overnight at 4 ℃, adding HRP-goat anti-mouse IgG which is diluted by PBST containing 1% BSA in a gradient manner the next day, wherein the dilution times are 500, 1000, 2000, 4000, 8000 and 16000 in turn, and adding one dilution in each row into a reaction hole of the enzyme label plate respectively.

The results are shown in tables 5 and 6. And taking the OD value closest to 1 as a determination standard of the optimal working concentration of the enzyme-labeled secondary antibody. Thus, when the primary antibody is a complete antigenWhen the monoclonal antibody is used, the optimal dilution multiple of the enzyme-labeled secondary antibody is 1000, and when the primary antibody is α_s1When the monoclonal antibody is casein, the optimal dilution factor of the enzyme-labeled secondary antibody is 2000.

TABLE 5 optimal working concentration of enzyme-labeled Secondary antibody reacting with complete antigen monoclonal antibody

TABLE 6 optimal working concentration of enzyme-labeled Secondary antibody reacted with α s1-Casein monoclonal antibody

4. Determination of working concentration of coating antigen and monoclonal antibody

The method adopts a matrix titration method for determination and comprises the following steps:

(1) the coating antigen is diluted in a gradient way, the concentration is respectively 1.25 mug/ml, 2.5 mug/ml, 5 mug/ml, 10 mug/ml and 20 mug/ml, each line has one dilution degree, 100 mug/ml, the mixture is kept stand overnight at 4 ℃, and the next day is sealed by a sealing solution.

(2) The monoclonal antibody is diluted in a gradient way, the dilution times are 2500, 5000, 10000, 20000, 40000, 80000, 160000 and 320000 respectively, and each column has one dilution degree and 100 mul/hole. The last column was blank without antibody. Plates were washed after 2h incubation at 37 ℃.

The rest steps are the same as the procedure of the indirect competition E L ISA method, the OD value of each hole is compared, and the condition that the OD value is about 1.0 corresponds to the optimal working concentration of the antigen and the monoclonal antibody is selected.

α on the basis of the optimal working concentration of the enzyme-labeled secondary antibody_s1Casein as antigen, complete antigen and α, respectively_s1The monoclonal antibody corresponding to casein is primary antibody, the OD values of the reaction of the monoclonal antibody with different dilutions and the antigen with different concentrations are determined by indirect competition E L ISA method, and the results are shown in the table4.7 and 4.8.

As shown in Table 7, the OD value decreased with the increase in the dilution of the complete antigen monoclonal antibody, and the OD value closest to 1.0 was used as the standard for determining the optimal working concentration of the antigen antibody, so that the optimal coating concentration of the antigen was 2.5. mu.g/m L, and the optimal working concentration of the complete antigen monoclonal antibody was 10000.

TABLE 7 determination of antigen coating concentration and working concentration of monoclonal antibody for complete antigen

As can be seen from Table 8, the following α_s1The optimal coating concentration of the antigen is 2.5 mu g/m L by taking the OD value closest to 1.0 as the standard for judging the optimal working concentration of the antigen and the antibody_s1The optimal working concentration of monoclonal antibodies to casein is 5000.

TABLE 8 antigen coating concentration and α_s1Determination of working concentration of monoclonal antibody to Casein

5. Establishment of Indirect competitive E L ISA inhibition curve

On the basis of optimizing the optimal working concentration of the envelope antigen and the monoclonal antibody and the optimal working concentration of the enzyme-labeled secondary antibody, respectively using the complete antigen monoclonal antibody and α according to an indirect competition E L ISA program_s1Casein monoclonal antibody as primary antibody, α_s1The common logarithm of the mass concentration of casein is the abscissa and the competitive inhibition rate is the ordinate, and an indirect competitive E L ISA standard curve is established, wherein the competitive inhibition rate is calculated by the OD value, and the formula is shown as (2).

Competitive inhibition ratio (%) ═ B/B₀(2)

B is each phaseα of corresponding concentration_s1OD value at competitive inhibition of Casein, B₀Is α free_s1OD in competition for inhibition by casein.

Detection limit, randomly selecting 10 holes to perform zero standard indirect competition E L ISA detection, and calculating D₄₅₀Average value of values (D)₀) And Standard Deviation (SD), L OD was calculated according to equation (3).

LOD＝(D₀-2SD)/D₀×100％ (3)

The corresponding antigen mass concentration is calculated on the standard curve as the detection lower limit (L OD) of the method (the specific operation refers to the establishment of the indirect competitive E L ISA detection method of bovine serum albumin of animal allergen of Zhu-Bao culture, Wang-Rev, Lu-Qing \39566, Qian-Xunxiang, Xunxiang-Honghua [ J ]. Nanjing university of agriculture proceedings 2016,39(02):305-311 ]).

Establishing an indirect competitive enzyme-linked immunosorbent assay (E L ISA) inhibition curve on the basis of optimizing the optimal working concentration of the antigen and the monoclonal antibody and the optimal working concentration of the enzyme-labeled secondary antibody, and then α_s1Casein standards were serially diluted at 1280ng/m L, 640ng/m L, 320ng/m L, 160ng/m L, 80ng/m L, 40ng/m L,20 ng/m L, 10ng/m L, 0ng/m L at α ng/m_s1When the primary antibody is a monoclonal antibody of complete antigen, the antigen concentrations are 640ng/m L, 320ng/m L, 160ng/m L, 80ng/m L, 40ng/m L,20 ng/m L, 10ng/m L and 0ng/m L, the standard curve is established to show a good linear relation, as shown in FIG. 9, and the linear regression equation is that y is-9.22 x +100.78(R is the linear regression equation)²0.9806), detection limit is 10.49ng/m L, when primary antibody is α_s1When the concentration of the antigen is 1280ng/m L, 640ng/m L, 320ng/m L, 160ng/m L, 80ng/m L and 40ng/m L in the monoclonal antibody of casein, a standard curve is established to show a better linear relation, as shown in figure 10, the linear regression equation is that y is-23.44 x +135.77(R is shown in figure 10)²0.9697), detection limit of 60.50ng/m L.

6. Accuracy and reproducibility of the method

The accuracy and repeatability of the method for establishing the competition inhibition curve are evaluated according to the intra-batch error and the inter-batch error, and the accuracy and repeatability of the indirect competition E L ISA method are evaluated according to the intra-batch error and the inter-batch error.

When the monoclonal antibody of the complete antigen is used as the primary antibody, α is added_s1 Casein design 5 dilution gradients, 3 replicates for each concentration, the E L ISA test performed under identical conditions of reagents, reaction conditions and detection equipment used, standard deviations were calculated, and the inter-well variation coefficients represent the batch error, see table 9, with variation coefficients in the range 1.159% to 4.406%.

Repeat 3 times with different elisa plates at different times. The standard deviation and the coefficient of variation are calculated, and the inter-batch error is expressed by the inter-batch coefficient of variation. The results are shown in Table 10, with a coefficient of variation in the range of 0.826% to 4.362%.

TABLE 9 in-batch precision test of the complete antigen monoclonal antibodies

TABLE 10 batch precision test of the complete antigen monoclonal antibodies

At α_s1When the monoclonal antibody of casein is used as the primary antibody, α is added_s1 Casein design 5 dilution gradients, 3 replicates for each concentration, the E L ISA test performed under identical conditions of reagents, reaction conditions and detection equipment used, standard deviations calculated and expressed as the inter-well variation coefficient for the batch error, see table 11, with variation coefficients in the range 0.828% to 5.191%.

Repeat 3 times with different elisa plates at different times. The standard deviation and the coefficient of variation are calculated, and the inter-batch error is expressed by the inter-batch coefficient of variation. The results are shown in Table 12, with a coefficient of variation in the range of 0.099% to 4.849%.

TABLE 11 α_s1In-batch precision testing of Casein monoclonal antibodies

TABLE 12 α_s1Batch precision test of Casein monoclonal antibodies

The above results were obtained with the complete antigen monoclonal antibody and α_s1The monoclonal antibodies of casein as primary antibodies indicate good accuracy and good reproducibility of the method, but relatively speaking, the accuracy and reproducibility of the method are better.

7. Method for verifying accuracy of indirect competition E L ISA method through reversed-phase high performance liquid chromatography

In order to further evaluate the accuracy of the detection method, the 6 proteases were hydrolyzed α by reversed-phase high performance liquid chromatography_s1The amount of casein hydrolysate allergen residues is determined and compared with the results of the test using the indirect competition E L ISA method.

Preparation of stock solutions of standards

α_s1Preparation of Casein Standard stock solution A stock solution of 2mg/m L was prepared by accurately weighing 20mg of α_s1Casein standard, volume fixed. Storing at-20 deg.C for use.

Chromatographic conditions

The chromatographic column is an Agilent Zorbax 300SB-C8 chromatographic column (4.6 × 150mm, 3.5 mu m), wherein the mobile phase A is 0.1% TFA/water solution, the mobile phase B is 0.1% TFA/ACN solution, the mobile phase is subjected to suction filtration and degasification for later use, linear gradient elution is adopted, the initial concentration of the mobile phase B is 33%, the initial concentration is increased to 35% within 5min, the initial concentration is increased to 37% within 5-9 min, the initial concentration is increased to 40% within 9-18 min, the initial concentration of the mobile phase B is increased to 41% from 40% within 18-22 min, then the concentration is kept for 5.5min, the concentration is continuously increased to 43% within 27.5-28 min, the concentration is increased to 45% within 28-36 min, the concentration is increased to 33% within 1min, the flow rate is kept to 8min, the flow rate is 0.5m L/min, the detection wavelength is 214nm, the column temperature is 45 ℃, and the sample feeding amount is 10 mu m L.

α measured by reversed phase high performance liquid chromatography_s1The casein standard map is shown in figure 11. It can be known that，α_s1The retention time of casein is 20.28 min.

Drawing of standard curve

α_s1Drawing a casein standard curve α_s1Casein stock dilutions 0.05, 0.1, 0.2, 0.4, 0.8, 1, 2mg/m L.

α according to the above chromatographic conditions_s1And (4) detecting a casein standard product, and drawing a standard curve by taking the mass concentration of the standard product as an abscissa and taking a peak area corresponding to each mass concentration as an ordinate.

α was measured for each mass concentration based on the chromatographic conditions_s1Casein standards, the peak area values of which are shown in table 13.

TABLE 13 quantitative determination of α for different mass concentrations by reversed phase HPLC_s1Casein standards

According to α_s1The mass concentrations of the casein standards and their corresponding peak areas, as α_s1Mass concentration of casein as abscissa and corresponding peak area as ordinate, α is plotted_s1Standard curve for casein, see fig. 12, y 9193.1x-165.92, R²＝0.9997。

α measurement by reversed phase high performance liquid chromatography_s1Residual amount of antigens of casein hydrolysate

Respectively measuring 6 α by reverse phase high performance liquid chromatography_s1Antigenic residual quantity of Casein hydrolysate (hydrolysates with protease addition of 1500U/g each), hydrolysate α was calculated_s1Peak area of casein and quantitative determination of α in each sample according to the standard curve established in FIG. 12_s1Casein content, see table 14. Each sample was run in 3 replicates, 3 replicates and averaged.

TABLE 14 determination of α in hydrolysates by reversed phase high performance liquid chromatography and indirect competitive E L ISA method_s1Residual amount of casein (mg/m L)

Protease enzyme

Protease M

Neutral protease

Compound protease

Pepsin

Alkaline 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

Complete 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

Evaluation of accuracy of Indirect competitive E L ISA method

The method of paired sample t test is adopted to verify that the established method is used for detecting α in hydrolysate_s1The accuracy of the residual casein quantity (table 14), i.e. the value of the residual quantity of antigen determined by the indirect competition E L ISA method established with the monoclonal antibody prepared with the complete antigen, was subjected to a t-test with the values determined in the liquid phase, respectively, and the p-values are shown in table 15.

α in hydrolysate Table 15_s1T-test p-value of the amount of casein remaining

Protease enzyme	Protease M	Neutral protease	Compound protease	Pepsin	Alkaline protease	Papain
							Complete antigen monoclonal antibody	0.301	-0.205	-0.310	0.132	0.213	0.331

As can be seen from the data in Table 15, the value of p is greater than0.05 that the reversed phase high performance liquid chromatography method and the indirect competitive E L ISA method did not significantly differ from the measured results of the same sample, therefore, α was established_s1The determination result of the casein indirect competition E L ISA method is accurate and can be used as a theoretical basis for developing an allergen kit.

8. Determining residual quantity of antigen in hydrolysate

α will be mixed_s1Freeze-dried powder of different enzymatic hydrolysates of casein, adding an antigen diluent (50 mmol/L carbonate solution with pH 9.6) to dilute to a suitable concentration, i.e. the antigen concentration is within the range of the established inhibition curve of indirect competition E L ISA and within the detection limit, applying α according to the procedure of indirect competition E L ISA method_s1Two different antibodies, namely a casein monoclonal antibody and a complete antigen monoclonal antibody, are used as primary antibodies, and the antigen residual quantity of different enzymes and hydrolysate with different enzyme adding amounts is detected.

Non-hydrolyzed α_s1Casein solution diluted to appropriate concentration, i.e. antigen concentration within the range of established inhibition curve of indirect competition E L ISA and detection limit, α was applied following procedure of indirect competition E L ISA method_s1Two different antibodies, casein monoclonal antibody and complete antigen monoclonal antibody, were used as primary antibodies, and the detected antigen content is shown in table 16.

TABLE 16 unhydrolyzed α_s1The antigen content of the casein solution

When the complete antigen monoclonal antibody is primary antibody, six kinds of α_s1The case where the amount of the antigen remaining in the casein hydrolysate decreased with the increase of the amount added is shown in FIG. 13. The antigen residual quantity of the enzymolysis liquid of the Protease M is the largest, and the residual quantity is obviously higher than that of other enzymes (p) under the condition of the same Protease adding quantity<0.05) and between 1.281 and 0.364mg/M L, the antigen residual quantity of Protease M enzymolysis liquid is obviously reduced along with the increase of the adding quantity of the enzyme (p)<0.05)。

When the addition amount of the neutral protease is the same, the antigen residue in the hydrolysate is obviously higher than that of the compound protease, the pepsin, the alkaline protease and the papain (p <0.05), the antigen residue in the neutral protease hydrolysate is between 0.887 and 0.189mg/m L, the residue is obviously reduced along with the increase of the enzyme addition amount at 1500-3500U/g, and the reduction of the antigen residue tends to be smooth at 2500-3500U/g.

When the added enzyme amount of the compound protease is 1500U/g, the antigen residual amount in the hydrolysate is obviously higher than 2 hydrolysates of alkaline protease and papain, but when the added enzyme amount is 3500U/g of 2000-2000, the antigen residual amount is obviously higher than 3 hydrolysates of pepsin, alkaline protease and papain (p <0.05), the residual amount of the compound hydrolysate is obviously reduced along with the increase of the added enzyme amount (p <0.05) and is between 0.447 and 0.088mg/m L.

The antigen residual quantity in the pepsin hydrolysate is greater than that of alkaline protease and papain when the protease addition quantity is the same, the difference is significant (p is less than 0.05) when the addition quantity is 1500U/g, but the difference is not significant when the addition quantity is 2000-3500U/g, and the residual quantity is not significantly reduced along with the increase of the enzyme addition quantity, and the antigen residual quantity of the pepsin hydrolysate is between 0.410 and 0.020mg/m L.

The antigen residual quantity of the alkaline protease hydrolysate is greater than that of the papain with the same enzyme adding quantity, but the difference is not significant (p is more than 0.05). The residual quantity of the alkaline protease hydrolysate is reduced with the increase of the enzyme adding quantity and is between 0.113 and 0.013mg/m L.

The antigen residual quantity of the papain hydrolysate is minimum compared with other enzymes when the enzyme dosage is the same, the antigen residual quantity is reduced along with the increase of the enzyme dosage and is between 0.058 and 0.010mg/m L, the antigen reduction rate of the papain hydrolysate, namely (the unhydrolyzed antigen residual quantity-the determination hydrolysate residual quantity)/the unhydrolyzed antigen residual quantity, is respectively 99.80 percent, 99.89 percent, 99.91 percent, 99.96 percent and 99.97 percent when the enzyme dosage is 1500U/g to 3500U/g.

When α_s16 α species when the monoclonal antibody of casein is primary antibody_s1The case where the amount of the antigen remaining in the casein hydrolysate decreased with the increase in the amount of the added enzyme is shown in FIG. 14. When the enzyme addition amount of the zymose M hydrolysate is 1500U/g, the antigen residual amount in the hydrolysate is obviously higher than that of other 5 kinds of hydrolysate, and the antigen residual amount is the largest. But when the enzyme adding amount is 2000-3500U/g,the antigen residual quantity is obviously less than that of pepsin and alkaline Protease and more than that of 3 enzymolysis liquids of compound Protease neutral Protease and papain, the antigen residual quantity of the Protease M enzymolysis liquid is 8.280-0.934mg/M L, and when the enzyme addition quantity is 1500-2500U/g, the antigen residual quantity is obviously reduced along with the increase of the enzyme addition quantity (p)<0.05), the residue was not significantly reduced at 2500-.

When the same protease is added, the original residue in the hydrolysate of the pepsin is obviously higher than that of alkaline protease, compound protease, neutral protease and papain (p < 0.05). The residue of the pepsin hydrolysate is obviously reduced along with the increase of the added enzyme amount (p <0.05) and is between 6.603 and 1.310mg/m L.

When the same protease is added, the original residue in the hydrolysate is obviously higher than that of compound protease, neutral protease and papain (p < 0.05). The residue of the alkaline protease hydrolysate is obviously reduced along with the increase of the added enzyme amount (p <0.05), and is 4.077-1.195mg/m L.

The antigen residual quantity of the compound protease hydrolysate is greater than that of neutral protease and papain when the same protease addition quantity is added, the residual quantity of the compound protease hydrolysate is obviously reduced along with the increase of the enzyme addition quantity (p is less than 0.05) and is between 1.925 and 0.616mg/m L.

The antigen residual quantity of the neutral proteolytic enzyme solution is obviously greater than that of the papain when the same protease addition amount is added, the residual quantity of the neutral proteolytic enzyme solution is obviously reduced along with the increase of the enzyme addition amount (p is less than 0.05), and the residual quantity is between 1.729 and 0.310mg/m L.

The antigen residual quantity of the papain hydrolysate is obviously less than that of other enzymes (p is less than 0.05) under the same enzyme adding quantity, the antigen residual quantity is reduced along with the increase of the enzyme adding quantity and is between 0.420 and 0.172mg/m L, the antigen reduction rate of the papain hydrolysate, namely (the residual quantity of unhydrolyzed antigen-the residual quantity of measured hydrolysate)/the residual quantity of unhydrolyzed antigen is respectively 98.60 percent, 99.08 percent, 99.21 percent, 99.30 percent and 99.43 percent when the enzyme adding quantity is 1500U/g to 3500U/g.

Monoclonal antibodies to complete antigens α_s1The reduction effect ranking of antigen residue in each enzymatic hydrolysate was also different for casein monoclonal antibody. When the complete antigen monoclonal antibody isWhen the addition amount of the Protease is the same, the antigen residual quantity of the primary antibody 6 protein enzymolysis liquid is Protease M from large to small>Neutral protease>Compound protease>Pepsin>Alkaline protease>Papain, Moschus α_s1When the monoclonal antibody of the casein is primary antibody, the antigen residual quantity of the Protease M enzymolysis liquid is obviously higher than that of other 5 enzymolysis liquids when the enzyme addition amount of the 6 protein enzymolysis liquid is 1500U/g. However, when the enzyme adding amount is 2000-3500U/g, the antigen residual quantity of the 6-protein enzymolysis solution is pepsin from large to small>Alkaline protease>Protease M>Compound protease>Neutral protease>Papain.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Beijing university of Industrial and commercial

<120> α s 1-monoclonal antibody prepared from antigenic epitope of casein and method for detecting cow milk allergen

<160>1

<170>Patent-In 3.3

<210>1

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequence α s 1-epitope of Casein

<400>1

SEEIVPNSVE QKHIQKEDVP SER 23

Claims (5)

1.α_s1An epitope peptide of casein having the amino acid sequence SEEIVPNSVEQKHIQKEDVPSER.

2.α the method of claim 1_s1-caseinThe complete antigen is obtained by coupling the white epitope peptide and bovine serum albumin.

3. The complete antigen of claim 2, which is coupled by the glutaraldehyde method.

4. The method for preparing complete antigen according to claim 2, wherein α is first synthesized by solid phase synthesis_s1-epitope peptides of casein and prepared as complete antigens by coupling with bovine serum albumin.

5. The method of claim 4, wherein the coupling is by the glutaraldehyde method.

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