CN115724982B - Monoclonal antibody for detecting adulterated common cow milk in buffalo milk, and preparation method and application thereof - Google Patents
Monoclonal antibody for detecting adulterated common cow milk in buffalo milk, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody for detecting adulterated common cow milk in buffalo milk, a preparation method and application thereof. The invention further discloses a test strip for quantitatively detecting the adulterated common cow milk in the 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 an anti-bovine IgG monoclonal antibody or an anti-bovine alpha-S1 monoclonal antibody marked by a detectable marker; t line and C line are set on the reaction film, bovine IgG antigen or bovine alpha-S1 antigen is coated on the T line, and goat anti-mouse IgG antibody is coated on the C line. The method for detecting the adulterated common cow milk in the buffalo milk by adopting the test strip has the characteristics of high accuracy, good precision, short detection time and simple and convenient operation, has low requirements on operators, does not need large-scale detection equipment support, and is very suitable for the on-site rapid detection of a basic layer.
Description
Technical Field
The invention belongs to the field of food quality safety detection, and particularly relates to a monoclonal antibody for detecting adulterated common cow milk in buffalo milk, and a preparation method and application thereof.
Background
The buffalo milk has higher nutritive value, is popular with consumers in the reputation of folk 'precious in milk'. However, the production of buffalo milk is relatively low, resulting in a price that is also higher than that of normal cow's milk, and the price of buffalo milk on the market is about 3 times that of normal cow's milk. Due to the long-term lack of effective supervision means and detection methods, under the driving of benefits, the phenomenon that other buffalo milk is mixed in buffalo milk at a breeding end or the buffalo milk is dug by a low-price Holstein milk or a buffalo milk source, the acquisition quality of the buffalo milk source and the brand reputation of a processing end are seriously influenced, the market order is disturbed, the benefits of consumers are infringed, and the healthy development of the whole industry chain is not facilitated. Therefore, there is a need for a simple, easy to operate and suitable for on-site law enforcement technique for monitoring the phenomenon of buffalo milk adulteration.
The existing detection technology for detecting the common cow milk components can be divided into immunological methods (immunochromatography, enzyme-linked immunosorbent, immune spots and the like) and non-immunological methods (polypropylene gel electrophoresis or isoelectric focusing electrophoresis, PCR, infrared spectroscopy, chromatography, mass spectrometry and the like). The isoelectric focusing electrophoresis is a reference method for detecting the components of common cow milk by European Union, and the PCR method is also written into qualitative detection PCR method for the incorporation of cow milk into the food safety local standard DBS45/023-2015 buffalo milk and products thereof in China.
However, the non-immune method, the enzyme-linked immunosorbent assay, the immune spots and the like involve complicated professional operations and equipment, and are not suitable for wide-range popularization and use. Second, the immunological detection method of most dairy adulteration is to detect alpha-casein, beta-casein, gamma-casein, and beta-globulin and other whey proteins, etc. by using antibodies, but these proteins are difficult to distinguish certain species of dairy products, such as common cow milk and buffalo milk. Therefore, it is highly desirable to develop a simple, easy to operate, and suitable for on-site law enforcement rapid test strip for detecting whether plain milk is adulterated in buffalo milk.
Patent document CN202010661984.3 discloses a characteristic peptide for detecting milk doped in buffalo milk and a detection method, wherein the method obtains specific peptide fragments of buffalo milk and milk through high-resolution mass spectrum screening, and can realize detection of milk adulteration in buffalo milk. Although the detection method has higher sensitivity, the requirements on instruments and equipment and operators are higher, and the requirements on rapid detection are not met.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention takes bovine IgG as a target, prepares and purifies to obtain bovine IgG Fc fragments, and takes the bovine IgG Fc fragments as immunogens to immunize mice to prepare anti-bovine IgG monoclonal antibodies; alternatively, the invention uses bovine alpha-S1 as immunogen to prepare anti-bovine alpha-S1 monoclonal antibody by immunizing mice. Furthermore, an immunochromatography detection method suitable for on-site use of a basic layer is established based on the anti-bovine IgG monoclonal antibody and/or the anti-bovine alpha-S1 monoclonal antibody, and the method is applied to detection of buffalo milk adulterated with common cow milk, and has good accuracy and precision.
The invention comprises the following technical scheme:
in a first aspect, the invention provides a monoclonal antibody for detecting adulterated common cow milk in buffalo milk, wherein the monoclonal antibody is one of an anti-cow IgG monoclonal antibody and an anti-cow alpha-S1 monoclonal antibody. The light chain variable region of the anti-bovine IgG monoclonal antibody has the amino acid sequence shown as SEQ ID NO. 1, and the heavy chain variable region has the amino acid sequence shown as SEQ ID NO. 2; the light chain variable region of the anti-bovine alpha-S1 monoclonal 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 normal cow's milk in buffalo milk, comprising the steps of:
(1) Preparing bovine IgG antigen or bovine alpha-S1 antigen;
(2) Immunized mice
Periodically injecting bovine IgG antigen or bovine alpha-S1 antigen subcutaneously or intraperitoneally to immunize Balb/c mice, taking blood after immunization, separating serum, and detecting polyclonal titer in the serum by an ELISA method;
(3) Hybridoma cell line fusion
Taking spleen cells of immunized mice with high serum titer and high sensitivity to fuse with myeloma cells, and adding feeder cells for culture;
the myeloma cells are SP2/0 cells, and the cell fusion method is a PEG method.
(4) Subcloning and screening
Sucking out hybridoma cell supernatant, subcloning the culture well with high titer by using bovine IgG Fc or bovine alpha-S1 polypeptide antigen as coating antigen and adopting an indirect ELISA method, repeating subcloning until the positive rate of the cell strain in the well is 100%, performing expanded culture on positive hybridoma cells, collecting supernatant, measuring titer by using an indirect ELISA method, and freezing;
(5) Monoclonal antibody acquisition and purification
Obtaining monoclonal cell strain, injecting immune mouse into abdomen to obtain ascites, purifying the produced ascites by Protein A/G affinity column to obtain bovine IgG monoclonal antibody or bovine alpha-S1 monoclonal antibody.
Preferably, the bovine IgG antigen in step (1) is a bovine IgG Fc fragment and the bovine alpha-S1 antigen is a bovine alpha-S1 polypeptide antigen.
The bovine IgG Fc fragment is prepared by the following method:
(1) Enzymolysis of bovine IgG by papain, ultrafiltration to obtain a mixture of bovine IgG Fab fragment and Fc fragment;
(2) The mixture was purified using Protein A affinity column to give the bovine IgG Fc fragment.
The bovine alpha-S1 polypeptide antigen is prepared by the following method:
(1) Synthesizing an alpha-S1 polypeptide fragment with an amino acid sequence of NQELAYFYPEL (SEQ ID NO: 5) by referring to the bovine alpha-S1 complete sequence;
(2) Coupling the alpha-S1 polypeptide fragment with carrier protein, and purifying to obtain the bovine alpha-S1 polypeptide antigen.
The carrier protein is selected from any one carrier protein or fusion protein formed by more than two carrier proteins of bovine serum albumin, ovalbumin, hemocyanin, diphtheria toxoid, nontoxic mutant of diphtheria toxin, tetanus toxoid and bacteria expressed protein.
In a third aspect, the invention provides 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 an anti-cow IgG monoclonal antibody or an anti-cow alpha-S1 monoclonal antibody 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 or bovine alpha-S1 antigen, and the quality control line (C line) is coated with goat anti-mouse IgG antibody.
The detectable marker is selected from one of fluorescent microsphere, latex microsphere, resin microsphere, magnetic microsphere, colloidal metal particle, fluorescein and quantum dot.
In a preferred embodiment of the invention, the detectable label is selected from fluorescent microspheres, in particular fluorescent microsphere-labelled anti-bovine IgG monoclonal antibodies, or fluorescent microsphere-labelled anti-bovine α -S1 monoclonal antibodies.
More preferably, the concentration of the fluorescent microsphere marked anti-bovine IgG monoclonal antibody is 2-3mg/mL, and specifically 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0mg/mL; the concentration of the fluorescent microsphere marked anti-bovine alpha-S1 monoclonal antibody is 3-4mg/mL, specifically 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 and 4.0mg/mL.
In a most preferred embodiment of the invention, the fluorescent microsphere-labeled anti-bovine IgG monoclonal antibody concentration is 2.3mg/mL; the concentration of the fluorescent microsphere marked anti-bovine alpha-S1 monoclonal antibody is 3.7mg/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 nitrocellulose membrane, T line (0.8. Mu.L/cm) and C line (0.8. Mu.L/cm) are coated with a membrane-drawing instrument, bovine IgG antigen coating concentration is 1.6mg/mL, bovine alpha-S1 antigen coating concentration is 1.4mg/mL, and goat anti-mouse IgG antibody concentration is 0.75mg/mL (0.7. Mu.L/cm).
The test strip is assembled by the following method: 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 and mutually overlapped (1-3 mm) with the water absorption pad and the release pad, the release pad is pressed with the sample pad (overlapped 1-3 mm), and a test strip with the width of 3.5-4.0mm is cut by a cutting machine, so that the test strip capable of quantitatively detecting the adulterated common cow milk in the water milk is obtained.
In a fourth aspect, the present invention provides a method for quantitatively detecting adulterated normal cow milk in buffalo milk, the method comprising the steps of:
(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, incubating at constant temperature, detecting by using a fluorescence immunoassay quantitative analyzer, exciting the sample at 360-365nm and detecting the sample at 600-610nm 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 for 5min at the constant temperature of 40 ℃, detecting by using a fluorescent immunity quantitative analyzer, exciting the sample with the wavelength of 360-365nm and detecting the sample with the wavelength of 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.
The test strip capable of quantitatively detecting the adulterated common cow milk in the buffalo milk provided by the invention has the detection principle that: taking an anti-bovine IgG monoclonal antibody as an example, a buffalo milk sample is dripped into a sample pad, bovine IgG in the sample (when common bovine milk is doped) is combined with the anti-bovine IgG monoclonal antibody marked by the fluorescent microsphere in the release pad, when the buffalo milk sample is chromatographed to a reaction membrane, bovine IgG fixed on a T line (only) is combined with free (namely, bovine IgG in the sample is not combined) fluorescent marked antibody, the fluorescent marked antibody combined with the bovine IgG in the sample is continuously chromatographed downwards, and is combined with goat anti-mouse IgG antibody at a C line. Therefore, the amount of fluorescent microsphere labelled antibody aggregated on the T-line is inversely proportional to the concentration of bovine IgG in the sample, i.e.: the higher the proportion of buffalo milk to adulterate common cow milk, the weaker the fluorescent signal on the T line, and the stronger the reverse.
The test strip capable of quantitatively detecting the adulterated common cow milk in the buffalo milk and the method for quantitatively detecting the adulterated common cow milk in the buffalo milk by adopting the test strip have the characteristics of high accuracy and good precision, and the detection method is reliable. In addition, the method has the advantages of simple operation and short detection time, has lower requirements on operators, does not need large-scale detection equipment to support, has simple steps, is very suitable for field detection of a base layer, and is a rapid detection test strip with very strong practicability.
Drawings
FIG. 1 is a schematic diagram of a test strip for quantitatively detecting adulterated common cow milk in buffalo milk;
FIG. 2 example 5 Standard curve of buffalo milk adulterated common cow milk detection method;
FIG. 3 example 6 Standard curve of buffalo milk adulterated normal cow milk detection method.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of bovine IgG monoclonal antibodies
1. Obtaining bovine IgG antigen
S1: bovine IgG powder (sigma, I5506) was dissolved in 0.01M PBS (pH 7.4) at a concentration of about 5mg/mL, dialyzed in 2L 0.01M PBS (pH 7.4) for 3 hours, and the final concentration was adjusted to 1.0mg/mL;
s2: papain (sigma, P4762) was dissolved in 0.01M PBS (pH 7.4, containing 0.2M EDTA and 0.01M cysteine) at a concentration of 1.0mg/mL and activated at 37℃for 30min;
s3: fully mixing the dialyzed bovine IgG solution and the activated papain solution according to the ratio of 10:1, and reacting for 6 hours in a 37 ℃ water bath;
s4: ultrafiltering the IgG and papain reaction solution at 4000rpm with 10KD ultrafilter tube to remove papain while replacing solution with 0.01M PBS (pH 7.4), wherein the dissolved solution is mixture of bovine IgG Fab fragment and Fc fragment; the mixed solution of the above bovine IgG Fab fragment and Fc fragment was filtered using a 0.22 μm filter to further purify the Fc fragment;
s5: protein A affinity column was equilibrated with 20mL of 0.01M PBS (pH 7.4), and the flow rate was controlled between 0.5mL/min and 1.0mL/min;
s6: the filtered mixed solution of the bovine IgG Fab fragment and the Fc fragment is subjected to a Protein A column after being subjected to overbalance, the flow rate is controlled to be 0.5mL/min-1.0mL/min, then 20mL of 0.01M PBS (pH 7.4) is used for passing the column, and the impurity Protein adsorbed on the column material is removed;
s7: taking a plurality of 1.5mL centrifuge tubes, numbering, adding 0.3mL of 1M Tris-HCL (pH 9.0) respectively, eluting Fc fragments by using 0.1M sodium citrate solution (pH 3.0), collecting 0.9mL of eluent from each centrifuge tube, measuring the concentration of Fc in each tube by using Nanodrop, and combining the solutions in the tubes with the concentration of more than 0.1 mg/mL;
s8: dialysis was performed 3 times with 2L of 0.01M PBS (pH 7.4), 3 hours each, to a final Fc concentration of 2-3mg/mL, and stored at-20deg.C for use.
2. Immunized mice
The prepared bovine IgG Fc fragment was diluted to 1mg/mL with sterile physiological saline as antigen, and was added with equivalent Freund's complete adjuvant (sigma, F5881) for the first immunization, and after complete emulsification (the mixture was not dispersed in water, i.e., was considered to be fully emulsified), 10 healthy Balb/c female mice (product of Beijing velari laboratory animal technologies Co., ltd.) of 8 weeks old were immunized by subcutaneous, multipoint injection on the back of the neck, and the immunization dose was 100. Mu.g/mouse. The total immunization was 6 times, each time was 2 weeks apart, and the specific immunization procedure is shown in Table 1.
Table 1 immunization program for monoclonal antibodies (mice)
| Number of immunizations | Immunogens | Dosage of | Immunization mode |
| First immunization | Immunogen+fca | 100 mug/g only | Subcutaneous multipoint injection of neck and back |
| Two-way valve | immunogen+FICA | 100 mug/g only | As above |
| Three-free | As above | As above | As above |
| Four-free | As above | As above | As above |
| Five-free food | As above | As above | As above |
| Six-free (strengthen immunity) | Immunogens | As above | Intraperitoneal injection |
Note that: FCA, freunds complete adjuvant (sigma, F5881); FICA, freund's incomplete adjuvant (sigma, F5506)
After the four-day period, carrying out orbital blood collection on the mice, standing for 2h at room temperature, centrifuging for 10min at 4000rpm, and taking serum for detection; the optimal working concentration of the coating antigen (bovine IgG Fc) and the antibody (serum) is determined by adopting an indirect ELISA square titration method, and then the sensitivity of the antibody is detected by adopting an indirect competition ELISA method.
3. Hybridoma cell line fusion
Mixing spleen cells of immunized mice with high serum titer and high sensitivity with mouse myeloma cells (SP 2/0) in logarithmic phase, performing immune fusion with 50% PEG, suspending uniformly with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate, culturing at 37deg.C and 5% CO 2 Culturing in an incubator, half-changing liquid with HAT medium after 5 days, and full-changing liquid at 9 days.
4. Subcloning and screening
After the cells are fused, sucking out the supernatant of the hybridoma cells when the cells grow to 1/4 of the area of the culture holes, and screening the culture holes with high titers by adopting an indirect ELISA method by taking bovine IgG Fc as a coating source to perform subcloning. Subcloning for several times until the positive rate of the cell strain in the hole is 100%, performing amplification culture on the positive hybridoma cells, collecting supernatant, measuring titer by using indirect ELISA, and freezing.
5. Antibody acquisition
Taking 8-10 week old Balb/c mice, and injecting into the abdominal cavity 0.3 mL/1.3X10 only 6 Monoclonal cell suspensions of individual cells. Observing the mice after 6 days, when the abdomen of the mice expands, extracting ascites, observing the mice every 2 days, and timely extracting the ascites; centrifuging the extracted ascites 10000r/min for 5min, collecting supernatant, packaging, and storing in a refrigerator at-20deg.C.
6. Purification of antibodies and variable region sequencing
S1: protein A affinity column was equilibrated with 20mL of 0.01M PBS (pH 7.4), and the flow rate was controlled between 0.5mL/min and 1.0mL/min;
s2: the Protein A column after the ascites produced in the step (3) is subjected to overbalance is controlled to have the flow rate of 0.5mL/min-1.0mL/min, and then 20mL of 0.01M PBS (pH 7.4) is used for passing through the column to remove the impurity Protein adsorbed on the column material;
s3: taking a plurality of 1.5mL centrifuge tubes, numbering, adding 0.3mL of 1M Tris-HCL (pH 9.0) respectively, eluting by using 0.1M sodium citrate solution (pH 3.0), collecting 0.9mL of eluent from each centrifuge tube, measuring the concentration of the monoclonal antibody in each tube by using Nanodrop, and combining the solutions in the tubes with the concentration of more than 0.1 mg/mL;
s4: dialysis was performed 3 times with 2L of 0.01M PBS (pH 7.4), 3h each time, and the final concentration of the monoclonal antibody was adjusted to 2-3mg/mL, and stored at-20deg.C for use.
Extracting total RNA in hybridoma cells used for preparing monoclonal antibody, obtaining cDNA by reverse transcription, then utilizing light chain variable region primer V L -F and V L R and heavy chain variable region primer V H -F and V H PCR amplification (Table 2) is carried out on R, TA clone screening is further carried out on the amplified products, positive clones obtained by screening are sent to Beijing Bomaide biotechnology Co., ltd for gene sequencing, and the positive clones are obtained according to the result of the gene sequencing: the amino acid sequence of the heavy chain variable region of the anti-bovine IgG monoclonal antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region of the anti-bovine IgG monoclonal antibody is shown as SEQ ID NO. 2.
TABLE 2 variable region Gene amplification primers for murine monoclonal antibodies
Note that: and (3) combining primers: s: C/G; m: A/C; r: A/G; w: A/T
SEQ ID NO. 1 amino acid sequence of the light chain variable region of an anti-bovine IgG monoclonal antibody
Gly Thr Lys Leu Glu Ile Tyr Pro Cys Gln Ala Asp Gly Phe Thr Phe Glu Asp Ala Tyr Met Ser Ile Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Asp Thr Ile Ser Asp Gly Arg Asp Tyr Thr Lys Val Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Leu Tyr Leu Val Phe Ser Asp Leu Ile Asp Glu Asp Thr Ala Met Tyr Trp Cys Phe Arg His Phe Glu Tyr Leu Ser Tyr Ala Met 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 Glu Thr Gly Thr Ser Ala Ile Val Ala Gln Gly Pro Thr Leu Ala Ala Pro Ser Glu Gly Ala Val Gly Gly Ala Cys Arg Pro Ser Ala Gly Ala Val Ala Ile Pro Asn Tyr Glu Asn Trp Val Gln Gly Lys Ser Asp His Leu Phe Ala Gly Leu Ile Glu Ile Ala Asn Asn Arg Thr Ser Gly Val Ser Thr Arg Phe Pro Glu Ser Leu Ile Glu Asp Lys Thr Ile Leu Ala Ile Ala Gly Thr Gln Ala Gly Asp Gly Thr Ile Tyr Phe Cys Arg Ser Asn Cys Thr Val Arg
EXAMPLE 2 preparation of anti-bovine alpha-S1 monoclonal antibody
1. Obtaining bovine alpha-S1 antigen
S1: the truncated polypeptide sequence NQELAYFYPEL and the molecular weight 1386.5Da of the bovine alpha-S1 complete sequence (GenBank: ACG 63494.1) are entrusted to be synthesized by Nanjing Jinsri biotechnology limited company;
s2: the carrier protein KLH (keyhole limpet hemocyanin) was dissolved using 0.1M carbonate buffer (containing 0.15M NaCl,pH 8.5) to a final concentration of 2.0mg/mL;
s3: the synthesized alpha-S1 polypeptide fragment was added to KLH solution at a final concentration of 10. Mu.g/mL, alpha-S1: KLH molar ratio of about 30:1;
s4: fresh glutaraldehyde is added into the solution to a final concentration of 1%, and the solution is reacted for 4 hours on a magnetic stirrer at the temperature of 4 ℃;
s5: adding sodium borohydride into the solution to a final concentration of 10mg/mL, and reacting for 1h on a magnetic stirrer at 4 ℃;
s6: the reaction product was dialyzed 6 times against 2L of 0.01M PBS (pH 7.4), 3 hours each time, adjusted to a final concentration of 2-3mg/mL, and stored at-20℃until use.
Extracting total RNA in hybridoma cells used for preparing the monoclonal antibody, obtaining cDNA through reverse transcription, then carrying out PCR amplification by utilizing light chain variable region primers VL-F and VL-R and heavy chain variable region primers VH-F and VH-R (table 3), further carrying out TA clone screening on amplified products, and sending positive clones obtained by screening to Beijing Bomaide biotechnology Co., ltd for gene sequencing, wherein the positive clones are obtained according to the result of gene sequencing: the amino acid sequence of the heavy chain variable region of the anti-bovine alpha-S1 monoclonal antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region of the anti-bovine alpha-S1 monoclonal antibody is shown as SEQ ID NO. 4.
TABLE 3 variable region Gene amplification primers for murine monoclonal antibodies
Note that: and (3) combining primers: s: C/G; m: A/C; r: A/G; w: A/T
SEQ ID NO. 3 amino acid sequence of anti-bovine alpha-S1 monoclonal antibody light chain variable region
Gly Thr Lys Leu Glu Ile Tyr Pro Cys Gln Ala Asp Gly Phe Thr Phe Glu Asp Ala Tyr Met Ser Ile Lys Tyr Val Met Asp Lys Asn Phe Ile His Ser Val Asp Thr Ile Ser Asp Gly Arg Asp Tyr Thr Lys Val Pro Asp Ser Phe Glu Ala Ile Tyr Val Met Ser Arg Asp Asp Ala Lys Asn Thr Leu Tyr Leu Val Phe Ser Asp Leu Ile Asp Glu Asp Thr Ala Met Tyr Trp Cys Phe Arg His Phe Glu Phe Gly Ala Pro Glu His 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 alpha-S1 monoclonal antibody
Leu Gly Pro Arg Asp His Gly His Arg Leu Leu Val Leu Ser Pro Glu Glu Thr Gly Thr Ser Ala Ile Val Ala Gln Gly Pro Thr Leu Asn Arg Glu Ile Ser Gly Ala Val Gly Thr Ala Cys Arg Pro Ser Ala Gly Ala Val Ala Tyr Ser Asn Tyr Phe Arg Asp Val Ser Arg Lys Pro Asp His Leu Lys Ala Gly Leu Ile Glu Ile Ala Asn Phe Arg Thr Ser Gly Val Ser Thr Arg Phe Pro Glu Ser Leu Ile Glu Asp Lys Thr Ile Leu Ala Ile Ala Arg Val Glu Lys Leu Asp Asn His Ile Tyr Phe Cys Arg Ser Asn Cys Arg Val Arg
Example 3 antibody labelling of fluorescent microspheres
S1: 50. Mu.L of fluorescent microspheres were added to 450. Mu.L of MES (0.05M, pH 5.0) activation buffer and sonicated for 5min; then, EDC and NHS solutions are sequentially added into the microsphere solution to ensure that the final concentration is 1mmol/L, and the mixture is subjected to oscillation reaction at room temperature for 0.5h;
s2: centrifuging at 12000g for 5min, and discarding supernatant; the precipitate was redissolved with 500. Mu.L PB (0.04M, pH 8.0), sonicated for 5min, added with an appropriate volume (5. Mu.L) of either 2.3mg/mL of anti-bovine IgG monoclonal antibody or 3.7mg/mL of anti-bovine alpha-S1 monoclonal antibody, reacted for 2h with shaking at room temperature, and centrifuged for 5min at 15000 g;
s3: to the pellet was added 500. Mu.L of blocking buffer (0.01M PB,2% BSA, pH 8.0) and reacted overnight with shaking at 4 ℃;
s4: and (3) centrifuging 15000g of the reaction solution for 5min, discarding the supernatant, using 50 mu L of 20mM PB redissolved microspheres again, performing ultrasonic 2min, and keeping the microspheres away from light at 4 ℃ for standby to obtain fluorescent microsphere marked anti-bovine IgG monoclonal antibodies or fluorescent microsphere marked anti-bovine alpha-S1 monoclonal antibodies.
Example 4 preparation of test strip for quantitative detection of adulterated common cow's milk in buffalo milk
Bovine IgG antigen (1.6 mg/mL, 0.8. Mu.L/cm) or bovine alpha-S1 antigen (1.4 mg/mL, 0.8. Mu.L/cm) obtained in example 1 and goat anti-mouse IgG (0.75 mg/mL, 0.7. Mu.L/cm) were coated on nitrocellulose membrane (NC membrane) with a membrane as detection line (T line) and quality control line (C line), respectively.
Spraying the fluorescent microsphere marked anti-bovine IgG monoclonal antibody or the fluorescent microsphere marked anti-bovine alpha-S1 monoclonal antibody prepared in the embodiment 3 on a release pad, wherein the spraying amount is 2.2 mu L/cm and 2.8 mu L/cm respectively, and drying the release pad in a drying oven at 37 ℃ for 2 hours to obtain the release pad containing the fluorescent microsphere marked anti-bovine IgG monoclonal antibody or the anti-bovine alpha-S1 monoclonal antibody.
Sequentially adhering a sample pad, a release pad containing fluorescent microsphere marked antibody, a nitrocellulose membrane containing a detection line T and a quality control line C and a water absorption pad to a PVC base plate, wherein one end of the base plate is the water absorption pad, the other end of the base plate is the sample pad, two ends of the nitrocellulose membrane are respectively overlapped (1-3 mm) with the water absorption pad and the release pad containing fluorescent microsphere marked antibody, the release pad containing fluorescent microsphere marked antibody is pressed with the sample pad (overlapped 1-3 mm), and a cutting machine is used for cutting into test strips with the width of 3.90mm to obtain the test strips for detecting the adulterated milk of common cow milk.
According to different types of monoclonal antibodies in the release pad and the coating antigen on the T line, the test strip 1 (anti-bovine IgG monoclonal antibody) and the test strip 2 (anti-bovine alpha-S1 monoclonal antibody) are respectively obtained.
Example 5 quantitative detection method of adulterated common cow milk in buffalo milk
(1) Drawing of a Standard Curve
S1: taking a negative buffalo milk sample (the PCR detection does not contain common cow milk), and respectively adding the common cow milk into the negative buffalo milk sample to ensure that the common cow milk comprises the following volume percentages: 0.0%, 0.05%, 0.15%, 0.45%, 1.35% and 4.05%;
s2: dripping 100 mu L of each sample onto a sample pad of the test strip 2, incubating at a constant temperature of 40 ℃ for 5min, detecting by using a fluorescence immunoassay quantitative analyzer, and obtaining a T line fluorescence signal value and a C line fluorescence signal value by using an excitation wavelength of 365nm and a detection wavelength of 610 nm;
s3: repeating the test for 5 times for each concentration, taking an average value, taking the percentage of common cow milk added in a sample as an X axis, taking the ratio (T/C) of a detection line T fluorescent signal value to a quality control line C fluorescent signal value as a Y axis, and carrying out four-parameter nonlinear fitting analysis by using origin8.0 to obtain a standard curve, wherein Y=0.1354+1.9649/(1+ (X/0.3023) 1.4134 )。
The results are shown in FIG. 2, and the fitting of the test data shows that the theoretical detection limit (IC) of the established method for detecting the water-milk adulterated common cow milk 20 ) 0.113%, IC 50 0.325％，R 2 0.9991.
(2) Sample detection to be tested
Dripping 100 mu L of sample liquid onto a sample pad of a buffalo milk adulterated common cow milk detection test strip 2, accurately reacting for 5min in a constant temperature incubator of a 40 ℃ detection card, taking out and detecting by using a fluorescent immunity quantitative analyzer to obtain a T line fluorescent signal value and a C line fluorescent signal value; substituting the ratio of the T line fluorescence signal value to the C line fluorescence signal value into a standard curve to obtain the percentage content of the common cow milk in each sample.
Methodological verification
(1) Minimum detection limit and quantitative limit of test paper strip
According to the method described in the above "sample detection to be detected", 20 water negative milk samples (negative in PCR detection) were detected using the water milk adulterated normal cow milk detection test strip 1, and the average value and standard deviation of the percentage content of normal cow milk were calculated, respectively: the average value is added with 3 times of standard deviation to obtain the detection limit; the average value is added with 10 times of standard deviation, and the quantitative limit is obtained.
TABLE 4 detection limit verification of buffalo milk adulterated common cow milk test strip (%)
| Sample of | Average value of | Standard deviation of | Detection limit | Quantitative limit |
| Water milk | 0.064 | 0.011 | 0.097 | 0.174 |
The results are shown in Table 4, and it can be seen that the standard curve was established using normal cow's milk as a control to give a minimum detection limit of 0.097% and a quantitative limit of 0.174%. In order to ensure the accuracy and reliability of the results, the detection of common cow milk in the buffalo milk is limited to 0.10 percent, and the quantification is limited to 0.20 percent.
(2) Method accuracy and precision
20 parts of buffalo milk negative samples are respectively added into common cow milk, the concentration of the added medicines of the samples is corresponding quantitative limit and 2 times of quantitative limit, 5 parallel concentration gradients are added into each concentration gradient, and the sample addition recovery rate and the intra-batch and inter-batch variation coefficients are calculated.
And analyzing the accuracy and precision of the established fluorescence immunochromatography analysis method according to the addition recovery data.
TABLE 5 accuracy and precision of test strips for detecting buffalo milk adulterated with common cow's milk
Recovery = (average/addition concentration) ×100%
Coefficient of variation= (standard deviation/average value) ×100%;
the results are shown in Table 5, the recovery rate of the method for adding common cow milk in the water milk is 94.30-107.77%, and the intra-batch and inter-batch variation coefficients are less than 10%, which shows that the method has good accuracy and precision.
(3) Test strip and PCR detection result comparison
The test strip and the detection method for quantitatively detecting the adulterated common cow milk in the buffalo milk provided by the invention are adopted to detect the buffalo milk sample (50 parts in total), and then the test strip and the detection method are respectively used for carrying out confirmation comparison on PCR detection results. The results are shown in Table 6.
TABLE 6 comparison of test strips for detecting buffalo milk adulterated with common cow milk and PCR detection results
Note that: "ND" means that normal cow milk was not detected
The result shows that the content of the buffalo milk adulterated common cow milk detected by the test paper provided by the invention is basically consistent with the PCR result, which proves that the detection test paper strip provided by the invention has higher accuracy in detecting the buffalo milk adulterated common cow milk.
Example 6 method for quantitative detection of adulterated common cow milk in buffalo milk
Samples with percentage contents of 0.0%, 0.05%, 0.15%, 0.45%, 1.35% and 4.05% of common cow's milk were prepared according to the method disclosed in example 5, and the samples were dropped onto the sample pad of test strip 1 to obtain a T-line fluorescence signal value and a C-line fluorescence signal value, and a standard curve was drawn, and the lowest detection limit and the lowest quantitative limit of the test strip, the accuracy and precision were verified and compared with the PCR method.
Fitting to obtain a standard curve: y=0.1148+1.8875/(1+ (x/0.2921) 1.5814 ) As shown in FIG. 3, the test data fitting shows that the theoretical detection limit of the established method for detecting the buffalo milk adulterated with common cow milk is 0.12%, and the IC 50 0.308％，R 2 0.9991.
(1) Minimum detection limit and quantitative limit of test paper strip
TABLE 7 detection limit verification of buffalo milk adulterated common cow milk test strip (%)
| Sample of | Average value of | Standard deviation of | Detection limit | Quantitative limit |
| Water milk | 0.049 | 0.015 | 0.094 | 0.199 |
The results are shown in Table 7, and it can be seen that the standard curve was established using normal cow's milk as a control to give a minimum detection limit of 0.094% and a quantitative limit of 0.199%. In order to ensure the accuracy and reliability of the results, the detection of common cow milk in the buffalo milk is limited to 0.10 percent, and the quantification is limited to 0.20 percent.
(2) Method accuracy and precision
TABLE 8 accuracy and precision of test strips for detecting buffalo milk adulterated with common cow's milk
The results are shown in Table 8, and the recovery rate of the method for adding common cow milk in the water milk is 89.30-110.37%, the intra-batch variation coefficient is less than 10%, and the inter-batch variation coefficient is less than 15%, which shows that the method has good accuracy and precision.
(3) Test strip and PCR detection result comparison
TABLE 9 comparison of test strips for detecting buffalo milk adulterated with common cow milk and PCR detection results
Note that: "ND" means that normal cow milk was not detected
The result shows that the content of the buffalo milk adulterated common cow milk detected by the test paper provided by the invention is basically consistent with the PCR result, which proves that the detection test paper strip provided by the invention has higher accuracy in detecting the buffalo milk adulterated common cow milk.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (11)
1. A monoclonal antibody for detecting adulterated common cow milk in buffalo milk is an anti-bovine alpha-S1 monoclonal antibody, wherein the light chain variable region of the anti-bovine alpha-S1 monoclonal antibody has an amino acid sequence shown as SEQ ID NO. 3, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 4.
2. The monoclonal antibody for detecting the adulterated common cow's milk in the buffalo milk according to claim 1, which is prepared by the following steps:
(1) Preparing bovine alpha-S1 antigen;
(2) Immunized mice
Periodically injecting bovine alpha-S1 antigen subcutaneously or intraperitoneally to immunize Balb/c mice, taking blood after immunization, separating serum, and detecting the polyclonal titer in the serum by an ELISA method;
(3) Hybridoma cell line fusion
Taking spleen cells of immunized mice with high serum titer and high sensitivity to fuse with myeloma cells, and adding feeder cells for culture;
(4) Subcloning and screening
Sucking out hybridoma cell supernatant, screening culture holes with high titer by using bovine alpha-S1 polypeptide antigen as a coating source through an indirect ELISA method for subcloning, repeating subcloning until the positive rate of cell strains in the holes is 100%, performing expanded culture on positive hybridoma cells, collecting supernatant, measuring titer through an indirect ELISA method, and freezing;
(5) Monoclonal antibody acquisition and purification
Obtaining monoclonal cell strain, injecting immune mouse into abdomen to obtain ascites, purifying produced ascites by Protein A/G affinity column to obtain bovine alpha-S1 monoclonal antibody.
3. The monoclonal antibody of claim 2, wherein the bovine α -S1 antigen of step (1) is a bovine α -S1 polypeptide antigen;
the bovine alpha-S1 polypeptide antigen is prepared by the following method:
(1) Synthesizing an alpha-S1 polypeptide fragment with an amino acid sequence of NQELAYFYPEL by referring to the whole sequence of bovine alpha-S1;
(2) Coupling the alpha-S1 polypeptide fragment with carrier protein, and purifying to obtain the bovine alpha-S1 polypeptide antigen.
4. A monoclonal antibody according to claim 3, wherein the carrier protein is selected from the group consisting of bovine serum albumin, ovalbumin, hemocyanin, diphtheria toxoid, a non-toxic mutant of diphtheria toxin, tetanus toxoid, and a fusion protein of two or more carrier proteins.
5. A test strip for quantitatively detecting adulterated common cow milk in water milk, which comprises a sample pad, a release pad, a reaction film 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-cow alpha-S1 monoclonal antibody 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 alpha-S1 antigen, and the quality control line (C line) is coated with goat anti-mouse IgG antibody.
6. The test strip of claim 5, wherein the detectable label is selected from one of fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal metal particles, and luciferin.
7. The test strip of claim 6, wherein the detectable label is selected from the group consisting of fluorescent microspheres.
8. The test strip of claim 7, wherein the concentration of the fluorescent microsphere-labeled anti-bovine α -S1 monoclonal antibody is 3-4 mg/mL.
9. The test strip of claim 5, 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.
10. The test strip of claim 5, 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 test strip with the width of 3.5-4.0mm is cut by a cutting machine to obtain the test strip for quantitatively detecting the adulterated common cow milk in the water milk.
11. 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 5-10, 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.
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| CN110018311A (en) * | 2019-03-22 | 2019-07-16 | 新疆大学 | Immunity detection reagent and its application of pseudo- cow's milk are mixed in a kind of detection bactrian camel milk |
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