CN113049828A - Double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen - Google Patents
Double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen Download PDFInfo
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- CN113049828A CN113049828A CN202110274530.5A CN202110274530A CN113049828A CN 113049828 A CN113049828 A CN 113049828A CN 202110274530 A CN202110274530 A CN 202110274530A CN 113049828 A CN113049828 A CN 113049828A
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Abstract
The invention relates to a double-antibody sandwich ELISA detection method for quantitatively detecting cow milk casein allergen, which comprises the steps of taking cow milk allergen casein as an immunizing antigen to respectively immunize a rabbit polyclonal antibody and a mouse monoclonal antibody which are respectively obtained by a New Zealand white rabbit and a BALB/c mouse, respectively identifying and evaluating the performances of the rabbit polyclonal antibody and the mouse monoclonal antibody, and then carrying out sandwich pairing on the rabbit polyclonal antibody and the mouse monoclonal antibody, thereby establishing the double-antibody sandwich ELISA detection method for quantitatively detecting cow milk casein allergen. Compared with the prior art, the method has higher sensitivity, specificity, precision and accuracy, and can meet the detection requirement of the bovine casein allergen.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen.
Background
Food allergy (food allergy) refers to an adverse reaction to food, which is usually caused by a specific component of food. Allergens that cause food allergy generally take three forms: contact allergens such as cosmetics and paints; inhalational allergens such as fungi, pollen, and dust mites; ingesting allergens such as peanuts, fish, eggs, milk, and the like. In general, food allergy is caused by milk, fish, eggs, peanuts, wheat, soybeans, nuts and crustaceans, namely ingestion type allergens, is easy to occur in sensitive people, and has the characteristics of difficult radical cure, easy repeated attack, long incubation period in vivo, various clinical symptoms and the like. According to one epidemiological investigation, developed countries experience food allergies in more than 8% of minors and 2% of adults, and in particular in the united states, about 5.3% of adults and about 6% of infants and children suffer from food allergies. In some cases, life-threatening allergic reactions can result. In recent years, with the development of science and technology, the living environment and food types of people have changed greatly, and the number of people suffering from food allergy diseases has increased greatly. At present, food allergy diseases have become a global and public health problem.
Milk has a high protein content and balanced nutrition and is therefore widely used in the food industry. Cow's milk allergy clinically refers to an abnormal immune response of the body to milk proteins, generally thought to be caused by the interaction between one or more milk proteins and IgE-mediated reactions. Reactions not involving the immune system are defined as milk protein intolerance. The major proteins of cow's milk are 80% casein and 20% whey protein. Caseins are classified as alpha-casein, beta-casein, kappa-casein and gamma-casein; whey proteins include: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), Bovine Serum Albumin (BSA), immunoglobulin and lactoferrin with a very small content. The study showed that cow milk contains more than 20 proteins capable of causing allergic reactions, of which casein, beta-lactoglobulin and alpha-lactalbumin are the most major allergenic substances.
Casein is the major protein in milk and an important component of cheese. Milk contains about 3% casein. It is isoelectric at pH4.6 and has a solubility in water of only 0.01%. It is amphoteric: at a pH below 4.6, casein forms moderately soluble salts, such as casein chloride; casein having a pH of 4.6 or higher forms a salt with a base. Sodium caseinate and other alkali metal salts are infinitely soluble, while calcium caseinate, other alkaline earth metal salts and heavy metal salts are almost insoluble.
Worldwide, people with allergic diseases account for about 25% of the global general population, and the World Health Organization (WHO) has listed "food allergy" as one of the major health problems in the 21 st century at the end of the 20 th century. National laws and regulations for allergens are gradually perfected, and aims to effectively protect the health and safety of people suffering from allergic diseases. Cow's milk allergy is one of the problems of food allergy, and there is no effective cow's milk major allergen diagnosis measure, and cross contamination may occur during the processing and production of food and beverage. Therefore, a convenient, quick, sensitive and specific method for detecting the main allergens of the cow milk is established, has great significance for guaranteeing public health and safety, and provides theoretical basis for an allergen identification system in international connection.
Disclosure of Invention
Enzyme-linked immunoassays are based on biological evolution. Enzyme-linked immunosorbent assay (ELISA), also known as Enzyme immunoassay, is a biochemical procedure by which a signal generated by an enzymatic reaction is used to detect and quantify the amount of a particular substance in a solution. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for the detection of antigens, but may also be used for the detection of other substances, including antibodies, hormones, and drugs. ELISA has the advantages of high sensitivity, strong specificity, low cost and the like.
The principle of enzyme-linked immunoassay is as follows: firstly, combining an antigen or an antibody with polystyrene under the action of physical adsorption to ensure the integrity of the immunocompetence of the antigen or the antibody; secondly, the antibody (antigen) and the enzyme-labeled antigen (enzyme-labeled antibody) are coupled under the action of chemical bonds, so that the enzyme activity and the immunocompetence integrity of the enzyme-labeled antibody (enzyme-labeled antigen) are ensured; and finally, combining Horse Radish Peroxidase (HRP) with a surface binding site on an enzyme-labeled antibody (enzyme-labeled antigen), adding a developing solution, and judging the result according to the developing condition. OD can also be performed at the microplate reader450And (4) establishing a standard curve, and carrying out quantitative detection analysis. The enzyme is used as a catalyst and plays a role in catalyzing chemical reaction and amplifying chemical signals, thereby greatly improving the detection sensitivity.
The invention aims to provide a double-antibody sandwich ELISA detection method for quantitatively detecting bovine milk casein allergen. Has higher sensitivity, specificity, precision and accuracy, and can meet the detection requirement of the bovine casein allergen.
The invention takes cow milk main allergen casein as immunogen to immunize New Zealand white rabbit and BALB/c mouse respectively, prepares rabbit source polyclonal antibody and mouse source monoclonal antibody, identifies and evaluates the performance of the rabbit source polyclonal antibody and the mouse source monoclonal antibody, and then carries out sandwich pairing on the rabbit source polyclonal antibody and the mouse source monoclonal antibody, thereby establishing a double-antibody sandwich ELISA detection method capable of detecting cow milk main allergen, and providing theoretical basis for the development and development of commercial kits.
The purpose of the invention can be realized by the following technical scheme:
a double-antibody sandwich ELISA detection method for quantitatively detecting cow milk casein allergen is characterized in that cow milk allergen casein is used as an immunizing antigen to respectively immunize a rabbit polyclonal antibody and a mouse monoclonal antibody which are respectively obtained by a New Zealand white rabbit and a BALB/c mouse, the performances of the rabbit polyclonal antibody and the mouse monoclonal antibody are respectively identified and evaluated, and then the rabbit polyclonal antibody and the mouse monoclonal antibody are subjected to sandwich pairing, so that the double-antibody sandwich ELISA detection method for quantitatively detecting cow milk casein allergen is established.
Preferably, the double antibody sandwich ELISA detection method comprises the following steps: screening an optimal casein capture antibody, optimizing the working concentration of an enzyme-labeled secondary antibody, optimizing the working concentration of a casein rabbit polyclonal antibody and establishing a standard curve;
the standard curve takes the concentration of a casein standard substance as an abscissa and takes the absorbance OD450As an ordinate, a casein standard concentration (ng/mL) was used as an abscissa, and the abscissa was set to a logarithmic form, a four-parameter fitting was performed using a logistic model in Origin software, a standard curve was drawn, and LOD of casein was calculated by P/N (positive/negative) > 2.1.
Preferably, the following methods are adopted for screening the optimal casein capture antibody, optimizing the working concentration of the enzyme-labeled secondary antibody and optimizing the working concentration of the casein rabbit polyclonal antibody:
(1) wrapping a plate: microtiter wells of ELISA plates were coated with 100. mu.L of capture-coated antibody, mouse monoclonal antibody, at 5. mu.g/mL, incubated overnight at 4 ℃ or in a 37 ℃ oven for 2 h;
(2) washing the plate: followed by three repeated washes with PBST to remove unbound antibody, patted dry;
(3) and (3) sealing: add 200. mu.L of blocking buffer to each well;
(4) adding a sample: adding the diluted casein standard substance into a 96-hole enzyme label plate by using a 12-hole arraying gun, wherein the liquid adding amount of each hole is 100 mu L, and placing the plate in a constant-temperature oven at 37 ℃ for incubation for 30 min;
(5) adding rabbit antiserum: washing the plate according to the operation step of the step (2), drying the plate, taking 100 mu L of a detection antibody-rabbit polyclonal antibody, diluting the detection antibody-rabbit polyclonal antibody in a diluted buffer solution PBS by a certain multiple, adding the diluted detection antibody-rabbit polyclonal antibody into each hole, and incubating the diluted detection antibody-rabbit polyclonal antibody in a constant-temperature oven at 37 ℃ for 30 min;
(6) HRP-labeled goat anti-rabbit IgG antibody: washing the plate according to the operation step of the step (2), after drying by beating, diluting the goat anti-rabbit IgG antibody-enzyme labeled secondary antibody marked by the HRP to a certain multiple according to the same method as the previous step, adding the diluted secondary antibody into the holes, wherein the liquid adding amount of each hole is 100 mu L, and incubating in a constant-temperature oven at 37 ℃ for 30 min;
(7) color development: washing the plate according to the operation step of the step (2), drying by beating, finally adding a freshly prepared TMB substrate chromogenic solution into each hole, performing incubation for 15min in a constant-temperature oven at 37 ℃ in a 100-mu L/well manner;
(8) and (4) terminating: reaction 2M H2SO4The reaction was terminated, and the amount of solution added to each well was 50. mu.L, and the absorbance value was measured at 450 nm.
Preferably, during the screening of the casein optimal capture antibody and the optimization of the working concentration of the enzyme-labeled secondary antibody:
using a mouse monoclonal antibody as a capture antibody to coat an enzyme label plate, controlling the coating concentration, casein standard dilution and rabbit polyclonal antibody dilution concentration to be unchanged, using a phosphate buffer solution to dilute the goat anti-rabbit IgG antibody marked by the HRP into three gradients of 1:5000, 1:10000 and 1:15000, using a phosphate buffer solution without a standard as a negative control, and measuring OD (optical density) by measuring450Determining the optimal working concentrations of a casein capture antibody and an enzyme-labeled secondary antibody;
preferably, during the optimization process of the casein rabbit polyclonal antibody working concentration:
performing operation according to the determined working concentrations of the casein optimal capture antibody and the enzyme-labeled secondary antibody, determining the optimal dilution multiple of the rabbit polyclonal antibody, diluting the rabbit polyclonal antibody to three gradients of 1:10000, 1:15000 and 1:45000, taking phosphate buffer solution without a standard as negative control, and determining OD (optical density) by measuring450And determining the working concentration of the casein rabbit polyclonal antibody.
Preferably, the working concentration of rabbit polyclonal antibody is 1: 10000.
Preferably, the optimal working concentration of the HRP-labeled goat anti-rabbit IgG antibody (enzyme-labeled secondary antibody) is 1: 10000.
Preferably, the linear range of the standard curve is 80-1280ng/mL, and the limit of detection (LOD) is 80 ng/mL; the method has intra-batch coefficient of variation CV of 2.72-5.97% and inter-batch coefficient of variation CV of 7.99-12.33%, and has high precision; the method only has cross reaction with casein, and has good specificity; the method has a casein recovery rate of 80.4-92.25%, has high accuracy, and can meet the detection requirements of cow milk casein allergen.
Preferably, the rabbit polyclonal antibody is obtained by the following method:
the rabbit serum is prepared by taking cow milk allergen casein as an immunizing antigen, mixing the cow milk allergen casein with Freund's adjuvant in equal amount, and immunizing healthy New Zealand white rabbits by adopting an immunization mode of neck and back subcutaneous injection.
Preferably, the performance characterization evaluation of the rabbit polyclonal antibody comprises:
purifying and separating the prepared rabbit serum by an octanoic acid-saturated ammonium sulfate precipitation method; performing purity identification on the purified rabbit serum by adopting an SDS-PAGE gel electrophoresis method to obtain a purified rabbit polyclonal antibody; four immunizations of casein were performed, each immunization at a dose of 500. mu.g/mouse; the titer of the purified rabbit polyclonal antibody is measured by an indirect ELISA method.
Preferably, the murine monoclonal antibody is obtained by the following method:
immunizing healthy female BALB/c mice with proper ages by adopting an immunization mode and an immune antigen which are the same as those of rabbit polyclonal antibody preparation, immunizing casein for six times, wherein the primary immunization dose is 100 mu g/mouse, the boosting immunization dose is 50 mu g/mouse, the spurt immunization dose is 25 mu g/mouse, detecting immune serum of the mice by using an indirect ELISA method, and selecting the mice with the highest six-immune serum titer;
detecting and screening positive holes by cell fusion and an indirect ELISA method, and screening to obtain a hybridoma cell strain stably secreting the casein monoclonal antibody after subcloning by adopting a limiting dilution method;
mass production of murine monoclonal antibodies was carried out by in vivo ascites induction.
Preferably, the performance of the murine monoclonal antibody is characterized by the following methods:
purifying the obtained ascites monoclonal antibody by an octanoic acid-saturated ammonium sulfate precipitation method; identifying the purity of the purified ascites monoclonal antibody by an SDS-PAGE gel electrophoresis method; the titer of the monoclonal antibody is measured by adopting an indirect ELISA method, and the monoclonal antibody subtype secreted by the casein cell strain is IgG1 according to the identification of a commercial monoclonal antibody subtype kit.
Compared with the prior art, the invention has the advantages that the intra-batch coefficient of variation CV of the casein measured by the established double-antibody sandwich method is between 2.72 and 5.97 percent, the inter-batch coefficient of variation CV is between 7.99 and 12.33 percent, the precision is higher, the linear range of the established standard curve is 80 to 1280ng/mL, the detection limit is 80ng/mL, the recovery rate of the casein is 80.4 to 92.25 percent, the accuracy is higher, and the detection requirement of the casein allergen of the cow milk can be met.
Drawings
Figure 1 casein tetra-immune rabbit serum titers.
FIG. 2 SDS-PAGE electrophoresis of casein mab.
FIG. 3Western-blotting to test the specificity of casein monoclonal antibody.
FIG. 4 shows a standard curve for detecting casein by a double antibody sandwich ELISA method.
Detailed Description
The invention provides a double-antibody sandwich ELISA detection method for quantitatively detecting cow milk casein allergen, which is characterized in that cow milk allergen casein is taken as an immune antigen to respectively immunize a rabbit polyclonal antibody and a mouse monoclonal antibody obtained by a New Zealand white rabbit and a BALB/c mouse, the performances of the rabbit polyclonal antibody and the mouse monoclonal antibody are respectively identified and evaluated, and then the rabbit polyclonal antibody and the mouse monoclonal antibody are subjected to sandwich pairing, so that the double-antibody sandwich ELISA detection method for quantitatively detecting cow milk casein allergen is established.
The method comprises the following steps:
A. immunizing a rabbit with cow milk allergen casein as an antigen to obtain a rabbit polyclonal antibody;
B. identifying the purity, titer, specificity and affinity of the rabbit polyclonal antibody;
C. immunizing a mouse by taking cow milk allergen casein as an antigen to obtain a mouse monoclonal antibody;
D. identifying the purity, titer, specificity and affinity of the monoclonal antibody;
E. and (3) establishing a casein double-antibody sandwich ELISA detection method.
The invention adopts enzyme-linked immunosorbent assay, which combines antigen or antibody with polystyrene under physical adsorption to ensure the integrity of immunocompetence; secondly, the antibody (antigen) and the enzyme-labeled antigen (enzyme-labeled antibody) are coupled under the action of chemical bonds, so that the enzyme activity and the immunocompetence integrity of the enzyme-labeled antibody (enzyme-labeled antigen) are ensured; and finally, combining Horse Radish Peroxidase (HRP) with a surface binding site on an enzyme-labeled antibody (enzyme-labeled antigen), adding a developing solution, and judging the result according to the developing condition. OD can also be performed at the microplate reader450And (4) establishing a standard curve, and carrying out quantitative detection analysis. The enzyme is used as a catalyst and plays a role in catalyzing chemical reaction and amplifying chemical signals, thereby greatly improving the detection sensitivity. At present, enzyme-linked immunoassay is widely applied to the rapid detection of biomedicine, pesticide and veterinary drug residues, aquatic products, food and the like. Meanwhile, enzyme-linked immunosorbent assay (ELISA) is widely applied in food allergen detection (qualitative and quantitative analysis). With the development of ELISA technology, commercial kits corresponding to the ELISA technology are produced, so that the aims of rapidness, simplicity and convenience are fulfilled.
The detection object of the invention is casein of cow milk, and the casein is the main protein component (about 23mg/mL) of cow milk and accounts for about 80 percent of the total protein content of cow milk. According to the invention, a new Zealand white rabbit and a BALB/c mouse are immunized respectively by taking casein serving as a main cow milk allergen as immunogen to prepare a rabbit-derived polyclonal antibody and a mouse-derived monoclonal antibody, the performances of the rabbit-derived polyclonal antibody and the mouse-derived monoclonal antibody are identified and evaluated, and then the rabbit-derived polyclonal antibody and the mouse-derived monoclonal antibody are subjected to sandwich pairing, so that a double-antibody sandwich ELISA detection method capable of detecting the main cow milk allergen is established, the standard casein curve has good linearity in the range of 80-1280ng/mL, and the detection limit is 80 ng/mL. The method only has cross reaction with casein, and shows that the method has better specificity; the recovery rate of casein by the method is 80.4-92.25%, which shows that the method has higher accuracy and can meet the detection requirement of the casein allergen of cow milk.
In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1: preparation and identification of casein rabbit polyclonal antibody
1. Preparation of Casein Rabbit antiserum
(1) Preparation of Rabbit polyclonal antibody
Taking casein as immunogen, adopting an immunization mode of subcutaneous multipoint small injection at neck and back to immunize healthy male New Zealand white rabbits of about 2.0kg, and preparing rabbit polyclonal antibodies, wherein the specific operation steps are as follows:
accurately weighing 500 mu g of purified protein as immunogen (casein), diluting the casein to a certain volume by using sterile physiological saline, and adding equivalent volume of Freund's complete adjuvant (only primary immunization uses Freund's complete adjuvant); then using a silver mercury mixer to react the solution and the adjuvant for about 90s, repeating for four times to fully mix the solution and the adjuvant uniformly to form a water-in-oil state, namely forming nondispersed oil drops on the water surface (the water-in-oil state is very important), carrying out back subcutaneous injection immunization on the thoroughly emulsified immunogen by using a 1mL disposable sterile syringe, beating for 8-10 points, and immunizing two rabbits with each protein respectively;
secondly, after the New Zealand white rabbits are immunized for 14d for the first time, the second immunization is carried out, and the operation steps are the same as the first step;
carrying out the third immunization after 7d of the second immunization, wherein the operation steps are the same as the second immunization;
fourthly, the fourth immunization and the third immunization;
before the third immunization, collecting whole blood from ear vein or artery of rabbit, placing the collected whole blood in a 37 deg.C oven, standing for about 1h (sealed by sealing membrane to prevent odor volatilization), allowing the blood to coagulate, centrifuging at 4 deg.C and 5000rpm for 10min by using a high-speed refrigerated centrifuge, collecting supernatant, repeating the operation, centrifuging for 2 times, and detecting titer;
sixthly, when the serum titer meets the requirement, blood can be taken through the carotid artery.
(2) Rabbit carotid artery blood sampling
Firstly, carrying out auricular intravenous injection anesthesia on a New Zealand white rabbit by using an anesthetic (3 percent sodium pentobarbital) according to the dose of 1mL/kg body weight, and then fixing the new Zealand white rabbit in a supine posture on an operation console;
secondly, lowering the head of the white rabbit to enable the neck fur of the white rabbit to be completely exposed in the visual field, picking out fur by taking the neck midline as the center, and then disinfecting the exposed skin by using alcohol;
using a scalpel to directly cut an incision with the length of about 10cm at a critical position 5-6cm away from the head and neck along the central line of the neck, and cleaning subcutaneous tissues until sternocleidomastoid muscles on two sides of a trachea are obviously exposed in the visual field;
separating the tissue of the triangular area of the neck in the gap between the trachea and the sternocleidomastoid muscle, wherein three blood vessels can be seen, the red blood vessel is the carotid artery, the two dark flowers are veins, and the carotid artery is separated by using the dentate hemostatic forceps, but the veins are not damaged;
embedding two thin threads below the carotid artery, respectively placing the two thin threads at the proximal end and the distal end, and clamping the artery at the proximal end by using a hemostatic clamp;
cutting a small opening on the wall of the artery vessel between the two thin lines by using a 10cm elbow surgical scissors with an inclination angle of 30-45 degrees, and inserting into an intravenous injection plastic tube. The vein injection plastic tube at the far-end and the near-end and the artery vessel wall are fastened by two thin lines to fix the blood vessel so that the blood vessel can not slip under the action of pressure;
seventhly, loosening the hemostatic forceps to enable blood to flow into the sterile triangular flask;
eighthly, placing the collected blood in a blast drying oven at 37 ℃, coagulating the blood after about 0.5-1h, then placing the blood in a triangular flask in a refrigerator at 4 ℃, and shrinking blood clots after 3-4h, wherein serum exudes. After blood coagulation and complete contraction of blood clots, the exuded serum is further set in a high speed refrigerated centrifuge at 4 deg.c and 3000rpm for 20min before eliminating precipitate. Mixing the serum and glycerol at a ratio of 1:1, adding 0.05% sodium azide, mixing, packaging, and storing at-20 deg.C.
2. Determination of rabbit immune serum titer
Antiserum titer is one of the important indicators of immune efficacy. The method for measuring the titer of the polyclonal antibody adopts an indirect ELISA method, and comprises the following specific operation steps:
(1) coating: diluting the coating source to 1, 0.5 and 100 mu L/well coating 96-hole enzyme label plate, and incubating for 2h at 4 ℃ overnight or in a constant-temperature air-blast drying oven at 37 ℃;
(2) washing: pouring off the solution in the enzyme-labeled pores, spin-drying, washing with PBST for 3 times, and placing on a horizontal decoloring shaking table for 3 minutes each time;
(3) and (3) sealing: adding 200 mu L of sealing liquid PBST into each hole, and sealing for 2h at 37 ℃ in a constant-temperature air-blast drying oven;
(4) primary anti-dilution: primary antiserum was initially diluted 100-fold (2 μ L primary antiserum +998 μ L antibody dilution), then diluted 1000-fold, followed by 3-fold dilution on dilution plates; (5) adding a primary antibody: after the ELISA plate is washed, adding a primary antibody diluted in a gradient manner, 100 mu L/well, and incubating for 30min in a constant-temperature air-blast drying box at 37 ℃;
(6) adding a secondary antibody: after the enzyme label plate is washed, diluting the goat anti-rabbit IgG antibody (rabbit secondary antibody) marked by HRP by 2500 times and adding the sample by 5000 times, and incubating for 30min in a constant-temperature oven at 37 ℃ in a 100 mu L/well manner;
(7) color development: after the enzyme label plate is washed, adding a freshly prepared TMB substrate developing solution, incubating for 15min in a constant-temperature oven at 37 ℃ in a 100 mu L/well manner; (8) and (4) terminating: 2M H2SO4The reaction was stopped, 50. mu.L/well;
(9) reading a plate: reading OD at microplate reader450The value of (c).
The titer of rabbit antiserum was determined as the dilution factor of the antibody at P/N (positive/negative wells) > 2.1.
After the second immunization, the white rabbits are subjected to auricular vein blood collection (for example, auricular artery blood collection can be carried out in cold days), the titer of the white rabbits is measured by adopting an indirect ELISA method, and rabbits with high titer are selected in principle to collect rabbit polyclonal antibody serum. Due to the lower primary immunization titers, 2 rabbit serum titers of immunocasein were detected starting from the second immunization (fig. 1). As can be seen from FIG. 1, the serum titer of the fourth time of immunizing the 1# rabbit with the immune casein antigen is the highest and can reach 1: 729000.
3. Purification of rabbit polyclonal antiserum
(1) Accurately sucking 5mL of prepared rabbit antiserum by using a pipette, centrifuging at 12000rpm at 4 ℃ for 10min, sucking out the supernatant, adding the supernatant into a centrifuge tube, adding 10mL of 60mmol/L sodium acetate solution with pH of 5.0, uniformly stirring, adjusting the pH to about 4.5, placing the mixture on a magnetic stirrer at room temperature, stirring for 30min, simultaneously dropwise adding n-octanoic acid, and standing for not less than 2h to fully precipitate;
(2) centrifuging the obtained supernatant at 4 deg.C and 12000rpm for 30min, transferring the centrifuged supernatant into a dialysis bag, and dialyzing in 0.01mol/L PBS (phosphate buffer solution) with pH of 8.0 for 24 h;
(3) adding 0.277g/mL ammonium sulfate solution into the dialyzed supernatant, stirring while adding, standing at 4 deg.C for 12h, centrifuging at 4 deg.C and 12000rpm for 30min, and discarding the supernatant;
(4) dissolving the obtained precipitate with 0.01moL/L PBS (pH 7.4), transferring into dialysis bag, dialyzing for 72 hr, changing solution every 6 hr, centrifuging at 12000rpm at 4 deg.C for 30min after dialysis is complete, discarding insoluble precipitated protein, and concentrating with PEG 20000.
4. Determination of Rabbit polyclonal antibody concentration
The determination of the concentration of the polyclonal antibody adopts a BCA method, a 96-well enzyme label plate is taken, and the reagents are added according to the following table 1:
TABLE 1 BCA assay for protein concentration
After the reagent is added, accurately sucking 20 mu L of sample solution to be detected, adding the sample solution into the holes of the ELISA plate, then adding 200 mu L of fresh BCA developing solution, slightly shaking the ELISA plate, placing the ELISA plate in a 37 ℃ air-blast drying oven for incubation for 30min, taking PBS as a blank control hole, and measuring the absorbance at 590nm at an ELISA reader. By light absorption OD590As ordinate, protein concentration (mg/mL) as abscissa, and standard is plottedAnd (3) calculating the content of the protein in the sample according to the light absorption value of the sample and a linear equation.
5. Determination of Rabbit polyclonal antibody purity
The purity of the polyclonal antibodies produced was characterized by electrophoresis on SDS-PAGE gels according to standard procedures.
(1) The specific experimental conditions are as follows: 10% of separation gum and 5% of concentrated gum; the electrode buffer solution is pH 8.3, 0.1% SDS, 0.384mol/L glycine, 0.05mol/L Tris, voltage 80V, electrophoresis time 1.5 h.
(2) The specific operation steps are as follows: the sample is dissolved and diluted to 1-2mg/mL by using sample buffer solution containing 2% SDS, 5% beta-mercaptoethanol, Tris-HCl (pH 8.0) and the like, and is subjected to centrifugation for 10min at 8000rpm in a boiling water bath for about 3min, and 10 mu L of sample is absorbed. After the electrophoresis, the gel was stained with Coomassie brilliant blue G-250 staining solution for 4 hours, and then the gel was placed on a horizontal shaker at room temperature and decolorized with decolorizing solution.
After the rabbit immune serum is purified by an octanoic acid-saturated ammonium sulfate method, the purity of the rabbit immune serum is identified by SDS-PAGE gel electrophoresis so as to determine that the obtained rabbit polyclonal antibody has higher purity. The results are shown in figure 2 below, the light chain and heavy chain of the rabbit polyclonal antibody against casein, alpha-lactalbumin and beta-lactoglobulin are clearly visible, and the molecular weight of the heavy chain protein is about 50kDa, which indicates that the rabbit polyclonal antibody against casein has high purity.
Example 2: preparation and identification of casein mouse monoclonal antibody
1. Immunization of BALB/c mice
Immunization protocol (BALB/c mice purchased need to be acclimated to new environment before injection immunization, pre-feeding time was set to 7 days).
TABLE 2 BALB/c mouse immunization protocol
2. Screening and establishment of hybridoma cell strain
(1) Recovery and culture of myeloma cell SP 2/0
In the case of non-use, the myeloma cells SP 2/0 were frozen in a liquid nitrogen tank or an ultra-low temperature refrigerator at-150 ℃. About one week before cell fusion, myeloma cells SP 2/0 frozen in a liquid nitrogen tank or an ultra-low temperature refrigerator at-150 ℃ need to be revived and expanded for culture. The specific operation steps are as follows: firstly, carrying out ultraviolet sterilization treatment on a superclean bench and cells, simultaneously preheating a constant-temperature water bath kettle to 37 ℃ in advance, taking out a cryopreservation tube from a liquid nitrogen tank or a-150 ℃ ultra-low temperature refrigerator, quickly transferring the cryopreservation tube into the constant-temperature water bath kettle preheated to 37 ℃ for melting, controlling the time within 1min, then transferring the cells melted in the cryopreservation tube into 10mL of 20% complete culture medium prepared in advance in a superclean bench, placing the cells into a centrifugal machine, centrifuging the cells for 10min at 500rpm, discarding supernatant, adding 20% complete culture medium, gently blowing the cells uniformly by using a liquid transfer gun, transferring the cells into a cell culture dish, and culturing the cells in a cell culture box (37 ℃, 5% CO (37 ℃) (5%)2)。
During the process of culturing the myeloma cell SP 2/0, the liquid is changed every other day, and when the cells grow to 70-80% of the whole culture dish, subculture is carried out. One day before the fusion, the myeloma cell SP 2/0 was subjected to a medium changing treatment, and its growth period was adjusted to a logarithmic growth state. On the day of cell fusion, cell supernatant was discarded, fresh incomplete medium DMEM was added, cells were blown off from a petri dish using a pipette gun, transferred to a 50mL centrifuge tube, centrifuged at 1000rpm for 10min, supernatant was discarded, the operation was repeated once, centrifuged cells were flicked, resuspended in 10mL incomplete medium DMEM, and a small amount of suspension was diluted 10-fold and 100-fold in a gradient and counted using a hemocytometer.
(2) Preparation of Positive splenocytes
And selecting the mouse with the highest antiserum titer after the 6 th immunization as a spleen source, and performing the sprint immunization by intraperitoneal injection 3 days before fusion, wherein the dosage of the sprint immunization is halved and no adjuvant is added. On the day of fusion, carrying out eyeball picking and blood sampling, taking eye blood of a mouse by using a 1.5mL PE tube until the eye blood is completely sampled, then placing the mouse in an oven at 37 ℃ for about 10min, layering the mouse, centrifuging the mouse at 4 ℃ and 1000rpm for 10min, collecting supernatant by using a pipette, subpackaging the supernatant, and placing the supernatant at-20 ℃ for later use, wherein collected serum is used as a positive control in cell fusion positive detection; immediately putting the mouse killed by adopting a neck dislocation method into a beaker with 75% ethanol for disinfection, controlling the time within 5min, quickly transferring the disinfected mouse into a sterilized clean bench, taking out the spleen under the aseptic condition, fully grinding the mouse on a 200-mesh screen by using a 5mL syringe rubber head, collecting spleen cell suspension, and centrifuging the spleen cell suspension for 10min at 1000 rpm. Adding incomplete culture medium DMEM again, repeating the operation for three times, after the last centrifugation, slightly loosening the cells at the bottom of the centrifuge tube, suspending the cells in 10mL of incomplete culture medium DMEM, taking a small amount of suspension for gradient dilution by 10, 100 and 1000 times, and counting by using a blood counting chamber.
(3) Cell fusion
DMEM medium, 20% complete medium and 25% HAT selection medium were pre-warmed in a 37 ℃ water bath. 1mL of induction fusion agent PEG1450 and 15mL of DMEM medium were placed in a 37 ℃ incubator and preheated in advance. Respectively collecting SP 2/0 myeloma cells and mouse spleen cells, adding the SP 2/0 myeloma cells and mouse spleen cells into a 50mL centrifuge tube according to the ratio of 1:5-15, uniformly mixing, centrifuging at 1000rpm for 10min, discarding supernatant, adding a DMEM culture medium for resuspension, and repeating the operation once. The cells at the bottom of the centrifuge tube were gently loosened and distributed as uniformly as possible at the bottom of the 50mL centrifuge tube. The method comprises the steps of sucking 1mL of PEG1450 preheated in advance by using a pipette gun, adding the PEG1450 into a centrifuge tube, rotating the centrifuge tube while adding, ensuring uniform dropping in the whole process as much as possible, ensuring that the bottom of the centrifuge tube is always held at the palm of the hand and completed within 60-90s, then placing the centrifuge tube on a centrifuge tube frame, standing for about 30s, taking 15mL of DMEM culture medium preheated in advance out of a cell incubator in the process, sucking the DMEM culture medium by using the 1mL of pipette gun and dropping into the centrifuge tube, stopping the fusion reaction, wherein the operation is the same as that of dropping the PEG1450, and completing within 10 min. And (3) centrifuging the fused cell suspension at the speed of 1000rpm for 10min, discarding the supernatant, and resuspending the cells by using HAT selection medium, wherein the action needs to be gentle. The resuspended cell suspension was plated in 96-well cell culture plates at 200. mu.L/well, a total of 8 plates were plated, and transferred toCarbon dioxide cell incubator (37 ℃, 5% CO)2) The culture is carried out.
(4) Screening of hybridoma cells
The positive property of cell supernatant is detected by an indirect ELISA method, the specific operation steps refer to the determination of rabbit immune serum titer, and an HRP-labeled goat anti-rabbit IgG antibody (rabbit secondary antibody) is changed into an HRP-labeled goat anti-mouse IgG antibody (mouse secondary antibody), and antiserum is changed into cell supernatant.
(5) Cryopreservation of hybridoma cells
In the process of preparing monoclonal antibodies, the freezing storage of cells is crucial, and SP 2/0, hybridoma cells and subcloned hybridoma cells need to be frozen in time. The cell freezing method comprises the following specific steps: collecting cells to be cryopreserved, centrifuging, removing cell supernatant, adding 1mL of cell cryopreservation liquid, gently blowing and uniformly beating by using a pipette gun, transferring into a cryopreservation tube, sealing the tube opening by using a sealing film, and marking (name, cell strain number, cryopreservation date and the like). And (3) placing the freezing tube at 4 ℃ for 1h, taking out, placing at-20 ℃ for 2h, and finally, after overnight at-80 ℃, transferring into liquid nitrogen for long-term freezing storage for later use.
(6) Mass production of monoclonal antibodies
Sterile paraffin oil was injected into the abdominal cavity of healthy BALB/c mice 0.5 mL/mouse a week or so earlier. Injecting the prepared hybridoma cell strain capable of stably secreting casein monoclonal antibody into abdominal cavity of BALB/c mouse, and injecting 1 × 10 antibody into each mouse6About one cell. Observing the state of the mouse every day after 7 days, collecting ascites with a needle if the abdomen of the mouse is obviously enlarged and the belly is tightly touched with hands, and extracting ascites at 4 deg.C and 1 × 104Centrifuging at rpm for 10min, discarding upper lipid and lower precipitate, collecting middle clear ascites, packaging, and freezing at-20 deg.C.
(7) Identification of monoclonal antibody specificity
The specificity of the monoclonal antibody can be identified by adopting a cross reaction experiment and Western-blotting, the invention adopts Western-blotting to identify the specificity of the three prepared monoclonal antibodies, and the result is shown in the following figure, as shown in figure 3, casein has a clear immunoblotting strip at about 36kDa, alpha-lactalbumin has a clear immunoblotting strip at about 14.4kDa, and beta-lactoglobulin has a clear immunoblotting strip at about 18.3 kDa. This further demonstrates that the monoclonal antibodies produced have good specificity for casein.
(8) Monoclonal antibody subtype identification
The invention adopts a commercial mouse antibody Ig class/subclass kit to identify the prepared casein monoclonal antibody subtype, and the operation is carried out according to the instruction, and the specific operation steps are as follows:
coating: the coating antigen is diluted to 1 mu g/mL, coated on an enzyme label plate by 100 mu L/well, and incubated in a constant temperature oven for 2h at 4 ℃ overnight or 37 ℃.
Washing: pouring out the liquid in the enzyme-labeled holes, spin-drying, washing with PBST for 3 times, 3min each time, and repeating the operation for 3 times.
Sealing: add 200. mu.L of blocking solution to each well and block for 2h at 37 ℃.
Fourthly, primary anti-dilution: the purified mab was initially diluted 1000-fold (10 μ L mab +990 μ L antibody dilution) and subsequently diluted 3-fold on dilution plates.
Adding primary antibody: the primary antibody diluted in the gradient is added into a well-washed enzyme label plate, and incubated in a constant-temperature oven of 100 mu L/well at 37 ℃ for 30 min.
Sixthly, adding a secondary antibody: after washing the plate, secondary antibodies (IgA, IgM, IgG1, IgG2a, IgG2b, IgG3, Kappa, Lambda) diluted 1000 times in advance were added to the wells of the plate and incubated at 100. mu.L/well in a constant temperature oven at 37 ℃ for 30 min.
And color development: after the ELISA plate is washed, a freshly prepared TMB developing solution is added, and incubation is carried out in a constant-temperature oven at 37 ℃ for 15min in a 100 mu L/well manner.
And stopping the step of: 2M H2SO4The reaction was stopped, 50. mu.L/well.
Ninthly, reading a board: reading OD at microplate reader450The value of (c). According to the color depth and OD450And judging the subtype of the monoclonal antibody.
(9) Monoclonal antibody affinity identification
Detection by noncompetitive indirect ELISADetermining antibody affinity. The specific method comprises the following steps: casein concentration was diluted with PBS (0.01M, pH 7.4) gradient to 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.25, 0. mu.g/mL, 100. mu.L/well, coated with 96-well plate and incubated at 37 ℃ for 2 h. The plate was washed 3 times with PBST for 3min each time, blotted dry, 200. mu.L of blocking solution was added to each well and blocked at 37 ℃ for 2 h. The monoclonal antibody is diluted by 3 times from 1000 times, 100 mu L/well is added into a closed enzyme label plate, and the mixture is incubated for 30min at 37 ℃. After washing the plate 3 times, 100. mu.L of HRP-labeled goat anti-mouse IgG was added to each well and incubated at 37 ℃ for 30 min. Finally 2M of H was added per well2SO4The reaction is stopped by the solution, and OD is measured at the place of the microplate reader450The value is obtained.
In a rectangular coordinate system, the abscissa is the logarithm of the antibody concentration (mol/L), the ordinate is the corresponding absorbance, reaction curves are made in the coordinate system, the antibody concentration corresponding to ODmax of each curve is found, and the antibody concentration corresponding to 50% ODmax is calculated. The affinity constant K of the antibody was calculated. Obtaining a casein affinity standard curve, and calculating to obtain a casein monoclonal antibody with an affinity constant of 3.1 × 10 as shown in FIG. 47L/mol, it is generally accepted that when the affinity constant K is 107-1012In the L/mol range, the affinity of the antibody is high, and therefore, the affinity of the monoclonal antibody obtained is high.
Example 3: establishment of casein double-antibody sandwich ELISA method
1. Casein double-antibody sandwich ELISA method
The specific operation steps are as follows:
(1) wrapping a plate: microtiter wells of ELISA plates were coated with 100. mu.L of capture-coated antibody (monoclonal antibody) at 5. mu.g/mL and incubated overnight at 4 ℃ or in a 37 ℃ oven for 2 h;
(2) washing the plate: followed by three repeated washes with PBST to remove unbound antibody, patted dry;
(3) and (3) sealing: add 200. mu.L of blocking buffer to each well;
(4) adding a sample: adding the diluted sample (standard) into a 96-well enzyme label plate by using a 12-well arraying gun, wherein the liquid adding amount of each well is 100 mu L, and placing the plate in a constant-temperature oven at 37 ℃ for incubation for 30 min;
(5) adding rabbit antiserum: washing the plate according to the operation step of the step (2), drying the plate, taking 100 mu L of detection antibody (polyclonal antibody) to dilute the detection antibody in PBS (buffer solution) by a certain multiple, adding the detection antibody into each hole, and incubating the detection antibody in a constant-temperature oven at 37 ℃ for 30 min;
(6) HRP-labeled goat anti-rabbit IgG antibody: washing the plate according to the operation step of the step (2), drying the plate, diluting goat anti-rabbit IgG marked by HRP to a certain multiple in the same way as the previous step, adding the diluted goat anti-rabbit IgG into the holes, wherein the liquid adding amount of each hole is 100 mu L, and incubating the holes in a constant-temperature oven at 37 ℃ for 30 min;
(7) color development: washing the plate according to the operation step of the step (2), drying by beating, finally adding a freshly prepared TMB substrate chromogenic solution into each hole, performing incubation for 15min in a constant-temperature oven at 37 ℃ in a 100-mu L/well manner;
(8) and (4) terminating: reaction 2M H2SO4The reaction was terminated, and the amount of solution added to each well was 50. mu.L, and the absorbance value was measured at 450 nm.
2. Screening of optimal casein capture antibody and optimization of enzyme-labeled secondary antibody working concentration
Respectively coating the enzyme label plate with 2 screened casein murine monoclonal antibodies as capture antibodies, wherein the coating concentration is 5 mug/mL, the casein standard substance is diluted to two gradients of 100ng/mL and 1 mug/mL for sample loading, goat anti-rabbit IgG antibody (enzyme-labeled secondary antibody) marked by horseradish peroxidase is diluted to three gradients of 1:5000, 1:10000 and 1:15000 by using phosphate buffer solution, the rabbit polyclonal antibody is diluted to 1:10000, and phosphate buffer solution without the added standard substance is used as negative control.
In the experiment, 2 screened casein murine monoclonal antibodies are used as capture antibodies to respectively coat an enzyme label plate, the coating concentration, the dilution of a casein standard product and the dilution of rabbit polyclonal antibodies are controlled to be unchanged, goat anti-rabbit IgG antibody (enzyme-labeled secondary antibody) marked by horseradish peroxidase is diluted by phosphate buffer solution and is released into three gradients of 1:5000, 1:10000 and 1:15000, the phosphate buffer solution without the added standard product is used as a negative control, and OD (optical density) is measured450As shown in Table 3 below, it can be seen that the optimal dilution ratio of the goat anti-rabbit enzyme-labeled secondary antibody was selected as10000, optimal capture antibody selection 2 A8.
TABLE 3 screening of Capture antibody and optimization of working concentration of enzyme-labeled Secondary antibody
3. Optimization of casein rabbit polyclonal antibody working concentration
The optimal working concentration of the casein coated antibody and the rabbit secondary antibody is operated according to the optimal conditions determined by the experiments, the optimal dilution multiple of the casein rabbit polyclonal antibody is determined, the casein rabbit polyclonal antibody is diluted to three gradients of 1:10000, 1:15000 and 1:45000, phosphate buffer solution without a standard is used as negative control, and OD (optical density) is measured450As shown in table 4 below, it can be seen that the optimal dilution factor for rabbit polyclonal antibody was chosen to be 1: 10000.
TABLE 4 optimal working concentration optimization results for rabbit polyclonal antibody
4. Establishment of standard curve for casein detection by double-antibody sandwich ELISA method
According to the conditions of the optimal capture antibody, the optimal enzyme-labeled secondary antibody working concentration and the rabbit polyclonal antibody working concentration determined above, the concentration of the casein standard substance is set to be 12 concentration gradients of 0, 2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1280 and 2560ng/mL, and the absorbance OD is calculated450As an ordinate, the concentration of casein standard (ng/mL) was used as an abscissa, the abscissa was set to a logarithmic form, four-parameter fitting was performed using a logistic model in Origin software, and then a double-antibody sandwich ELISA standard curve was drawn. The LOD of casein was calculated by P/N (positive/negative) > 2.1. As shown in FIG. 5, the casein concentration was in the range of 80-1280ng/mLThe LOD can reach 80 ng/mL.
5. Determination of specificity of casein double-antibody sandwich ELISA method
The invention selects 5 main proteins (bovine serum albumin BSA, alpha-lactalbumin, beta-lactoglobulin, casein and ovalbumin OVA) in cow milk, dilutes 5 cross-reactant proteins to three gradients of 10, 20 and 40ng/mL, and detects OD of the cross-reactant proteins by the established casein double-antibody sandwich ELISA method450Value, phosphate buffered saline PBS as negative control; the cross sample wells are designated P and the negative control wells are designated N, and P/N (positive/negative) values are calculated to determine whether casein cross-reacts with 5 cross-reactant proteins, if P/N > 2.1, the protein cross-reacts with the cross-reactant protein, otherwise, no cross-reaction is detected. The results of P/N values obtained by calculation are shown in Table 5, and it can be seen that the casein double antibody sandwich ELISA detection method established by the invention only has cross reaction with casein, and other proteins do not have cross reaction, which indicates that the casein double antibody sandwich ELISA detection method established by the invention has high specificity.
TABLE 5 Cross-reaction results (P/N)
6. Determination of precision of casein double-antibody sandwich ELISA method
In ELISA, there are many factors that affect its precision, such as: the number and time of plate washing, plate beating, sample dilution, etc. The precision of the established casein double antibody sandwich ELISA method is evaluated by two factors, namely, batch difference and batch-to-batch difference, but the precision must be evaluated under the condition of operating enough standards and external environment enough to be stable, and the batch difference and the batch-to-batch difference need to be represented by a coefficient of variation CV.
Coefficient of variation CV (%) ═ SD/X × 100% formula (1)
Wherein: SD-standard deviation; x-average number
The specific determination method is as follows:
a. intra-batch difference: casein standards were diluted to three gradient concentrations of 40, 80 and 160ng/mL, 5 replicates of each concentration in the same experiment were run, and the absorbance OD was compared at the same concentration450And calculating the coefficient of variation CV according to the variation condition.
b. The difference between batches: casein standards were diluted to three gradient concentrations of 40, 80 and 160ng/mL, each concentration being assayed twice daily for 5 consecutive days, based on the absorbance OD determined for 5 days450And calculating the coefficient of variation CV according to the variation condition.
TABLE 6 Casein Standard batch inter-batch coefficient of variation CV determination results (n ═ 5)
7. Casein double-antibody sandwich ELISA method standard adding recovery rate determination
The method comprises the steps of purchasing biscuits which are not marked with cow milk allergen casein on an outer packaging bag in a supermarket, diluting a casein standard solution into three gradient concentrations of 40, 80 and 160ng/mL, and adding and recovering the purchased biscuits. Each concentration was performed in 3 replicates, the casein content of the samples was determined, and the recovery rate of the spiked samples was calculated.
Recovery ═ recovery (detection of spiked sample-detection of non-spiked sample) ÷ spiked quantity × 100% formula (2)
The results are shown in table 7, and it can be seen that the range of the recovery rate of the casein spiked by the casein double antibody sandwich ELISA detection method established herein is 80.4-92.25%, which can meet the detection requirement of allergen casein in milk actual samples.
TABLE 7 Casein double antibody sandwich ELISA spiked recovery results (n ═ 3)
The embodiments described above are intended to facilitate the understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A double-antibody sandwich ELISA detection method for quantitatively detecting cow milk casein allergen is characterized in that cow milk allergen casein is taken as an immune antigen to respectively immunize a rabbit polyclonal antibody and a mouse monoclonal antibody obtained by a New Zealand white rabbit and a BALB/c mouse, the performances of the rabbit polyclonal antibody and the mouse monoclonal antibody are respectively identified and evaluated, and then the rabbit polyclonal antibody and the mouse monoclonal antibody are subjected to sandwich pairing, so that the double-antibody sandwich ELISA detection method for quantitatively detecting cow milk casein allergen is established.
2. The method for quantitatively detecting bovine casein allergen according to claim 1, wherein the method comprises: screening an optimal casein capture antibody, optimizing the working concentration of an enzyme-labeled secondary antibody, optimizing the working concentration of a casein rabbit polyclonal antibody and establishing a standard curve;
the standard curve takes the concentration of a casein standard substance as an abscissa and takes the absorbance OD450As an ordinate, a casein standard concentration (ng/mL) was used as an abscissa, and the abscissa was set to a logarithmic form, a four-parameter fitting was performed using a logistic model in Origin software, a standard curve was drawn, and LOD of casein was calculated by P/N (positive/negative) > 2.1.
3. The double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen as claimed in claim 2, wherein the following methods are adopted for screening of the optimal casein capture antibody, optimization of the working concentration of the enzyme-labeled secondary antibody and optimization of the working concentration of the casein rabbit polyclonal antibody:
(1) wrapping a plate: microtiter wells of ELISA plates were coated with 100. mu.L of capture-coated antibody, mouse monoclonal antibody, at 5. mu.g/mL, incubated overnight at 4 ℃ or in a 37 ℃ oven for 2 h;
(2) washing the plate: followed by three repeated washes with PBST to remove unbound antibody, patted dry;
(3) and (3) sealing: add 200. mu.L of blocking buffer to each well;
(4) adding a sample: adding the diluted casein standard substance into a 96-hole enzyme label plate by using a 12-hole arraying gun, wherein the liquid adding amount of each hole is 100 mu L, and placing the plate in a constant-temperature oven at 37 ℃ for incubation for 30 min;
(5) adding rabbit antiserum: washing the plate according to the operation step of the step (2), drying the plate, taking 100 mu L of a detection antibody-rabbit polyclonal antibody, diluting the detection antibody-rabbit polyclonal antibody in a diluted buffer solution PBS by a certain multiple, adding the diluted detection antibody-rabbit polyclonal antibody into each hole, and incubating the diluted detection antibody-rabbit polyclonal antibody in a constant-temperature oven at 37 ℃ for 30 min;
(6) HRP-labeled goat anti-rabbit IgG antibody: washing the plate according to the operation step of the step (2), after drying by beating, diluting the goat anti-rabbit IgG antibody-enzyme labeled secondary antibody marked by the HRP to a certain multiple according to the same method as the previous step, adding the diluted secondary antibody into the holes, wherein the liquid adding amount of each hole is 100 mu L, and incubating in a constant-temperature oven at 37 ℃ for 30 min;
(7) color development: washing the plate according to the operation step of the step (2), drying by beating, finally adding a freshly prepared TMB substrate chromogenic solution into each hole, performing incubation for 15min in a constant-temperature oven at 37 ℃ in a 100-mu L/well manner;
(8) and (4) terminating: reaction 2M H2SO4The reaction was terminated, and the amount of solution added to each well was 50. mu.L, and the absorbance value was measured at 450 nm.
4. The double-antibody sandwich ELISA detection method for quantitatively detecting bovine milk casein allergen as claimed in claim 3, wherein in the process of screening of the casein optimal capture antibody and optimization of the working concentration of the enzyme-labeled secondary antibody:
using a mouse monoclonal antibody as a capture antibody to coat an enzyme label plate, controlling the coating concentration, casein standard substance dilution and rabbit polyclonal antibody dilution concentration to be unchanged, and using phosphate buffer solution to label goat anti-rabbit IgG marked by HRPAntibody dilution was performed in three gradients of 1:5000, 1:10000 and 1:15000, and OD was measured by using phosphate buffer without standard as negative control450And determining the optimal working concentration of the casein capture antibody and the enzyme-labeled secondary antibody.
5. The double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen according to claim 4, wherein in the optimization process of the working concentration of casein rabbit polyclonal antibodies:
performing operation according to the determined working concentrations of the casein optimal capture antibody and the enzyme-labeled secondary antibody, determining the optimal dilution multiple of the rabbit polyclonal antibody, diluting the rabbit polyclonal antibody to three gradients of 1:10000, 1:15000 and 1:45000, taking phosphate buffer solution without a standard as negative control, and determining OD (optical density) by measuring450And determining the working concentration of the casein rabbit polyclonal antibody.
6. The double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen of claim 2, wherein the linear range of the standard curve is 80-1280ng/mL, and the detection limit is 80 ng/mL; the method has intra-batch coefficient of variation CV of 2.72-5.97% and inter-batch coefficient of variation CV of 7.99-12.33%, and has high precision; the method only has cross reaction with casein; the method has a casein recovery rate of 80.4-92.25%.
7. The double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen as claimed in claim 1, wherein the rabbit polyclonal antibody is obtained by the following method:
the rabbit serum is prepared by taking cow milk allergen casein as an immunizing antigen, mixing the cow milk allergen casein with Freund's adjuvant in equal amount, and immunizing healthy New Zealand white rabbits by adopting an immunization mode of neck and back subcutaneous injection.
8. The double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen of claim 7, wherein the performance identification and evaluation of rabbit polyclonal antibody comprises:
purifying and separating the prepared rabbit serum by an octanoic acid-saturated ammonium sulfate precipitation method; performing purity identification on the purified rabbit serum by adopting an SDS-PAGE gel electrophoresis method to obtain a purified rabbit polyclonal antibody; four immunizations of casein were performed, each immunization at a dose of 500. mu.g/mouse; the titer of the purified rabbit polyclonal antibody is measured by an indirect ELISA method.
9. The double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen of claim 1 is characterized in that the murine monoclonal antibody is obtained by the following method:
immunizing healthy female BALB/c mice with proper ages by adopting an immunization mode and an immune antigen which are the same as those of rabbit polyclonal antibody preparation, immunizing casein for six times, wherein the primary immunization dose is 100 mu g/mouse, the boosting immunization dose is 50 mu g/mouse, the spurt immunization dose is 25 mu g/mouse, detecting immune serum of the mice by using an indirect ELISA method, and selecting the mice with the highest six-immune serum titer;
detecting and screening positive holes by cell fusion and an indirect ELISA method, and screening to obtain a hybridoma cell strain stably secreting the casein monoclonal antibody after subcloning by adopting a limiting dilution method;
mass production of murine monoclonal antibodies was carried out by in vivo ascites induction.
10. The double-antibody sandwich ELISA detection method for quantitatively detecting bovine casein allergen of claim 9 is characterized in that the performance of the murine monoclonal antibody is identified by the following method:
purifying the obtained ascites monoclonal antibody by an octanoic acid-saturated ammonium sulfate precipitation method; identifying the purity of the purified ascites monoclonal antibody by an SDS-PAGE gel electrophoresis method; the titer of the monoclonal antibody is measured by adopting an indirect ELISA method, and the monoclonal antibody subtype secreted by the casein cell strain is IgG1 according to the identification of a commercial monoclonal antibody subtype kit.
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| 朱永智: "牛乳中三种主要过敏原双抗夹心ELISA检测方法的建立", 《中国硕博士学位论文全文数据库（硕士）工程科技Ⅰ辑》 * |
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| CN114751974A (en) * | 2022-04-21 | 2022-07-15 | 胡巍 | IgG binding epitopes of the major allergen alpha-lactalbumin from bovine milk whey |
| CN115253736A (en) * | 2022-07-21 | 2022-11-01 | 苏州逻晟生物医药有限公司 | Novel antigen emulsification method |
| CN115558027A (en) * | 2022-12-05 | 2023-01-03 | 北京万泰生物药业股份有限公司 | Nuclease monoclonal antibody and application thereof |
| CN115558027B (en) * | 2022-12-05 | 2023-02-28 | 北京万泰生物药业股份有限公司 | Nuclease monoclonal antibody and application thereof |
| CN117147836A (en) * | 2023-10-30 | 2023-12-01 | 湖南冠牧生物科技有限公司 | ELISA detection method, kit, use method and application thereof |
| CN117147836B (en) * | 2023-10-30 | 2024-05-07 | 湖南冠牧生物科技有限公司 | ELISA detection method, kit, use method and application thereof |
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Application publication date: 20210629 |