阅读说明：本技术 一种β-乳球蛋白兔多克隆抗体的制备方法及免疫分析方法 (Preparation method and immunoassay method of beta-lactoglobulin rabbit polyclonal antibody ) 是由 王世杰 朱宏 于 2019-10-10 设计创作，主要内容包括：本发明提供了一种β-乳球蛋白兔多克隆抗体的制备方法及免疫分析方法,所述制备方法主要过程为β-乳球蛋白直接免疫新西兰大耳白兔,经过五次免疫后股动脉取全血,获得β-乳球蛋白抗体血清；通过优化抗体包被量,抗体的稀释倍数,样品缓冲溶液的pH值建立β-乳球蛋白间接竞争ELISA方法。本发明制备的抗体效价较高,建立的方法灵敏度和特异性较好。(The invention provides a preparation method and an immunoassay method of a beta-lactoglobulin rabbit polyclonal antibody, wherein the preparation method mainly comprises the steps of directly immunizing a New Zealand big ear white rabbit with beta-lactoglobulin, and taking whole blood from femoral artery after five times of immunization to obtain beta-lactoglobulin antibody serum; the beta-lactoglobulin indirect competition ELISA method is established by optimizing the antibody coating amount, the dilution multiple of the antibody and the pH value of a sample buffer solution. The antibody prepared by the invention has higher titer, and the established method has better sensitivity and specificity.)

1. A preparation method of a beta-lactoglobulin rabbit polyclonal antibody is characterized by comprising two steps of an immunization process and an antibody purification process;

the specific method of the immune process comprises the following steps: selecting rabbit as immune animal, the immunogen is beta-lactoglobulin, adopting subcutaneous and intramuscular injection method as immune method, carrying out 4 times of boosting immunity after primary immunization, respectively locating at 2, 4, 6 and 8 weeks after primary immunization, taking whole blood 7-10 days after 5 times of immunization, collecting serum, and freezing for later use;

the antibody purification process specifically comprises the following steps:

1) all buffers and solvents required in the experiment are subjected to ultrasonic treatment for 10-30min to remove bubbles;

2) and (3) balancing and purifying the column: the line was first flushed with pH 7.4 phosphate buffer for 1-2min, with the flow rate set to 5.0-8.0 mL/min. After the pipeline is washed, the ethanol in the purification column automatically flows out, then the purification column is connected with a protein purifier, the column is washed by phosphate buffer solution with pH of 7.4, and the flow rate is set to be 0.5-1.5 mL/min; stopping flushing the column when the ultraviolet and conductance baselines in the program screen are observed to be parallel lines;

3) loading: taking 0.5-1.5mL of beta-lactoglobulin polyclonal antibody serum to be purified and phosphate buffer solution for equal-volume dilution, and then loading the serum onto a column, and adjusting the flow rate to be 0.3-0.8 mL/min. The purification instrument shows a hetero-protein peak in the ultraviolet conductance diagram;

4) and (3) elution: after the heteroprotein peak appeared, the column was continued to be washed with phosphate buffer until the baseline reached equilibrium and stopped. Then washing the column with glycine-hydrochloric acid buffer solution with pH 2.7 at a flow rate of 0.3-0.8mL/min, at which time the immunoglobulin bound to Protein A-Sepharose 4B is eluted;

5) collecting: after adding the eluent, paying attention to the change of the ultraviolet conductance graph of the instrument, starting to collect the eluent when the base line begins to change, collecting about 1mL of liquid by each collecting pipe until the curve does not change, and stopping collecting; reading the collected liquid at 280nm of a spectrophotometer, discarding the collected liquid with the absorbance value less than 0.4, uniformly mixing the liquid with the absorbance value greater than 0.4, and immediately rapidly adjusting the antibody by using Tris-HCl to ensure that the pH value is 7.0;

6) treating a purification column: after the antibody purification is finished, rapidly washing the column with 0.05-0.20mol/L acetic acid for 1-3min at a flow rate of 3-8mL/min, then equilibrating the column with phosphate buffer solution, and simultaneously measuring the pH value of the effluent buffer solution with pH test paper until the pH value is neutral. Finally, washing the purification column with 20% ethanol solution for 20min, filling the column with 20% ethanol solution, and sealing at 4 deg.C;

7) the antibody preservation method comprises the following steps: pouring the purified beta-lactoglobulin antibody into an ultrafiltration device for ultrafiltration, repeating for 2-3 times, stopping ultrafiltration when the liquid is remained for 4-5mL for the last time, taking out the liquid, measuring the absorbance by using a spectrophotometer, uniformly mixing the liquid with glycerol with the same volume, and storing the mixture in a refrigerator at the temperature of-20 ℃ for later use;

the immunization process specifically comprises the following steps:

1) primary immunization: mixing immunogen and Freund's complete adjuvant at a volume of 1:1, emulsifying by a glass syringe with good sealing property, and immunizing animals with an immunizing dose of 1 mg/mouse;

2) and (3) boosting immunity: mixing and emulsifying the immunogen and Freund's incomplete adjuvant in equal volume, and immunizing animals, wherein the immunizing dose is 0.5 mg/animal;

3) and (3) the whole blood collected after the immunization is finished is firstly put to the room temperature for coagulation, then put into a refrigerator at 4 ℃, after the serum is separated out, the supernatant is collected and is subpackaged and stored at-20 ℃ for later use.

2. An immunoassay method for a beta-lactoglobulin polyclonal antibody prepared by the preparation method according to claim 1, comprising the steps of:

1) coating: diluting beta-lactoglobulin with coating solution, coating the beta-lactoglobulin on a 96-well polystyrene enzyme label plate, incubating at 4 ℃ for 12-16h at 100 mu L/well, discarding the liquid in the well, and washing the plate with PBST washing solution for 3 times, each time for 2 min;

2) and (3) sealing: adding 200 μ L of blocking solution into enzyme well of enzyme labeling plate, blocking at 37 deg.C or room temperature for 1h, discarding the liquid in the well, washing the plate with PBST washing solution for 3 times, each time for 2 min;

3) adding antiserum: adding 100 μ L rabbit antiserum diluted with PBS into each well, incubating at 37 deg.C for 1h, washing the plate with PBST washing solution for 2min each time for 4 times;

4) adding an enzyme-labeled secondary antibody: diluting goat-anti-rabbit secondary antibody labeled by horseradish peroxidase by 20000 times with PBS buffer solution, adding 100 μ L/hole into an enzyme label plate, reacting at room temperature for 30min, and washing the plate with PBST washing solution for 5 times, each time for 2 min;

5) color development: sucking the substrate A15 min ahead of time to recover the room temperature, adding the substrate B, and uniformly mixing; adding 100 μ L into each well, and developing at 37 deg.C in dark for 15-20 min;

6) and (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction;

7) reading: reading the absorbance value by an enzyme-linked immunosorbent assay (ELISA) instrument in a mode of 450-650nm dual wavelength;

8) and drawing a standard curve.

The formulation of the solution used was as follows:

coating liquid: 0.05mol/L sodium carbonate-sodium bicarbonate buffer, pH 9.6: respectively and accurately weighing Na₂CO₃ 1.60g，NaHCO₃2.91g, adding double distilled water to reach the constant volume of 1000mL, and adjusting the pH value to 9.6.

A substrate system solution which is a TMB-carbamide peroxide solution,

substrate solution A: accurately weighing 8.20g of anhydrous sodium acetate and 2.50g of beta-dextrin, dissolving the anhydrous sodium acetate and the beta-dextrin in 1000mL of double distilled water, then accurately weighing 3.23g of citric acid, adjusting the pH to 5.0, accurately weighing 428.6mg of carbamide peroxide after all medicines are completely dissolved, adding the carbamide peroxide into the prepared solution, storing the carbamide peroxide in a refrigerator at 4 ℃, taking out the carbamide peroxide in advance for 15min when in use, and recovering the carbamide peroxide to room temperature for use;

substrate solution B: accurately weighing 100mg of TMB, dissolving in 10mL of DMSO, and storing in a dark place in a brown bottle at normal temperature;

the substrate solution A and the substrate solution B need to be uniformly mixed before use: respectively taking 10.95mL of substrate A solution and 0.3375mL of substrate B solution;

stopping liquid: 1.25mol/L of H₂SO₄And (3) solution.

3. The immunoassay method according to claim 2, characterized in that: in the step 1), the coating amount of the beta-lactoglobulin is 0.03-0.08 mu g/hole.

Technical Field

The invention belongs to the technical field of macromolecular protein immunization, and particularly relates to a preparation method and an immunoassay method of a beta-lactoglobulin rabbit polyclonal antibody.

Background

Beta-lactoglobulin is currently the highest proportion of allergens in whey protein and is present in milk at about 4.0 mg/mL. It is a polypeptide chain, exists as a dimer with the relative molecular weight of 36000 in fresh milk, and a monomer of the polypeptide chain comprises 162 amino acid residues, has the molecular weight of about 18kDa and the isoelectric point of 5.1-5.3.

Approximately 80% of the population will develop food allergies associated with beta-lactoglobulin. It is not present in breast milk and is not digested and hydrolyzed by pepsin, so it can enter blood circulation through gastrointestinal tract, has strong sensitization, and can be used as the primary allergic component in cow milk to stimulate the hypersensitivity of infant immune system. Most of the existing beta-lactoglobulin is monoclonal antibody, and the research on polyclonal antibody is less, so that a polyclonal antibody needs to be developed to improve the sensitivity and specificity of the antibody.

Disclosure of Invention

In view of this, the present invention is directed to a method for preparing a rabbit polyclonal antibody against beta-lactoglobulin and an immunoassay method for improving the sensitivity and specificity of the antibody.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a preparation method of a beta-lactoglobulin rabbit polyclonal antibody comprises two steps of an immunization process and an antibody purification process;

in the immune process, an immune animal is selected from rabbits, the immunogen is beta-lactoglobulin, the immune method adopts subcutaneous and intramuscular injection methods, a plurality of times of boosting immunity are carried out after primary immunity, blood is taken 7-10 days after each boosting immunity to measure the titer of antiserum, whole blood is taken from the animal 7-10 days after the last immunization, the serum is collected, and the animal is frozen for later use. The antibody purification process is realized by using a purification column and a protein purifier.

Preferably, in the immunization process, four boosts are performed after the primary immunization, and the boosts are respectively performed at 2, 4 and 6 and 8 weeks after the primary immunization, and whole blood is taken by using femoral artery and stored after 7-10 days of 5 times of immunization.

Preferably, the immunization process specifically comprises the following steps:

1) primary immunization: the immunogen was mixed with freund's complete adjuvant in a ratio of 1:1, mixing in equal volume, and fully emulsifying by a glass syringe with good sealing property until immunogen and adjuvant are completely mixed and are in a milky white state, wherein the dose of the immunogen is 1mg per mouse;

2) and (3) boosting immunity: mixing and emulsifying the immunogen and Freund's incomplete adjuvant in equal volume, and immunizing animals, wherein the immunizing dose is 0.5 mg/animal;

3) after the immunization is finished, the collected blood is coagulated for 2 hours at room temperature, then stored in a refrigerator at 4 ℃ to shrink the blood clot, and after the serum is separated out, collected, subpackaged and stored at-20 ℃ for later use.

Preferably, the antibody purification process specifically comprises the following steps:

1) all buffers and solvents required in the experiment were sonicated for 20min to eliminate air bubbles;

2) and (3) balancing and purifying the column: the tube was flushed with pH 7.4 phosphate buffer for 1-2min, with the flow rate set to 5-8 mL/min. After allowing the ethanol in the purification column to flow out by itself, the purification column was connected to a protein purifier, and the column was washed with a phosphate buffer solution of pH 7.4 at a flow rate of 0.5 to 1.5 mL/min. Observing that two baselines of ultraviolet and electric conductance in a program screen become parallel lines, and stopping column flushing;

3) loading: centrifuging 0.5-1.5mL antiserum to be purified at 10000rpm for 10min, and setting the temperature at 4 ℃; passing the supernatant through a 0.45 μm water membrane, diluting the serum after passing the membrane with phosphate buffer solution in equal volume, and loading on the column, wherein when the serum sample is about to enter the column, the flow rate is adjusted to 0.2-0.8 mL/min. Immunoglobulin in antiserum is specifically adsorbed on a binding site of Protein A-Sepharose 4B, while other Protein fat and other impurities are not adsorbed and flow out along with the buffer solution, and a hetero-Protein peak is displayed in an ultraviolet conductance chart of a purifier;

4) and (3) elution: after the impurity protein peak appears, continuing to wash the column with phosphate buffer until the baseline reaches equilibrium, and stopping; washing the column with glycine-hydrochloric acid buffer solution of pH 2.7 at a flow rate of 0.3-0.8mL/min, at which time the immunoglobulin bound to Protein A-Sepharose 4B is eluted;

5) collecting: after adding the eluent, paying attention to the change of the ultraviolet conductance graph of the instrument, collecting the eluent when the base line begins to change, collecting about 1mL of liquid in each collecting pipe until the curve does not change, and stopping collecting; reading the collected liquid at 280nm of a spectrophotometer, discarding the collected liquid with the absorbance value less than 0.4, uniformly mixing the liquid with the absorbance value greater than 0.4, immediately and rapidly adjusting the antibody by using Tris-HCl to ensure that the pH value of the antibody is 7.0 so as to prevent the protein in the antibody from being denatured under the acidic condition;

6) treating a purification column: after the antibody purification is finished, rapidly washing the column for 1-3min by 0.05-0.20mol/L acetic acid at the flow rate of 3-8mL/min, then balancing the column by using a phosphate buffer solution, and simultaneously measuring the pH value of an outflow buffer solution by using a pH test paper until the outflow buffer solution is neutral; finally, washing the purification column with 20% ethanol solution for 20min, filling the column with 20% ethanol solution, and sealing at 4 deg.C;

7) antibody preservation: and pouring the purified antibody into an ultrafiltration device for ultrafiltration, pouring a PBS (phosphate buffer solution) with the pH of 7.4 when 5mL of liquid remains in an ultrafiltration container, repeating the process for 2-3 times, stopping ultrafiltration when 4-5mL of liquid remains for the last time, taking out the liquid, measuring the absorbance of the liquid by using a spectrophotometer to calculate the concentration, mixing the liquid with glycerol with the same volume, and storing the mixture in a refrigerator at the temperature of-20 ℃ for later use.

The invention also provides an immunoassay method of the beta-lactoglobulin rabbit polyclonal antibody prepared by the preparation method, which comprises the following steps:

1) coating: diluting the beta-lactoglobulin standard solution with a coating solution, coating the diluted solution in a 96-well polystyrene enzyme label plate, incubating at the temperature of 4 ℃ for 12-16h at a rate of 100 mu L/well, discarding the liquid in the well, and washing the plate with PBST washing liquor for 3 times, each time for 2 min;

2) and (3) sealing: adding 200 μ L of blocking solution into each well, blocking at 37 deg.C or room temperature for 1 hr, discarding the liquid in the well, washing the plate with PBST lotion for 3 times, each time for 2 min;

3) sample adding: adding 50 μ L of beta-lactoglobulin standard solution and 50 μ L of beta-lactoglobulin rabbit polyclonal antibody into each well, performing competitive reaction in an oven at 37 ℃ for 1h, taking out, washing the plate with PBST washing solution for 4 times, and each time for 2 min;

4) adding an enzyme-labeled secondary antibody: adding horseradish peroxidase-labeled goat anti-rabbit secondary antibody diluted by PBS (phosphate buffer solution), fixing the dilution multiple at 1:20000 and 100 μ L/well, reacting at 37 deg.C for 30min, washing the plate with PBST (basic-positive-negative-potential) washing solution for 5 times, each time for 2 min;

5) color development: preparing the substrate solution in advance for 15min, standing at room temperature, adding 100 μ L of the substrate solution into each well, and developing at 37 deg.C in dark for 15-20 min;

6) and (4) terminating: adding 50 mu L of stop solution into each hole;

7) reading: the absorbance was read with a microplate reader using a dual wavelength method of 450 and 650 nm.

8) Drawing a standard curve: the X axis of the inhibition rate curve is beta-lactoglobulin concentration value, the Y axis is corresponding inhibition rate, the standard curve is typical S-shaped curve, IC₅₀Defined as the corresponding beta-lactoglobulin concentration at an inhibition of 50%.

Further, the formulation of the solution used above is as follows:

coating liquid: 0.05mol/L sodium carbonate-sodium bicarbonate buffer, pH 9.6: respectively and accurately weighing Na₂CO₃1.60g，NaHCO₃2.91g, adding double distilled water to reach the volume of 1000mL, and finally adjusting the pH value to 9.6.

Substrate system solution (TMB-Urea hydroperoxide solution)

Substrate solution A: 8.20g of anhydrous sodium acetate and 2.50g of beta-dextrin are accurately weighed and dissolved in 1000mL of double distilled water, then 3.23g of citric acid is accurately weighed and the pH is adjusted to 5.0, and 428.6mg of carbamide peroxide is accurately weighed and added into the prepared solution after all the medicines are completely dissolved. The application method comprises taking out the product 15min before, and recovering to room temperature.

Substrate solution B: 100mg of TMB was accurately weighed and dissolved in 10mL of DMSO, and stored in a brown bottle at room temperature in a dark place.

The substrate solution A and the substrate solution B need to be uniformly mixed before use: 10.95mL of substrate A solution and 0.3375mL of substrate B solution were taken, respectively.

Stopping liquid: 1.25mol/L of H₂SO₄And (3) solution.

Compared with the prior art, the preparation method and the immunoassay method of the beta-lactoglobulin rabbit polyclonal antibody have the following advantages:

(1) the antibody prepared by the preparation method of the beta-lactoglobulin rabbit polyclonal antibody has high titer and high inhibition rate;

(2) the antibody prepared by the preparation method of the beta-lactoglobulin rabbit polyclonal antibody has better specificity and reproducibility.

(3) The immunoassay method, namely the indirect competitive ELISA method, provided by the invention is simple, convenient, rapid and specific to operate, has higher sensitivity and specificity, the specificity of the immunoassay method depends on the specificity of immunological reaction, and the sensitivity of the immunoassay method depends on the affinity of antibodies.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a standard curve plotted in the immunoassay method according to the first embodiment of the present invention.

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The present invention will be described in detail with reference to examples.

Example 1

A preparation method of a beta-lactoglobulin rabbit polyclonal antibody is characterized by comprising the following steps: comprises two steps of an immunization process and an antibody purification process;

in the immune process, an immune animal selects a rabbit, the immunogen is beta-lactoglobulin, the immune method adopts subcutaneous and intramuscular injection methods, 4 times of boosting immunity is carried out after primary immunity, the boosting immunity is respectively carried out 2 weeks, 4 weeks, 6 weeks and 8 weeks after the primary immunity, and whole blood is taken by femoral artery and stored after 7-10 days of five times of immunity.

The antibody purification process specifically comprises the following steps:

1) all reagents required in the experiment were sonicated for 20min to eliminate air bubbles;

2) and (3) balancing and purifying the column: the tube was flushed with pH 7.4 phosphate buffer for 1-2min, with the flow rate set to 6.5 mL/min. After allowing the ethanol in the purification column to flow out by itself, the purification column was connected to a protein purifier, and the column was washed with a phosphate buffer solution of pH 7.4 at a flow rate of 1 mL/min. Observing that two baselines of ultraviolet and electric conductance in a program screen become parallel lines, and stopping column flushing;

3) loading: taking 1mL beta-lactoglobulin rabbit polyclonal antibody serum to be purified, centrifuging at 10000rpm for 10min, setting the temperature at 4 ℃, passing the supernatant through a 0.45 mu m water film, diluting the serum after passing the film with phosphate buffer in equal volume, putting the diluted serum on a column, and adjusting the flow rate to be 0.5mL/min when a serum sample is about to enter the column. Immunoglobulin in antiserum is specifically adsorbed on a binding site of Protein A-Sepharose 4B, while other Protein fat and other impurities are not adsorbed and flow out along with the buffer solution, and a hetero-Protein peak is displayed in an ultraviolet conductance chart of a purifier;

4) and (3) elution: after the impurity protein peak appears, continuing to wash the column with phosphate buffer until the baseline reaches equilibrium, and stopping; washing the column with glycine-hydrochloric acid buffer solution of pH 2.7 at a flow rate of 0.5mL/min, at which time the immunoglobulin bound to Protein A-Sepharose 4B was eluted;

5) collecting: after the eluent is added, the change of the ultraviolet conductance graph of the instrument is noticed, the eluent is collected when the base line begins to change, about 1mL of liquid is collected in each collecting pipe until the curve does not change, and the collection is stopped. Reading the collected liquid at 280nm of a spectrophotometer, discarding the collected liquid with the absorbance value less than 0.4, uniformly mixing the liquid with the absorbance value greater than 0.4, and immediately rapidly adjusting the pH value of the antibody to 7.0 by using Tris-HCl so as to prevent the protein in the antibody from being denatured under the acidic condition;

6) treating a purification column: after the antibody purification was completed, the column was rapidly washed with 0.1mol/L acetic acid at a flow rate of 5mL/min for 2min, and then equilibrated with phosphate buffer while measuring the pH of the effluent buffer with pH paper until it was neutral. Finally, washing the purification column with 20% ethanol solution for 20min, filling the column with 20% ethanol solution, and sealing at 4 deg.C;

7) antibody preservation: and pouring the purified antibody into an ultrafiltration device for ultrafiltration, pouring a PBS (phosphate buffer solution) with the pH of 7.4 into the ultrafiltration container for continuous ultrafiltration when 5mL of the liquid remains in the ultrafiltration container, repeating the process for 2-3 times, stopping ultrafiltration when 4-5mL of the liquid remains in the last time, taking out the liquid, measuring the absorbance of the liquid by using a spectrophotometer to calculate the concentration, mixing the liquid with glycerol with the same volume, and storing the mixture in a refrigerator at the temperature of-20 ℃ for later use.

An immunoassay method for a rabbit polyclonal antibody against beta-lactoglobulin prepared using the preparation method as described above, comprising the steps of:

1) coating: diluting the beta-lactoglobulin standard solution with coating solution, coating on 96-well polystyrene enzyme-labeled plate, incubating at 4 deg.C for 12-16h with 100 μ L/well, discarding the liquid in the well, and washing the plate with PBST washing solution for 3 times, each time for 2 min.

2) And (3) sealing: adding 200 μ L of blocking solution into each well, blocking at 37 deg.C or room temperature for 1 hr, discarding the blocking solution, washing the plate with PBST lotion for 3 times, each time for 2 min;

3) sample adding: mu.L of beta-lactoglobulin standard solution and 50. mu.L of rabbit antibody diluted with PBS were added to each well, and the reaction was performed for 1 hour at 37 ℃ and the plate was washed 4 times with PBST wash for 2min each.

4) Adding an enzyme-labeled secondary antibody: adding horseradish peroxidase-labeled goat anti-rabbit secondary antibody diluted by PBS, fixing the dilution multiple at 1:20000, 100 μ L/well, reacting at 37 deg.C for 30min, and washing the plate with PBST washing solution for 5 times, each time for 2 min.

5) Color development: mixing the substrate solution at room temperature 15min earlier, adding 100 μ L of 37 deg.C solution into each well

Developing in dark for 15-20 min.

6) And (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction;

7) reading: and reading the absorbance value by an enzyme-linked immunosorbent assay (ELISA) instrument in a dual-wavelength mode of 450-650 nm.

8) Drawing a standard curve: the X-axis is the protein concentration value and the Y-axis is the corresponding inhibition rate, the standard curve is a typical S-shaped curve, IC₅₀Defined as the concentration of milk allergen protein corresponding to an inhibition of 50%, see fig. 1.

The formulation of the solution used was as follows:

coating liquid: 0.05mol/L sodium carbonate-sodium bicarbonate buffer, pH 9.6: respectively and accurately weighing Na₂CO₃1.60g，NaHCO₃2.91g, adding double distilled water to constant volumeTo 1000mL and finally to pH 9.6.

Substrate system solution (TMB-Urea hydroperoxide solution)

Substrate solution A: 8.20g of anhydrous sodium acetate and 2.50g of beta-dextrin are accurately weighed and dissolved in 1000mL of double distilled water, then 3.23g of citric acid is accurately weighed and the pH is adjusted to 5.0, 428.6mg of carbamide peroxide is accurately weighed and added into the prepared solution after all the medicines are completely dissolved. Storing in 4 deg.C refrigerator, taking out 15min in advance, and recovering to room temperature.

Substrate solution B: 100mg of TMB was accurately weighed and dissolved in 10mL of DMSO, and stored in a brown bottle at room temperature in a dark place.

The substrate solution A and the substrate solution B need to be uniformly mixed before use: 10.95mL of substrate A solution and 0.3375mL of substrate B solution were taken, respectively.

Stopping liquid: 1.25mol/L of H₂SO₄And (3) solution.

Sensitivity: under certain optimal conditions, a standard curve of the beta-lactoglobulin indirect competition ELISA method as shown in figure 1 was established, from which the sensitivity of the method, its half inhibitory concentration IC, was derived₅₀The value was 3.08. + -. 0.24. mu.g/mL.

Specificity: in the experiment, the cross reaction detection is carried out on 10 daily proteins of easily sensitized foods by using the ovomucoid antibody, the purpose is to check the specificity of the antibody, and the smaller the cross reaction rate is, the better the specificity of the antibody is. As shown in table 1, β -lactoglobulin does not cross-react with several other common allergen proteins.

TABLE 1 beta-lactoglobulin antibodies Cross-reactive with others

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, so that any modifications, equivalents, improvements and the like, which are within the spirit and principle of the present invention, should be included in the scope of the present invention.