Screening method of lactoferrin detection antibody pair and application of screening method in lactoferrin detection and dairy product process authenticity evaluation

Abstract

The invention provides a screening method of lactoferrin detection antibody pairs and application of the screening method in lactoferrin detection and dairy product process authenticity evaluation, and relates to the technical field of dairy product process detection. The screening method of the lactoferrin detection antibody pair comprises the following steps: immunizing an animal with lactoferrin, then obtaining a monoclonal antibody through a hybridoma technology, obtaining a polyclonal antibody through affinity purification, carrying out cross combination on the monoclonal antibody and the polyclonal antibody to form a candidate antibody pair, and screening the candidate antibody pair to obtain the lactoferrin detection antibody pair. And carrying out primary screening, medium screening and final screening on the candidate antibody pair to obtain a detection antibody pair. The detection antibody pair screened by the method provided by the invention can specifically identify lactoferrin, can avoid cross reaction of other proteins in a milk matrix, and ensures the reliability of actual detection. The invention also utilizes the screened detection antibody to construct a hot processing parameter-lactoferrin antigen recognition relationship, and provides an efficient technical means for rapid traceability of hot processing technologies such as milk pasteurization, UHT technology and the like.

Description

Technical Field

[0001] This invention belongs to the field of dairy processing testing technology, specifically relating to a method for screening antibody pairs for lactoferrin detection and its application in lactoferrin detection and dairy processing authenticity evaluation. Background Technology

[0002] Milk heat processing is a crucial step in ensuring food safety and extending shelf life. Different heat processing techniques, such as pasteurization (60-85℃ at normal pressure), medium-high temperature processing (90-100℃), and ultra-high temperature sterilization (UHT) (≥135℃, or equivalent long-term treatment at 105-120℃), directly affect the retention of milk's nutrients (such as lactoferrin activity), sensory quality, and market positioning. Currently, the methods for identifying milk heat processing techniques have significant shortcomings: traditional methods rely on detecting microbial residues (e.g., pasteurized milk contains a small number of live bacteria, UHT milk is sterile), but the detection cycle is long (requiring 24-48 hours of incubation), and sensory evaluation (such as flavor and color) is highly subjective and easily affected by differences in raw milk.

[0003] Lactoferrin is a key functional active protein in milk, and its activity is directly related to the integrity of its antigenic structure. Milk heat processing (such as pasteurization, high-temperature processing, and ultra-high-temperature sterilization) is a core factor affecting the structure of the lactoferrin antigen. ELISA (Enzyme-Linked Immunosorbent Assay) is currently the mainstream method for detecting lactoferrin, relying on the specific binding of antibodies to lactoferrin epitopes, i.e., antigen recognition performance. For specific antibodies, the ELISA antigen recognition performance of lactoferrin shows a significant quantitative relationship with heat processing temperature, and the natural lactose in milk can mitigate the damage to its antigenic structure caused by heat processing. Therefore, to prevent cross-reactivity of proteins in dairy products and the effects of heat processing on the denaturation of lactoferrin epitopes, and to accurately determine the heat processing technology of dairy products, it is urgent to develop a method that uses lactoferrin as an antigen to accurately determine dairy processing technology. Summary of the Invention

[0004] To address the aforementioned technical problems, the primary objective of this invention is to provide a method for screening antibody pairs for lactoferrin detection. The antibody pairs screened by this method can highly specifically recognize lactoferrin, avoiding cross-reactivity with other proteins in the milk matrix and ensuring the reliability of actual detection.

[0005] A second objective of this invention is to provide a detection antibody pair obtained by the above-described screening method.

[0006] A third objective of this invention is to provide a kit for detecting lactoferrin content.

[0007] A fourth objective of this invention is to provide the application of the above-described detection antibody pairs or the above-described kit in detecting different heat-processed lactoferrin contents.

[0008] The fifth objective of this invention is to provide an ELISA method for detecting lactoferrin content.

[0009] The sixth objective of this invention is to provide an application of the above-described detection antibody pair or the above-described kit in the evaluation of dairy product authenticity processes.

[0010] The seventh objective of this invention is to provide a method for evaluating the authenticity of dairy product processes using the above-mentioned detection antibody pairs or the above-mentioned kits.

[0011] To achieve the above-mentioned objectives, the technical solution of this invention is as follows: This invention provides a method for screening antibody pairs for lactoferrin detection, comprising the following steps: immunizing animals with lactoferrin, then obtaining monoclonal antibodies through hybridoma technology, obtaining polyclonal antibodies through affinity purification, cross-combining monoclonal antibodies and polyclonal antibodies to form candidate antibody pairs, and obtaining detection antibody pairs through primary screening, intermediate screening and final screening of candidate antibody pairs; The initial screening method includes: using candidate antibody pairs to detect unprocessed lactoferrin samples and BSA interference solution samples respectively, and screening antibody pairs with P / N ≥ 2.1 for unprocessed lactoferrin samples and P / N < 1.5 for BSA interference solution samples as qualified antibody pairs for initial screening; The intermediate screening method includes: using antibody pairs that passed the initial screening to detect unprocessed lactoferrin samples, moderately heat-processed lactoferrin samples, and heavily heat-processed lactoferrin samples; calculating the level of deficiency (LOD); and screening antibody pairs with LOD ≤ 100 μg / L for unprocessed lactoferrin samples, LOD ≤ 1000 μg / L for moderately heat-processed lactoferrin samples, and LOD ≥ 20000 μg / L for heavily heat-processed lactoferrin samples as qualified antibody pairs for intermediate screening. The temperature of the moderate heating process is 80~100℃, and the temperature of the heavily heat-processed process is 100-105℃. The final screening method includes: detecting lactoferrin with antibodies that passed the intermediate screening, fitting a detection curve of lactoferrin treatment temperature versus absorbance, and screening the curve R. 2 Antibody pairs with an absorbance value ≥0.990 and a negative correlation between heat treatment intensity and absorbance value were used as detection antibody pairs.

[0012] As one implementation method, the preparation method of the monoclonal antibody includes: immunizing mice with lactoferrin, then fusing mouse spleen cells with myeloma cells, and obtaining the antibody from the supernatant after expansion culture.

[0013] As one implementation method, the preparation method of the polyclonal antibody includes: immunizing rabbits with lactoferrin, and then obtaining polyclonal antibodies through affinity purification from rabbit serum.

[0014] The present invention also provides a detection antibody pair obtained by the above screening method.

[0015] The present invention also provides a kit for detecting lactoferrin content, comprising the above-mentioned detection antibody pair.

[0016] The present invention also provides the application of the above-described detection antibody pair or the above-described kit in the detection of heat-processed lactoferrin content.

[0017] The present invention also provides an ELISA method for detecting lactoferrin content, wherein the above-mentioned detection antibody is mixed with the heat-processed lactoferrin sample and incubated, followed by color development and measurement to obtain the lactoferrin content in the sample.

[0018] In one implementation, the monoclonal antibody in the detection antibody pair is used for coating, and the polyclonal antibody in the detection antibody pair is used as the primary antibody.

[0019] The present invention also provides the application of the above-mentioned detection antibody pairs or the above-mentioned kits in the evaluation of dairy product authenticity processes.

[0020] This invention also provides a method for evaluating the authenticity of dairy product processes using the aforementioned detection antibody or kit. The detection antibody is used to determine the dairy product process by detecting the concentration of lactoferrin in the tested dairy product. The criteria for determination are: a concentration >100 μg / mL indicates pasteurization; 20~100 μg / mL indicates medium-high temperature processing; and <20 μg / mL indicates ultra-high temperature sterilization or high-pressure assisted heat processing.

[0021] The advantages of this invention compared to existing technologies are as follows: The detection antibody pairs screened by the method of this invention can highly specifically recognize lactoferrin, avoid cross-reactivity with other proteins in the milk matrix, and ensure the reliability of actual detection.

[0022] This invention uses lactoferrin as the core target. By screening antibody pairs that can accurately capture the quantitative relationship between lactoferrin antigen recognition and thermal processing parameters, and combining them with ELISA detection technology, it achieves simultaneous detection of milk thermal processing technology and lactoferrin content. This solves the problems of lack of traditional process discrimination methods and strong subjectivity, while achieving accurate detection of lactoferrin content. It can be used for quality control and market supervision in dairy product enterprises. Attached Figure Description

[0023] Figure 1 The changes in immunodetection signals when monoclonal antibodies and polyclonal antibodies are used as capture antibodies and detection antibodies, respectively: System 1: Monoclonal antibody is used as capture antibody and polyclonal antibody is used as detection antibody; System 2: Polyclonal antibody is used as capture antibody and monoclonal antibody is used as detection antibody. Figure 2 This is a standard curve for the detection of unprocessed lactoferrin. Figure 3 Standard curve for detecting lactoferrin processed at 75℃; Figure 4 Standard curve for detecting lactoferrin processed at 95℃; Figure 5 Standard curve for detecting lactoferrin processed at 105℃; Figure 6 The image shows the results of LF content detection in milk samples. Figure 7 The graph shows the detection results of lactoferrin content in milk samples. The outer bar graph represents the detection results of a commercially available reagent kit, while the inner bar graph represents the detection results of the detection method in Example 2. Detailed Implementation

[0024] This invention provides a method for screening antibodies for lactoferrin detection, comprising the following steps: immunizing animals with lactoferrin, then obtaining monoclonal antibodies through hybridoma technology, obtaining polyclonal antibodies through affinity purification, cross-combining monoclonal antibodies and polyclonal antibodies to form candidate antibody pairs, and obtaining detection antibody pairs through primary screening, intermediate screening and final screening of candidate antibody pairs.

[0025] In this invention, the method for preparing monoclonal antibodies includes the following steps: injecting emulsion A into mice for primary immunization, then injecting emulsion B into mice for booster immunization. After the immunization program is completed, whole blood is collected from the mice, and serum is obtained after standing. Emulsion A is unprocessed lactoferrin and Freund's complete adjuvant in a volume ratio of 1:0.5-1.5, and emulsion B is unprocessed lactoferrin and Freund's incomplete adjuvant in a volume ratio of 1:0.5-1.5. The concentration of unprocessed lactoferrin 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 first injection in this invention is primary immunization, and subsequent injections are booster immunizations; the injection dose for each mouse is 45-55 μg, and the injection site is intraperitoneal injection. The injection cycle of this invention is two weeks / injection, and the number of injections is 2-5 times, preferably 3, 4, or 5 times. The lactoferrin is purchased from Sigma-Aldrich and is of analytical grade. One week after the immunization program ends, 0.5-1.5 mg / mL of unprocessed lactoferrin is injected into mice at the injection dose for a sprint immunization. The antibody titer of this invention is >1:100,000. Preferably, in this invention, blood is collected from the orbital cavity of mice 8-10 days after the second to fourth immunizations, diluted 1000-500,000 times with PBS, and the antibody titer is detected by indirect competitive ELISA. The immunization program ends when the antibody titer is >1:100,000. In this invention, mice with the highest titer are selected, and their spleen cells are fused with myeloma cells, screened for positive cell wells, cloned, and cultured to obtain monoclonal antibodies.

[0026] In this invention, the preparation method of polyclonal antibodies includes the following steps: injecting emulsion A into rabbits for primary immunization, then injecting emulsion B into rabbits for booster immunization. After the immunization program is completed, whole blood from the rabbits is collected, allowed to stand, and then separated to obtain serum. Emulsion A is unprocessed lactoferrin and Freund's complete adjuvant in a volume ratio of 1:0.5-1.5, and emulsion B is unprocessed lactoferrin and Freund's incomplete adjuvant in a volume ratio of 1:0.5-1.5. The concentration of unprocessed lactoferrin 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 first injection in this invention is primary immunization, and subsequent injections are booster immunizations; the injection dose for each rabbit is 1-2 mg, and the injection sites are multiple subcutaneous injections. The injection cycle of this invention is two weeks / injection, and the number of injections is 2-5 times, preferably 3, 4, or 5 times. One week after the immunization program ends, 0.5-1.5 mg / mL of unprocessed lactoferrin is injected into rabbits at the injection dose for a sprint immunization. The antibody titer of this invention is >1:100,000. Preferably, blood is collected from rabbits 8-10 days after the 2nd to 4th immunizations, diluted 1000-500,000 times with PBS, and the antibody titer is detected by indirect competitive ELISA. The immunization program ends when the antibody titer is >1:100,000. The serum obtained from the rabbits is subjected to affinity purification. As one embodiment, the affinity purification method includes Protein A affinity purification to obtain polyclonal antibodies. The monoclonal antibodies and polyclonal antibodies of this invention are cross-combined as candidate antibody pairs. In this invention, the indirect competitive ELISA detection of antibody titer and the Protein A purification column purification are performed according to conventional procedures.

[0027] In this invention, the initial screening method includes detecting unprocessed lactoferrin samples and BSA interference solutions using candidate antibody pairs, respectively. The protein concentration of the unprocessed lactoferrin samples is 10 μg / L to 40000 μg / L, preferably 20 μg / L, 40 μg / L, 80 μg / L, 150 μg / L, 300 μg / L, 650 μg / L, 1250 μg / L, 2500 μg / L, 5000 μg / L, 10000 μg / L, 20000 μg / L, or 40000 μg / L. The concentration of the BSA interference solution samples is 10 μg / L to 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. μg / L; then calculate the P / N value for each sample, and screen qualified antibody pairs based on the P / N value. This invention screens antibody pairs with P / N ≥ 2.1 for unprocessed lactoferrin samples, P / N < 1.5 for BSA interference solutions samples, and RSD ≤ 5% for parallel detection, retaining antibody pairs that meet specificity and repeatability requirements as qualified antibody pairs for initial screening.

[0028] In this invention, the intermediate screening method includes detecting unprocessed lactoferrin samples, moderately heat-processed lactoferrin samples, and heavily heat-processed lactoferrin samples with qualified antibody pairs from the initial screening, fitting detection curves of different heat-processed lactoferrin treatment concentrations and absorbance, and then calculating the limit of detection (LOD) for each antibody pair. The LOD is calculated based on a signal-to-noise ratio of 2 to 4, preferably 3. Qualified antibody pairs from the intermediate screening are then selected based on the LOD. In this invention, the protein concentration of the unprocessed lactoferrin sample is 10 μg / L to 40000 μg / L, preferably 20 μg / L, 40 μg / L, 80 μg / L, 150 μg / L, 300 μg / L, 650 μg / L, 1250 μg / L, 2500 μg / L, 5000 μg / L, 10000 μg / L, 20000 μg / L, or 40000 μg / L. The temperature of the moderate heat treatment is 80~100℃; the protein concentration of the moderately heat-treated lactoferrin sample is 10 μg / L~40000 μg / L, preferably 20 μg / L, 40 μg / L, 80 μg / L, 150 μg / L, 300 μg / L, 650 μg / L, 1250 μg / L, 2500 μg / L, 5000 μg / L, 10000 μg / L, 20000 μg / L or 40000 μg / L. The temperature of the heavy heat processing is 100~105℃, and the protein concentration of the heavily heat-processed lactoferrin sample is 10 μg / L~40000 μg / L, preferably 20 μg / L, 40 μg / L, 80 μg / L, 150 μg / L, 300 μg / L, 650 μg / L, 1250 μg / L, 2500 μg / L, 5000 μg / L, 10000 μg / L, 20000 μg / L, or 40000 μg / L. Antibody pairs with a LOD ≤ 100 μg / L for unprocessed lactoferrin samples, LOD ≤ 1000 μg / L for moderately heat-processed lactoferrin samples, and LOD ≥ 20000 μg / L for heavily heat-processed lactoferrin samples are selected as qualified antibody pairs for the intermediate screening. This invention retains antibody pairs that meet the sensitivity standards through the intermediate screening.

[0029] In this invention, the final screening method includes: detecting lactoferrin with antibodies that passed the intermediate screening, and fitting a detection curve of lactoferrin treatment temperature versus absorbance; the treatment temperature is 25℃~150℃, and as an optional embodiment, the treatment temperature is 25℃, 75℃, 95℃, or 105℃; the treatment pressure of the temperature gradient group is atmospheric pressure, and the treatment time is 15~25 min, preferably 20 min. Then, the screening curve R is selected. 2Antibody pairs with an absorbance value ≥0.990 and a negative correlation between heat treatment intensity and absorbance value are selected as detection antibody pairs. This invention can accurately capture the quantitative relationship between heat treatment process parameters and lactoferrin antigen recognition performance through final screening, and select antibody pairs that meet the rule that the higher the heat treatment intensity, the lower the absorbance value and the weaker the antigen recognition performance, as the core antibody pairs for subsequent ELISA detection.

[0030] The present invention also provides a detection antibody pair obtained by the above screening method.

[0031] This invention also provides a kit for detecting lactoferrin content, comprising the aforementioned detection antibody pair. The kit further includes a coating solution, a blocking solution, and goat anti-rabbit IgG.

[0032] The present invention also provides the application of the above-described detection antibody pair or the above-described kit in the detection of heat-processed lactoferrin content.

[0033] This invention also provides an ELISA method for detecting lactoferrin content, comprising the following steps: mixing and incubating the above-mentioned detection antibody pair with a heat-processed lactoferrin sample, followed by color development and measurement to obtain the lactoferrin content in the sample. In this invention, the sample is first coated with 0.1~10 µg / mL monoclonal antibody from the detection antibody pair, preferably at a concentration of 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. Then, it is incubated at 2~8℃ for 12~16 h, preferably at 3℃, 4℃, 5℃, 6℃, or 7℃; the incubation time is preferably 13 h, 14 h, or 15 h. In this invention, after discarding the coating solution, a blocking agent is added, comprising 1% gelatin, 1% fish skin glue, 1% fetal bovine serum, 1% skim milk powder, or 1% BSA; then incubated at 35-38°C for 0.5-1.5 h, preferably at 36°C or 37°C; 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 lactoferrin sample to be tested and the polyclonal antibody in the detection antibody pair diluted 800-1200 times are added, and incubated at 35-38°C for 0.5-1.5 h, preferably at 36°C or 37°C; 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, add HRP-labeled goat anti-rabbit IgG diluted 4000-6000 times, and incubate at 35-38℃ for 0.5-1.5 h, preferably at 36℃ or 37℃; the preferred incubation time is 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, add TMB chromogenic reagent and react at room temperature in the dark for 10-20 min. After adding stop solution, measure the absorbance value and calculate the lactoferrin content in the sample.

[0034] Based on the ability of this invention to detect the content of heat-processed lactoferrin, this invention also provides the application of the above-mentioned detection antibody pairs or the above-mentioned kits in the evaluation of dairy product authenticity processes.

[0035] This invention also provides a method for evaluating the authenticity of dairy product processing using the aforementioned detection antibody or kit. The detection antibody is used to determine the dairy product processing by detecting the concentration of lactoferrin in the tested dairy product. The criteria for determination are: concentration >100 μg / mL indicates pasteurization; 20-100 μg / mL indicates medium-high temperature processing; and <20 μg / mL indicates ultra-high temperature sterilization or high-pressure assisted heat processing. In this invention, five sets of lactoferrin standards with different processing parameters are constructed. A standard curve of absorbance values ​​versus lactoferrin concentration is obtained using the aforementioned ELISA detection method. The absorbance value is the absorbance at 450 nm measured by a microplate reader. This invention determines the processing technology of the tested dairy product based on its lactoferrin content. As an optional implementation, the tested dairy product includes fresh milk, pasteurized milk, UHT milk, or dairy processing intermediates.

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0037] Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available. Unless otherwise specified, they are generally used under conventional conditions or as recommended by the company.

[0038] Example 1 A screening method for lactoferrin detection antibody pairs, the specific procedure is as follows: Figure 1 As shown: 1. Preparation of candidate antibody pairs (1) Immunization: Five Balb / c mice were injected with emulsion A at a dose of 50 µg / mouse for primary immunization. Emulsion A was prepared by thoroughly emulsifying 1 mg / mL unprocessed LF and Freund's complete adjuvant at a volume ratio of 1:1. Subsequently, Balb / c mice were immunized every two weeks with emulsion B at a dose of 50 µg / mouse. Emulsion B was prepared by thoroughly emulsifying 1 mg / mL unprocessed LF and Freund's incomplete adjuvant at a volume ratio of 1:1. Eight to ten days after the second to fourth immunizations, tail vein blood was collected from the mice. The blood was diluted 1000-500000 times with PBS and the antibody titer was detected by indirect competitive ELISA. Immunization was terminated when the antibody titer was >1:100000. One week after the immunization, Balb / c mice were injected with 1 mg / mL unprocessed LF at a dose of 50 µg / mouse for sprint immunization. Balb / c mice with the highest titer after immunization were euthanized by cervical dislocation, and spleen cells were collected. After fusing with myeloma cells, anti-lactoferrin hybridoma cells were screened, cloned, and expanded cultured to obtain 15 anti-lactoferrin monoclonal antibodies (mAb1-mAb15) from the supernatant.

[0039] Five New Zealand white rabbits were injected with emulsion A (1.5 mg / rabbit) for primary immunization. Emulsion A was prepared by thoroughly emulsifying 1 mg / mL unprocessed lactoferrin with Freund's complete adjuvant at a 1:1 volume ratio. Subsequently, rabbits were immunized every two weeks with emulsion B (1.5 mg / rabbit) for booster immunization. Emulsion B was prepared by thoroughly emulsifying 1 mg / mL unprocessed lactoferrin with Freund's incomplete adjuvant at a 1:1 volume ratio. Eight to ten days after the second to fourth immunizations, ear vein blood was collected from the rabbits. The blood was diluted 1000-500000 times with PBS, and antibody titers were detected using an indirect competitive ELISA. Immunization was terminated when the antibody titer was >1:100000. One week after the initial immunization, rabbits were injected with 1 mg / mL unprocessed lactoferrin (1.5 mg / rabbit) for a wash immunization. Whole blood was collected from immunized rabbits, allowed to stand, and separated to obtain serum. Serum was then purified using a Protein A purification column with Protein A-Sepharose 4B as the packing material. The specific steps are as follows: Equilibrate the column: Rinse the tubing with phosphate buffer (pH 7.4) at a flow rate of 6.5 mL / min for 2 min. After packing the column, equilibrate it again with phosphate buffer (pH 7.4) at a flow rate of 1 mL / min until baseline is reached.

[0040] Sample loading: After reaching baseline levels, dilute the separated serum with an equal volume of pH 7.4 phosphate buffer (1:1, V / V) and load it onto the column at a flow rate of 0.5 mL / min. After loading, flush the column with pH 7.4 binding buffer at a flow rate of 1 mL / min. The antibody is specifically adsorbed onto the packing site, while other proteins elute with the buffer until baseline levels are reached.

[0041] Elution: Elute the antibody bound to the column with elution buffer at pH 2.7 to obtain the target protein at a flow rate of 0.5 mL / min.

[0042] Determination: The protein concentration of the eluent was detected under 280 nm ultraviolet light. When the absorbance A 280 When the pH is >0.2, collect the eluent and immediately adjust the pH to 7.0 with 1 mol / L Tris.

[0043] Antibody dialysis and storage: The obtained antibody was dialyzed with pH 7.4 antibody dialysis buffer (phosphate buffer, PB), dialyzed at 4℃ for three days, with the dialysis solution changed three times a day. After dialysis, the antibody was removed, 0.1% (w / v) NaN3 was added, and the antibody was stored at 4℃ for later use.

[0044] Preparation of the purification column: After antibody collection, quickly wash the column with 0.1 M acetic acid solution for 2 min at a flow rate of 2 mL / min, then equilibrate the column with phosphate buffer (pH 7.4) until the pH of the eluent is neutral. Finally, wash the column with 20% ethanol solution, fill it with water after 20 min, and store it at 4°C.

[0045] After purification, five anti-lactoferrin polyclonal antibodies (pAb1-pAb5) were obtained.

[0046] (2) The 15 monoclonal antibodies (mAb1-mAb15) and 5 polyclonal antibodies (pAb1-pAb5) obtained were cross-combined to form 75 candidate antibody pairs.

[0047] 2. Step-by-step screening The tiered screening process narrows down the candidate pool step by step in the order of "initial screening - intermediate screening - final screening" to ensure that the final selected antibodies meet the full range of requirements of "specificity - sensitivity - linear correlation".

[0048] (1) Initial screening: Sample testing: The obtained candidate antibody pairs were used to test 100 µg / L unprocessed LF and 100 µg / L BSA samples, respectively. The P / N value of each candidate antibody pair was calculated, and antibody pairs with P / N < 2.1 and RSD > 5% for unprocessed LF and P / N ≥ 1.5 for BSA were eliminated. This invention eliminated 38 candidate antibody pairs through initial screening, retaining 37 candidate antibody pairs (such as mAb2-pAb1, mAb3-pAb2, etc.) as qualified antibody pairs in the initial screening.

[0049] (2) Medium sieve: Using antibody pairs that passed the initial screening, we tested unprocessed lactoferrin standards, lactoferrin samples heated at 95°C for 20 min, and lactoferrin samples heated at 105°C for 20 min, at gradient concentrations of 20 μg / L, 40 μg / L, 80 μg / L, 150 μg / L, 300 μg / L, 650 μg / L, 1250 μg / L, 2500 μg / L, 5000 μg / L, 10000 μg / L, 20000 μg / L, and 40000 μg / L, as well as lactoferrin test samples heated at 105°C for 20 min. We calculated the limit of detection (LOD) for the antibody pairs that passed the initial screening, based on a signal-to-noise ratio of 3. We discarded samples with an LOD > 100 μg / L for unprocessed lactoferrin, an LOD > 1000 μg / L for lactoferrin samples heated at 95°C for 20 min, and an LOD < 20000 μg / L for lactoferrin test samples heated at 105°C for 20 min. Antibody pairs with a concentration of μg / L were selected, and those that met the sensitivity criteria were ultimately retained. This invention eliminated 22 antibody pairs that passed the initial screening through intermediate screening, retaining 15 antibody pairs that passed the initial screening (including mAb3-pAb2 and mAb5-pAb3) as antibody pairs that passed intermediate screening.

[0050] (3) Final screening: First, thermally processed LF samples were prepared at temperature gradients (25℃, 75℃, 95℃, 105℃, treated for 20 min). Then, 15 antibody pairs that passed the intermediate screening were used to detect the samples at these temperature gradients and the absorbance values ​​were recorded. The antibody pairs that passed the intermediate screening and conformed to the rule of "higher thermal processing intensity - lower absorbance value - weaker antigen recognition performance" were considered the optimal antibody pairs. Experimental results showed that the mAb3-pAb2 antibody pair accurately captured the quantitative relationship between "thermal processing parameters - lactoferrin antigen recognition performance," showing significant sensitivity to temperature changes and meeting all requirements, making it the core antibody pair for subsequent ELISA detection. Other antibody pairs (such as mAb7-pAb4) did not conform to the rule of "higher thermal processing intensity - lower absorbance value - weaker antigen recognition performance" and were therefore unsatisfactory.

[0051] Example 2 An ELISA method for detecting LF content in heat-processed products: 1. Reagent preparation: Diluent: 0.05 M carbonate buffer at pH 9.6; Coating solution: Dilute monoclonal antibody mAb3 to 1 µg / mL with diluent; Primary antibody dilution buffer: Dilute the polyclonal antibody pAb2 1000 times with the dilution buffer; Secondary antibody dilution buffer: Dilute HRP (horseradish peroxidase) labeled goat anti-rabbit IgG 5000 times with dilution buffer; Blocking agent: 1% skim milk powder (prepared with PBS); TMB colorimetric reagent: TMB (3,3',5,5'-tetramethylbenzidine) + H2O2.

[0052] 2. Testing process: Coating: Add 100 µL of monoclonal antibody dilution to the wells of the microplate, incubate overnight at 4°C, discard the solution, and wash the plate three times with PBST. Blocking: Add 200 µL of 1% skim milk powder to each well, incubate at 37°C for 1 h, and wash the plate 3 times; Add 100 µL of the sample diluted 10-fold to each well, incubate at 37°C for 1 h, and wash the plate 3 times. Add primary antibody: Add 100 µL of polyclonal antibody dilution buffer to each well, incubate at 37°C for 1 h, and wash the plate 3 times; Add secondary antibody: Add 100µL of secondary antibody dilution buffer to each well, incubate at 37℃ for 1 h, and wash the plate 3 times; Color development and detection: Add 100 μL of TMB colorimetric reagent to each well, react at room temperature in the dark for 15 min, add 50 μL of 2MH2SO4 to stop the reaction, and measure the absorbance at 450 nm using an ELISA reader.

[0053] This invention is based on the LF standard curve (R 2 =0.993) Calculate the LF concentration in the sample to be tested.

[0054] Example 3 A method for evaluating the authenticity of dairy product processes: 1. Preparation of lactoferrin standards with different processing parameters Unprocessed milk LF with a purity ≥95% was used to prepare a 10000 µg / L LF stock solution using PBS buffer at pH 7.4. The LF stock solution was then divided into 5 groups, and each group was treated with different heat processing parameters for 20 min according to the actual milk processing procedure, resulting in 5 groups of LF standards with different processing parameters. Group 1 was unprocessed LF (25℃, atmospheric pressure) as a blank control; Group 2 was pasteurized LF (75℃, atmospheric pressure); Group 3 was LF processed at medium-high temperature under atmospheric pressure (95℃); and Group 4 was ultra-high temperature sterilized LF (105℃). All samples were rapidly cooled to 25℃ after treatment.

[0055] The mother liquors from the five groups were diluted to 12 concentration gradients: 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. Following the ELISA detection procedure in Example 2, the LF standards from groups 1 to 5 were used as the dairy products to be tested. The absorbance at 450 nm and the P / N ratio were measured. A standard curve was plotted with the LF standard concentration on the x-axis and the absorbance value on the y-axis. Figure 2 This is the detection standard curve for Group 1. Figure 3 The standard curve for Group 2 is shown. Figure 4 This is the detection standard curve for Group 3. Figure 5 This is the detection standard curve for Group 4. The higher the absorbance value, the stronger the antigen recognition performance; P / N ≥ 2.1 indicates effective recognition, and a higher value indicates stronger specificity.

[0056] Comparative Example 1 The difference from Example 2 is that the polyclonal antibody pAb2 was diluted to 1 µg / mL with diluent as the coating solution, and the monoclonal antibody mAb3 was diluted 1000 times with diluent as the primary antibody diluent. All other steps are the same as in Example 2.

[0057] Experimental Example 1 1. Detection was performed using two detection systems, Example 2 and Comparative Example 1, respectively. The relationship between the immunodetection signal (P / N value) and the dilution of the detection antibody was analyzed. Figure 1In the experimental results, for the detection system of Example 2 (orange-pink gradient column), the P / N value showed a characteristic of first increasing and then decreasing with the increase of the detection antibody dilution factor. At low dilutions (16000-64000), due to the high concentration of detection antibody, the sandwich structure was easily saturated. When the dilution reached 128000-256000, the P / N value reached its peak (3.4-3.7). At this time, the binding of detection antibody concentration with antigen and capture monoclonal antibody reached the optimal balance. The polyclonal antibody can recognize multiple antigenic epitopes of LF and synergize with the single epitope of monoclonal antibody to maximize the sandwich signal. At high dilutions (512000-1024000), the detection antibody concentration was too low, the binding probability decreased, and the P / N value decreased significantly (3.2-2.0). The P / N value of the detection system of Comparative Example 1 (green-purple gradient column) was generally lower than that of the system of Example 2, and the change trend was gradual. Within the full dilution range (16,000-1,024,000), the P / N value remained stable in the range of 1.2-1.8, with a maximum value of only 1.8 (far lower than the peak value of 3.7 in Example 2). This is because when Comparative Example 1 used polyclonal antibodies for capture, the LF antigen epitope was partially masked (polyclonal antibodies recognize multiple epitopes, which may overlap with the epitopes detected by monoclonal antibodies), reducing the sandwich efficiency; at the same time, the signal of this system fluctuated little with dilution, limiting the detection sensitivity.

[0058] 2. Sensitivity Validation: Based on the detection method in Example 2, this invention established optimal sELISA working conditions with a coating concentration of 0.1 µg / mL, a polyclonal antibody titer of 1:512000, and a blocking buffer of 1% skim milk powder. Under these optimal sELISA conditions, unprocessed lactoferrin (LF) standards were serially diluted from 40 µg / mL to 19 µg / L. A standard curve for the double-antibody sandwich ELISA was plotted using a logistic model in Origin software, with absorbance as the ordinate and protein standard concentration as the abscissa, for parameter fitting (see [link to Origin software]). Figure 2 The curve has a background signal value (N value) of 0.282, a detection limit (LOD, 3σ) of 51.2 µg / L, and a linear range of 156–10000 µg / L (correlation coefficient R). 2 =0.993), showing a good linear correlation, proving that the method of the present invention is accurate in quantification and linearly stable.

[0059] This invention, under optimal conditions for the sELISA method, serially diluted lactoferrin (LF) standard processed at 75℃ from 40 µg / mL up to 19 µg / L. Using absorbance as the ordinate and protein standard concentration as the abscissa, a logistic model from Origin software was used for parameter fitting to plot the double-antibody sandwich ELISA standard curve (see [link to sELISA method]). Figure 3The background signal value (N value) of the curve is 0.282, and the detection limit (LOD, 3σ) reaches 325 µg / L.

[0060] This invention, under optimal conditions for the sELISA method, serially diluted lactoferrin (LF) standard processed at 95℃ from 40 µg / mL up to 19 µg / L. Using absorbance as the ordinate and protein standard concentration as the abscissa, a logistic model from Origin software was used for parameter fitting to plot the double-antibody sandwich ELISA standard curve (see [link to sELISA method]). Figure 4 The background signal value (N value) of the curve is 0.282, and the detection limit (LOD, 3σ) reaches 1200 µg / L.

[0061] This invention, under optimal sELISA conditions, serially diluted lactoferrin (LF) standards processed at 105°C from 40 µg / mL up to 19 µg / L. Parameters were fitted using a logistic model in Origin software, with absorbance as the ordinate and protein standard concentration as the abscissa (see [link to Origin software]). Figure 5 This sELISA cannot recognize lactoferrin processed at 105°C.

[0062] 2. Specificity Validation: Five proteins were selected: bovine serum albumin (BSA), ovalbumin (OVA), casein (CN), β-lactoglobulin (β-LG), and α-lactalbumin (α-LA). Each protein was supplemented with 1 μg / mL of standard, and the P / N values ​​were determined using the ELISA method described in Example 2 (see [link to Example 2]). Figure 6 The results showed that only LF had a significantly higher P / N value than other proteins (close to 5), while the P / N values ​​of BSA, OVA, β-LG, α-LA, and CN were all below 2. This fully demonstrates that the double-antibody sandwich ELISA method of the present invention has good specificity for LF and can effectively distinguish LF from other common milk-derived proteins.

[0063] Experimental Example 2 Commercially available pasteurized milk, UHT milk, and reconstituted milk were selected as models. Casein was removed using isoelectric point precipitation, and the milk was diluted several times before ELISA detection. Results showed that this method only had a significant ability to identify pasteurized milk, but could not identify UHT milk or pasteurized milk. Furthermore, the detection results of LF concentration were compared with commercial ELISA kits to verify this method. The lactoferrin content in milk ranges from 31.78 to 485.63 µg / mL. The method developed in this invention detected slightly lower levels of lactoferrin in pasteurized milk (types 1-3) than the ELISA kit, but the overall results were consistent. However, the method of this invention could not detect LF in UHT milk and reconstituted milk, while the detection results of the ELISA kits were not significantly different from those of pasteurized milk types 1 and 4. This is because commercial kits use methanol as a pretreatment reagent, which alters the structure of lactoferrin after heat processing, reducing some epitopes.

[0064] Samples 1-6 of heat-processed milk were selected for testing (sample 1 is medium-high temperature milk, samples 2-3 are pasteurized milk, samples 4-5 are UHT milk, and sample 6 is reconstituted milk). The lactoferrin detection method described in Example 2 was used to detect the samples, and the lactoferrin concentration in the samples was calculated. The results are as follows: Figure 7 As shown in the figure, milk sample 1 had a lactoferrin concentration between 20 and 100 µg / mL, indicating medium-high temperature processing; milk samples 2 and 3 had lactoferrin concentrations higher than 100 µg / mL, indicating pasteurization; and milk samples 4 and 6 had lactoferrin concentrations lower than 20 µg / mL, indicating medium-UHT processing. This further verifies that the method of this invention can effectively distinguish dairy products processed by different methods, especially UHT milk and reconstituted milk that have undergone ultra-high temperature treatment and have severely damaged LF structure, demonstrating good specificity. It also shows that the method has certain reliability and application potential in actual sample testing.

[0065] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for screening pairs of lactoferricin antibodies, characterized in that, It comprises the following steps: The lactoferrin immunizes animals, and then obtains the monoclonal antibody through the hybridoma technology, obtains the polyclonal antibody through the affinity purification, cross-combines the monoclonal antibody with the polyclonal antibody to form the candidate antibody pair, and obtains the detection antibody pair through the preliminary screening, the intermediate screening and the final screening; The method of the preliminary screening comprises the following steps: the candidate antibody pair is used for detecting the unprocessed lactoferrin sample and the BSA interference liquid sample respectively, and the antibody pair with the unprocessed lactoferrin sample P / N≥2.1 and the BSA interference liquid sample P / N<1.5 is screened as the preliminary screening qualified antibody pair; The method of the intermediate screening comprises the following steps: the preliminary screening qualified antibody pair is used for detecting the unprocessed lactoferrin sample, the moderately heat-processed lactoferrin sample and the severely heat-processed lactoferrin sample, the LOD is calculated, and the antibody pair with the unprocessed lactoferrin sample LOD≤100 μg / L, the moderately heat-processed lactoferrin sample LOD≤1000 μg / L and the severely heat-processed lactoferrin sample LOD≥20000 μg / L is screened as the intermediate screening qualified antibody pair, the temperature of the moderately heat-processed lactoferrin sample is 80-100 DEG C, and the temperature of the severely heat-processed lactoferrin sample is 100-105 DEG C; The method of the final screening comprises: detecting lactoferrin with the middle screening qualified antibody pair, fitting the detection curve of lactoferrin treatment temperature and absorbance, screening the antibody pair with R 2 ≥0.990, and the heat processing strength is negatively correlated with the absorbance value of the antibody pair as the detection antibody pair.

2. The screening method according to claim 1, characterized in that, The preparation method of the monoclonal antibody comprises the following steps: lactoferrin is used for immunizing mice, then the mouse spleen cells are fused with myeloma cells, and the monoclonal antibody is obtained from the supernatant after expansion.

3. The screening method according to claim 1, characterized by, The preparation method of the polyclonal antibody comprises the following steps: lactoferrin is used for immunizing big white rabbits, and then the polyclonal antibody is obtained from the serum of the big white rabbits through affinity purification.

4. A detection antibody pair obtained by the screening method according to any one of claims 1-3.

5. A kit for detecting lactoferrin content, characterized by, It comprises the detection antibody pair according to claim 4.

6. The application of the detection antibody pair according to claim 4 or the kit according to claim 5 in detecting the heat-processed lactoferrin content.

7. An ELISA method for the detection of lactoferrin content, characterized in that, It comprises the following steps: The detection antibody pair according to claim 4 is mixed with the heat-processed lactoferrin sample to be detected, incubation is carried out, color development and determination are carried out, and the content of lactoferrin in the sample to be detected is obtained.

8. The ELISA detection method according to claim 7, characterized in that, The monoclonal antibody in the detection antibody pair is used for coating, and the polyclonal antibody in the detection antibody pair is used as the primary antibody.

9. The application of the detection antibody pair according to claim 4 or the kit according to claim 5 in the evaluation of the dairy authenticity process.

10. A method for evaluating the authenticity of a dairy product process using the antibody of claim 4 or the kit of claim 5, characterized in that, The detection antibody pair is used for detecting the concentration of lactoferrin in the sample to be detected to determine the dairy process; the determination standard is that the concentration>100 μg / mL determines that the process of the sample to be detected is pasteurization, the concentration 20-100 μg / mL determines that the process of the sample to be detected is moderate high-temperature processing, and the concentration<20 μg / mL determines that the process of the sample to be detected is ultra-high-temperature sterilization.

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