CN105085676A - Rabbit anti-bovine lactoferrin polyclonal antibody and preparation method and application thereof - Google Patents
Rabbit anti-bovine lactoferrin polyclonal antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a rabbit anti-bovine lactoferrin polyclonal antibody and a preparation method and application thereof. The preparation method includes the steps that 1, lactoferrin and an immunologic adjuvant are mixed to obtain a lactoferrin solution with a composite adjuvant; 2, the lactoferrin solution with the composite adjuvant is used for immunizing a New Zealand white rabbit, antiserum is obtained, centrifugation is performed, and supernatant is taken; 3, the prepared antiserum is purified through a magnetic-activated cell sorting method and a protein A-sepharose affinity chromatography method in sequence, and the purified rabbit anti-bovine lactoferrin polyclonal antibody is obtained. The prepared polyclonal antibody is high in purity, titer and specificity and can serve as a detection antibody for qualitatively or quantitatively detecting lactoferrin in milk products. A lactoferrin ELISA detection method established through the lactoferrin polyclonal antibody has the advantages that the cost is low, the detection range is wide and reliable results can be rapidly obtained, and can be widely applied to qualitatively or quantitatively detecting lactoferrin in cow lacteal glands, milk and milk products.
Description
Technical field
The invention belongs to technical field of immunoassay.The present invention relates to a kind of polyclonal antibody, particularly relate to the anti-Bovinelactoferrin polyclonal antibody of rabbit, the invention further relates to the application of the anti-Bovinelactoferrin polyclonal antibody of this rabbit in quantitative and qualitative analysis detection breast, milk-product or mammary gland in lactoferrin.
Background technology
In recent years, the requirement of people to milk-quality is more and more higher, and milk-protein is as the important indicator weighing milk-quality, and the nutrition regulation of its synthesis has become the focus of dairy cow nutrition research.The multiple efficacies such as lactoferrin, also referred to as lactotransferrin (lactoferrin, Lf), is one of important biomolecule active substance in milk, and have and regulate iron to absorb, broad spectrum antibiotic infects, and regulates organism immune response, anti-oxidant, anticancer, antiviral.Just Ruzhong content is higher people for lactoferrin, and concentration is 6-8mg/mL; In Bovine Colostrum, concentration is 1-2mg/mL, and lower at the content in normal Ruzhong, and people normal Ruzhong concentration is only 1-2mg/mL, and milk cow normal Ruzhong concentration is only 0.02-0.35mg/mL.Due to human milk limited source, therefore cow's milk obtain the most direct way of lactoferrin for people.For detecting breast and the content of lactoferrin in dairy products, effectively reducing the loss caused in milk processing, needing to set up a kind of method measuring fresh milk and lactoferrin in dairy products content fast and accurately.
So far, the quantitative and qualitative analysis detection method of lactoferrin mainly comprises random immunodiffusion method (RID), radio immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis method (SDS-PAGE) and high performance liquid chromatography (HPLC) etc.Wherein, RID and RIA method sensing range is limited, poor accuracy, and there is certain radiologic hazard.SDS-PAGE method is relatively consuming time longer, and is not easy to accurate quantitative analysis, should not detect batch samples.Though HPLC method can accurate quantitative analysis, its detected object is limited, is only applicable to detecting liquid state milk and powdered milk sample, and sample purity be there are certain requirements, be only applicable to high purity sample determination, sample pretreatment process relative complex, plant and instrument is expensive simultaneously, and requires higher for the operating skill of instrument.
By contrast, enzyme-linked immunosorbent assay (ELISA) method detects the effective ways of specific protein, has quick, sensitive, easy, carrier and be easy to the advantages such as stdn.In conjunction with SDS-PAGE and Western-blot, the quantitative and qualitative analysis that can be used for for specific antigen detects.But, utilize enzyme-linked immunosorbent assay for measuring to detect lactoferrin content in fresh milk and milk products, need to use corresponding lactoferrin antibody.
The many existence in various degree of existing lactoferrin polyclonal antibody are tired lower, the defect such as purity is not high, specificity is not strong, limiting it detecting the application in the lactoferrin content in fresh milk and milk products, having much room for improvement.
Summary of the invention
Technical problem to be solved by this invention overcomes the deficiencies in the prior art, there is provided a kind of height of tiring, purity rabbit that is high, high specificity anti-Bovinelactoferrin polyclonal antibody, for the lactoferrin synthesized in detection by quantitative milk and milk products and in cow mammary gland provides effective research material.
Technical problem to be solved by this invention is achieved through the following technical solutions:
The anti-Bovinelactoferrin polyclonal antibody of a kind of rabbit, it prepares by the following method:
(1) lactoferrin is mixed with immunological adjuvant, obtain the lactoferrin solution containing composite adjuvant;
(2) with the lactoferrin solution immunize rabbit containing composite adjuvant, antiserum(antisera) is obtained; Centrifugal, get supernatant, obtain antiserum(antisera), for subsequent use;
(3) antiserum(antisera) is carried out purifying with immunomagnetic beads method and protein A-sepharose affinity chromatography successively, obtain the anti-Bovinelactoferrin polyclonal antibody of rabbit of purifying.
Wherein, in step (1), lactoferrin and CpG adjuvant are dissolved in PBS and obtain mixing solutions, then with Al (OH)
3mixing, obtains the lactoferrin solution containing composite adjuvant; Preferably, 0.5mg lactoferrin and 50 μ gCpG adjuvants are dissolved in 1mLPBS obtain mixing solutions; By mixing solutions and Al (OH)
3mix according to the volume ratio of 1:5, obtain the lactoferrin solution containing composite adjuvant.
Described immunization ways comprises: adopt the mode of subcutaneous multi-point injection method by the lactoferrin solution immunize New Zealand white rabbits containing composite adjuvant, and the number of times of immunity is 4 times; Wherein, the 1st immunity, after 4 weeks, carries out the 2nd immunity; Every 2 weeks later repeat immunity 1 time, carry out the 3rd time and the 4th immunity; Within the 7th day after the 4th immunity, collect antiserum(antisera).In order to reach better technique effect, when the 1st time is immune, every rabbit immunity 500 μ g lactoferrins; 2nd time, the 3rd time and the 4th immunity time, every rabbit immune 250 μ g lactoferrins at every turn.
Antiserum(antisera) is carried out purifying by different modes, there is larger difference in the purity of the lactoferrin polyclonal antibody obtained; The present invention is found by shaker test, the purity of the lactoferrin polyclonal antibody adopting saturated ammonium sulphate method purifying antiserum(antisera) to obtain is only 62%, the purity of the lactoferrin polyclonal antibody adopting immunomagnetic beads method associating protein A-sepharose affinity chromatography purifying antiserum(antisera) to obtain reaches more than 87%, far away higher than this way of purification of saturated ammonium sulphate method; Therefore, the present invention preferably adopts immunomagnetic beads method to combine protein A-sepharose affinity chromatography purifying antiserum(antisera).
Described immunomagnetic beads method purifying antiserum(antisera) comprises: joined by magnetic bead in centrifuge tube, magnetic separator is placed and sucks supernatant liquor, add the antiserum(antisera) of dilution, leave standstill, suck supernatant liquor; Resuspended magnetic bead, repeated washing, sucks supernatant liquor; Add sodium citrate buffer, vortex shakes, and leaves standstill, Aspirate supernatant, repeated washing, is mixed by gained supernatant liquor, obtains purified antibodies;
Preferably, getting 100 μ L magnetic beads adds in 2mL centrifuge tube, supernatant liquor is sucked after magnetic separator is placed 3min, add the antiserum(antisera) of 300 μ L according to the dilution proportion of 1:100, inclination slow circumvolve 10min is kept under 4 DEG C of conditions, supernatant liquor is sucked after then leaving standstill 3min, with the resuspended magnetic bead of the PBS of pH=7.4, after repeated washing 3 times, centrifuge tube is again placed 3min and is sucked supernatant liquor on magnetic separator, add the 0.1mol/L sodium citrate buffer of pH3.0, vortex concussion 3min, and then on magnetic separator, place Aspirate supernatant after 2min, repeated washing 3 times, gained supernatant liquor is mixed, obtain purified antibodies.
Described mode of antiserum(antisera) protein A-sepharose affinity chromatography being carried out purifying comprises: first balance Protein A-agarose-4B with combination/lavation buffer solution before loading and fill post, washing away the impurity such as contained protein, waiting to flow out elutriant OD
280during <0.02; Antiserum(antisera) after being diluted by combination/lavation buffer solution adds in post, enters after post bed until whole serum sample, rinses pillar, treat elutriant OD with combination/lavation buffer solution
280during <0.02, with the 3.5% glacial acetic acid wash-out of pH=2.8, the elutriant containing antibody is collected in the container containing level pad, concentrated, to obtain final product;
Preferably, described combination/lavation buffer solution is the 0.1mol/LTris-HCl damping fluid containing 1MNaCl, its pH=8.6;
In order to reach better effect, first balancing Protein A-agarose-4B with combination/lavation buffer solution before loading and filling post, washing away impurity with 1mL/min flow velocity; Wait to flow out elutriant OD
280during <0.02, serum sample (1:4, v/v) after being diluted by combination/lavation buffer solution adds in post, enters after post bed until whole serum sample, rinse pillar with combination/lavation buffer solution and make damping fluid take-off rate be about 1mL/min, treating elutriant OD
280during <0.02, with the 3.5% glacial acetic acid wash-out of pH=2.8, the elutriant containing antibody is collected in the container containing level pad with in and elutriant pH to 7.3;
Described concentrating preferably concentrates with 10kDa super filter tube.
Anti-for prepared rabbit Bovinelactoferrin polyclonal antibody and the anti-Bovinelactoferrin polyclonal antibody (Sigma Co., USA) of existing commercialization have been carried out potency ratio comparatively by the present invention, the anti-Bovinelactoferrin polyclonal antibody of rabbit of the present invention is tired as 1:128000, and the anti-Bovinelactoferrin polyclonal antibody (Sigma Co., USA) of commercialization is tired and is only 1:32000.
Western-blot result shows the anti-Bovinelactoferrin polyclonal antibody of the rabbit after purifying of the present invention can specific recognition lactoferrin.ELISA test-results is visible, and the anti-Bovinelactoferrin polyclonal antibody of rabbit prepared by the present invention only combines lactoferrin specificity, and with casein without association reaction, shows that it has good specificity.
The present invention is by obtaining its antiserum(antisera) to new zealand white rabbit immune milk ferritin, purifying is carried out by immunomagnetic beads method and protein A-sepharose affinity chromatography antagonistic Serum, obtain purity and the anti-Bovinelactoferrin polyclonal antibody of high rabbit of tiring, its antibody concentration is 11.02mg/mL, and its antibody titer is 1:128000.The anti-Bovinelactoferrin polyclonal antibody of rabbit of the present invention can specific recognition lactoferrin, only combines lactoferrin specificity, and with casein without association reaction, shows that it has good specificity.
The lactoferrin extracted in the anti-Bovinelactoferrin polyclonal antibody of rabbit prepared by the present invention and cow mammary gland tissue has reactivity, also there is atopy to the lactoferrin in milk powder simultaneously, can be used for detecting the lactoferrin in cow mammary gland tissue and milk preparation.
Therefore, the invention provides and anti-for prepared rabbit Bovinelactoferrin polyclonal antibody is applied to lactoferrin in qualitative or detection by quantitative milk-product or mammary gland; For example, anti-for rabbit prepared by the present invention Bovinelactoferrin polyclonal antibody is adopted enzyme-linked immunosorbent assay (ELISA) methods combining SDS-PAGE and Western-blot as detection antibody, qualitative or detection by quantitative is carried out to the lactoferrin in milk-product or mammary gland, there is quick, sensitive, easy, carrier and be easy to the advantages such as stdn.
Invention further provides a kind of ELISA kit of qualitative or detection by quantitative lactoferrin, comprising: detect antibody, antibody diluent, bag be buffered liquid, confining liquid, PBS damping fluid, lavation buffer solution and stop buffer; Wherein, described detection antibody is the anti-Bovinelactoferrin polyclonal antibody of rabbit prepared by the present invention.
Described bag is buffered liquid preferably: solvent is ultrapure water, and solute is Na
2cO
3and NaHCO
3, described solute Na
2cO
3and NaHCO
3be 0.05M, pH be 9.6 in the bag concentration be buffered in liquid;
Described confining liquid is preferably: solvent is ultrapure water, and solute is oralbumin, and the concentration of described solute oralbumin in Block buffer is 1%;
Described PBS damping fluid is preferably: solvent is ultrapure water, and solute is NaCl, KCl, KH
2pO
4and Na
2hPO
4, described solute NaCl, KCl, KH
2pO
4and Na
2hPO
4concentration in described PBS damping fluid is respectively 8g/L, 0.2g/L, 0.2g/L and 0.46g/L, and pH value is 7.4;
Described lavation buffer solution is preferably: solvent is described PBS damping fluid, and solute is tween 20, and the concentration of described solute tween 20 in described lavation buffer solution is 50mL/L;
Described antibody diluent is preferably: solvent is described PBS damping fluid, and solute is tween 20 and gelatin, and described solute tween 20 and the concentration of gelatin in described antibody diluent are respectively 10mL/L and 0.01M;
Described stop buffer is preferably the aqueous sulfuric acid of 2M;
Described substrate solution A is preferably: solvent is ultrapure water, and solute is Urea Peroxide, Na
2hPO
4, citric acid and tween 20, Urea Peroxide, Na
2hPO
4, citric acid and tween 20 concentration in described substrate solution A is respectively 1g/L, 14.2g/L, 9.42g/L and 0.1mL/L;
Described substrate solution B is preferably: solvent is ultrapure water, and solute is tetramethyl benzidine, citric acid and Sulfothiorine, and tetramethyl benzidine, citric acid and the Sulfothiorine concentration in described substrate solution B is respectively 0.4g/L, 1.83g/L and 0.1g/L.
Antibody of the present invention can lactoferrin in specific binding milk-product and in mammary tissue.The competitive Enzyme linked immunosorbent assay method detection lactoferrin that the present invention sets up for detecting antibody with the anti-Bovinelactoferrin polyclonal antibody of prepared rabbit, the IC50 value of competitive ELISA is 24.5 μ g/mL, detection is limited to 11.2 μ g/mL, and detecting linearity range is 8-40 μ g/mL.The anti-Bovinelactoferrin polyclonal antibody of rabbit of the present invention may be used for identifying the lactoferrin in milk-product, for the lactoferrin studied further in the lactoferrin of cow mammary gland synthesis and detection by quantitative milk-product provides effective scientific research material.
Rabbit provided by the invention anti-Bovinelactoferrin polyclonal antibody has that purity is high, antibody titer is high, high specificity, with low cost, sensing range is wide, easy and simple to handle, the result advantage such as reliable rapidly, and the ELISA detection kit containing this antibody can be widely used in the qualitative or detection by quantitative of lactoferrin in cow mammary gland, milk and milk products.
Accompanying drawing explanation
Fig. 1 purified antibodies electrophorogram; 1 is protein Marker, and 2 is polyclonal antibody after purifying.
The anti-Bovinelactoferrin polyclonal antibody of Fig. 2 rabbit is tired curve.
The competitive ELISA working curve of polyclonal antibody prepared by Fig. 3 the present invention and lactoferrin; The OD value that protein concentration is 0 (negative control), the lactoferrin of 8 μ g/mL, 12 μ g/mL, 16 μ g/mL, 20 μ g/mL, 24 μ g/mL, 28 μ g/mL, 32 μ g/mL, 36 μ g/mL and 40 μ g/mL is corresponding is respectively: 2.300,2.120,1.825,1.537,1.322,1.166,1.045,0.916,0.830 and 0.714, inhibiting rate is respectively 100%, 92.19%, 79.37%, 66.84%, 57.48%, 50.71%, 45.46%, 39.83%, 36.11% and 31.05%.
Fig. 4 Western-blot method qualification lactoferrin antibodies specific (M: protein molecular weight standard; 1-2: lactoferrin).
Fig. 5 Western-blot method identifies the reactivity of the lactoferrin extracted in antibody prepared by the present invention and cow mammary gland tissue and milk preparation; M: protein molecular weight standard; 1-2: sample of breast tissue; 3: powdered milk sample.
The competitive ELISA working curve of Fig. 6 Sigma antibody and lactoferrin.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.It should be understood that described embodiment is only exemplary, any restriction is not formed to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments or replacement all fall into protection scope of the present invention.
The preparation of the anti-Bovinelactoferrin polyclonal antibody of embodiment 1 rabbit and qualification
1 material and test method
1.1 test materials
Test materials comprises lactoferrin standard substance (Sigma Co., USA, purity>=85%), Al (OH)
3adjuvant (American I nvivoGen company), CpGODN adjuvant (Sangon Biotech's synthesis), Protein A-agarose-4B (ProteinA-Sepharose4B, Sigma Co., USA), magnetic bead (
m-280SheepAnti-RabbitIgG, Dynal company of the U.S.), the immunoglobulin G (IgG-HRP, Wuhan doctor's moral company) of goat-anti rabbit horseradish peroxidase-labeled, radioimmuno-precipitation assay (RIPA) lysate (the green skies company in Shanghai), BCA quantification of protein test kit (Kang Run Cheng Ye bio tech ltd, Beijing), DAB horseradish peroxidase colouring reagents box (Kang Run Cheng Ye bio tech ltd, Beijing).
1.2 experimental animals and feeding and management
4 purebred new zealand white rabbits of health are chosen in test, and single cage is raised, every day fed granules feed, free choice feeding and drinking-water.To thoroughly cleaning rabbit home and sterilizing before raising, adapt to feeding environment after 1 week until rabbit, ear edge vein exploitating blood, measure its serum and lactoferrin reactionless, therefore carry out immunity by immune programme for children.
1.3 immune programme for children and sero-fast preparation
The 1st week on-test, 0.5mg lactoferrin and 50 μ gCpG adjuvants are dissolved in 1mLPBS, with Al (OH)
3(1:5, v/v) after mixing, adopt subcutaneous multi-point injection method, every rabbit injection 1mL contains the protein solution (containing 500 μ g lactoferrins) of composite adjuvant, after 4 weeks, same method dorsal sc multi-point injection contains the lactoferrin (containing 250 μ g lactoferrins) of this composite adjuvant, and later every 2 weeks repeat 1 time, the 7th day ear edge vein exploitating blood after each immunity, ELISA method detects antiserum titre.Within the 7th day after the 4th immunity, carry out Culling heart blood.After coagulation of blood, in the centrifugal 20min of 3000 × g/min, get rearmounted-80 DEG C of supernatant packing and save backup.
1.4 immunomagnetic beads method purifying antiserum(antisera)s
Getting 100 μ L magnetic beads adds in 2mL centrifuge tube, supernatant liquor is sucked after magnetic separator is placed 3min, add 300 μ L and dilute antiserum(antisera) (1:100), inclination slow circumvolve 10min is kept under 4 DEG C of conditions, supernatant liquor is sucked after then leaving standstill 3min, with PBS (pH=7.4) resuspended magnetic bead, after repeated washing 3 times, centrifuge tube is again placed 3min and is sucked supernatant liquor on magnetic separator, add 0.1mol/L sodium citrate buffer (pH=3.0), vortex concussion 3min, and then on magnetic separator, place Aspirate supernatant after 2min, repeated washing 3 times, gained supernatant liquor is mixed, obtain purified antibodies.
1.5 protein A-sepharose affinity chromatographies are further purified antibody
In conjunction with the 0.1mol/LTris-HCl damping fluid (pH=8.6) that/lavation buffer solution is containing 1MNaCl, first balance Protein A-agarose-4B with combination/lavation buffer solution before loading and fill post, wash away the impurity such as contained protein and sanitas with 1mL/min flow velocity, wait to flow out elutriant OD
280can loading during <0.02.Serum sample (1:4, v/v) after being diluted by combination/lavation buffer solution adds in post, enters after post bed until whole serum sample, rinses pillar and makes damping fluid take-off rate be about 1mL/min, treat elutriant OD with combination/lavation buffer solution
280elution requirement is changed during <0.02, with 3.5% glacial acetic acid (pH=2.8) wash-out, elutriant containing antibody is collected in the beaker containing level pad with in and elutriant pH to 7.3, wash-out antibody-solutions 10kDa super filter tube out concentrates.
Antibody purity after 1.6 sodium dodecyl sulfate-polyacrylamide gel electrophoresis purification Identification
The preparation separation gel of 12% and the concentrated glue of 5%, get 80 μ L antibody samples and 20 μ L5 × sample-loading buffer mixes, in boiling water, boil 5min.Every hole loading 12 μ L, voltage is adjusted to 80V and starts electrophoresis, after indicating dye enters separation gel, voltage is adjusted to 120V, and continue electrophoresis until dyestuff arrives at be about 1cm place stopping electrophoresis apart from separation gel lower end, stationary liquid is fixed, coomassie brilliant blue staining, destainer decolours, until band is clear and legible, to take pictures observation with gel imaging system.
1.7ELISA detects tiring of purified antibodies
Lactoferrin standard substance coating buffer is diluted to 0.31 μ g/mL, every hole 100 μ L coated elisa plate, 4 DEG C are spent the night, and do negative control with PBS, discard liquid in hole next day, every hole adds 200 μ L confining liquids (1% oralbumin (OVA)), put 37 DEG C of thermostat container 1h, after the liquid that inclines, wash 4 times with lavation buffer solution (PBST), incline rapidly after each standing 3min washings, pats dry.Get different dilution purified antibodies (1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 and 1:128000), each extent of dilution 3 repetition, 37 DEG C of incubation 1h, then incline liquid, PBST washs, and incline liquid, pats dry.Every hole adds the goat anti-rabbit igg-HRP antibody that 100 μ L dilute by 1:5000, and 37 DEG C of incubation 1h, PBST wash, and incline liquid, pats dry.Every hole adds the freshly prepared substrate solution A of 100 μ L and substrate solution B, incubated at room 20min.Every hole adds 50 μ L stop buffer (H
2sO
4, 2mol/L), in microplate reader, measure the absorbance under 450nm after 5min.If treat gaging hole OD
450be more than or equal to 2.1 times of negative control hole, namely think positive value, thus draw tiring of antibody.
The mensuration of 1.8 lactoferrin working curves and detection limit
The polyclonal antibody prepared with the present invention detects lactoferrin
Detect the foundation of the typical curve of lactoferrin
(1) envelope antigen: get the coating buffer containing lactoferrin 0.50 μ g/mL; Add in enzyme plate by this coating buffer by the amount in 0.1mL/ hole, 4 DEG C are spent the night, and discard liquid in hole next day;
(2) close: add confining liquid 200 μ L/ hole, in 37 DEG C of thermostat containers, close 1h; Abandon liquid, wash 3 times with washings, each 3min, pats dry.
(3) the adding of the polyclonal antibody prepared of the present invention and competition antigen: polyclonal antibody the present invention prepared is done after proper concn dilutes (1:8000), after mixing with different concns lactoferrin (40 μ g/mL, 36 μ g/mL, 32 μ g/mL, 28 μ g/mL, 24 μ g/mL, 20 μ g/mL, 16 μ g/mL, 12 μ g/mL, 8 μ g/mL) standard solution respectively with equal-volume, room temperature reaction 15min, every hole adds the above-mentioned mixed solution of 0.1mL, and it is parallel that each standard substance do 3 holes; 37 DEG C of incubation 1h, then incline liquid, and PBST washs, and incline liquid, pats dry.
(4) the adding of ELIAS secondary antibody: every hole adds the goat anti-rabbit igg-HRP antibody that 100 μ L antibody diluents dilute by 1:5000, and 37 DEG C of incubation 1h, after abandoning liquid, wash 4 times with PBST, incline liquid, pats dry.
(5) develop the color: get substrate solution (substrate solution A, B being mixed according to the ratio of 1:1), every hole adds 100 μ L, 37 DEG C of lucifuge reaction 20min.
(6) stop: every hole adds 50 μ L stop buffer (H
2sO
4, 2mol/L), in microplate reader, measure the absorbance under 450nm after 5min.
Result calculates: with the Ig value of the concentration of lactoferrin standardized solution (ng/mL) for X-coordinate, take inhibiting rate as ordinate zou, production standard working curve, the inhibiting rate of calculating antibody and lactoferrin.
The mensuration of 1.9 mammary tissues and lactoferrin in dairy products concentration
The cow mammary gland tissue sample gathered from slaughterhouse is put into Liquid nitrogen storage rapidly.During mensuration, after sample of breast tissue is taken out, pour liquid nitrogen into grind, take a certain amount of sample of breast tissue, fully be dissolved in the PBS of 5 times of quality, add RIPA lysate and proteinase inhibitor simultaneously, the centrifugal 10min of 12000 × g, get supernatant liquor, detect the content of total protein in measuring samples with BCA determination of protein concentration test kit.Get 1mL liquid state milk, add after distilled water dilutes, the centrifugal 10min of 3000 × g, removes upper-layer fat.Adding dilute hydrochloric acid adjust ph is 4.6, removes casein, obtains lactoferrin crude product.Get 3g powdered milk sample, add water and be settled to 100mL, centrifugal segregation fat and casein after, obtain lactoferrin crude product.Utilize lactoferrin to measure working curve, detect the content of lactoferrin in mammary tissue, liquid state milk and milk powder crude extract.
Antibodies specific and reactivity after 1.10 immunoblottings (Western-blot) purification Identification
Be dissolved in PBS solution by lactoferrin sterling, cow mammary gland tissue, liquid state milk and milk powder pretreatment mode are with 1.9.Using total protein concentration as SDS-PAGE applied sample amount foundation, prepare 12% separation gel and 5% concentrated glue, to lactoferrin sterling, cow mammary gland tissue and powdered milk sample carry out electrophoresis, after electrophoresis terminates, protein transduction is moved on on the PVDF membrane (PVDF) in advance with methyl alcohol activation, the chicken serum of 0.5% is utilized to close, after lactoferrin Anti-TNF-α liquid solution being pressed 1:6000 dilution, incubated at room 2h, goat-anti rabbit horseradish peroxidase-labeled IgG antibody (1:5000 dilution) is added after PBST washes, PBST washes film, scanning result after DAB chromogenic reagent.
2 test-results
2.1 lactoferrin polyclonal antibody purity
As shown in Figure 1, the lactoferrin polyclonal antibody heavy chain obtained after immunomagnetic beads method and protein A-sepharose affinity chromatography 2 step purifying is high-visible for SDS-PAGE result, shows to obtain the higher lactoferrin polyclonal antibody of purity by purifying.
2.2 lactoferrin polyclonal antibodies are tired
Fig. 2 is the graphic representation of tiring of antibody, and antibody is after purifying, and recording its concentration with BCA determination of protein concentration test kit is 11.02mg/mL, and ELISA method detects tiring as 1:128000 of antibody.Chessboard method determines the optimal concentration of envelope antigen and antibody purification simultaneously.With coating buffer dilution lactoferrin standard substance to 5,2.5,1.25,0.625,0.3125 μ g/mL, dilution antibody is to 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 and 1:128000.Chessboard method result shows, when envelope antigen concentration is 0.625 μ g/mL and antibody dilution multiple is 1:64000, and OD
450value is closest to 1.
2.3 polyclonal antibody detection limit measurement results
As shown in Figure 3, linear equation is Y=88.329X-338 (R to test-results
2=0.9985);
Inhibiting rate (%)=(absorbance (B0) of absorbance (the B)/negative control hole in 1-standard solution hole) × 100%.
The OD value that in Fig. 3, protein concentration is 0 (negative control), the lactoferrin of 8 μ g/mL, 12 μ g/mL, 16 μ g/mL, 20 μ g/mL, 24 μ g/mL, 28 μ g/mL, 32 μ g/mL, 36 μ g/mL and 40 μ g/mL is corresponding is respectively: 2.300,2.120,1.825,1.537,1.322,1.166,1.045,0.916,0.830 and 0.714; Inhibiting rate is respectively 100%, 92.19%, 79.37%, 66.84%, 57.48%, 50.71%, 45.46%, 39.83%, 36.11% and 31.05%.
Test-results shows, the IC50 value of the competitive ELISA of the detection lactoferrin set up with polyclonal antibody prepared by the present invention is 24.5 μ g/mL, detects and is limited to 11.2 μ g/mL, and detecting linearity range is 8-40 μ g/mL.
The mensuration of 2.4 mammary tissues and lactoferrin in dairy products concentration
Working curve is utilized to measure sample of breast tissue and lactoferrin in dairy products concentration, to record in 5 parts of sample of breast tissue lactoferrin concentration scope at 14.72 μ g/g total protein to 18.64 μ g/g total proteins, mean value is 16.13 μ g/g total proteins, relative standard deviation is 11.05%, in liquid state milk, lactoferrin concentration is close to 0, in milk powder, lactoferrin concentration scope is 4.91 μ g/g to 5.73 μ g/g, and mean value is 5.28 μ g/g, and relative standard deviation is 6.42%.
2.5 lactoferrin antibodies specifiies
As shown in Figure 4, the antibody after purifying can be combined with lactoferrin standard substance specificity Western-blot result, shows that the antibody that the present invention obtains can specific recognition lactoferrin.
The Western-blot result display of Fig. 5, the lactoferrin extracted in antibody of the present invention after purifying and cow mammary gland tissue has reactivity, also there is atopy to the lactoferrin in milk powder simultaneously, can be used for detecting the lactoferrin in cow mammary gland tissue and milk preparation.
The anti-Bovinelactoferrin polyclonal antibody of test example 1 rabbit of the present invention and commercial antibody performance comparison test
1 tires comparison test
1.1 test method
Lactoferrin standard substance coating buffer is diluted to 5,2.5,1.25,0.625,0.31 μ g/mL, every hole 100 μ L coated elisa plate, 4 DEG C are spent the night, and do negative control with PBS, discard liquid in hole next day, every hole adds 200 μ L confining liquids (1% oralbumin (OVA)), put 37 DEG C of thermostat container 1h, after the liquid that inclines, wash 4 times with lavation buffer solution (PBST), incline rapidly after each standing 3min washings, pats dry.Get different dilution purified antibodies and commercial antibody (purchased from American Sigma company) (1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 and 1:128000), each extent of dilution 3 repetition, 37 DEG C of incubation 1h, then incline liquid, PBST washs, incline liquid, pats dry.Every hole adds the goat anti-rabbit igg-HRP antibody that 100 μ L dilute by 1:5000, and 37 DEG C of incubation 1h, PBST wash, and incline liquid, pats dry.Every hole adds the freshly prepared substrate solution A of 100 μ L and substrate solution B, incubated at room 20min.Every hole adds 50 μ L stop buffer (H
2sO
4, 2mol/L), in microplate reader, measure the absorbance under 450nm after 5min.If treat gaging hole OD
450be more than or equal to 2.1 times of negative control hole, namely think positive value, thus draw tiring of antibody.
1.2 test-results
The anti-Bovinelactoferrin polyclonal antibody of rabbit prepared by the embodiment of the present invention 1 and Sigma sell antibody titer results contrast in table 1 and table 2.The anti-Bovinelactoferrin polyclonal antibody of rabbit prepared by the present invention is tired as 1:128000, and the anti-Bovinelactoferrin polyclonal antibody that Sigma sells is tired and is only 1:32000.
Polyclonal antibody prepared by table 1 the present invention tires (OD value)
| 1 | 2 | 3 | 4 | 5 | |
| A | 2.737 | 2.957 | 2.723 | 2.75 | 2.826 |
| B | 2.506 | 2.846 | 2.449 | 2.42 | 2.186 |
| C | 3.041 | 3.013 | 2.718 | 2.435 | 1.951 |
| D | 3.184 | 2.827 | 2.514 | 1.864 | 1.353 |
| E | 2.73 | 2.451 | 2.205 | 1.214 | 0.858 |
| F | 2.091 | 1.89 | 1.327 | 1.011 | 0.55 |
| G | 1.308 | 1.206 | 0.822 | 0.406 | 0.331 |
| H | 0.169 | 0.119 | 0.136 | 0.146 | 0.126 |
Note: lactoferrin is diluted to 5,2.5,1.25,0.625,0.3125 μ g/mL, add in 1-5 row successively, the antibody purification (1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 and 1:128000) of the different Dilution ratio of A-G behavior, H behavior PBS negative control, in table, data are 3 mean values repeated.
Table 2Sigma sells the titration (OD value) of Bovinelactoferrin antibody
| 1 | 2 | 3 | |
| A | 3.296 | 3.373 | 2.537 |
| B | 2.301 | 2.121 | 1.186 |
| C | 1.167 | 1.019 | 0.492 |
| D | 0.561 | 0.452 | 0.236 |
| E | 0.403 | 0.228 | 0.141 |
| F | 0.157 | 0.132 | 0.097 |
| G | 0.115 | 0.093 | 0.082 |
| H | 0.093 | 0.073 | 0.074 |
Note: lactoferrin is diluted to 5,2.5,1.25 μ g/mL, add in 1-3 row successively, the Sigma of the different Dilution ratio of A-G behavior sells antibody (1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 and 1:128000), H behavior PBS negative control, in table, data are 3 mean values repeated.
Sigma antibody concentration is 15.8mg/mL, and chessboard method determines the optimal concentration of envelope antigen and Sigma antibody simultaneously.With coating buffer dilution lactoferrin standard substance to 5,2.5 and 1.25 μ g/mL, dilution antibody is to 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000 and 1:128000.Chessboard method result shows, when envelope antigen concentration is 2.5 μ g/mL and antibody dilution multiple is 1:8000, and OD
450value is closest to 1.
2 working curve comparison tests
2.1 test method
The inhibiting rate between Sigma antibody and lactoferrin is measured with competitive ELISA.Step is with 1.8 of embodiment 1.According to OD
450value drawing standard curve, calculates the inhibiting rate of this antibody and lactoferrin.
2.2 test-results
The inhibiting rate of the anti-Bovinelactoferrin polyclonal antibody of rabbit prepared by the present invention and lactoferrin is shown in Fig. 6, suppress antigen and the anti-Bovinelactoferrin polyclonal antibody of rabbit prepared by the present invention in conjunction with 50% time lactoferrin concentration (IC
50) be 33.5 μ g/mL.
Competitive ELISA the results are shown in Figure 6, and (standard specimen establishes 8 concentration gradients, from the initial doubling dilution of 40 μ g/mL, each concentration establishes 3 repetitions), visible according to Fig. 6, suppress antigen and the Bovinelactoferrin polyclonal antibody that Sigma sells in conjunction with 50% time lactoferrin concentration (IC
50) be 33.5 μ g/mL.
3 specificity comparison tests
3.1 test method
Wrap by enzyme mark version respectively with 0.625 μ g/mL casein and 2.5 μ g/mL lactoferrin sterlings, every hole 100 μ L, 4 DEG C are spent the night, and do negative control with PBS, discard liquid in hole next day, every hole adds 200 μ L confining liquids (1% oralbumin (OVA)), put 37 DEG C of thermostat container 1h, after the liquid that inclines, wash 4 times with lavation buffer solution (PBST), incline rapidly after each standing 3min washings, pats dry.To make purified antibodies (1:64000) and Sigma sale antibody (1:8000) by oneself, each extent of dilution 3 repetition, 37 DEG C of incubation 1h, then incline liquid, and PBST washs, and incline liquid, pats dry.Every hole adds the goat anti-rabbit igg-HRP antibody that 100 μ L dilute by 1:5000, and 37 DEG C of incubation 1h, PBST wash, and incline liquid, pats dry.Every hole adds the freshly prepared substrate solution A of 100 μ L and substrate solution B, incubated at room 20min.Every hole adds 50 μ L stop buffer (H
2sO
4, 2mol/L), in microplate reader, measure the absorbance under 450nm after 5min.
3.2 test-results
From the ELISA test-results of table 3, the embodiment of the present invention 1 prepare the anti-Bovinelactoferrin polyclonal antibody of rabbit and from Sigma buy Bovinelactoferrin polyclonal antibody only lactoferrin specificity is combined, and with casein without association reaction, all have and have good specificity.
Table 3ELISA test-results
| Casein bag quilt | Lactoferrin bag quilt | |
| Antibody prepared by the present invention | 0.131 | 1.009 |
| Sigma antibody | 0.136 | 1.007 |
Test example 2 antigen immune optimum program shaker test
1.1 antigen immune dose screenings
Select 6 purebred new zealand white rabbits of health, be divided into 3 groups at random.First week on-test, respectively 0.1mg, 0.5mg and 1.0mg lactoferrin and 50 μ gCpG adjuvants are dissolved in 1mLPBS, with Al (OH)
3(1:5, v/v) after mixing, adopt subcutaneous multi-point injection method, every rabbit injection 1mL contains the protein solution (respectively containing 100,500 and 1000 μ g lactoferrins) of composite adjuvant, after 4 weeks, same method dorsal sc multi-point injection contains the lactoferrin (respectively containing 50,250 and 500 μ g lactoferrins) of this composite adjuvant, and later every 2 weeks repeat 1 time, the 7th day ear edge vein exploitating blood after each immunity, ELISA method detects antiserum titre.The results are shown in Table 4.
As shown in Table 4, antibody titer raises to some extent with the raising of immunizing dose, consider that immunizing dose is too high and be unfavorable for animal health and antigen absorption, therefore in the present invention, adopt 500 μ g lactoferrins to carry out primary immune, apply 250 μ g lactoferrins and carry out booster immunization.
Table 4 antigen immune dosage different gained antibody titer detected result
| Antigen injection dosage | 100μg | 500μg | 1000μg |
| Latter 7 days of first time immunity | 1：500 | 1：600 | 1：800 |
| Latter 7 days of second time immunity | 1：20000 | 1：36000 | 1：42000 |
| Latter 7 days of third time immunity | 1：48000 | 1：64000 | 1：70000 |
1.2 antigen immune adjuvant screenings
Select 4 purebred new zealand white rabbits of health, be divided into 2 groups at random.The 1st week on-test, 0.5mg lactoferrin and 50 μ gCpG adjuvants are dissolved in 1mLPBS, with Al (OH)
3(1:5, v/v) after mixing, adopt subcutaneous multi-point injection method to wherein 1 group of New Zealand white rabbit immunity, every rabbit immunity 1mL contains the protein solution (containing 500 μ g lactoferrins) of composite adjuvant, after 4 weeks, same method dorsal sc multi-point injection contains the lactoferrin (containing 250 μ g lactoferrins) of this composite adjuvant, and later every 2 weeks repeat 1 time, the 7th day ear edge vein exploitating blood after each immunity, ELISA method detects antiserum titre.The 1st week on-test, injection contained the emulsion of equivalent lactoferrin and Freund's complete adjuvant to other one group of New Zealand white rabbit, the subcutaneous multi-point injection method of same employing, every rabbit injection 1mL emulsion (containing 500 μ g lactoferrins), after 4 weeks, same method dorsal sc multi-point injection is through Freund's incomplete adjuvant emulsification lactoferrin (containing 250 μ g lactoferrins) completely, every 2 weeks later repeat 1 time, the 7th day ear edge vein exploitating blood after each immunity, ELISA method detects antiserum titre.
Test-results is in table 5.From test-results, adopt mixing adjuvant can significantly improve tiring of antibody than freund's adjuvant.
Table 5 immunological adjuvant different gained antibody titer detected result
Test example 3 antibody purification mode shaker test
1 saturated ammonium sulphate method purifying antiserum(antisera)
Get 500 μ L serum and add isopyknic phosphate buffered saline buffer (PBS, 0.01mol/L, pH7.4), dropwise add 250 μ L saturated ammonium sulphate solution under ice bath, vortex mixes, 4 DEG C of standing 30min, 4 DEG C, the centrifugal 15min of 4000 × g/min, get supernatant liquor.In supernatant liquor, dropwise add 1000 μ L saturated ammonium sulphate solution, vortex mixes, and 4 DEG C of standing 30min centrifugally abandon supernatant.Precipitation is dissolved with 500 μ LPBS, dropwise adds 250 μ L saturated ammonium sulphate solution in the solution, and limit edged stirs, 4 DEG C of standing 30min, and 4 DEG C centrifugal, retains precipitation.Precipitation continues to dissolve with PBS, drips saturated ammonium sulphate solution, leaves standstill, centrifugal, retains precipitation.Precipitation is dissolved in PBS, loads 4 DEG C of dialysed overnight in dialysis tubing.
2 immunomagnetic beads method associating protein A-sepharose affinity chromatography purifying antiserum(antisera)s
Getting 100 μ L magnetic beads adds in 2mL centrifuge tube, supernatant liquor is sucked after magnetic separator is placed 3min, add 300 μ L and dilute antiserum(antisera) (1:100), inclination slow circumvolve 10min is kept under 4 DEG C of conditions, supernatant liquor is sucked after then leaving standstill 3min, with PBS (pH=7.4) resuspended magnetic bead, after repeated washing 3 times, centrifuge tube is again placed 3min and is sucked supernatant liquor on magnetic separator, add 0.1mol/L sodium citrate buffer (pH=3.0), vortex concussion 3min, and then on magnetic separator, place Aspirate supernatant after 2min, repeated washing 3 times, gained supernatant liquor is mixed, obtain purified antibodies.
In conjunction with the 0.1mol/LTris-HCl damping fluid (pH=8.6) that/lavation buffer solution is containing 1MNaCl, first balance Protein A-agarose-4B with combination/lavation buffer solution before loading and fill post, wash away the impurity such as contained protein with 1mL/min flow velocity, wait to flow out elutriant OD
280can loading during <0.02.Serum sample (1:4, v/v) after being diluted by combination/lavation buffer solution adds in post, enters after post bed until whole serum sample, rinses pillar and makes damping fluid take-off rate be about 1mL/min, treat elutriant OD with combination/lavation buffer solution
280during <0.02 change elution requirement, with 3.5% glacial acetic acid (pH=2.8) wash-out, the elutriant containing antibody is collected in the beaker containing level pad with in elutriant pH to 7.3.Wash-out antibody-solutions 10kDa super filter tube out concentrates.
By adopting above two kinds of method gained purification antibody to detect purity through SDS-PAGE method, adopt BCA kit measurement antibody concentration.
The purity of above-mentioned two kinds of method gained purification antibody and concentration are in table 6.
Table 6 different method of purification gained antibody compares
| Content | Purity | Concentration |
| Saturated ammonium sulphate method | 62％ | 9.25mg/mL |
| Immunomagnetic beads method associating protein A-sepharose affinity chromatography | 87％ | 11.02mg/mL |
From test-results, the antibody purity that immunomagnetic beads method associating protein A-sepharose affinity chromatography purifying antiserum(antisera) obtains reaches 87%, and the antibody purity adopting saturated ammonium sulphate method purifying antiserum(antisera) to obtain is only 62%, far below the antibody purity that immunomagnetic beads method associating protein A-sepharose affinity chromatography purifying antiserum(antisera) obtains; The antibody concentration that immunomagnetic beads method associating protein A-sepharose affinity chromatography purifying antiserum(antisera) obtains is 11.02mg/mL, and the antibody concentration degree adopting saturated ammonium sulphate method purifying antiserum(antisera) to obtain is only 9.25mg/mL, far below the antibody concentration that immunomagnetic beads method associating protein A-sepharose affinity chromatography purifying antiserum(antisera) obtains.
Claims (10)
1. a preparation method for the anti-Bovinelactoferrin polyclonal antibody of rabbit, is characterized in that, comprise the following steps:
(1) lactoferrin is mixed with immunological adjuvant, obtain the lactoferrin solution containing composite adjuvant;
(2) with the lactoferrin solution immunize New Zealand white rabbits containing composite adjuvant, antiserum(antisera) is obtained; Centrifugal, get supernatant, obtain antiserum(antisera);
(3) antiserum(antisera) is carried out purifying with immunomagnetic beads method and protein A-sepharose affinity chromatography successively, obtain the anti-Bovinelactoferrin polyclonal antibody of rabbit of purifying.
2. according to preparation method according to claim 1, it is characterized in that: lactoferrin and CpG adjuvant are dissolved in PBS and obtain mixing solutions, then with Al (OH)
3mixing, obtains the lactoferrin solution containing composite adjuvant; Preferably, 0.5mg lactoferrin and 50 μ gCpG adjuvants are dissolved in 1mLPBS obtain mixing solutions; By mixing solutions and Al (OH)
3mix according to the volume ratio of 1:5, obtain the lactoferrin solution containing composite adjuvant.
3. according to preparation method according to claim 1, it is characterized in that, described immunization ways comprises: adopt the mode of subcutaneous multi-point injection method by the lactoferrin solution immunize New Zealand white rabbits containing composite adjuvant, and the number of times of immunity is 4 times.
4. according to preparation method according to claim 3, it is characterized in that: wherein, after the 1st time immune 4 weeks, carry out the 2nd immunity; Every 2 weeks later repeat immunity 1 time, carry out the 3rd time and the 4th immunity; Within the 7th day after the 4th immunity, collect antiserum(antisera); Preferably, the 1st time immune time, every rabbit immunity 500 μ g lactoferrins; 2nd time, the 3rd time and the 4th immunity time, every rabbit immune 250 μ g lactoferrins at every turn.
5. according to preparation method according to claim 1, it is characterized in that, described immunomagnetic beads method purifying antiserum(antisera) comprises: joined by magnetic bead in centrifuge tube, magnetic separator is placed, suck supernatant liquor, add the antiserum(antisera) of dilution, leaves standstill, sucks supernatant liquor; Resuspended magnetic bead, repeated washing, sucks supernatant liquor; Add sodium citrate buffer, vortex shakes, and leaves standstill, Aspirate supernatant, repeated washing, is mixed by gained supernatant liquor, obtains purified antibodies;
Preferably, getting 100 μ L magnetic beads adds in 2mL centrifuge tube, supernatant liquor is sucked after magnetic separator is placed 3min, add the antiserum(antisera) of 300 μ L according to the dilution proportion of 1:100, inclination slow circumvolve 10min is kept under 4 DEG C of conditions, supernatant liquor is sucked after then leaving standstill 3min, with phosphate buffered saline buffer (PBS) the resuspended magnetic bead of pH=7.4, after repeated washing 3 times, centrifuge tube is again placed 3min and is sucked supernatant liquor on magnetic separator, add the 0.1mol/L sodium citrate buffer of pH3.0, vortex concussion 3min, and then on magnetic separator, place Aspirate supernatant after 2min, repeated washing 3 times, gained supernatant liquor is mixed, obtain purified antibodies.
6. according to preparation method according to claim 1, it is characterized in that, described mode of antiserum(antisera) protein A-sepharose affinity chromatography being carried out purifying comprises:
First balance Protein A-agarose-4B with combination/lavation buffer solution before loading and fill post, washing away the impurity such as contained protein, waiting to flow out elutriant OD
280during <0.02; Antiserum(antisera) after being diluted by combination/lavation buffer solution adds in post, enters after post bed until whole serum sample, rinses pillar, treat elutriant OD with combination/lavation buffer solution
280during <0.02, with the 3.5% glacial acetic acid wash-out of pH=2.8, the elutriant containing antibody is collected in the container containing level pad, concentrated, to obtain final product;
Preferably, described combination/lavation buffer solution is the 0.1mol/LTris-HCl damping fluid containing 1MNaCl, its pH=8.6;
First balance Protein A-agarose-4B with combination/lavation buffer solution before loading and fill post, washing away impurity with 1mL/min flow velocity; Wait to flow out elutriant OD
280during <0.02, serum sample (1:4, v/v) after being diluted by combination/lavation buffer solution adds in post, enters after post bed until whole serum sample, rinse pillar with combination/lavation buffer solution and make damping fluid take-off rate be about 1mL/min, treating elutriant OD
280during <0.02, with the 3.5% glacial acetic acid wash-out of pH=2.8, the elutriant containing antibody is collected in the container containing level pad with in and elutriant pH to 7.3;
Described concentrated be concentrate with 10kDa super filter tube.
7. the anti-Bovinelactoferrin polyclonal antibody of the rabbit obtained by any one preparation method of claim 1-6.
8. rabbit according to claim 7 anti-Bovinelactoferrin polyclonal antibody is qualitative or detection by quantitative is newborn, application in milk-product or mammary gland in lactoferrin.
9. an ELISA kit for qualitative or detection by quantitative lactoferrin, comprising: detect antibody, antibody diluent, bag be buffered liquid, confining liquid, PBS damping fluid, lavation buffer solution, stop buffer; It is characterized in that: described detection antibody is the anti-Bovinelactoferrin polyclonal antibody of rabbit according to claim 7.
10. test kit according to claim 9, is characterized in that:
Described bag is buffered liquid: solvent is ultrapure water, and solute is Na
2cO
3and NaHCO
3, described solute Na
2cO
3and NaHCO
3be 0.05M, pH be 9.6 in the bag concentration be buffered in liquid;
Described confining liquid is: solvent is ultrapure water, and solute is oralbumin, and the concentration of described solute oralbumin in Block buffer is 1%;
Described PBS damping fluid is: solvent is ultrapure water, and solute is NaCl, KCl, KH
2pO
4and Na
2hPO
4, described solute NaCl, KCl, KH
2pO
4and Na
2hPO
4concentration in described PBS damping fluid is respectively 8g/L, 0.2g/L, 0.2g/L and 0.46g/L, and pH value is 7.4;
Described lavation buffer solution is: solvent is described PBS damping fluid, and solute is tween 20, and the concentration of described solute tween 20 in described lavation buffer solution is 50mL/L;
Described antibody diluent is: solvent is described PBS damping fluid, and solute is tween 20 and gelatin, and described solute tween 20 and the concentration of gelatin in described antibody diluent are respectively 10mL/L and 0.01M;
Described stop buffer is: the aqueous sulfuric acid of 2M;
Described substrate solution A: solvent is ultrapure water, solute is Urea Peroxide, Na
2hPO
4, citric acid and tween 20, described Urea Peroxide, Na
2hPO
4, citric acid and tween 20 concentration in described substrate solution A is respectively 1g/L, 14.2g/L, 9.42g/L and 0.1mL/L;
Described substrate solution B: solvent is ultrapure water, solute is tetramethyl benzidine, citric acid and Sulfothiorine, and tetramethyl benzidine, citric acid and the Sulfothiorine concentration in described substrate solution B is respectively 0.4g/L, 1.83g/L and 0.1g/L.
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