CN111848792A - Preparation method and detection method of antibody of bovine serum albumin and other impurities in vaccine - Google Patents
Preparation method and detection method of antibody of bovine serum albumin and other impurities in vaccine Download PDFInfo
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- CN111848792A CN111848792A CN202010684592.9A CN202010684592A CN111848792A CN 111848792 A CN111848792 A CN 111848792A CN 202010684592 A CN202010684592 A CN 202010684592A CN 111848792 A CN111848792 A CN 111848792A
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention provides a preparation method of antibodies of impurities such as bovine serum albumin in vaccines, which relates to the following steps: the bovine serum albumin antibody is obtained by directly immunizing animals, the impurity neomycin antibody is obtained by preparing neomycin immunogen and further immunizing animals, and the obtained antibody is applied to an enzyme-linked immunoassay kit.
Description
Technical Field
The invention relates to an antibody preparation technology, in particular to a preparation method of an antibody for detecting bovine serum albumin and neomycin.
Background
Bovine serum albumin is widely used in relevant laboratories such as basic laboratories, biochemistry laboratories, teaching laboratories, scientific researches, medicines, biological products and the like, and especially in the production process of biological products such as vaccines and the like (such as cell culture, tissue culture and the like), a certain amount of bovine serum is generally used, and although final products of various varieties are removed by a certain process, a small amount of bovine serum can still remain. Bovine serum, as a sensitizing substance, may have serious consequences after vaccination. Therefore, the detection of residual bovine serum is an important index in the quality control of biological products, Bovine Serum Albumin (BSA) is used as a main component in the bovine serum, and the residual content of the bovine serum in the products is strictly limited by the World Health Organization (WHO) and the regulations of biological products of all countries in the world. The detection of the BSA content is related to the qualification of the product, thereby affecting the health of human body and even directly related to the safety of life. Additionally, antibiotics such as neomycin may be present in vaccines and are impurities that need to be detected.
The present application develops methods for the preparation of antibodies to bovine serum albumin and neomycin for use in rapid detection methods.
Disclosure of Invention
The invention aims to provide an antibody preparation technology, in particular to an antibody preparation method for detecting bovine serum albumin and neomycin, and the prepared antibody can be applied to a rapid detection method, such as an enzyme linked immunosorbent assay kit.
The enzyme linked immunosorbent assay kit comprises: the enzyme-linked immunosorbent assay kit comprises an ELISA plate coated with a coating antigen, a standard solution, an antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate chromogenic solution, a stop solution and a washing solution, wherein the coating antigen is a neomycin coupling antigen and bovine serum albumin, and the enzyme conjugate is an enzyme-labeled neomycin antibody and a bovine serum albumin antibody.
The neomycin coupling antigen is obtained by coupling neomycin hapten and carrier protein, the neomycin hapten is obtained by carrying out a series of chemical reactions on neomycin sulfate, 6-maleimide-hexanal and other substances, and the carrier protein is mouse serum protein, thyroid protein, pig urine albumin, rabbit serum protein, human serum protein, ovalbumin, hemocyanin or fibrinogen.
The neomycin antibody is prepared by taking a neomycin coupling antigen as an immunogen, and the neomycin specific antibody can be a neomycin monoclonal antibody or a neomycin polyclonal antibody, wherein the neomycin monoclonal antibody is preferred.
The bovine serum albumin antibody is prepared by taking bovine serum albumin as an immunogen, and can be a bovine serum albumin monoclonal antibody or a bovine serum albumin polyclonal antibody, wherein the bovine serum albumin monoclonal antibody is preferred.
The labeled enzyme of the enzyme conjugate is horseradish peroxidase or alkaline phosphatase extracted from bacteria, wherein horseradish peroxidase is preferred; the enzyme conjugate is obtained by coupling enzyme and antibody.
In order to facilitate field monitoring and large-scale sample screening, the kit further comprises a standard solution, a substrate developing solution, a stopping solution and a washing solution.
The concentration of the neomycin standard solution in 6 bottles is 0 mug/L, 0.5 mug/L, 1.5 mug/L, 4.5 mug/L, 13.5 mug/L and 40.5 mug/L respectively.
The bovine serum albumin standard solution 6 bottles respectively have the concentrations of 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L.
When the labeled enzyme is horseradish peroxidase, the substrate color development solution consists of a substrate solution A and a substrate solution B, wherein the A is hydrogen peroxide or carbamide peroxide, the B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2 mol/L sulfuric acid solution or hydrochloric acid buffer solution; when the marker enzyme is bacteria extracted alkaline phosphatase, the substrate color developing solution is a para-nitrophosphate buffer solution, and the stop solution is 1-2 mol/L sodium hydroxide solution.
The washing liquid preferably has a pH value of 7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages.
The coating buffer solution used in the preparation process of the ELISA plate is carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, and the confining solution is carbonate buffer solution with the pH value of 7.1-7.5, contains 1-3% of casein and 0.1-0.3 mol/L of phosphate buffer solution, and the percentage is weight volume percentage.
The preparation process of the ELISA plate comprises the following steps: diluting the coating source into 20 mu g/mL by using a coating buffer solution, adding 100 mu l into each hole, incubating for 2h in a dark place at 25 ℃ or overnight at 4 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 150-200 mu l of a sealing solution into each hole, incubating for 1-2 h in a dark place at 25 ℃, pouring off liquid in the holes, patting to dry, drying, and performing vacuum sealing and storage by using an aluminum film.
The detection principle of the invention is as follows:
the bovine serum albumin kit adopts a direct competition ELISA method to pre-coat immunogen on an ELISA plate micropore strip, the residual bovine serum albumin in the sample and the pre-coated immunogen on the ELISA plate micropore strip compete for enzyme conjugate resisting bovine serum albumin, TMB substrate is used for color development, the absorbance value of the sample is in negative correlation with the content of the residual bovine serum albumin, the absorbance value is compared with a standard curve, and the corresponding dilution multiple is multiplied, so that the residual amount of the bovine serum albumin in the sample can be obtained.
The neomycin kit adopts an indirect competitive ELISA method, conjugate antigens are pre-coated on an enzyme label plate microporous strip, the residual neomycin in a sample and the conjugate antigens pre-coated on the enzyme label plate microporous strip compete for anti-neomycin antibodies, after an enzyme-labeled secondary antibody is added, a TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of the residual neomycin contained in the sample, the absorbance value is compared with a standard curve, and the absorbance value is multiplied by the corresponding dilution multiple, so that the residual quantity of the neomycin in the sample can be obtained.
The ELISA kit provided by the invention mainly adopts an ELISA method to qualitatively or quantitatively detect the content of an object to be detected in a sample; the pretreatment requirement on the sample is low, the pretreatment process of the sample is simple, and a large amount of samples can be detected quickly; the main reagent is provided in the form of working solution, and the detection method is convenient and easy to implement and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like. The enzyme linked immunosorbent assay kit has the advantages of simple structure, convenient use, low price, convenient carrying, high efficiency, accuracy, simplicity and convenience of the detection method, and is suitable for qualitative and quantitative screening of large-batch samples.
Drawings
FIG. 1: neomycin hapten synthesis roadmap
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of kit Components
1. Preparation of neomycin hapten
680mg of neomycin sulfate is taken and dissolved in 25ml of water, 6ml of potassium carbonate with the concentration of 0.2mol/L is added, the mixture is fully stirred, 1ml of methanol solution containing 151mg of 6-maleimide-hexanal is added, the mixture is stirred for 8h at room temperature, 55.2mg of sodium borohydride is added, the mixture is continuously stirred for 2h, the pH value is adjusted to 7 by 6mol/LHCl, the mixture is evaporated to dryness by rotary evaporation, the water is removed, 50ml of pyridine is added, the mixture is fully stirred and dissolved, the salt is removed, the organic phase is evaporated to dryness by rotary evaporation, 5ml of absolute ethyl alcohol is added for recrystallization, 470mg of the hapten product maleimide-neomycin is obtained, and the yield is.
2. Preparation of antigens
Immunogen preparation-coupling of a neomycin hapten and Bovine Serum Albumin (BSA) to obtain an immunogen.
Taking 100mg of bovine blood albumin (BSA), adding 0.05mol/L PB8.06ml for dissolving, adding 7.8mg of dithiothreitol (DDT), fully dissolving, stirring overnight to obtain solution A, taking 68mg of maleimide-neomycin, adding 2ml of ethanol for dissolving, dropwise adding into the solution A, reacting at room temperature for 6h, dialyzing with 0.02mol/L PBS for three days, changing the solution for 3 times every day to obtain a neomycin-BSA conjugate which is an immunogen, subpackaging and storing at-20 ℃ for later use.
Preparation of coating antigen-coupling of neomycin hapten and Ovalbumin (OVA) to obtain immunogen.
Taking 100mg of Ovalbumin (OVA), adding 0.05mol/L PB8.06ml for dissolving, adding 5.3mg of dithiothreitol (DDT), fully dissolving, stirring overnight to obtain solution A, taking 35mg of maleimide-neomycin, adding 2ml of ethanol for dissolving, dropwise adding into the solution A, reacting at room temperature for 6h, dialyzing with 0.02mol/L PBS for three days, changing the solution for 3 times every day to obtain a neomycin-OVA conjugate, namely a coating antigen, subpackaging and storing at-20 ℃ for later use.
3. Preparation of neomycin monoclonal antibody and bovine serum albumin monoclonal antibody
Animal immunization: neomycin immunogen/bovine serum albumin was injected into different Balb/c mice at an immunization dose of 150. mu.g/mouse to generate antiserum.
Cell fusion and cloning: after the serum determination result of the mouse is higher, spleen cells are taken and fused with SP2/0 myeloma cells according to the ratio of 8:1 (quantitative ratio), indirect competitive ELISA is adopted to determine cell supernatant, and positive holes are screened. Cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain secreting a neomycin monoclonal antibody/bovine serum albumin monoclonal antibody.
Freezing and recovering cells: preparation of monoclonal hybridoma cell line into 1 × 10 frozen stock solution 6Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
Of monoclonal antibodiesProduction and purification: injecting sterilized paraffin oil 0.5 mL/mouse into the abdominal cavity of Balb/c mouse, and injecting stable monoclonal hybridoma cell strain 5X 10 into the abdominal cavity after 7 days5Ascites were collected 7 days later. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.
4. Preparation of enzyme conjugates
Taking a goat as an immune animal and taking a monoclonal antibody as an immunogen to immunize the goat without the pathogen to obtain a corresponding antibody. The antibody is coupled with horseradish peroxidase (HRP) to obtain an enzyme conjugate.
5. Preparation of ELISA plates
Diluting the coating source to 20 mu g/mL by using a coating buffer solution, adding 100 mu l of the coating source into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 200 mu l of a sealing solution into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, patting to dry, and performing vacuum sealing and storage by using an aluminum film after drying.
EXAMPLE 2 construction of enzyme-linked immunoassay kit
An enzyme linked immunosorbent assay kit for detecting neomycin/bovine serum albumin is constructed, and comprises the following components:
(1) An ELISA plate coated with a coating antigen;
(2) 6 bottles of neomycin standard solution with the concentrations of 0 mug/L, 0.5 mug/L, 1.5 mug/L, 4.5 mug/L, 13.5 mug/L and 40.5 mug/L respectively; 6 bottles of bovine serum albumin standard solution with the concentrations of 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L respectively;
(3) an antibody labeled with horseradish peroxidase;
(4) the substrate color development liquid consists of a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(5) the stop solution is 2mol/L sulfuric acid;
(6) the washing liquid has a pH value of 7.4, and contains 0.5-1.0% of tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages;
EXAMPLE 3 detection of analyte in biological products
1. Detection with a kit
And numbering the corresponding micropores of the samples and the standard products in sequence, making 2 holes in parallel for each sample and standard product, and recording the positions of the standard holes and the sample holes. Adding 20-80 μ l of standard substance/sample into corresponding micro-hole, adding 20-80 μ l of enzyme conjugate working solution into corresponding micro-hole, gently shaking, mixing, covering with cover plate, and reacting at 25 deg.C in dark environment for 30 min. Spin-drying the liquid in the holes, adding 250 mul/hole of washing working solution, fully washing for 4-5 times at intervals of 10s each time, splashing the washing solution in the holes of the plate, and patting the plate dry by using absorbent paper. Adding 50 mul/hole of the substrate solution A, adding 50 mul/hole of the substrate solution B, lightly shaking and mixing, covering with a cover plate, and reacting for 15min in a dark environment at 25 ℃. Adding 50 mul/well of stop solution, gently oscillating and mixing, setting an enzyme-linked immunosorbent assay (ELISA) instrument at 450nm, and measuring the OD value of each well.
2. Analysis of detection results
The percent absorbance of the standard or sample is equal to the average of the absorbance values of the standard or sample (double well) divided by the average of the absorbance values of the first standard (0 standard) and multiplied by 100% to obtain the percent absorbance value of the standard or sample. The standard curve is plotted with the percent absorbance of the standard as the ordinate and the logarithm of the concentration of the standard (μ g/L) as the abscissa. And substituting the percent absorbance of the sample into the standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the substance to be detected in the sample.
EXAMPLE 4 Key technical parameters of the neomycin kit
1. Linear range of the kit
The linear range of the kit is 0.5-40.5 mu g/L.
2. Accuracy of kit
The accuracy of the kit is 70-130%.
3. Precision of the kit
Precision of the kit: the variation coefficient between the plates is less than 10%.
4. Specificity of the kit
Streptomycin is less than 0.1%;
kanamycin is less than 0.1%;
apramycin less than 0.1%;
gentamicin less than 0.7%;
the tobramycin content is less than 0.1%.
Example 5 Critical technical parameters of the bovine serum Albumin kit
1. Linear range of the kit
The linear range of the kit is 0.1-8.1 mu g/L.
2. Accuracy of kit
The accuracy of the kit is 80% -100%.
3. Precision of the kit
Precision of the kit: the variation coefficient between the plates is less than 10%.
Claims (4)
1. A preparation method of antibodies of impurities such as bovine serum albumin in vaccines is characterized in that: the bovine serum albumin antibody is obtained by directly immunizing animals, the impurity neomycin antibody is obtained by preparing neomycin immunogen and further immunizing animals, the neomycin immunogen is obtained by coupling neomycin hapten with protein, and the preparation method of the neomycin hapten is as follows:
680mg of neomycin sulfate is taken and dissolved in 25ml of water, 6ml of potassium carbonate with the concentration of 0.2mol/L is added, the mixture is fully stirred, 1ml of methanol solution containing 151mg of 6-maleimide-hexanal is added, the mixture is stirred for 8h at room temperature, 55.2mg of sodium borohydride is added, the stirring is continued for 2h, the pH value is adjusted to 7 by 6mol/L HCl, the mixture is evaporated to dryness by rotary evaporation, the water is removed, 50ml of pyridine is added, the mixture is fully stirred and dissolved, the salt is removed, the organic phase is evaporated to dryness by rotary evaporation, 5ml of absolute ethyl alcohol is added for recrystallization, 470mg of the hapten product maleimide-neomycin is obtained, and the yield is 35.
2. The method of claim 1, wherein the neomycin conjugate antigen is obtained by conjugating a neomycin hapten with a carrier protein, and the neomycin hapten is obtained by reacting neomycin sulfate with 6-maleimido-hexanal through a series of chemical reactions.
3. The method of claim 1, wherein the neomycin hapten has the formula:
4. the method of claim 1, wherein the bovine serum albumin antibody is prepared by the following steps:
injecting bovine serum albumin into different Balb/c mice with the immune dose of 150 mug/mouse to generate antiserum; after the serum determination result of the mouse is higher, taking splenocytes of the mouse, fusing the splenocytes with SP2/0 myeloma cells according to the ratio of 8:1 (quantitative ratio), determining cell supernatant by adopting indirect competitive ELISA, and screening positive holes; cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain secreting a bovine serum albumin monoclonal antibody; preparation of monoclonal hybridoma cell line into 1 × 10 frozen stock solution6Cell suspension per mL, and storing in liquid nitrogen for a long time; taking out the cryopreservation tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast thawing, centrifuging to remove the cryopreservation liquid, and transferring the tube into a culture bottle for culture; injecting sterilized paraffin oil 0.5 mL/mouse into the abdominal cavity of Balb/c mouse, and injecting stable monoclonal hybridoma cell strain 5X 10 into the abdominal cavity after 7 days 5Ascites were collected 7 days later; purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114591430A (en) * | 2022-05-10 | 2022-06-07 | 翌圣生物科技(上海)股份有限公司 | BSA (bovine serum albumin) monoclonal antibody, application thereof and BSA (bovine serum albumin) detection kit containing antibody |
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| EP0513778A2 (en) * | 1991-05-17 | 1992-11-19 | Takeda Chemical Industries, Ltd. | Hybrid monoclonal antibodies and compositions containing them |
| AU2001249609A1 (en) * | 2000-03-28 | 2001-10-08 | Department Of Veterans Affairs | Methods for increasing a cytotoxic T lymphocyte response in vivo |
| WO2002008288A2 (en) * | 2000-07-20 | 2002-01-31 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
| CN101021536A (en) * | 2007-03-12 | 2007-08-22 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunological kit for detecting neomycin drug and method |
| AU2015202447A1 (en) * | 2008-07-18 | 2015-06-04 | Bristol-Myers Squibb Company | Compositions monovalent for cd28 binding and methods of use |
| CN106526167A (en) * | 2016-10-31 | 2017-03-22 | 中国农业大学 | Penitrem A combined antigen as well as preparation and application of antibody of antigen |
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2020
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| EP0513778A2 (en) * | 1991-05-17 | 1992-11-19 | Takeda Chemical Industries, Ltd. | Hybrid monoclonal antibodies and compositions containing them |
| AU2001249609A1 (en) * | 2000-03-28 | 2001-10-08 | Department Of Veterans Affairs | Methods for increasing a cytotoxic T lymphocyte response in vivo |
| WO2002008288A2 (en) * | 2000-07-20 | 2002-01-31 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
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| AU2015202447A1 (en) * | 2008-07-18 | 2015-06-04 | Bristol-Myers Squibb Company | Compositions monovalent for cd28 binding and methods of use |
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| CN114591430A (en) * | 2022-05-10 | 2022-06-07 | 翌圣生物科技(上海)股份有限公司 | BSA (bovine serum albumin) monoclonal antibody, application thereof and BSA (bovine serum albumin) detection kit containing antibody |
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