CN116333151B - A monoclonal antibody for detecting adulterated ordinary milk in buffalo milk and its preparation method and application - Google Patents

Abstract

The invention discloses a monoclonal antibody for detecting adulterated ordinary milk in buffalo milk, a preparation method and application thereof. Secondly, the present invention discloses a test strip for quantitatively detecting adulterated ordinary milk in buffalo milk, which includes a sample pad, a release pad, a reaction film, and a water-absorbing pad that are sequentially connected and fixed on a PVC bottom plate. Anti-bovine IgG monoclonal antibody labeled with the marker to be detected; T line and C line are set on the reaction membrane, the T line is coated with bovine IgG antigen, and the C line is coated with goat anti-mouse IgG antibody. The method of using the test strip to detect adulterated ordinary milk in buffalo milk has the characteristics of high accuracy, good precision, short detection time, and easy operation, and has low requirements for operators and does not require the support of large-scale inspection equipment, so it is very applicable Rapid on-site inspection at the grassroots level.

Description

A monoclonal antibody for detecting adulterated ordinary milk in buffalo milk and its preparation method and use

technical field

The invention belongs to the field of food quality and safety detection, and in particular relates to a monoclonal antibody for detecting adulterated common cow milk in buffalo milk, a preparation method and application thereof.

Background technique

The present invention is a divisional application of CN202210965744.1.

Buffalo milk has a high nutritional value, and is known as the "treasure in milk" among the people, and is generally welcomed by consumers. However, the production of buffalo milk is relatively low, resulting in a higher price than ordinary milk. The price of raw buffalo milk on the market is about three times that of ordinary milk. Due to the long-term lack of effective supervision methods and detection methods, driven by profit, the breeding side frequently mixes buffalo milk with other bovine milk, or uses lower-priced Holstein milk or yellow cow milk as buffalo milk. Seriously affect the quality of buffalo milk source purchases and the brand reputation of the processing end, disrupt market order, infringe on the interests of consumers, and are not conducive to the healthy development of the entire industry chain. . Therefore, a simple, easy-to-operate technical means suitable for on-site law enforcement is needed to supervise the adulteration of buffalo milk.

Existing detection techniques for detecting common milk components can be divided into immunological methods (immunochromatography, ELISA, immunospot, etc.) and non-immunological methods (polypropylene gel electrophoresis or isoelectric focusing electrophoresis, PCR, infrared spectroscopy, etc.) chemistry, chromatography, mass spectrometry, etc.). Among them, isoelectric focusing electrophoresis is the reference method for the detection of common milk components in the European Union, and the PCR method has also been written into the local food safety standard of my country "DBS45/023-2015 PCR method for the qualitative detection of buffalo milk and its products mixed with bovine milk". ".

However, non-immune methods, enzyme-linked immunosorbent assays, and immunospots all involve cumbersome professional operations and equipment, and are not suitable for widespread use. Secondly, most immunological detection methods for adulteration of dairy products use antibodies to detect α-casein, β-casein, γ-casein, and β-globulin and other whey proteins, but these proteins are difficult to distinguish certain Species of dairy products, such as ordinary cow's milk and buffalo milk. Therefore, it is very necessary to develop a simple, easy-to-operate and rapid detection test strip suitable for on-site law enforcement to detect whether buffalo milk is adulterated with ordinary milk.

Patent document CN202010661984.3 discloses a characteristic peptide and detection method for detecting milk mixed in buffalo milk. The method obtains specific peptides of buffalo milk and milk through high-resolution mass spectrometry screening, which can realize the detection of buffalo milk. Detection of milk adulteration. Although the detection method has high sensitivity, it has high requirements for equipment and operators, which does not meet the requirements of rapid detection.

Contents of the invention

In order to improve the deficiencies in the prior art, the present invention uses bovine IgG as the target, prepares and purifies bovine IgG Fc fragments, and uses it as an immunogen to immunize mice to prepare anti-bovine IgG monoclonal antibodies; or, the present invention uses bovine α- S1 is the immunogen, and the anti-bovine α-S1 monoclonal antibody is prepared by immunizing mice. Further, based on anti-bovine IgG monoclonal antibody and/or anti-bovine α-S1 monoclonal antibody, an immunochromatographic detection method suitable for grass-roots field use is established, and the method is applied to the detection of buffalo milk adulterated ordinary milk , the method has good accuracy and precision.

The present invention includes following technical solutions:

In the first aspect, the present invention provides a monoclonal antibody for detecting adulterated ordinary cow milk in buffalo milk. The monoclonal antibody is one of anti-bovine IgG monoclonal antibody and anti-bovine α-S1 monoclonal antibody. The light chain variable region of the anti-bovine IgG monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 2; anti-bovine α-S1 monoclonal The light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:4.

In a second aspect, the present invention provides a method for preparing a monoclonal antibody for detecting adulterated ordinary milk in buffalo milk, comprising the steps of:

(1) preparing bovine IgG antigen or bovine α-S1 antigen;

(2) Immunized mice

Regular subcutaneous or intraperitoneal injection of bovine IgG antigen or bovine α-S1 antigen to immunize Balb/c mice. After immunization, blood was collected and serum was separated, and the polyantibody titer in serum was detected by ELISA method;

(3) Hybridoma cell line fusion

Splenocytes from immunized mice with high serum titer and high sensitivity were fused with myeloma cells, and fed with feeder cells for culture;

The myeloma cells are SP2/0 cells, and the cell fusion method is PEG method.

(4) Subcloning and screening

Aspirate the supernatant of hybridoma cells, use bovine IgG Fc or bovine α-S1 polypeptide antigen as the coating source, and use indirect ELISA method to screen the culture wells with high titer for subcloning, repeat subcloning until the positive rate of cell lines in the wells is 100% 1. Expand the culture of positive hybridoma cells, collect the supernatant to measure the titer by indirect ELISA, and freeze it;

(5) Monoclonal antibody acquisition and purification

Monoclonal cell lines were obtained, ascites was obtained by intraperitoneal injection of immunized mice, and the resulting ascites was purified with Protein A/G affinity column to obtain bovine IgG monoclonal antibody or bovine α-S1 monoclonal antibody.

Preferably, the bovine IgG antigen described in step (1) is a bovine IgG Fc fragment, and the bovine α-S1 antigen is a bovine α-S1 polypeptide antigen.

The bovine IgG Fc fragment is prepared by the following method:

(1) using papain to enzymatically hydrolyze bovine IgG, and obtain a mixture of bovine IgG Fab fragments and Fc fragments by ultrafiltration;

(2) The mixture was purified using a Protein A affinity column to obtain bovine IgG Fc fragments.

The bovine α-S1 polypeptide antigen is prepared by the following method:

(1) Synthesizing an α-S1 polypeptide fragment whose amino acid sequence is NQELAYFYPEL (SEQ ID NO: 5) with reference to the complete sequence of bovine α-S1;

(2) Coupling and purifying the α-S1 polypeptide fragment with carrier protein to obtain the bovine α-S1 polypeptide antigen.

The carrier protein is selected from bovine serum albumin, ovalbumin, hemocyanin, diphtheria toxoid, avirulent mutant of diphtheria toxoid, tetanus toxoid and any carrier protein or two or more of proteins expressed by bacteria Fusion protein formed by carrier protein.

In a third aspect, the present invention provides a test strip for quantitatively detecting adulterated ordinary milk in buffalo milk, the test strip includes a sample pad, a release pad, a reaction film, and a water-absorbent pad that are sequentially connected and fixed on a PVC base plate , the release pad is coated with an anti-bovine IgG monoclonal antibody or an anti-bovine α-S1 monoclonal antibody labeled with a detectable marker; a detection line (T line) and a quality control line (C line) are arranged on the reaction membrane, The detection line (T line) is coated with bovine IgG antigen or bovine α-S1 antigen, and the quality control line (C line) is coated with goat anti-mouse IgG antibody.

The detectable label is selected from one of fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal metal particles, fluorescein, and quantum dots.

In a preferred embodiment of the present invention, the detectable marker is selected from fluorescent microspheres, specifically anti-bovine IgG monoclonal antibodies labeled with fluorescent microspheres, or anti-bovine α-S1 monoclonal antibodies labeled with fluorescent microspheres. Clonal Antibody.

More preferably, the concentration of the anti-bovine IgG monoclonal antibody labeled with fluorescent microspheres is 2-3 mg/mL, specifically 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 mg/mL mL; the concentration of anti-bovine α-S1 monoclonal antibody labeled with fluorescent microspheres is 3-4 mg/mL, specifically 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 mg/mL.

In the most preferred embodiment of the present invention, the concentration of the anti-bovine IgG monoclonal antibody labeled with fluorescent microspheres is 2.3 mg/mL; the concentration of anti-bovine α-S1 monoclonal antibody labeled with fluorescent microspheres is 3.7 mg/mL.

The reaction membrane is made of any one of the following materials: nitrocellulose membrane, cellulose acetate membrane, mixed cellulose ester membrane, polyvinylidene fluoride membrane, nylon membrane.

In a preferred embodiment of the present invention, the reaction membrane material is a nitrocellulose membrane, and the T line (0.8 μL/cm) and the C line (0.8 μL/cm) are coated with a film-drawing instrument, and the bovine IgG antigen coating concentration is The coating concentration of bovine α-S1 antigen was 1.4mg/mL, and the concentration of goat anti-mouse IgG antibody was 0.75mg/mL (0.7μL/cm).

The test strip of the present invention is assembled by the following method: stick the sample pad, the release pad, the reaction film, and the water-absorbing pad on the PVC bottom plate in sequence, one end of the bottom plate is a water-absorbing pad, the other end is a sample pad, and the reaction film is The two ends are respectively connected with the absorbent pad and the release pad overlapping each other (1-3mm), and the sample pad is pressed on the release pad (1-3mm overlapping), and cut into test strips with a width of 3.5-4.0mm by a cutting machine to obtain A test strip that can quantitatively detect adulterated ordinary milk in buffalo milk.

In a fourth aspect, the present invention provides a method for quantitatively detecting adulterated ordinary milk in buffalo milk, said method comprising the following steps:

(1) Draw a standard curve

a: Take a negative sample of buffalo milk, and add ordinary cow’s milk with different volume percentage or mass percentage to it to prepare the test sample;

b: Take an appropriate amount of test sample and drop it on the sample pad of the above test strip, incubate at constant temperature, and use a fluorescent immunoassay analyzer to detect, the excitation wavelength is 360-365nm, and the detection wavelength is 600-610nm, and the T-line fluorescence signal value and C-line fluorescence are obtained Signal value, each concentration was repeatedly detected 5 times, and the average value was taken;

c: Take the percentage of added ordinary milk as the X-axis, and the ratio (T/C) of the fluorescent signal value of the detection line T to the fluorescent signal value of the quality control line C as the Y-axis to obtain a standard curve;

(2) Take an appropriate amount of the sample to be tested and drop it on the sample pad of the above test strip, incubate at a constant temperature of 40°C for 5 minutes, and use a fluorescent immunoassay analyzer to detect, the excitation wavelength is 360-365nm, the detection wavelength is 610-615nm, and the T-line fluorescence signal is obtained. Value and C-line fluorescence signal value, the ratio of T-line fluorescence signal value to C-line fluorescence signal value is substituted into the standard curve, and the percentage content of ordinary milk in the sample to be tested is obtained.

The detection principle of the test strip that can quantitatively detect adulterated ordinary milk in buffalo milk provided by the present invention is as follows: taking the anti-bovine IgG monoclonal antibody as an example, the buffalo milk sample is dropped on the sample pad, and the bovine IgG (existing adulterated bovine IgG) in the sample fake ordinary milk) combined with the anti-bovine IgG monoclonal antibody labeled with fluorescent microspheres in the release pad, and when it was chromatographed to the reaction membrane, the bovine IgG immobilized on the T line (only) and the free (that is, bovine IgG in the unbound sample) IgG) fluorescently labeled antibody combined, and the fluorescently labeled antibody combined with bovine IgG in the sample continued to be chromatographically downward, and combined with goat anti-mouse IgG antibody at line C. Therefore, the amount of fluorescent microsphere-labeled antibody aggregated on the T line is inversely proportional to the concentration of bovine IgG in the sample, that is, the higher the proportion of buffalo milk adulterated with ordinary milk, the weaker the fluorescent signal on the T line, and vice versa.

The test strip that can quantitatively detect adulterated common milk in buffalo milk provided by the present invention, and the method for quantitatively detecting adulterated common cow milk in buffalo milk by using the test strip has the characteristics of high accuracy and good precision, and the detection method reliable. In addition, it also has the advantages of simple operation and short detection time. It has low requirements for operators and does not require the support of large-scale inspection equipment. The steps are simple and it is very suitable for grass-roots field inspection. It is a very practical rapid detection test paper strip.

Description of drawings

Fig. 1 quantitatively detects the adulterated ordinary cow's milk test paper schematic diagram in buffalo milk;

The typical curve of Fig. 2 embodiment 5 buffalo milk adulteration ordinary cow's milk detection method;

Fig. 3 is the standard curve of the detection method of buffalo milk adulterated ordinary milk in Example 6.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below, obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The preparation of embodiment 1 bovine IgG monoclonal antibody

1. Obtain bovine IgG antigen

S1: Dissolve bovine IgG powder (sigma, I5506) in 0.01M PBS (pH 7.4) at a concentration of about 5 mg/mL, dialyze in 2L of 0.01M PBS (pH 7.4) for 3 hours, and adjust the final concentration to 1.0 mg/mL ;

S2: Dissolve papain (sigma, P4762) in 0.01M PBS (pH 7.4, containing 0.2M EDTA and 0.01M cysteine) at a concentration of 1.0mg/mL, and activate at 37°C for 30min;

S3: Fully mix the dialyzed bovine IgG solution and the activated papain solution at a ratio of 10:1, and react in a water bath at 37°C for 6 hours;

S4: Use a 10KD ultrafiltration tube to ultrafilter the above IgG and papain reaction solution at 4000 rpm to remove papain, and replace the solution with 0.01M PBS (pH 7.4). The dissolved solution is a mixture of bovine IgG Fab fragments and Fc fragments ; Use a 0.22 μm filter to filter the mixed solution of the above-mentioned bovine IgG Fab fragment and Fc fragment, and further purify the Fc fragment;

S5: Use 20mL 0.01M PBS (pH 7.4) to equilibrate the Protein A affinity column, and control the flow rate to 0.5mL/min-1.0mL/min;

S6: Pass the filtered mixed solution of bovine IgG Fab fragments and Fc fragments through the equilibrated Protein A column, control the flow rate at 0.5mL/min-1.0mL/min, and then pass through the column with 20mL 0.01M PBS (pH 7.4) to remove Miscellaneous proteins adsorbed on the column material;

S7: Take multiple 1.5mL centrifuge tubes, add 0.3mL 1M Tris-HCL (pH9.0) after numbering, use 0.1M sodium citrate solution (pH 3.0) to elute the Fc fragment, and collect the eluate in each centrifuge tube 0.9mL, use Nanodrop to measure the concentration of Fc in each tube, and combine the solutions in the tubes with a concentration greater than 0.1mg/mL;

S8: Use 2L of 0.01M PBS (pH 7.4) to dialyze 3 times for 3 hours each time, adjust to a final Fc concentration of 2-3 mg/mL, and store at -20°C for later use.

2. Immunization of mice

Dilute the above-prepared bovine IgG Fc fragment as an antigen to 1 mg/mL with sterile saline, add an equal amount of Freund's complete adjuvant (sigma, F5881) for the first immunization, and emulsify completely (the mixture will not disperse when dropped into water) considered fully emulsified), immunized ten 8-week-old healthy Balb/c female mice (products of Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) by subcutaneous and multi-point injection on the back of the neck, and the immune dose was 100 μg/ Only. A total of 6 immunizations were performed, and the interval between each immunization was 2 weeks. The specific immunization procedures are shown in Table 1.

Table 1 Immunization program of monoclonal antibody (mouse)

Number of immunizations immunogen dose way of immunization first immunization Immunogen+FCA 100μg/piece Multiple subcutaneous injections on the back of the neck Second exemption Immunogen+FICA 100μg/piece ditto Three exemptions ditto ditto ditto Four exemptions ditto ditto ditto Five free ditto ditto ditto Six immunizations (boost immunization) immunogen ditto intraperitoneal injection

Note: FCA, Freund's complete adjuvant (sigma, F5881); FICA, Freund's incomplete adjuvant (sigma, F5506)

One week after four immunizations, blood was collected from the eyes of the mice, placed at room temperature for 2 hours, centrifuged at 4000rpm for 10 minutes, and serum was taken for testing; the optimal working concentration of the coating source (bovine IgG Fc) and antibody (serum) was determined by indirect ELISA square array titration , and then use the indirect competition ELISA method to detect the sensitivity of the antibody.

3. Hybridoma cell line fusion

Spleen cells from immunized mice with high serum titer and high sensitivity were mixed with mouse myeloma cells (SP2/0) in the logarithmic growth phase, then immunofused with 50% PEG, suspended evenly in HAT medium, Then add appropriate amount of feeder cells, culture in 96-well culture plate, culture in 37°C, 5% CO ₂ incubator, half change the medium with HAT medium after 5 days, and fully change the medium after 9 days.

4. Subcloning and screening

After cell fusion, when the cells grew to 1/4 of the area of the culture well, the supernatant of the hybridoma cells was aspirated, and the culture well with high titer was screened by indirect ELISA with bovine IgG Fc as the coating source, and then subcloned. Subcloning several times until the positive rate of the cell line in the well is 100%, the positive hybridoma cells are expanded and cultured, and the supernatant is collected to measure the titer by indirect ELISA, and then frozen.

5. Antibody acquisition

Take 8-10 week-old Balb/c mice and inject 0.3mL/monoclonal cell suspension containing 1.3×10 ⁶ cells intraperitoneally. Observe the mice after 6 days. When the abdomen of the mice swells, extract the ascites, observe the mice every 2 days, and extract the ascites in time; centrifuge the extracted ascites at 10,000 r/min for 5 minutes, collect the supernatant, and store in -20°C refrigerator .

6. Antibody purification and variable region sequencing

S1: Use 20mL 0.01M PBS (pH 7.4) to equilibrate the Protein A affinity column, and control the flow rate to 0.5mL/min-1.0mL/min;

S2: Pass the ascites produced in step (3) through the balanced Protein A column, control the flow rate to 0.5mL/min-1.0mL/min, and then use 20mL 0.01M PBS (pH 7.4) to pass through the column to remove impurities adsorbed on the column material. protein;

S3: Take multiple 1.5mL centrifuge tubes, add 0.3mL 1M Tris-HCL (pH9.0) after numbering, use 0.1M sodium citrate solution (pH 3.0) to elute, and collect 0.9mL eluate in each centrifuge tube , use Nanodrop to measure the concentration of monoclonal antibody in each tube, and combine the solutions in the tubes with a concentration greater than 0.1mg/mL;

S4: Dialyze 3 times with 2L 0.01M PBS (pH 7.4), 3 hours each time, adjust to the final concentration of monoclonal antibody 2-3 mg/mL, and store at -20°C for later use.

Extract the total RNA in the hybridoma cells used for the prepared monoclonal antibody, obtain cDNA by reverse transcription, and then use the light chain variable region primers V _L -F and V _L -R and the heavy chain variable region primer V _H -F and _VH -R for PCR amplification (Table 2), further TA clone screening of the amplified products, and the positive clones obtained by screening were sent to Beijing Biomed Biotechnology Co., Ltd. for gene sequencing. According to the gene sequencing results: The amino acid sequence of the light chain variable region of the anti-bovine IgG monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region of the anti-bovine IgG monoclonal antibody is shown in SEQ ID NO:2.

Table 2 Primers for gene amplification of mouse monoclonal antibody variable region

Note: Merger primers: S: C/G; M: A/C; R: A/G; W: A/T

SEQ ID NO: 1 Amino acid sequence of light chain variable region of anti-bovine IgG monoclonal antibody

Gly Thr Lys Leu Glu Ile Tyr Pro Cys Gln Ala Asp Gly Phe Thr Phe GluAsp Ala Tyr MetSer Ile Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val AspThr Ile Ser Asp Gly Arg Asp TyrThr Lys Val Pro Asp Ser Val Lys Gly Arg PheThr Ile Ser Arg Asp Asp Ala Lys Asn Thr LeuTyr Leu Val Phe Ser Asp Leu IleAsp Glu Asp Thr Ala Met Tyr Trp Cys Phe Arg His Phe GluTyr Leu Ser Tyr AlaMet Asp Tyr Trp Gly Gln Asp Ile Gln Leu Thr Glu Ser Pro

SEQ ID NO:2 Amino acid sequence of heavy chain variable region of anti-bovine IgG monoclonal antibody

Leu Gly Pro Arg Asp His Gly His Arg Leu Leu Val Leu Ser Pro Glu GluThr Gly Thr SerAla Ile Val Ala Gln Gly Pro Thr Leu Ala Ala Pro Ser Glu GlyAla Val Gly Gly Ala Cys Arg ProSer Ala Gly Ala Val Ala Ile Pro Asn Tyr GluAsn Trp Val Gln Gly Lys Ser Asp His Leu Phe AlaGly Leu Ile Glu Ile Ala AsnAsn Arg Thr Ser Gly Val Ser Thr Arg Phe Pro Glu Ser Leu Ile GluAsp Lys ThrIle Leu Ala Ile Ala Gly Thr Gln Ala Gly Asp Gly Thr Ile Tyr Phe Cys Arg SerAsn Cys Thr Val Arg

Embodiment 2 Preparation of anti-bovine α-S1 monoclonal antibody

1. Obtain bovine α-S1 antigen

S1: Refer to the complete sequence of bovine α-S1 (GenBank: ACG63494.1), entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize its truncated polypeptide sequence NQELAYFYPEL, with a molecular weight of 1386.5Da;

S2: Dissolve the carrier protein KLH (keyhole limpet hemocyanin) in 0.1M carbonate buffer (containing 0.15M NaCl, pH 8.5), with a final concentration of 2.0 mg/mL;

S3: Add the synthesized α-S1 polypeptide fragment to the KLH solution, the final concentration is 10 μg/mL, and the molar ratio of α-S1:KLH is about 30:1;

S4: Add fresh glutaraldehyde to the above solution to a final concentration of 1%, and react on a magnetic stirrer at 4°C for 4 hours;

S5: Add sodium borohydride to the above solution to a final concentration of 10 mg/mL, and react on a magnetic stirrer at 4°C for 1 h;

S6: The above reaction product was placed in 2L 0.01M PBS (pH 7.4) and dialyzed 6 times, 3 hours each time, adjusted to a final concentration of 2-3mg/mL, and stored at -20°C for future use.

Step 2 immunization of mice, step 3 fusion of hybridoma cell lines, step 4 subcloning and screening, step 5 antibody acquisition, and step 6 antibody purification were all the same as in Example 1 to obtain purified anti-bovine α-S1 monoclonal antibody.

Extract the total RNA in the hybridoma cells used for the prepared monoclonal antibody, obtain cDNA by reverse transcription, and then use the light chain variable region primers VL-F and VL-R and the heavy chain variable region primers VH-F and VH- R was amplified by PCR (Table 3), and the amplified products were further screened by TA clones, and the screened positive clones were sent to Beijing Biomed Biotechnology Co., Ltd. for gene sequencing. According to the results of gene sequencing: anti-bovine α- The amino acid sequence of the light chain variable region of the S1 monoclonal antibody is shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region of the anti-bovine α-S1 monoclonal antibody is shown in SEQ ID NO:4.

Table 3 Mouse monoclonal antibody variable region gene amplification primers

Note: Merger primers: S: C/G; M: A/C; R: A/G; W: A/T

SEQ ID NO: 3 Amino acid sequence of light chain variable region of anti-bovine α-S1 monoclonal antibody

Gly Thr Lys Leu Glu Ile Tyr Pro Cys Gln Ala Asp Gly Phe Thr Phe GluAsp Ala Tyr MetSer Ile Lys Tyr Val Met Asp Lys Asn Phe Ile His Ser Val AspThr Ile Ser Asp Gly Arg Asp TyrThr Lys Val Pro Asp Ser Phe Glu Ala Ile TyrVal Met Ser Arg Asp Asp Ala Lys Asn Thr LeuTyr Leu Val Phe Ser Asp Leu IleAsp Glu Asp Thr Ala Met Tyr Trp Cys Phe Arg His Phe GluPhe Gly Ala Pro GluHis Ile Leu Trp Gly Gln Asp Ile Gln Leu Thr Glu Ser Pro

SEQ ID NO: 4 Amino acid sequence of heavy chain variable region of anti-bovine α-S1 monoclonal antibody

Leu Gly Pro Arg Asp His Gly His Arg Leu Leu Val Leu Ser Pro Glu GluThr Gly Thr SerAla Ile Val Ala Gln Gly Pro Thr Leu Asn Arg Glu Ile Ser GlyAla Val Gly Thr Ala Cys Arg ProSer Ala Gly Ala Val Ala Tyr Ser Asn Tyr PheArg Asp Val Ser Arg Lys Pro Asp His Leu LysAla Gly Leu Ile Glu Ile Ala AsnPhe Arg Thr Ser Gly Val Ser Thr Arg Phe Pro Glu Ser Leu IleGlu Asp Lys ThrIle Leu Ala Ile Ala Arg Val Glu Lys Leu Asp Asn His Ile Tyr Phe Cys ArgSerAsn Cys Arg Val Arg

Antibody labeling of embodiment 3 fluorescent microspheres

S1: Add 50 μL of fluorescent microspheres to 450 μL of MES (0.05M, pH5.0) activation buffer, sonicate for 5 min; Oscillation reaction 0.5h;

S2: Centrifuge at 12000g for 5min, discard the supernatant; redissolve the precipitate with 500μL PB (0.04M, pH 8.0), sonicate for 5min, add an appropriate volume (5μL) of 2.3mg/mL anti-bovine IgG monoclonal antibody or 3.7mg/mL anti-bovine IgG Bovine α-S1 monoclonal antibody, shake at room temperature for 2 hours, centrifuge at 15000g for 5 minutes;

S3: Add 500 μL of blocking buffer (0.01M PB, 2% BSA, pH 8.0) to the precipitate, shake and react overnight at 4°C;

S4: Centrifuge the reaction solution at 15,000 g for 5 min, discard the supernatant, reconstitute the microspheres with 50 μL of 20 mM PB, sonicate for 2 min, and store in the dark at 4°C for later use to obtain anti-bovine IgG monoclonal antibodies or fluorescent microspheres labeled with fluorescent microspheres anti-bovine α-S1 monoclonal antibody.

Example 4 Preparation of test strips for quantitative detection of adulterated common milk in buffalo milk

The bovine IgG antigen (1.6mg/mL, 0.8μL/cm) or bovine α-S1 antigen (1.4mg/mL, 0.8μL/cm) obtained in Example 1 was mixed with goat anti-mouse IgG (0.75mg/mL) with a scratcher. mL, 0.7μL/cm) were coated on nitrocellulose membrane (NC membrane) as detection line (T line) and quality control line (C line).

Spray the anti-bovine IgG monoclonal antibody labeled with fluorescent microspheres or the anti-bovine α-S1 monoclonal antibody labeled with fluorescent microspheres prepared in Example 3 on the release pad, and the spray volumes are 2.2 μL/cm and 2.8 μL/cm respectively, Dry in an oven at 37°C for 2 hours to obtain a release pad containing fluorescent microsphere-labeled anti-bovine IgG monoclonal antibody or anti-bovine α-S1 monoclonal antibody.

Stick the sample pad, the release pad containing the antibody labeled with fluorescent microspheres, the nitrocellulose membrane containing the test line T and the quality control line C, and the absorbent pad to the PVC bottom plate in sequence. One end of the bottom plate is an absorbent pad, and the other end is a As a sample pad, the two ends of the nitrocellulose membrane are respectively connected to the water-absorbing pad and the release pad of the fluorescent microsphere-labeled antibody (1-3 mm), and the sample pad (overlapping 1mm) is pressed on the release pad of the fluorescent microsphere-labeled antibody. ~3mm), cut into test strips with a width of 3.90mm with a cutting machine to obtain test strips for detecting adulteration of buffalo milk and ordinary milk.

In this experiment, test strip 1 (anti-bovine IgG monoclonal antibody) and test strip 2 (anti-bovine α-S1 monoclonal antibody) were obtained according to the different types of monoclonal antibody in the release pad and the antigen coated on the T line.

Example 5 Quantitative detection method for adulterated ordinary milk in buffalo milk

(1) Drawing of standard curve

S1: Take a negative buffalo milk sample (the PCR test does not contain ordinary milk), add ordinary milk to it, so that the volume percentages of ordinary milk are: 0.0%, 0.05%, 0.15%, 0.45%, 1.35% and 4.05% respectively;

S2: Take 100 μL of each of the above samples and drop them on the sample pad of test strip 2, incubate at 40°C for 5 minutes, and use a fluorescent immunoassay analyzer for detection, the excitation wavelength is 365nm, and the detection wavelength is 610nm, and the T-line fluorescence signal value and C Line fluorescence signal value;

S3: Repeat the test 5 times for each concentration, take the average value, take the percentage of ordinary milk added in the sample as the X-axis, and the ratio of the fluorescent signal value of the detection line T to the fluorescent signal value of the quality control line (T/C) as the Y-axis , using origin8.0 to do four-parameter nonlinear fitting analysis to obtain a standard curve: Y=0.1354+1.9649/(1+(x/0.3023) ^1.4134 ).

The results are shown in Figure 2. The fitting of test data shows that the theoretical detection limit (IC ₂₀ ) of the established buffalo milk detection method for adulterated ordinary milk is 0.113%, IC ₅₀ is 0.325%, and R ² is 0.9991.

(2) Detection of samples to be tested

Take 100 μL of the sample liquid and add it dropwise to the sample pad of the buffalo milk adulteration ordinary milk test strip 2, and accurately react in the 40°C test card constant temperature incubator for 5 minutes, then take it out and use the fluorescent immunoassay analyzer to detect it, and obtain the T-line fluorescence signal Value and C-line fluorescence signal value; Substitute the ratio of the T-line fluorescence signal value to the C-line fluorescence signal value into the standard curve to obtain the percentage content of ordinary milk in each sample.

Methodology Validation

(1) The minimum detection limit and quantification limit of the test strip

According to the method described in the above "testing of samples to be tested", use buffalo milk adulterated ordinary milk test strip 1 to detect 20 water-negative milk samples (negative by PCR test), and calculate the average and standard deviation of the percentage content of ordinary milk respectively : The average value plus 3 times the standard deviation is the detection limit; the average value plus 10 times the standard deviation is the quantification limit.

Table 4 Verification of detection limit of buffalo milk adulterated ordinary milk test strips (%)

sample average value standard deviation The detection limit The limit of quantitation buffalo milk 0.064 0.011 0.097 0.174

The results are shown in Table 4. It can be seen that the minimum detection limit of ordinary milk obtained by establishing a standard curve with ordinary milk as a control is 0.097%, and the limit of quantification is 0.174%. In order to ensure the accuracy and reliability of the results, the detection limit of ordinary cow's milk in buffalo milk was limited to 0.10%, and the quantitative limit was 0.20%.

(2) Method accuracy and precision

20 copies of buffalo milk negative samples were added to ordinary cow’s milk respectively. The concentration of the drug added to the sample was the corresponding limit of quantification and 2 times the limit of quantification. Five parallels were added to each concentration gradient, and the recovery rate of sample addition and the intra-assay and inter-assay variation coefficients were calculated.

The accuracy and precision of the established fluorescence immunochromatographic analysis method were analyzed according to the addition and recovery data.

Table 5 Accuracy and precision of test strips for buffalo milk adulteration of ordinary milk

Recovery = (average value/added concentration) × 100%

Coefficient of variation = (standard deviation/mean value) × 100%;

The results are shown in Table 5. The recovery rate of the present invention for the addition of common milk in buffalo milk is 94.30-107.77%, and the intra-assay and inter-assay coefficients of variation are both less than 10%, indicating that the method has good accuracy and precision.

(3) Comparison of test strips and PCR test results

The buffalo milk samples (50 copies in total) were detected by using the test strip and the detection method provided by the present invention, which can be used for quantitative detection of adulterated common milk in buffalo milk, and then confirmed and compared with the PCR detection results respectively. The results are shown in Table 6.

Table 6 Comparison of buffalo milk adulteration ordinary milk test strips and PCR detection results

Note: "ND" means no normal cow's milk was detected

The results show that the content of adulterated common milk in buffalo milk detected by the test paper provided by the invention is basically the same as the PCR result, indicating that the detection test strip provided by the invention has higher accuracy in detecting the adulterated common milk in buffalo milk.

Example 6 Quantitative detection method for adulterated ordinary milk in buffalo milk

According to the method disclosed in Example 5, samples of ordinary milk with a percentage content of 0.0%, 0.05%, 0.15%, 0.45%, 1.35% and 4.05% were prepared, and the samples were added dropwise to the sample pad of test strip 1 to obtain a T line Fluorescence signal value and C-line fluorescence signal value, draw a standard curve, and verify the lowest detection limit and quantification limit, accuracy and precision of the test strip, and the comparison with the PCR method.

Fitting obtains standard curve: Y=0.1148+1.8875/(1+(x/0.2921) ^1.5814 ), the result is as shown in Figure 3, shows by the fitting of test data, the buffalo milk adulteration common milk detection method established Theoretical detection limit was 0.12%, _IC50 was 0.308%, and ^R2 was 0.9991.

(1) The minimum detection limit and quantification limit of the test strip

Table 7 Verification of detection limit of buffalo milk adulterated ordinary milk test strips (%)

sample average value standard deviation The detection limit The limit of quantitation buffalo milk 0.049 0.015 0.094 0.199

The results are shown in Table 7. It can be seen that the minimum detection limit of ordinary milk obtained by establishing a standard curve with ordinary milk as a control is 0.094%, and the limit of quantification is 0.199%. In order to ensure the accuracy and reliability of the results, the detection limit of ordinary cow's milk in buffalo milk was limited to 0.10%, and the quantitative limit was 0.20%.

(2) Method accuracy and precision

Table 8 Accuracy and precision of test strips for buffalo milk adulteration of ordinary milk

Result is shown in table 8, and present embodiment is to the addition recovery rate of common cow's milk in buffalo milk 89.30-110.37%, and intra-assay coefficient of variation is less than 10%, and inter-assay coefficient of variation is less than 15%, shows that this method has better accuracy. accuracy and precision.

(3) Comparison of test strips and PCR test results

Table 9 Comparison of buffalo milk adulteration ordinary milk test strips and PCR detection results

Note: "ND" means no normal cow's milk was detected

The results show that the content of adulterated common milk in buffalo milk detected by the test paper provided by the invention is basically the same as the PCR result, indicating that the detection test strip provided by the invention has higher accuracy in detecting the adulterated common milk in buffalo milk.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims (7)

1. A monoclonal antibody for detecting adulterated common cow milk in buffalo milk is an anti-bovine IgG monoclonal antibody, wherein the light chain variable region of the anti-bovine IgG monoclonal antibody has an amino acid sequence shown as SEQ ID NO. 1, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 2.

2. A test strip for quantitatively detecting adulterated common cow milk in water milk, which comprises a sample pad, a release pad, a reaction membrane and a water absorption pad which are sequentially connected and fixed on a PVC bottom plate, wherein the release pad is coated with the anti-bovine IgG monoclonal antibody of claim 1 marked by a detectable marker; the reaction membrane is provided with a detection line (T line) and a quality control line (C line), the detection line (T line) is coated with bovine IgG antigen, and the quality control line (C line) is coated with goat anti-mouse IgG antibody.

3. The test strip of claim 2, wherein the detectable label is selected from one of fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal metal particles, and luciferin.

4. The test strip of claim 3, wherein the detectable label is selected from the group consisting of fluorescent microspheres, and further wherein the concentration of anti-bovine IgG monoclonal antibody labeled with fluorescent microspheres is 2-3 mg/mL.

5. The test strip of claim 2, wherein the reaction membrane is made of any one of the following materials: nitrocellulose membrane, cellulose acetate membrane, mixed cellulose ester membrane, polyvinylidene fluoride membrane, nylon membrane.

6. The test strip of claim 2, wherein the test strip is assembled by: the method comprises the steps of sequentially sticking a sample pad, a release pad, a reaction membrane and a water absorption pad to a PVC base plate, wherein one end head of the base plate is the water absorption pad, the other end head of the base plate is the sample pad, two ends of the reaction membrane are respectively overlapped with the water absorption pad and the release pad, the sample pad is pressed on the release pad, and a cutting machine is used for cutting the sample pad into test strips with the width of 3.5-4.0mm, so that the test strips capable of quantitatively detecting the adulterated common cow milk in the water milk are obtained.

7. A method for quantitatively detecting adulterated common cow milk in buffalo milk, comprising the following steps:

(1) Drawing a standard curve

a: taking a milk negative sample, and adding common cow milk with different volume percentages or mass percentages into the milk negative sample to prepare a detection sample;

b: dripping a proper amount of detection sample onto a sample pad of the test strip according to any one of claims 2-6, incubating at constant temperature, detecting by using a fluorescence immunoassay quantitative analyzer, exciting the sample at 360-365nm and detecting the sample at 610-615nm to obtain a T line fluorescence signal value and a C line fluorescence signal value, repeatedly detecting each concentration for 5 times, and taking an average value;

c: taking the percentage of the added common cow milk as an X axis, and taking the ratio (T/C) of the fluorescent signal value of the detection line T and the fluorescent signal value of the quality control line C as a Y axis to obtain a standard curve;

(2) And (3) dripping a proper amount of sample to be detected on a sample pad of the test strip, incubating at a constant temperature, detecting by using a fluorescent immunity quantitative analyzer, exciting the sample at 360-365nm, detecting the sample at 610-615nm to obtain a T-line fluorescent signal value and a C-line fluorescent signal value, and substituting the ratio of the T-line fluorescent signal value to the C-line fluorescent signal value into a standard curve to obtain the percentage content of the common cow milk in the sample to be detected.