CN112858695A - Preparation method of ELISA (enzyme-Linked immunosorbent assay) detection kit for bovine whey component content in formula goat milk powder - Google Patents
Preparation method of ELISA (enzyme-Linked immunosorbent assay) detection kit for bovine whey component content in formula goat milk powder Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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Abstract
The invention discloses a preparation method of an ELISA detection kit for detecting the content of a bovine whey component in formula goat milk powder. The invention adopts cow milk globulin polyclonal antibody which is prepared and purified by the conventional method as coating antibody, adopts hybridoma cell 1-beta which can secrete specificity recognition beta-lactoglobulin monoclonal antibody developed by the invention to prepare monoclonal antibody (as detection antibody), establishes sandwich ELISA detection method and assembles into kit, the minimum detection limit of the kit is 0.25 ng/ml.
Description
The technical field is as follows:
the invention relates to the technical field of purification and identification of hybridoma cell strains and monoclonal antibodies, in particular to a preparation method of an ELISA detection kit for detecting the content of bovine whey in formula goat milk powder.
Secondly, background art:
goat milk has excellent nutritive value and is easy to digest and absorb, and is called as the king of milk. Is also considered a nutritional good in some european countries. Goat milk powder is the product with the most share in the goat milk market in China, and dairy factories producing goat milk powder mainly concentrate on Shaanxi, Shandong, Liaoning and the like.
At present, goat milk powder products mainly comprise whole milk powder, skim milk powder, infant formula milk powder, whey protein powder and the like. The infant formula goat milk powder is far higher in selling price than goat milk powder and goat milk products, and the components of the infant formula goat milk powder are more complex. The infant formula goat milk powder is also called as mother emulsified goat milk powder, and in order to meet the nutritional requirements of infants, fresh goat milk is used as a main raw material, part of casein is removed on the basis of common milk powder, and whey powder is added to prepare a milk powder product.
The infant formula milk powder in China has large market demand and rapid industrial development, but has short development time. At present, 103 infant formula milk powder production enterprises in China have nearly 2000 formulas, and even more than 180 formulas of individual enterprises. The problems of excessive formula, excessive abuse, random formula formulation, frequent replacement and the like of the infant formula milk powder are prominent, and certain potential quality safety risks exist.
According to the requirement of newly revised food safety laws, on the basis of extensive comments, the national food and drug administration promulgated the registration management method of infant formula milk powder product formula in 2016 (6 months) and 6 days. In the thirty-third chapter of the infant formula milk powder product formula registration management method, it is clearly specified that "animal sources are included in the product names, and animal sources of milk raw materials such as raw milk, milk powder, whey (protein) powder and the like used should be faithfully shown in an ingredient table according to the product formula. From the aspects of guaranteeing the rights and benefits of consumers in China and food safety and strengthening the supervision ability of food safety supervision departments in China, the establishment of the rapid detection method for the bovine-derived components in the infant formula goat milk powder has great significance.
The milk and the milk powder also contain beta-lactoglobulin, so the purpose of detecting bovine-derived components in the goat milk powder can be achieved by detecting the components of the bovine beta-lactoglobulin.
Most of the existing detection methods are based on the protein difference between goat milk and cow milk, such as isoelectric focusing electrophoresis, high performance liquid chromatography and the like. Due to the complex production process of the milk powder, the protein property can be changed through a high-temperature process, and the complex operation of the method and the high requirement on the technique of personnel are added, so that the application of the methods in daily monitoring is limited.
The existing bovine beta-casein detection kit can only detect bovine beta-casein which is a bovine-derived component in the formula milk powder, but cannot detect bovine whey powder, whey protein and other components added in the formula goat milk powder and infant formula goat milk powder. The bovine whey powder is an important formula component in formula goat milk powder and infant formula goat milk powder, so that a detection kit is urgently needed.
Third, the invention
The invention provides a preparation method of an ELISA detection kit for detecting the bovine whey component content in formula goat milk powder, and the detection method is simple to operate, sensitive and stable in detection result, and suitable for large-scale sample detection.
In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of an ELISA detection kit for detecting the bovine whey component content in formula goat milk powder is characterized by comprising the following steps: the preparation method comprises the following steps:
establishing and assembling a bovine beta-lactoglobulin ELISA detection kit by using a hybridoma cell strain 1-beta of a monoclonal antibody for specifically recognizing bovine beta-lactoglobulin as a detection antibody;
the hybridoma cell strain 1-beta is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO. C2020210 in 10 months and 22 days of 2020.
The concentration of the detection antibody is 0.1-24 mug/hole, the incubation temperature after the detection antibody is added is 37 ℃, and the time is 30-120 min.
The incubation time for adding the detection sample into the plate hole of the enzyme label plate is 40min-120 min.
The minimum detection limit of the kit is 0.25 ng/ml.
Compared with the prior art, the invention has the following advantages and effects:
1) the monoclonal antibody provided by the invention can show higher titer, has no cross reaction with casein and the like, and has high specificity to bovine beta-lactoglobulin in bovine whey and bovine whey powder, so that the monoclonal antibody can be used as a detection antibody to prepare an ELISA detection kit;
2) the ELISA detection kit for the bovine beta-lactoglobulin can accurately detect the bovine beta-lactoglobulin content in the traditional goat milk powder, the formula goat milk powder and the infant formula goat milk powder, and further determine the bovine whey powder (or whole cow milk) content.
Fourthly, explanation of the attached drawings:
FIG. 1 is a standard graph for determining different concentrations and absorbance values of bovine beta-lactoglobulin;
FIG. 2 is a graph showing the determination of absorbance values of bovine whey powder at different concentrations;
FIG. 3 is a graph of the absorbance values of goat milk incorporating varying amounts of cow milk, whey powder;
FIG. 4 is a graph of absorbance values of goat milk powder blended with different amounts of cow whey powder;
FIG. 5 is a graph of absorbance values of goat milk powder incorporating bovine whey powder after heat treatment;
FIG. 6 is a graph of absorbance values of goat milk powder incorporating bovine whey powder after acid treatment.
Fifth, detailed description of the invention
The technical scheme of the invention is further defined by combining the specific implementation modes as follows:
the invention relates to an ELISA detection kit for bovine whey component content in formula goat milk powder, which is prepared by the following steps:
the bovine beta-lactoglobulin polyclonal antibody prepared and purified by the traditional conventional method is used as a coating antibody, the bovine beta-lactoglobulin monoclonal antibody developed by the invention is used as a detection antibody, and a bovine beta-lactoglobulin ELISA detection kit is established and assembled.
The concentration of the coating antibody is 4-20 mug/hole, and the coating time is 12-36 h;
the concentration of the detection antibody is 0.1-24 mug/hole, the incubation temperature after the detection antibody is added is 37 ℃, and the time is 30-120 min.
The ELISA detection method has high sensitivity, and the lowest detection limit reaches 0.25 ng/ml.
1. Preparation of bovine beta-lactoglobulin polyclonal antibody and monoclonal antibody
Beta-lactoglobulin is purchased from sigma company, and is used for immunizing rabbits by a conventional method, and a bovine beta-lactoglobulin polyclonal antibody is prepared and purified to be used as a coating antibody.
The hybridoma cell strain 1-beta for secreting the beta-lactoglobulin monoclonal antibody developed by the laboratory is preserved in China center for type culture collection (CCTCC NO. C2020210) within 10 months and 22 days of 2020, and is subjected to cell counting after being subjected to expanded culture. Taking BALB/c mice injected with liquid paraffin in the abdominal cavity for one week, injecting 1 hybridoma cell into the abdominal cavity of each mouse, and 105And (3) breeding one mouse per mouse conventionally for about 10 days, extracting the ascites of the mouse about 1-2ml when the abdomen of the mouse is enlarged and the activity of the mouse is limited, centrifuging at 1500-2000 rpm for 10min, removing the lower-layer precipitate and the upper-layer liquid paraffin, and taking clear liquid. Purifying the collected clear liquid by using a proteinG column affinity chromatography to obtain a large amount of monoclonal antibodies, dialyzing the obtained monoclonal antibodies at 4 ℃ overnight, and freeze-drying and storing.
2. Establishment of double-antibody sandwich ELISA detection method
1) The purified polyclonal antibody was formulated into a solution at a concentration of 1ug/ml to 10ug/ml, 100 ul/well, and left overnight at 4 ℃. After coating, washing with PBST, washing with 200 mu L/hole, slightly shaking for 10s, discarding PBST in the ELISA plate, repeatedly washing for 3 times, and after the last washing, residual PBST in a clean gauze drying hole;
2) adding the confining liquid into an ELISA plate, sealing at the temperature of 37 ℃ for 30-60 min at 100 ul/hole. Washing after closing, wherein the washing operation is the same as the step 1);
3) adding the antigen to be detected, incubating at 37 ℃ for 30-60 min at 100 ul/hole. Washing after the incubation is finished, wherein the washing operation is the same as that in the step 1);
4) and (3) preparing the purified freeze-dried monoclonal antibody into a solution with the concentration of 1ug/ml-5ug/ml, incubating at 100 ul/hole and incubating at 37 ℃ for 30-60 min. Washing after the incubation is finished, wherein the washing operation is the same as that in the step 1);
5) diluting the horse radish peroxidase labeled goat anti-mouse antibody with a reagent diluent at a ratio of 1:3000-1:5000, incubating at 37 ℃ for 30-60 min at 100 ul/hole, washing after incubation is finished, and performing washing operation in the same step 1);
6) adding TMB color development solution, 100 ul/hole, keeping out of the sun, and incubating for 15-20 min at 37 ℃;
7) adding stop solution, 100 ul/well, detecting OD of each well within 15min450Value in P/N>2.1 are positive wells.
Determination of sensitivity of ELISA detection method
Dissolving standard bovine beta-lactoglobulin in PBST to prepare solutions with concentrations of 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.5ng/ml, 0.25ng/ml, 0.1ng/ml and 0.05ng/ml, and detecting OD corresponding to different concentrations according to ELISA operation steps in step 2450Value, OD of negative control450Comparing the values to obtain P/N values corresponding to different concentrations, and taking the P/N values as P/N>2.1 corresponds to the minimal detection limit of the ELISA detection method. The ELISA detection method has the minimum detection limit of 0.25ng/ml, and the specific determination results are shown in the table 1:
TABLE 1 results of sensitivity measurements
Specific identification of ELISA detection method
The following components are used respectively: and (3) taking 10 diluted cow whey powder, goat milk powder and fresh goat milk as antigen to be detected to coat a 96-hole ELISA plate, detecting according to the ELISA detection process, and then determining the OD value of the goat milk to be detected to obtain the monoclonal antibody which does not recognize the goat milk and the goat milk powder and is a cow beta-lactoglobulin specific recognition antibody.
TABLE 2 results of specificity identification
| Detection of antigens | Goat milk powder | Goat milk | Bovine whey powder | Negative control (PBST) |
| OD450Value of | 0.069 | 0.074 | 1.168 | 0.049 |
| P/N value | 1.4 | 1.5 | 23.8 | 1.0 |
5. Repeatability test
(1) In-batch repeat type test: on the same enzyme label plate, 5 parts of beta lactoglobulin standard positive sample and negative sample are taken respectively, and OD is determined according to the ELISA operation steps in 1450Three replicates per sample were made and the Coefficient of Variation (CV) was calculated in batches, where CV is (SD/OD)450Average) × 100%.The results show that the intra-batch coefficient of variation is less than 5%. The specific test results are shown in table 3:
table 3: results of in-batch repeatability tests
(2) Batch to batch repeatability test: on the ELISA plates coated in different batches, 5 parts of standard positive samples and negative samples are taken respectively, and OD is determined according to the ELISA operation steps in step 1450Three replicates per sample were made and the Coefficient of Variation (CV) between batches was calculated, where CV is (SD/OD)450) X 100%. The results show that the inter-batch coefficient of variation is less than 10%. The specific test results are shown in the table:
table 4: results of batch to batch repeatability tests
6. Drawing of standard curve
Dissolving standard beta-lactoglobulin in carbonate solution to prepare solutions with the concentrations of 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml and 100ng/ml, and detecting OD corresponding to different concentrations according to the ELISA operation steps in step 2450The value is obtained. The standard curve was plotted with the standard beta-lactoglobulin concentration as the abscissa (y) and the OD450 value as the ordinate (x). The resulting standard curve is: Y0.01012X +0.09307, R2 0.9996, as shown in fig. 1.
Assembly of ELISA kits
The bovine beta-lactoglobulin ELISA detection kit comprises the following components:
(1) one blank 96-hole enzyme label plate;
(2) one of the detection antibodies was diluted with coating diluent before use.
(3) One positive sample and one negative sample of beta-lactoglobulin.
(4) The goat anti-mouse antibody marked by horseradish peroxidase is diluted by sample diluent 1:5000 before use;
(5) TMB developing solution, stop solution, confining solution, sample diluent and washing solution are respectively one.
Test example 1: bovine whey powder for detecting different concentrations
Mixing the following components in parts by weight: 10 bovine whey powder after dissolution was diluted with PBS solution to different proportions: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (v/v). The assay was performed as in test 2, and 3 parallel wells were set for each percentage of the standard samples, and their absorbance at 450nm was averaged. The percentage of milk and bovine whey powder spiked was then plotted as the abscissa and OD450nm as the ordinate, as shown in figure 2, for a single blind experimental verification of quantitative spiking.
Test example 2: detecting milk and whey powder mixed in goat milk powder
Mixing milk with a mixture of 1: 10, respectively adding the dissolved bovine whey powder into goat milk and a mixture of 1: 10 preparing and mixing standard products in the dissolved goat milk powder. The incorporation percentages are as follows: 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (v/v). The assay was performed as in test 2, and 3 parallel wells were set for each percentage of the standard samples, and their absorbance at 450nm was averaged. The percentage of milk and bovine whey powder incorporated was then plotted on the abscissa and OD450nm on the ordinate, as shown in fig. 3 and 4, as a curve from a single blind experimental verification chart for quantitative adulteration.
As a result: when the volume percentage of the milk and the cow whey powder mixed in the goat milk and the goat milk powder is 1-50 volume percent, A450nm is in a linear relation with the goat milk and the goat milk powder, the linear detection range of the experiment can be obtained to be 1-50 volume percent, and the linear correlation coefficient is-0.9982; the lowest detection limit was 1 vol%. The repeatability of the experimental detection sample is good, and the variation coefficient of A450nm (CV is sd/mean x 100%; sd is the standard deviation of several measured data, mean value thereof) (CV) of the ingredient of bovine whey powder mixed in the goat milk powder detected in a plate is less than 10%. The obtained detection curve is applied to a single blind experiment, and the method can be used for quantitatively detecting the incorporated bovine whey powder within the range of 1-80% according to the obtained experimental result, and the detection value has no significant difference between the known values (P ═ 0.184).
Test example 3: detecting goat milk powder doped with cow whey powder after heat treatment and acid treatment
After the bovine whey powder and goat milk powder samples are mixed according to the method of the test example 3, the mixture is respectively sterilized by a water area of 62 ℃ for 30min, heated by 95 ℃ for 10min and subjected to high temperature sterilization at 135-150 ℃ for 2-5s to obtain the heat-treated milk sample.
After mixing the bovine whey powder-goat milk powder samples according to the method of test example 3, each percentage of the samples was adjusted to pH 6.4, 5.7, 5 using 0.5mM hydrochloric acid to obtain acid-treated samples.
The resulting heat-and acid-treated samples were tested by indirect ELISA as in test 2, with triplicate per percentage. The results are shown in the figure.
FIGS. 5 and 6 show that the curves of the mixed samples treated with different heat and acid treatments are similar to the curves of the untreated samples, respectively, so that the heat and acid treatments do not affect the detection range and results of ELISA.
Test example 4:
different commercially available infant formula goat milk powder, common goat milk powder and raw whey powder are used as antigens to be detected according to different dilutions, and after detection by an ELISA kit, comparison of repeatability detection values and statistics of detection rates are carried out.
Detection application result of bovine beta-lactoglobulin kit
Note: cow whey powder, infant formula goat milk powder and common goat milk powder are commercially available products, and 83 parts of detected products are used as antigens to be detected
The data in the table show that the ELISA detection kit for the bovine beta-lactoglobulin has good repetition rate and detection rate, so that the kit can be used for detecting goat milk products to be detected, formula goat milk powder or infant formula goat milk powder samples in all areas of China, and can be popularized and used in a large range.
The monoclonal antibody provided by the experiment is a specific antibody capable of identifying the bovine beta-lactoglobulin, can be specifically combined with bovine beta-lactoglobulin antigens in formula goat milk powder, infant goat milk powder and raw material whey powder and whey protein powder, and can be used for detecting whether whey powder is added in the formula goat milk powder sold in the market of China or not and the content of the whey powder.
Compared with the common methods such as PCR, fluorescent quantitative PCR, capillary electrophoresis technology, loop-mediated isothermal amplification detection and the like, the antigen of the bovine beta-lactoglobulin monoclonal antibody is detected by adopting the ELISA technology, the specificity is strong, the sensitivity is high, the occurrence of false positive results can be prevented, meanwhile, the ELISA detection kit is simple to operate, excessive instruments and equipment are not needed, the detection cost is low, and the method is more beneficial to large-scale popularization and use.
The preferred embodiments of the present invention have been described in detail, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the technical features described in the above embodiments may be combined in any suitable manner without contradiction, and in order to avoid unnecessary repetition, various possible combinations of the features described in the embodiments are not described separately.
In addition, various different embodiments of the present invention can be combined arbitrarily, and the same should be regarded as the disclosure of the present invention as long as it does not depart from the idea of the present invention.
Claims (4)
1. The preparation method of the ELISA detection kit for the bovine whey component content in the formula goat milk powder is characterized by comprising the following steps: the preparation method comprises the following steps:
establishing and assembling a bovine beta-lactoglobulin ELISA detection kit by using a hybridoma cell strain 1-beta of a monoclonal antibody for specifically recognizing bovine beta-lactoglobulin as a detection antibody;
the hybridoma cell strain 1-beta is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO. C2020210 in 10 months and 22 days of 2020.
2. The method for preparing the ELISA kit for detecting the bovine whey content in the formula goat milk powder as claimed in claim 1, wherein the ELISA kit comprises:
the concentration of the detection antibody is 0.1-24 mug/hole, the incubation temperature after the detection antibody is added is 37 ℃, and the time is 30-120 min.
3. The method for preparing the ELISA kit for detecting the bovine whey content in the formula goat milk powder as claimed in claim 1, wherein the ELISA kit comprises:
the incubation time for adding the detection sample into the plate hole of the enzyme label plate is 40min-120 min.
4. The method for preparing the ELISA kit for detecting the bovine whey content in the formula goat milk powder as claimed in claim 1, wherein the ELISA kit comprises: the minimum detection limit of the kit is 0.25 ng/ml.
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