Screening method of alpha-lactalbumin detection antibody and application of screening method in alpha-lactalbumin detection and dairy product process authenticity evaluation

Abstract

The invention provides a screening method of an alpha-lactalbumin detection antibody and application of the screening method in alpha-lactalbumin detection and dairy product process authenticity evaluation, and relates to the technical field of dairy product process detection. The screening method of the detection antibody for recognizing the lactalbumin comprises the following steps: taking animal serum immunized by alpha-lactalbumin, carrying out affinity purification to obtain candidate antibodies, and carrying out primary screening and final screening on the candidate antibodies to obtain the detection antibody. The detection antibody screened by the method is highly specific to alpha-lactalbumin, cross reaction of other proteins in a milk matrix can be avoided, and the reliability of actual detection is ensured. According to the invention, a screened detection antibody is also utilized to construct a'hot processing parameter-ELISA antigen recognition performance 'correlation model, and an efficient technical means is provided for rapid traceability of hot processing technologies such as milk pasteurization and UHT technology.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of dairy product process detection, and particularly relates to a screening method of an alpha-lactalbumin detection antibody and application of the screening method in detection of alpha-lactalbumin and evaluation of authenticity of a dairy product process. BACKGROUND

[0002] Alpha-lactalbumin (a-LA) is a core nutritional functional protein in milk, which is rich in essential amino acids. The antigenic structural integrity of a-LA directly determines the nutritional value, safety attribute and market value of dairy products. Milk heat processing (such as pasteurization, medium-high temperature processing, ultra-high temperature sterilization (UHT) and high-pressure assisted heat processing) is a necessary link to ensure food safety. However, heat processing can destroy the secondary / tertiary structure of a-LA (such as a-helix uncoiling and disulfide bond breaking), induce the formation of molecular aggregates, and thus affect the antigen recognition performance in the detection process.

[0003] There are three key defects in the prior art: the ELISA detection is not adapted to the heat processing characteristics: the existing a-LA ELISA detection uses a fixed antibody pair and uniform parameters (such as a monoclonal antibody coating concentration of 1 µg / mL and a random selection of blocking agents), and does not consider the influence of heat processing (temperature, pressure) and the synergistic effect of lactose on the antigenic epitope of a-LA. For example, commercially available ELISA kits are only suitable for detecting a-LA in raw milk and pasteurized milk; the heat stability evaluation lacks a quantitative system and only determines the presence or absence of a-LA by "whether it is detected", without establishing a quantitative correlation between "heat processing parameters (temperature / pressure) and antigen recognition performance (inhibition rate, absorbance)", which cannot quantify the degree of damage to the stability of a-LA caused by heat processing and is difficult to guide the optimization of dairy product processes; the process traceability lacks protein characteristic support, and the traditional milk heat processing process is determined by microbial culture (24-48 h) or sensory evaluation (high subjectivity), without using the sensitivity of a-LA to heat / pressure to establish a corresponding relationship between "heat process parameters and protein antigen performance", which cannot quickly trace the processing history and is difficult to deal with the "process impersonation" chaos in the market (such as high-pressure heat-processed milk impersonating pasteurized milk).

[0004] Research has found that the ELISA antigen recognition performance of a-LA (with inhibition rate and absorbance as core indicators) is significantly correlated with the intensity of heat processing: high temperature (≥95℃) and high pressure can cause a decrease in inhibition rate and an increase in aggregates (SDS-PAGE shows that the proportion of bands above 100 KDa increases), while natural lactose in milk can form a "lactose-a-LA complex" by combining with the hydrophilic groups of a-LA, thereby alleviating the denaturation of antigenic epitopes. Therefore, in order to prevent the cross-reaction of proteins in dairy products, the influence of heat processing on the antigenic epitope of a-LA, and accurately determine the heat processing process of dairy products, it is urgent to develop a method for accurately determining the dairy product process using a-LA as the detection protein. SUMMARY

[0005] In order to solve the above technical problems, the first object of the present application is to provide a screening method of an α-lactalbumin detection antibody. The detection antibody screened by the method of the present application is highly specific to α-lactalbumin, and can avoid cross-reaction with other proteins in a milk matrix, thereby ensuring the reliability of actual detection.

[0006] The second object of the present application is to provide an α-lactalbumin detection antibody obtained by the above screening method.

[0007] The third object of the present application is to provide a kit for detecting α-lactalbumin.

[0008] The fourth object of the present application is to provide the use of the above α-lactalbumin detection antibody or the above kit in detecting the content of α-lactalbumin.

[0009] The fifth object of the present application is to provide an ELISA detection method of the content of α-lactalbumin.

[0010] The sixth object of the present application is to provide the use of the above α-lactalbumin detection antibody or the above kit in evaluating a dairy authenticity process.

[0011] The seventh object of the present application is to provide an evaluation method of a dairy authenticity process using the detection antibody obtained by the above screening method or the above kit.

[0012] In order to achieve the above objects, the technical solutions of the present application are as follows: The present application provides a screening method of an α-lactalbumin detection antibody, comprising the following steps: taking serum of an animal immunized with α-lactalbumin, performing affinity purification to obtain a candidate antibody, and obtaining a detection antibody through preliminary screening and final screening of the candidate antibody; the method of preliminary screening comprises: detecting an unprocessed α-lactalbumin sample, a heat-processed α-lactalbumin sample and a BSA interference liquid sample with the candidate antibody respectively, calculating LOD, and screening an antibody with LOD of the unprocessed α-lactalbumin sample ≤ 20 μg / L, LOD of the heat-processed α-lactalbumin sample ≥ 50 μg / L, and LOD of the BSA interference liquid sample ≥ 100 μg / L as a qualified candidate antibody; the method of final screening comprises: detecting α-lactalbumin with the qualified candidate antibody, fitting a detection curve of the treatment temperature of α-lactalbumin and absorbance, denoted as curve 1; detecting α-lactalbumin with the qualified candidate antibody, fitting a detection curve of the treatment pressure of α-lactalbumin and absorbance, denoted as curve 2; screening a qualified candidate antibody with R 450 ≥ 0.995 of curve 1, R 2 ≥ 0.990 of curve 2, and negative correlation between curve 1 and curve 2 as a detection antibody. 2 ≥ 0.990 of curve 2, and negative correlation between curve 1 and curve 2 as a detection antibody.

[0013] As an implementation form, the LOD is calculated according to a signal-to-noise ratio of 2-4 times.

[0014] As an implementation form, the treatment temperature is 25-150 DEG C, and the treatment pressure is 0-0.5 MPa.

[0015] The application further provides the alpha-lactalbumin detection antibody obtained by the screening method.

[0016] The application further provides a kit for detecting alpha-lactalbumin, comprising the alpha-lactalbumin detection antibody.

[0017] The application further provides application of the alpha-lactalbumin detection antibody or the kit in detecting the content of alpha-lactalbumin.

[0018] The application further provides an ELISA detection method for the content of alpha-lactalbumin, comprising the following steps: mixing the alpha-lactalbumin detection antibody with a heat-processed alpha-lactalbumin sample to be detected, developing color, and determining to obtain the content of alpha-lactalbumin in the sample to be detected.

[0019] As an implementation form, the pH value of the heat-processed alpha-lactalbumin sample is 7.0-8.0.

[0020] The application further provides application of the alpha-lactalbumin detection antibody or the kit in evaluating a dairy product authenticity process.

[0021] The application further provides an evaluation method for a dairy product authenticity process by using the alpha-lactalbumin detection antibody to detect the inhibition rate of alpha-lactalbumin in a dairy product to be detected to determine the process of the dairy product; the determination standard is that the inhibition rate of 55%-60% determines that the process of the dairy product to be detected is pasteurization, the inhibition rate of 15%-20% determines that the process of the dairy product to be detected is medium-high temperature processing, and the inhibition rate of less than 10% determines that the process of the dairy product to be detected is high-pressure assisted heat processing or UHT.

[0022] Compared with the prior art, the application has the following beneficial effects: The alpha-LA detection antibody screened by the screening method of the application is highly specific to alpha-LA, can avoid cross-reaction of other proteins in a milk matrix, and ensures reliability of actual detection.

[0023] The indirect competitive ELISA method prepared based on the screened alpha-LA specific antibody has a good linear relationship (R 2With a mean of 0.99, high sensitivity, and strong specificity, this ELISA method effectively avoids cross-interference from extraneous proteins such as β-lactoglobulin and casein in milk. It can accurately distinguish different heat processing processes based on α-LA antigenicity differences. α-LA treated at 75℃ (close to pasteurization temperature) is detectable with a signal close to that at room temperature, while α-LA treated at 105℃ and ultra-high temperature instantaneous sterilization (UHT, a synergistic effect of temperature and pressure) is undetectable. Actual sample testing shows that the α-LA concentration in pasteurized milk is significantly higher than that in UHT milk and reconstituted milk (P<0.001), further validating the practicality of this method. In summary, the "heat processing parameter-ELISA antigen recognition performance" correlation model established in this invention provides an efficient technical means for rapid traceability of milk pasteurization and UHT processes, and has significant practical application value for ensuring dairy product quality and maintaining market order. Attached Figure Description

[0024] Figure 1 The standard curve for the detection of unprocessed α-LA; Figure 2 Standard curve for detecting α-LA processed at 75℃; Figure 3 Standard curve for detecting α-LA processed at 95℃; Figure 4 For the screening and specificity analysis of α-LA antibodies, A is a heatmap of absorbance difference between antibody and α-LA binding (red represents high value, blue represents low value), B is a heatmap of the inhibition rate of α-LA binding to antibody (red represents high inhibition rate, blue represents low inhibition rate), C is a curve of the antibody inhibition rate of α-LA as a function of α-LA concentration, and D is a bar graph of the cross-reactivity inhibition rate of antibody against α-LA, ovalbumin (OVA), β-lactoglobulin (β-LG), casein (CN), bovine serum albumin (BSA), and lactoferrin (LF). Figure 5 To verify the accuracy of the casein precipitation method and detect the α-LA content in different milk samples, A is a graph showing the detection curves of α-LA standards at different concentrations after casein precipitation treatment, and B is a graph showing the detection results of α-LA content in milk samples. Detailed Implementation

[0025] This invention provides a method for screening antibodies for α-lactalbumin detection, comprising the following steps: taking serum from animals immunized with α-lactalbumin, performing affinity purification to obtain candidate antibodies, and obtaining detection antibodies by primary screening and final screening of the candidate antibodies; The preparation method of the animal serum comprises the following steps: injecting emulsion A into the animal to perform primary immunization, then injecting emulsion B into the animal to perform booster immunization, and taking the whole blood of the animal after the immunization procedure is completed, and then separating the serum after standing. The emulsion A is a volume ratio of 1:0.5-1.5 of unprocessed α-lactalbumin and Freund's complete adjuvant, the emulsion B is a volume ratio of 1:0.5-1.5 of unprocessed α-lactalbumin and Freund's incomplete adjuvant, and the concentration of the unprocessed α-lactalbumin is 0.5-1.5 mg / mL, preferably 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.3 mg / mL or 1.4 mg / mL. The injection is intraperitoneal injection, and the injection dose is 1-2 mg per animal. The injection cycle of the application is two weeks per time, the injection times are 2-5 times, preferably 3 times, 4 times or 5 times; the first injection is primary immunization, and then booster immunization; the animal is preferably a big white rabbit; and the unprocessed α-lactalbumin is purchased from Sigma Company (analytical pure).

[0026] In the application, the blood of the big white rabbit is collected from the tail artery 8-10 days after the second to fourth immunization, diluted 1000-500000 times with PBS, and the antibody titer is detected by indirect competitive ELISA. When the antibody titer is greater than 1:100000, the immunization procedure is completed, then the whole blood of the animal is taken, and the serum is separated after standing.

[0027] In the application, the separated serum is subjected to affinity purification to obtain the candidate antibody. As an embodiment, the method of affinity purification comprises Protein A affinity purification to obtain the antibody of α-lactalbumin as the candidate antibody. In the application, the Protein A affinity purification can be performed according to the conventional method.

[0028] In the present application, the method of the preliminary screening comprises: using the candidate antibody to detect the unprocessed alpha-lactalbumin sample, the heat-processed alpha-lactalbumin sample and the BSA interference solution sample respectively, the protein concentration of the unprocessed alpha-lactalbumin sample is 10 μg / L-1000 μg / L, preferably 20 μg / L, 50 μg / L, 100 μg / L, 300 μg / L, 500 μg / L, 700 μg / L or 900 μg / L; the protein concentration of the heat-processed alpha-lactalbumin sample is 10 μg / L-1000 μg / L, preferably 20 μg / L, 50 μg / L, 100 μg / L, 300 μg / L, 500 μg / L, 700 μg / L or 900 μg / L. Then the minimum detection limit (LOD) of each sample is calculated, the LOD is calculated according to 2-4 times the signal-to-noise ratio, preferably 3 times the signal-to-noise ratio, and the qualified candidate antibody is screened through the LOD. In the present application, the standard of the qualified candidate antibody is that the blank OD of the unprocessed alpha-lactalbumin sample is less than 0.2, the LOD of the unprocessed alpha-lactalbumin sample is less than or equal to 20 μg / L, the LOD of the heat-processed alpha-lactalbumin sample is greater than or equal to 50 μg / L, and the LOD of the BSA interference solution sample is greater than or equal to 100 μg / L. In the present application, the unqualified candidate antibodies are eliminated through the preliminary screening, and the antibodies with specificity and repeatability are reserved. 450 <0.2, the LOD of the unprocessed alpha-lactalbumin sample is less than or equal to 20 μg / L, the LOD of the heat-processed alpha-lactalbumin sample is greater than or equal to 50 μg / L, and the LOD of the BSA interference solution sample is greater than or equal to 100 μg / L. In the present application, the unqualified candidate antibodies are eliminated through the preliminary screening, and the antibodies with specificity and repeatability are reserved.

[0029] In the present application, the method of the final screening comprises: using the qualified candidate antibody to detect the alpha-lactalbumin, fitting the detection curve of the treatment temperature of the alpha-lactalbumin and the absorbance, which is recorded as curve 1; using the qualified candidate antibody to detect the alpha-lactalbumin, fitting the detection curve of the treatment pressure of the alpha-lactalbumin and the absorbance, which is recorded as curve 2. The treatment temperature in the present application is 25℃-150℃, as an optional embodiment, the treatment temperature is 25℃, 75℃, 95℃, 105℃; the treatment pressure of the curve 1 is normal pressure, and the treatment time is 15-25 min, preferably 20 min; the treatment pressure in the present application is 0-0.5 MPa, as an optional embodiment, the treatment pressure is 0 MPa, 0.1 MPa, 0.2 MPa, 0.3 MPa; the treatment temperature of the curve 2 is 75-95℃, and the treatment time is 15-25 min, preferably 20 min. Then the qualified candidate antibody with the curve 1 R 2 ≥0.995, the curve 2 R 2 ≥0.990, and the curve 1 and the curve 2 are negatively correlated is used as the detection antibody. In the present application, the final screening can accurately capture the quantitative relationship between the heat processing process parameters and the antigen recognition performance of the alpha-lactalbumin, and the antibody meeting the rule that the higher the heat processing strength is, the lower the absorbance value is, and the weaker the antigen recognition performance is is screened out as the core antibody for subsequent ELISA detection.

[0030] The application further provides an alpha-lactalbumin detection antibody obtained by the screening method.

[0031] The application further provides a kit for detecting alpha-lactalbumin, comprising the alpha-lactalbumin detection antibody.

[0032] The application further provides application of the alpha-lactalbumin detection antibody or the kit in detecting the content of alpha-lactalbumin.

[0033] The application further provides an ELISA method for detecting the content of alpha-lactalbumin, comprising the following steps: mixing the alpha-lactalbumin detection antibody with a heat-processed alpha-lactalbumin sample to be tested, developing color and determining to obtain the content of alpha-lactalbumin in the sample to be tested. In the application, 0.1-10 μg / mL of unprocessed alpha-lactalbumin is used as an antigen for coating, and the concentration of the unprocessed alpha-lactalbumin is preferably 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL or 9 μg / mL, and the coating liquid concentration can improve the sensitivity and save the cost. Then, 2-8℃ incubation is performed for 12-16 h, and the incubation temperature is preferably 3℃, 4℃, 5℃, 6℃ or 7℃, and the incubation time is preferably 13 h, 14 h or 15 h. In the application, after the coating liquid is discarded, a blocking agent is added, and the blocking agent comprises 1% gelatin, 1% fish skin glue, 1% fetal bovine serum, 1% skim milk powder or 1% BSA. Then, 35-38℃ incubation is performed for 0.5-1.5 h, and the incubation temperature is preferably 36℃ or 37℃, and the incubation time is preferably 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h or 1.4 h. After washing the plate, the heat-processed alpha-lactalbumin sample to be tested and the detection antibody diluted by 800-1200 times are added, 35-38℃ incubation is performed for 0.5-1.5 h, the incubation temperature is preferably 36℃ or 37℃, and the incubation time is preferably 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h or 1.4 h. Then, HRP-labeled goat anti-rabbit IgG is added, and 35-38℃ incubation is performed for 0.5-1.5 h. In the application, the pH value of the heat-processed alpha-lactalbumin sample to be tested is 7.0-8.0, and the pH value is preferably 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9. In the application, by adjusting the pH value of the detection system to neutral for the acidic or alkaline milk sample, the R 2From 0.985 to 0.999, avoid the influence of pH on antibody-antigen binding, improve accuracy. After washing the plate, add TMB color developing agent, react at room temperature for 10-20 min in the dark, add stop solution to determine the absorbance value, and calculate the content of α-lactalbumin in the sample to be measured.

[0034] Based on the ability to detect heat-processed α-lactalbumin content, the application also provides a use of the above-mentioned α-lactalbumin detection antibody or the above-mentioned kit in the evaluation of dairy authenticity process.

[0035] The application also provides an evaluation method of the above-mentioned α-lactalbumin detection antibody or the above-mentioned kit for the dairy authenticity process, which uses the detection antibody to detect the inhibition rate of α-lactalbumin in the dairy product to be measured to determine the dairy process, and the determination standard is that the inhibition rate of 55%-60% determines that the process of the dairy product to be measured is pasteurization, the inhibition rate of 15%-20% determines that the process of the dairy product to be measured is medium-high temperature processing, and the inhibition rate of <10% determines that the process of the dairy product to be measured is high-pressure assisted heat processing or UHT. In the application, five groups of lactalbumin standards with different processing parameters are constructed, the absorbance value is determined according to the above-mentioned ELISA detection method, and the inhibition rate is calculated, the absorbance value is the absorbance value at 450 nm detected by the enzyme label instrument, which directly reflects the amount of antibody-antigen binding, and the higher the A value, the stronger the antigen recognition performance. According to the absorbance value of α-lactalbumin, the inhibition rate of α-lactalbumin is calculated, the heat processing parameter-antigen recognition performance is obtained, and a correlation database is established. The calculation method of the inhibition rate is: inhibition rate (%) = (A0-A X ) / (A0-A b )×100%, wherein A0 is the detection absorbance value of the reaction cup when the solution without the measured substance is detected, A x is the detection absorbance value of the reaction cup when the solution containing the measured substance is detected, and A b is the detection absorbance value of the reaction cup when the standard solution or the sample to be measured is not added. The standard curve is drawn with the inhibition rate of the α-lactalbumin standard solution as the ordinate and the logarithm of the concentration of the α-lactalbumin standard as the abscissa. The concentration of each sample to be measured can be calculated by the sample determination absorbance value to calculate the inhibition rate, and the concentration thereof can be read from the standard curve according to the inhibition rate. As an optional embodiment, the dairy product to be measured includes fresh milk, pasteurized milk, UHT milk or dairy product intermediate.

[0036] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be described in detail below with examples, but they should not be understood as limiting the scope of protection of the application.

[0037] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources, such as the reagents, consumables, etc. related to the present application, and are generally used according to conventional conditions, or according to the conditions recommended by the company, unless otherwise specified.

[0038] Example 1 A screening method for an α-lactalbumin detection antibody: 1. Preparation of candidate antibodies (1) Animal immunization: 10 New Zealand white rabbits were injected with emulsion A at an immunization dose of 1 mg / rabbit for primary immunization, wherein emulsion A was prepared by mixing 1 mg / mL unprocessed α-LA (25°C, normal pressure, complete antigen epitope) and Freund's complete adjuvant at a volume ratio of 1:1. Then, 10 New Zealand white rabbits were continuously injected with emulsion B at an immunization dose of 1 mg / rabbit for booster immunization, wherein emulsion B was prepared by mixing 1 mg / mL unprocessed α-LA and Freund's incomplete adjuvant at a volume ratio of 1:1. Then, 8-10 days after the second to fourth immunization, the blood of the rabbits was collected from the tail artery, and diluted 40,000 times with PBS to obtain a rabbit blood diluent. Then, 100 μL of unprocessed α-LA (1 μg / mL) was added to a 96-well plate and incubated at 4°C overnight. After washing the plate 3 times with PBST, 200 μL of BSA (1%, w / w) was added and incubated at 37°C for 1 h. After washing the plate 3 times again, 100 μL of an α-LA standard solution was added to the wells, and 100 μL of the rabbit blood diluent was added to capture the antigen protein for 1 h. Finally, 100 μL of diluted enzyme-labeled antibody was added at 37°C for 30 min, and the absorbance was measured at 450 nm. The antibody titer was detected by indirect competitive ELISA, and when the antibody titer was >1:100,000, the immunization program was completed.

[0039] (2) Preparation of candidate antibodies: After the completion of the immunization program, the whole blood of the rabbits was taken, and the serum was obtained by standing and separation. The serum was affinity purified by a Protein A purification column filled with Protein A-Sepharose 4B, and the specific steps were as follows: Equilibrium column: rinse the pipeline with pH 7.4 phosphate buffer (Binding buffer) at a flow rate of 6.5 mL / min for 2 min. After column loading, equilibrate the column with pH 7.4 phosphate buffer (Binding buffer) at a flow rate of 1 mL / min until the baseline level.

[0040] Loading: After baseline, the separated serum was diluted (1:1, V / V) with equal volume of pH 7.4 Binding buffer, and then loaded into the column at a flow rate of 0.5 mL / min. After loading, the column was washed with pH 7.4 Binding buffer at a flow rate of 1 mL / min. The antibody was specifically adsorbed on the filler site, and other impurities flowed out with the buffer until the baseline level.

[0041] Elution: The antibody bound on the column was eluted with pH 2.7 Elution buffer, and the target protein was washed out at a flow rate of 0.5 mL / min.

[0042] Detection: The protein concentration of the eluate was detected at 280 nm ultraviolet, and when the absorbance A 280 >0.2, the eluate was collected, and after collection, the pH was quickly adjusted to 7.0 with 1 mol / L Tris.

[0043] Dialysis and storage of the antibody: The obtained antibody was dialyzed with pH 7.4 antibody dialysis solution (phosphate buffer, PB), and dialyzed at 4°C for three days, with three changes per day. After dialysis, it was taken out, 0.1% (W / V) NaN3 was added, and it was stored at 4°C for standby.

[0044] Treatment of the purification column: After the antibody was collected, the column was quickly washed with 0.1 M acetic acid solution for 2 min at a flow rate of 2 mL / min, and then the column was equilibrated with pH 7.4 phosphate buffer (Binding buffer) until the effluent pH was neutral. Finally, the column was washed with 20% ethanol solution, and after 20 min, it was filled and stored at 4°C.

[0045] After purification, 10 α-LA antibodies (pAb1~pAb10) were obtained as candidate antibodies.

[0046] 2. Ladder screening The ladder screening process gradually narrows the candidate range in the order of "primary screening-final screening", and ensures that the finally screened antibody meets the full-dimensional requirements of "specificity-sensitivity-linear correlation".

[0047] (1) Preliminary screening: The obtained candidate antibodies were used to detect 100 μg / L unprocessed α-LA, 100 μg / L BSA interference solution, and 100 μg / L heat-processed α-LA heated at 95°C for 20 min, respectively, and the lowest detection limit (LOD, calculated according to 3 times the signal-to-noise ratio) was calculated. The qualified candidate antibodies were finally screened out, which had a blank absorbance value of the α-LA sample of ≥0.2, an LOD of the unprocessed α-LA sample of >20 μg / L, an LOD of the heat-processed α-LA sample of <50 μg / L, and an LOD of the BSA interference sample of <100 μg / L. Through preliminary screening, 6 candidate antibodies were eliminated, and only 4 antibodies, pAb1, pAb2, pAb6, and pAb8, which had qualified specificity and repeatability, were reserved.

[0048] (2) Final screening: heat-processed α-LA sample-temperature gradient groups (25°C, 75°C, 95°C, 105°C, normal pressure, treatment for 20 min) and heat-processed α-LA sample-pressure gradient groups (95°C+0 MPa, 95°C+0.1 MPa, 95°C+0.2 MPa, 95°C+0.3 MPa, treatment for 20 min) were prepared, and the above gradient samples were detected by the 4 antibodies qualified in the preliminary screening, and the absorbance values were recorded. The temperature gradient group fitting curve and the pressure gradient group fitting curve were constructed with “temperature / pressure” as the horizontal coordinate and “absorbance value” as the vertical coordinate, and the linear correlation coefficient R of the fitting curve was calculated. 2 Finally, the optimal antibody was screened out, which had a temperature gradient group R 2 ≥0.995, a pressure gradient group R 2 ≥0.990, and a significantly negative correlation between the temperature gradient group fitting curve and the pressure gradient group fitting curve. The experimental results showed that the antibody pAb2 met the rule that the higher the heat processing intensity, the weaker the antigen recognition performance, and was obviously changed with temperature, and met the requirements and could accurately capture the quantitative relationship between “heat processing process parameters-α-LA antigen recognition performance”, and was used as the core antibody for subsequent ELISA detection; the other antibodies did not meet the rule that the higher the heat processing intensity, the lower the absorbance value, and the weaker the antigen recognition performance, and were not up to standard.

[0049] Example 2 An ELISA detection method for the content of heat-processed α-LA: 1. Preparation of reagents Lactalbumin solution: 0.05 M carbonate buffer solution with a pH of 9.6 was used as a coating buffer, and unprocessed α-LA was diluted to 1 µg / mL with the coating buffer.

[0050] Blocking agent: 1% skimmed milk powder (prepared with PBS); Incubation conditions: coating 4℃ incubation for 12~16 h (to ensure uniform coating of antibodies), blocking, primary antibody (pAb2 detection antibody, dilution ratio 1:1000), secondary antibody (HRP-labeled goat anti-rabbit IgG, dilution ratio 1:5000) are all 37℃ incubated for 1 h (to balance the reaction efficiency and specificity); pH adjustment: for acidic or alkaline milk samples (such as fermented milk), adjust the pH of the detection system to 7.4 (neutral), which can make the standard curve R 2 From 0.985 to 0.999 (to avoid the influence of pH on antibody-antigen binding); Color developing agent: TMB (3,3',5,5'-tetramethylbenzidine) + H2O2.

[0051] 2. Detection process Coating: add 1 μg / mL lactalbumin solution to the enzyme-labeled plate hole, 4℃ incubate overnight, discard the coating solution, and wash the plate 3 times with PBST; Blocking: add 200 μL 1% skimmed milk powder to each well, 37℃ incubate for 1 h, and wash the plate 3 times; Sample addition: add 100 μL of the sample to be tested to each well, then add 100 μL of pAb (diluted 1000 times), 37℃ incubate for 1 h, and wash the plate 3 times; Addition of secondary antibody: add 100 μL of HRP-labeled goat anti-rabbit IgG (diluted 5000 times) to each well, 37℃ incubate for 1 h, and wash the plate 3 times; Color development and detection: add 100 μL of TMB color developing agent to each well, react at room temperature for 15 min, add 50 μL of 2M H2SO4 to stop the reaction, and measure the absorbance at 450 nm.

[0052] According to the standard curve (R 2 ≥0.992) of α-LA, the concentration of α-LA in the sample to be tested is calculated.

[0053] Example 3 An evaluation method for a dairy authenticity process: The α-LA with purity ≥95% was prepared into 40000 μg / L α-LA mother liquor with PBS buffer solution with pH 7.4, and then the α-LA mother liquor was divided into 5 groups, and was treated for 20 min according to the actual milk processing procedure with different heat processing parameters to prepare 5 groups of α-LA standards with different processing parameters, wherein group 1 was the unprocessed α-LA (25°C, normal pressure) as a blank control; group 2 was the pasteurized α-LA (75°C, normal pressure); group 3 was the α-LA with normal pressure and medium-high temperature heat processing (95°C, normal pressure); group 4 was the α-LA with pressure-assisted heat processing (95°C, 0.01 MPa); and group 5 was the UHT α-LA (105°C, 0.02 MPa). After the treatment, the 5 groups were quickly cooled to 25°C.

[0054] The mother liquor in the 5 groups was respectively diluted into 12 concentration gradients of 20 μg / L, 39 μg / L, 78 μg / L, 156 μg / L, 312 μg / L, 625 μg / L, 1250 μg / L, 2500 μg / L, 5000 μg / L, 10000 μg / L, 20000 μg / L and 40000 μg / L, and according to the detection procedure of the ELISA detection method in Example 2, the α-LA standards of groups 1-5 were detected as the milk to be detected, the absorbance value at 450 nm was detected, and the inhibition rate was calculated, and the standard curve was drawn with the concentration of the α-LA standard as the abscissa and the inhibition rate as the ordinate, wherein Figure 1 the detection standard curve of group 1 is shown in FIG. 1, Figure 2 the detection standard curve of group 2 is shown in FIG. 2, Figure 3 the detection standard curve of group 3 is shown in FIG. 3, and groups 4 and 5 cannot be recognized by the antibody due to the large heat processing intensity, so that the standard curve cannot be established. Among them: the higher the inhibition rate, the stronger the antigen recognition performance; the inhibition rate ≥15% is effective recognition, and the higher the value, the stronger the specificity.

[0055] According to the absorbance value and the inhibition rate of the 5 groups of α-LA standards, the heat processing parameter-antigen recognition performance was determined, and a correlation database was established. If the inhibition rate of 1 μg / mL α-LA is 55%-60%, it is determined as “high stability” (corresponding to pasteurization); if the inhibition rate is <10%, it is determined as “extremely low stability” (corresponding to UHT or high-pressure assisted heating), as shown in Table 1: Table 1 Typical parameters of heat processing parameter-antigen recognition performance correlation model based on pAb2

[0056] Comparative Example 1 An ELISA detection method for the content of heat-processed α-LA: Compared with Example 2, the pAb2 detection antibody was replaced with the pAb3 antibody screened in Example 1, and the other steps were the same as in Example 2.

[0057] An evaluation method for a dairy authenticity process: Compared with Example 3, the detection process according to the ELISA detection method in Example 2 was replaced with the detection process according to the ELISA detection method in Comparative Example 1, and the α-LA standard samples in Groups 1 to 5 were detected as the dairy products to be detected, the absorbance values at 450 nm were detected, the inhibition rates were calculated, a correlation database was established, and the results are shown in Table 2: Table 2 Typical parameters of heat processing parameter-antigen recognition performance correlation model based on pAb3

[0058] As can be seen from Table 2, the absorbance values and inhibition rates of the five groups of α-LA standard samples in the heat processing parameter-antigen recognition performance results were all between 32% and 48%, and there was a problem of the same or similar inhibition rates, which could not be used to determine the authenticity of the dairy process.

[0059] Test Example 1 1. Optimization of the conditions of the indirect competitive ELISA method: The detection process of the ELISA detection method in Example 2 was used, and 0.05-1 μg / mL α-LA and 200-25.6 W pAb antibody titers were coated, respectively, to construct an indirect competitive ELISA (ic-ELISA) method. Through the coating antigen concentration x antibody titer matrix, the progressive optimization of the core reaction conditions was completed, and the experimental results are shown in Figure 4 A and B in the middle. As can be seen from the results, taking the basic binding signal (A-A0) as an indicator, when the coating antigen concentration is 0.1-0.2 μg / mL and the antibody titer is 1.6 W-3.2 W, the antigen-antibody binding is the most sufficient Figure 4 A in the middle, deep red high signal), which laid a stable baseline for the subsequent competition reaction; if the antigen or antibody concentration is unbalanced (such as too high coating antigen or too high antibody titer), the signal will drop sharply due to steric hindrance or insufficient binding Figure 4 A in the middle, blue low signal). On this basis, 1 μg / mL α-LA samples were introduced to verify the competition efficiency: when the coating antigen concentration is 0.05-0.2 μg / mL and the antibody titer is 6.4 W-25.6 W, the inhibition rate is significantly higher than that when the coating antigen concentration is 0.2-1 μg / mL and the antibody titer is 2000-3.2 W, and the inhibition rate reaches a peak Figure 4(Middle B, deep red high signal) indicates that the sample antigen can effectively compete with the antibody binding site; if the antigen or antibody concentration is inappropriate (e.g., the coating antigen is too high, the antibody titer is too low), the competition will fail because the coating antigen occupies too many sites or the antibody concentration is too high. Figure 4 (Blue low signal in B). Therefore, the optimal working conditions were ultimately selected as antibody titer 6.4 W and coating agent concentration 0.2 μg / mL.

[0060] 2. Sensitivity verification: Standard curves for 10–10000 μg / L α-LA were constructed under optimal conditions for the indirect competitive ELISA method, and the results are as follows: Figure 4 As shown in Figure C, the α-LA concentration and inhibition rate exhibit a typical S-shaped dose-response relationship, with a steep slope (high sensitivity) in the low concentration range (10~100 μg / L) and a flattening slope (approaching antibody saturation) in the high concentration range (above 10000 μg / L). 2 =0.99, the concentration at which the inhibition rate is 15% (IC50) 15 The concentration was 52.2 μg / L, proving that the method is accurate and linearly stable.

[0061] 3. Specificity Validation: Using the ELISA detection procedure described in Example 2, α-LA at a concentration of 0.5 μg / mL and OVA, β-LG, CN, BSA, and LF at a concentration of 5 μg / mL were measured. The experimental results are as follows: Figure 4 As shown in Figure D, the inhibition rate of α-LA (>50%) was significantly higher than that of other proteins such as OVA and β-LG (<10%), indicating that the pAb2 antibody is highly specific for α-LA and can avoid cross-reaction with other proteins in the milk matrix, thus ensuring the reliability of actual detection.

[0062] Experimental Example 2 Casein precipitation is commonly used for whey protein extraction, but because changes in the solution microenvironment during precipitation can affect the concentration of α-LA, α-LA standards are first processed and detected using this method. Figure 5 (A). The results showed that the detection concentration of α-LA standard in the test solution obtained after pH change and centrifugation was consistent with that of the untreated α-LA standard. Therefore, casein precipitation was used for sample pretreatment for this ELISA.

[0063] Four types of pasteurized milk, one type of UHT milk, and one type of reconstituted milk were selected for casein precipitation assay. The results are as follows: Figure 5The results of the left column chart are the detection results of the alpha-LA kit of Jiangsu Meizheng Factory, and the results of the right column chart are the detection results of the detection method of Example 2. The theoretical content of alpha-LA in milk is about 1000 mg / L, and according to the results of the left column chart of Figure 5 The results of the left column chart are the detection results of the alpha-LA kit of Jiangsu Meizheng Factory, and the results of the right column chart are the detection results of the detection method of Example 2. The theoretical content of alpha-LA in milk is about 1000 mg / L, and according to the results of the left column chart of

[0064] The indirect competitive ELISA method prepared based on the screened alpha-LA specific antibody has good linear relationship (R 2 =0.99), high sensitivity and strong specificity after optimization, and can effectively avoid the cross interference of beta-lactoglobulin, casein and other impurities in milk. The ELISA method can accurately distinguish different heat processing processes through the antigenic difference of alpha-LA. The alpha-LA treated at 75 DEG C (close to the pasteurization temperature) can be detected and the signal is close to that at room temperature. The alpha-LA treated by 105 DEG C and ultra-high temperature instantaneous sterilization (UHT, temperature and pressure synergistic effect) process cannot be detected. The detection of actual samples shows that the concentration of alpha-LA in pasteurized milk is significantly higher than that in UHT milk and reconstituted milk (P<0.001), which further verifies the practicability of the method. In summary, the correlation model of "heat processing parameter-ELISA antigen recognition performance" established by the present application provides an efficient technical means for the rapid tracing of pasteurization and UHT process of milk, and has important practical application value for protecting the quality of dairy products and maintaining the market order.

[0065] The above-mentioned only for the embodiment of the present application, and not therefore limit the patent range of the present application, any equivalent structure or equivalent process transformation, or direct or indirect application in other related technical fields, are also included in the patent protection range of the present application.

Claims

1. A method for screening antibodies for α-lactalbumin detection, characterized in that, Includes the following steps: Serum from animals immunized with α-lactalbumin was collected, and affinity purification was performed to obtain candidate antibodies. The candidate antibodies were then subjected to primary screening and final screening to obtain detection antibodies. The initial screening method includes: detecting unprocessed α-lactalbumin samples, heat-processed α-lactalbumin samples, and BSA interference solution samples with candidate antibodies, calculating the LOD, and screening blank OD of unprocessed α-lactalbumin samples. 450 Antibodies with a LOD of <0.2, LOD of unprocessed α-lactalbumin samples ≤20 µg / L, LOD of heat-processed α-lactalbumin samples ≥50 µg / L, and LOD of BSA interference solution samples ≥100 µg / L are considered as qualified candidate antibodies. The final screening method includes: detecting α-lactalbumin with qualified candidate antibodies, fitting a detection curve of α-lactalbumin treatment temperature versus absorbance, denoted as curve 1; detecting α-lactalbumin with qualified candidate antibodies, fitting a detection curve of α-lactalbumin treatment pressure versus absorbance, denoted as curve 2; and screening curve 1 R. 2 ≥0.995, Curve 2 R 2 Qualified candidate antibodies with a value ≥0.990 and a negative correlation between curve 1 and curve 2 are used as detection antibodies.

2. The screening method according to claim 1, characterized in that, The LOD is calculated based on a signal-to-noise ratio of 2 to 4.

3. The screening method according to claim 1, characterized in that, The processing temperature is 25℃~150℃, and the processing pressure is 0~0.5 MPa.

4. The α-lactalbumin detection antibody obtained by the screening method according to any one of claims 1 to 2.

5. A kit for detecting α-lactalbumin, characterized in that, Including the α-lactalbumin detection antibody as described in claim 3.

6. The use of the α-lactalbumin detection antibody of claim 4 or the kit of claim 5 in the detection of α-lactalbumin content.

7. An ELISA method for detecting α-lactalbumin content, characterized in that, The α-lactalbumin detection antibody according to claim 4 is mixed with a thermally processed α-lactalbumin sample and incubated, followed by color development and determination to obtain the content of α-lactalbumin in the sample.

8. The ELISA detection method according to claim 7, characterized in that, The pH value of the heat-processed α-lactalbumin test sample was 7.0~8.

0.

9. The application of the α-lactalbumin detection antibody of claim 4 or the kit of claim 5 in the evaluation of dairy product authenticity processes.

10. A method for evaluating the authenticity of dairy product processes using the α-lactalbumin detection antibody of claim 4 or the kit of claim 5, characterized in that... The inhibition rate of α-lactalbumin in the tested dairy products was determined by using the α-lactalbumin detection antibody to assess the dairy product process. The criteria for determination are as follows: an inhibition rate of 55%-60% indicates that the dairy product under test was processed by pasteurization; an inhibition rate of 15%-20% indicates that the dairy product under test was processed by medium-high temperature processing; and an inhibition rate of <10% indicates that the dairy product under test was processed by high-pressure assisted heat processing or UHT.

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