CN106290893B - A method of based on Platinum Nanoparticles probe in detecting cow's milk beta lactoglobulin and its sensitization residue - Google Patents
A method of based on Platinum Nanoparticles probe in detecting cow's milk beta lactoglobulin and its sensitization residue Download PDFInfo
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Abstract
A method of based on Platinum Nanoparticles probe in detecting cow's milk beta lactoglobulin and its sensitization residue, beta lactoglobulin IgE epitopes series connection body protein is prepared by recombination and expression techniques, again respectively using connect body protein and beta lactoglobulin as antigen, routinely technology prepares corresponding polyclonal antibody, then prepares specific antibody through affinity purification.It is capture antibody with concatermer polyclonal antibody, the Platinum Nanoparticles probe of biotinylation beta lactoglobulin polyclonal antibody label is that detection antibody establishes Sandwich ELISA, light absorption value is detected through microplate reader, by establishing standard curve, the quantitative detection to anaphylactogen beta lactoglobulin in food and its sensitization residue is realized.The present invention has many advantages, such as that easy to operate, high sensitivity, specificity is good, the range of linearity is wide, related allergen and its sensitization residue in numerous food are detected for highly sensitive, high throughput analysis and provides a kind of effective method, and there is good promotion and application foreground.
Description
Technical field
The invention belongs to food analysis technical fields, are related to the highly sensitive inspection of food allergen and its sensitization residue content
Survey method.
Background technology
Cow's milk also through being often added in other food is often taken the photograph in people's daily life with abundant nutriment
The substance entered, while cow's milk, as one of eight major class allergenic foods, incidence of the Milk allergy in crowd is 0.3%~7.5%,
And the incidence in children is up to 0.1~7.5%, wherein the incidence in one-year-old infant below reaches 2%~3%.Food
The code committee（Codex Alimentarius Commission）, the European Food label instructions committee（European
Food Labeling Directive）And Food and Drug Adminstration of the US（FDA）It must all be listed in regulation food labelling all kinds of
Allergen, wherein including just ox milk and milk products.Detection to food allergen is the key link of food hypersenstivity management, with
Just risk assessment, production and mark are carried out to allergenic foods.Anaphylactogen complicated component in cow's milk, wherein beta lactoglobulin account for about ox
The 10% of content of milk protein, accounts for about the 50% of lactalbumin total amount, and in the milk allergies that mediate of IgE 82% patient to β-milk-globule
Protein allergies.Therefore, the detection for carrying out beta lactoglobulin is the key that protection Milk allergy crowd's legitimate rights and interests behave, also has
Conducive to low sensitization food is produced, to improve the quality of life of Milk allergy patient.
Currently, the detection technique of food allergen has immunological technique, polymerase chain reaction technology, high performance liquid chromatography
Technology, liquid phase/mass spectrometric hyphenated technique etc., wherein immunological technique is most ripe, the most frequently used.Antibody detects food in immunological method
Key effect is played in anaphylactogen.Antibody includes monoclonal antibody and polyclonal antibody, and wherein monoclonal antibody can only identify one
There is apparent deficiency when detecting sensitization residue in a epitope, antibody production techniques are more demanding, expensive, and primary
Property be difficult to obtain the cell strains of more plants of identification different epitopes.And Anti-TNF-α physical efficiency identifies multiple epitopes, preparation method is simple,
Economy can make up monoclonal antibody in Antibody preparation and the deficiency of detection anaphylactogen peptide fragment well.
The structure of protein determines its function, and as the albumen of food allergen, allergen epitope is to cause allergic reaction
Material base, including linear epitope and conformational epitope.Food can usually pass through heating, hair in processing and commercial Application
The processing such as ferment, enzymolysis, this makes anaphylactogen conformational epitope easily be destroyed, but linear epitope is then stablized relatively, is not easy to be destroyed.Profit
With epiposition vaccine principle, cow's milk beta lactoglobulin IgE linear epitope concatermers are built, epitope concatermer is prepared by prokaryotic expression
Albumen prepares epitope concatermer polyclonal antibody further according to immunological method.
Since metal nanoparticle has the characteristics that large specific surface area, easily with antibody coupling, it is widely used in highly sensitive
In detection method.Currently, have colloidal gold, nano silver, immunomagnetic beads, quantum dot, Platinum Nanoparticles etc. using more nano metal, and
Platinum Nanoparticles also have catalysis H other than having the characteristics that metal nanoparticle2O2The characteristics of aoxidizing TMB colour developings.Therefore, life is utilized
Object element beta lactoglobulin polyclonal antibody prepares Platinum Nanoparticles probe with Platinum Nanoparticles, and establishes and exempted from based on the enzyme-linked of Platinum Nanoparticles probe
Epidemic disease absorption method（ELISA）It carries out detecting the content of beta lactoglobulin and its sensitization residue in food with sensitivity, while can comment
The power of valence foodstuff samples sensitization, assesses the height of food safety risk, is also provided for food allergen mark more reliable
Detection method.Currently, in China, in conjunction with Platinum Nanoparticles highly sensitive and IgE epitope polyclonal antibodies specificity to mistake in food
Quick former and its sensitization residue quantitative detection or blank.
Invention content
It is a kind of based on Platinum Nanoparticles probe in detecting cow's milk beta lactoglobulin and its sensitization it is a primary object of the present invention to propose
The method of property residue establishes β-milk-globule egg in a kind of IgE linear epitope recognition detection food highly sensitive based on Platinum Nanoparticles probe
The method of its sensitization residue of bletilla.This method is accurate, high sensitivity, specificity is good, the rate of recovery is high, the range of linearity is wide.
Technical scheme is as follows.
1, the preparation of beta lactoglobulin IgE linear epitopes concatermer polyclonal antibody.
Epiposition vaccine is the vaccine prepared based on epitope, by B cell epitope, t cell epitope and intervening sequence
Composition.Wherein B cell epitope is used for the humoral immune response of directional induction host, and by selecting the t cell epitope of specificity
Make the cellullar immunologic response of body orient to specific type to develop, intervening sequence, which has, preserves the independent immunogenicity of epitope, together
When avoid generating new epitope.
Based on epiposition vaccine design principle, with cow's milk beta lactoglobulin IgE linear epitopes（B：AA1~8、AA31~48、AA47
~ 60, AA67 ~ 78, AA75 ~ 86, AA127 ~ 144 and AA141 ~ 152）, t cell epitope（T：AA77~97）And intervening sequence（KK with
G）Based on.The antigenicity, surface accessibility and hydrophily of various combination concatermer are analyzed by DNAStar softwares,
The antigenicity of each epitope and hydrophily is set to be more than 0, while surface accessibility is more than 1；Recycle network server SOMPA to not
Concatermer secondary structure with combination is analyzed, and epitope is made to form β-bend and random coil structure.Believed by the above biology
The analysis of the epitope concatermer of method is ceased, finally obtained concatermer is combined as：T-K-K-B1-G-B6-G-B5-G-B3-G-
B7-G-B2-G-B4.Tandem gene is synthesized again, and cow's milk beta lactoglobulin epitope series connection weight is prepared by prokaryotic expression
Histone.Finally, using body protein of connecting as antigen, concatermer protein polyclone antibody is obtained by conventional method.
2, the purifying of concatermer polyclonal antibody.
First, beta lactoglobulin and CNBr-Sepharose 4B column materials are coupled, prepare immune affinity column.Again anti-blood
It is mixed with isometric PBS clearly, serum is slowly added in Sepharose 4B affinity columns, is incubated 30 min at room temperature, then
It is primary to recycle loading；Non-specific elution, then the 3 M MgCl with 10 times of column volumes are carried out with the PBS of 20 times of column volumes2It carries out
Specificity affords cow's milk beta lactoglobulin specific antibody.After purification, it is fought with the super filter tube that molecular cut off is 3 kDa
Body carries out desalination, and is concentrated into suitable volume.
3, identification of the concatermer polyclonal antibody to epitope peptide.
Because the amino acid number that the cow's milk beta lactoglobulin sensitization peptide fragment being detected in actual sample is included usually is wanted
More than the amino acid number of epitope itself.Therefore, in order to analyze prepared concatermer polyclonal antibody epitope recognition capability,
We devise epitope-blocking peptide, i.e., add at each IgE epitopes both ends and respectively add an amino acid, and synthesize this 7 IgE epitope peptides
And its blocking peptide.Recognition capability of the concatermer polyclonal antibody to epitope peptide and blocking peptide is analyzed using indirect competitive ELISA method.
After beta lactoglobulin standard items are diluted with carbonate buffer solution, 100 holes μ L/（Plate A）, overnight, PBST washes three to 4 °C of coatings
Secondary, 3 min buttons are dry every time.Separately take one piece of ELISA Plate（Plate B）, 3% gelatin solution 37 of 250 μ L is added in plate A and plate B per hole
It °C blockades.Plate B is cleaned after blockading 1 h, and the polypeptide of concatermer polyclonal antibody and concentration gradient is added per hole（PBS is control）Respectively
70 μ L, 37 °C of 1 h of incubation.Plate A is cleaned 3 times with PBST, and the reaction solution of 100 μ L antibody and polypeptide is taken to be added to plate from plate B
It in B, is cleaned after 37 °C of 1 h of incubation, 100 μ L OPD developing solutions is added per hole and add per hole after 37 °C are protected from light 15 min of incubation
Enter the H of 50 μ L, 2 M2SO4Reaction is terminated, surveys light absorption value with microplate reader immediately after.Various concentration peptide is calculated according to light absorption value
Inhibiting rate, concatermer polyclonal antibody can be reflected to different epitopes peptide and blocking peptide recognition capability according to inhibiting rate height
It is strong and weak.
4, the preparation of beta lactoglobulin polyclonal antibody.
Using cow's milk beta lactoglobulin as antigen, beta lactoglobulin polyclonal antibody is obtained by conventional method.
5, the biotinylation of beta lactoglobulin polyclonal antibody.
First, beta lactoglobulin polyclonal antibody is purified by step [0012].β-milk-globule that purifying is obtained again
Protein-specific polyclonal antibody presses 1 with long-chain biological element：20 molar ratio mixing, 2 h of ice bath, then it is extra with desalting column removing
Biotin.
6, the preparation of Platinum Nanoparticles probe.
Take 1 mL Platinum Nanoparticles solution（Grain size is about 20 nm）10,000 × g room temperatures centrifuge 15 min, remove supernatant, add 990
The PBS of μ L（pH 6.5）Platinum Nanoparticles precipitation is resuspended, adds the biotinylated beta lactoglobulin of 10 a concentration of 1mg/mL of μ L
Polyclonal antibody, it is 1 to make antibody and Platinum Nanoparticles mass ratio:100, overnight in 4 °C of couplings.Then 10,000 × g room temperatures centrifugation 15
Min removes the supernatant containing unmarked antibody, and 1 mL is added and preserves liquid（Containing 1% BSA, 0.1 % Triton X-100,5% sucrose
PBS, pH 6.5）The Platinum Nanoparticles probe of precipitation labelled antibody is resuspended.
7, the drafting based on the sandwich ELISA method detection beta lactoglobulin standard curve of Platinum Nanoparticles probe.
It is capture antibody with concatermer polyclonal antibody, the Platinum Nanoparticles probe of labelled antibody is detection antibody, β-milk-globule egg
White is detection antigen, determines capture antibody using chessboard method and detects antibody best effort concentration.100 are added in ELISA Plate per hole
The diluted capture antibody of μ L carbonate, overnight, PBST is washed three times 4 °C of coatings, 3 min and detains dry every time, 250 μ L are added per hole
2% gelatin solution, 37 °C are blockaded 0.5 h.After cleaning, the beta lactoglobulin of 100 μ L known concentration gradients of addition per hole, 37
°C be incubated 2 h.After cleaning, 100 μ L Platinum Nanoparticles probes, 37 °C of 1 h of incubation are added per hole.After cleaning, 100 μ L are added per hole
The Streptavidin of horseradish peroxidase-labeled, 37 °C of 0.5 h of incubation.After cleaning, 100 μ L TMB solution are added per hole,
After 37 °C are protected from light 15 min of incubation, the H of 50 μ L, 2 M is added per hole2SO4Reaction is terminated, then surveys light absorption value with microplate reader,
Beta lactoglobulin standard curve is drawn according to the relationship of light absorption value and antigen concentration.
8, sample pretreatment.
（1）Liquid diary product：Take a certain amount of liquid diary product in centrifuge tube, with 10,000g, 4 °C of refrigerated centrifuge
It centrifuges 15 min, removes fat deposit and precipitation, sampling, which is used, blockades liquid and be diluted in the detection range of linearity as measuring samples.
（2）Powdered dairy products：Claim a certain amount of sample to be diluted with PBS, is centrifuged after dissolving, removal precipitation and fat, supernatant
It uses and blockades liquid and be diluted in the detection range of linearity as measuring samples.
（3）Solid-state dairy products：Claim a certain amount of sample, wear into uniform powder, 1g powder is taken to be added to 20 mL extracting solutions
（20 mmol/L Tris-HCl, 2% Tween-20, pH 8.0）, 4 °C are stirred overnight extraction albumen.Again through 10,000 × g
Centrifuge 10 min, removal precipitation and fat in 4 °C, supernatant, which is used, to be blockaded liquid and be diluted in the detection range of linearity as measuring samples.
9, sample detection.
Food to be detected is pre-processed, detection sample is prepared.It is coated with according to the above method and captures antibody, closed and is clear
After washing, the 100 pretreated samples of μ L are added per hole, after being incubated cleaning, then is sequentially added by [0020] step and detects antibody, peppery
Streptavidin, the TMB solution of root peroxidase labelling, through H2SO4After terminating reaction, light absorption value is detected with microplate reader, suction
Light value brings the content that standard curve calculates beta lactoglobulin and its sensitization residue in test sample into.
It is an advantage of the invention that：1, the present invention is that antigen prepares Anti-TNF-α using cow's milk beta lactoglobulin epitope concatermer
Body has the advantages that specific recognition beta lactoglobulin IgE epitopes；2, the present invention utilizes Platinum Nanoparticles and Antibody preparation probe, tool
Have the advantages that highly sensitive；3, beta lactoglobulin and its sensitization in epitope antibodies and Platinum Nanoparticles probe quantitative detection milk product are based on
The sandwich ELISA method of residue has many advantages, such as that easy to operate, high sensitivity, specificity is good, the range of linearity is wide, is convenient for high pass
Amount detection；4, the method that the present invention is established will be that high-throughput High Sensitive Analysis detects related allergen and its cause in numerous food
Quick property residue provides a kind of effective method, has good promotion and application foreground.
Description of the drawings
Fig. 1 is recognition capability of the concatermer polyclonal antibody to epitope peptide and blocking peptide.B1-B7：Epitope peptide；b1-b7：Resistance
Disconnected peptide.
Fig. 2 detects the standard curve of beta lactoglobulin for built Sandwich ELISA.
Specific implementation mode
The present invention will be described further by example in detail below.
Embodiment 1：Quantitatively detect anaphylactogen beta-lactoglobulin content in sunlight plain chocolate.
1. sample pretreatment.
1 mL sunlight plain chocolate 10,000 × g, 4 °C are taken to centrifuge 15min, removal fat, supernatant dilutes 10 with liquid is blockaded,
As detection sample after 000 times.
2. sample detection.
（1）Coating：After concatermer polyclonal antibody is diluted to 3.5 μ g/mL with coating buffer, 100 μ L, 4 °C of packets are added per hole
It is stayed overnight.
（2）Washing：Use PBST（Containing 0.1% Tween-20）Washing 3 times, 3 min, button are dry every time.
（3）It blockades：Match 2% gelatin with PBS（Containing 0.1% Tween-20）Liquid is blockaded, 250 μ L are added per hole, 37 °C are incubated 0.5
h.After blockading, washed 3 times with PBST, 3 min, button are dry every time.
（4）Add antigen：It is with liquid is blockaded that beta lactoglobulin standard items are dilute in 0.49-16,000 ng/mL range inside gradients
It releases, 100 μ L standard items/sample is added per hole, in triplicate, 37 °C of 2 h of incubation.After incubation, washed 3 times with PBST,
3 min every time, button are dry.
（5）Add detection antibody：100 μ L after 200 times of Platinum Nanoparticles probe dilution, will be added per hole with liquid is blockaded, 37 °C are incubated 1
h.After incubation, washed 4 times with PBST, 3 min, button are dry every time.
（6）Add the Streptavidin of horseradish peroxidase-labeled：It will add horseradish peroxidase-labeled with liquid is blockaded
Streptavidin dilutes 60 times, and 100 μ L, 37 °C of 0.5 h of incubation are added per hole.After incubation, 4 times are washed with PBST, every time 3
Min, button are dry.
（7）Add developing solution：Add 100 μ L TMB solution per hole, 37 °C are protected from light 15 min of incubation.
（8）Survey light absorption value：After incubation, the H of 50 μ L, 2 M is added per hole2SO4Reaction is terminated, microplate reader is then used
Light absorption value is surveyed, absorbing wavelength is 450 nm, standard curve is drawn according to light absorption value, according to equation of linear regression and sample light absorption value
The content of beta lactoglobulin in sample is calculated, the average value detected three times is final detection level.
3. result.
Examination criteria curvilinear equation is y=0.26185x+0.1936, r2=0.99898, sensitivity is 0.12 ng/mL.Sun
The content of beta lactoglobulin is 3.50 mg/mL in light plain chocolate, and the content of beta lactoglobulin is about 3-4 mg/mL in cow's milk,
Illustrate that established detection method is feasible.
Embodiment 2：Quantitatively beta lactoglobulin sensitization residue in the super energy grace partial hydrolysis baby milk powder of detection nest
Content.
1. sample pretreatment.
The super energy grace partial hydrolysis baby milk powder of 0.2 g nest is weighed, addition is blockaded liquid and fully dissolved, and keeps albumen dense
Degree is 5 mg/mL.10,000 × g centrifuges 15min, removal fat and precipitation in 4 °C, and supernatant is diluted to 250 with liquid is blockaded
μ g/mL, 50 μ g/mL and 10 μ g/mL, as detection sample.
2. sample detection.
（1）Coating：After concatermer polyclonal antibody is diluted to 3.5 μ g/mL with coating buffer, 100 μ L, 4 °C of packets are added per hole
It is stayed overnight.
（2）Washing：Use PBST（Containing 0.1% Tween-20）Washing 3 times, 3 min, button are dry every time.
（3）It blockades：Match 2% gelatin with PBS（Containing 0.1% Tween-20）Liquid is blockaded, 250 μ L are added per hole, 37 °C are incubated 0.5
h.After blockading, washed 3 times with PBST, 3 min, button are dry every time.
（4）Add antigen：It is with liquid is blockaded that beta lactoglobulin standard items are dilute in 0.49-16,000 ng/mL range inside gradients
It releases, 100 μ L standard items/sample is added per hole, in triplicate, 37 °C of 2 h of incubation.After incubation, washed 3 times with PBST,
3 min every time, button are dry.
（5）Add detection antibody：100 μ L after 200 times of Platinum Nanoparticles probe dilution, will be added per hole with liquid is blockaded, 37 °C are incubated 1
h.After incubation, washed 4 times with PBST, 3 min, button are dry every time.
（6）Plus the Streptavidin of horseradish peroxidase-labeled：It will add horseradish peroxidase-labeled with liquid is blockaded
Streptavidin dilute 60 times, 100 μ L, 37 °C of 0.5 h of incubation are added per hole.After incubation, 4 times are washed with PBST, often
Secondary 3 min, button are dry.
（7）Add developing solution：Add 100 μ L TMB solution per hole, 37 °C are protected from light 15 min of incubation.
（8）Survey light absorption value：After incubation, the H of 50 μ L, 2 M is added per hole2SO4Reaction is terminated, microplate reader is then used
Light absorption value is surveyed, absorbing wavelength is 450 nm, standard curve is drawn according to light absorption value, according to equation of linear regression and sample light absorption value
The content of beta lactoglobulin in sample is calculated, the average value detected three times is final detection level.
3. result.
Examination criteria curvilinear equation is y=0.26185x+0.1936, r2=0.99898, sensitivity is 0.12 ng/mL.Sparrow
The content of beta lactoglobulin and its sensitization residue is 205.32 mg/kg in the super energy grace partial hydrolysis baby milk powder of nest.
Embodiment 3：Quantitatively detect beta-lactoglobulin content in Milka Choco Break biscuits.
1. sample pretreatment.
Milka Choco Break biscuits are clayed into power, 1.0 g powder are weighed, add 20 mL extracting solutions（20 mmol/L
Tris-HCl, 2% Tween-20, pH 8.0）, 4 °C are stirred overnight extraction albumen.By extracting solution in 4 °C of 10,000 g
15min, removal fat and precipitation are centrifuged, supernatant is diluted 1,000 times with liquid is blockaded, as detection sample.
2. sample detection.
（1）Coating：After concatermer polyclonal antibody is diluted to 3.5 μ g/mL with coating buffer, 100 μ L, 4 °C of packets are added per hole
It is stayed overnight.
（2）Washing：Use PBST（Containing 0.1% Tween-20）Washing 3 times, 3 min, button are dry every time.
（3）It blockades：Match 2% gelatin with PBS（Containing 0.1% Tween-20）Liquid is blockaded, 250 μ L are added per hole, 37 °C are incubated 0.5
h.After blockading, washed 3 times with PBST, 3 min, button are dry every time.
（4）Add antigen：It is with liquid is blockaded that beta lactoglobulin standard items are dilute in 0.49-16,000 ng/mL range inside gradients
It releases, 100 μ L standard items/sample is added per hole, in triplicate, 37 °C of 2 h of incubation.After incubation, washed 3 times with PBST,
3 min every time, button are dry.
（5）Add detection antibody：100 μ L after 200 times of Platinum Nanoparticles probe dilution, will be added per hole with liquid is blockaded, 37 °C are incubated 1
h.After incubation, washed 4 times with PBST, 3 min, button are dry every time.
（6）Plus the Streptavidin of horseradish peroxidase-labeled：It will add horseradish peroxidase-labeled with liquid is blockaded
Streptavidin dilute 60 times, 100 μ L, 37 °C of 0.5 h of incubation are added per hole.After incubation, 4 times are washed with PBST, often
Secondary 3 min, button are dry.
（7）Add developing solution：Add 100 μ L TMB solution per hole, 37 °C are protected from light 15 min of incubation.
（8）Survey light absorption value：After incubation, the H of 50 μ L, 2 M is added per hole2SO4Reaction is terminated, microplate reader is then used
Light absorption value is surveyed, absorbing wavelength is 450 nm, standard curve is drawn according to light absorption value, according to equation of linear regression and sample light absorption value
The content of beta lactoglobulin in sample is calculated, the average value detected three times is final detection level.
3. result.
Examination criteria curvilinear equation is y=0.26185x+0.1936, r2=0.99898, sensitivity is 0.12 ng/mL.
The content of beta lactoglobulin is 6.25 g/kg in Milka Choco Break biscuits.
Claims (1)
1. a kind of method based on Platinum Nanoparticles probe in detecting cow's milk beta lactoglobulin and its sensitization residue, it is characterized in that by following
Step：
（1）Based on epiposition vaccine design principle, with cow's milk beta lactoglobulin IgE linear epitopes AA1 ~ 8, AA31 ~ 48, AA47 ~ 60,
Based on AA67 ~ 78, AA75 ~ 86, AA127 ~ 144 and AA141 ~ 152, t cell epitope AA77 ~ 97 and intervening sequence KK and G；
The antigenicity, surface accessibility and hydrophily of various combination concatermer are analyzed by DNAStar softwares, make each epitope
Antigenicity and hydrophily be more than 0, while surface accessibility be more than 1；Recycle strings of the network server SOMPA to various combination
Conjuncted secondary structure is analyzed, and epitope is made to form β-bend and random coil structure；Pass through the above bioinformatics method
The analysis of epitope concatermer, finally obtained concatermer are combined as：T-K-K-B1-G-B6-G-B5-G-B3-G-B7-G-B2-G-
B4；Tandem gene is synthesized again, and cow's milk beta lactoglobulin epitope concatermer recombinant protein is prepared by prokaryotic expression；Most
Afterwards, using body protein of connecting as antigen, concatermer polyclonal antibody is obtained by conventional method；
（2）Beta lactoglobulin and CNBr-Sepharose 4B column materials are coupled, immune affinity column is prepared；According to a conventional method with exempting from
Beta lactoglobulin polyclonal antibody in epidemic disease is affine column purification antiserum；
（3）Add at each IgE epitopes both ends and respectively add an amino acid, and synthesizes this 7 IgE epitope peptides and its blocking peptide；Using
Indirect competitive ELISA method analyzes recognition capability of the concatermer polyclonal antibody to epitope peptide and blocking peptide；By beta lactoglobulin mark
After quasi- product are diluted with carbonate buffer solution, plate A is added in 100 holes μ L/, and overnight, PBST is washed three times 4 DEG C of coatings, and 3 min are detained every time
It is dry；It is another that one piece of ELISA Plate B, plate A is taken to be blockaded with 37 DEG C of 3% gelatin solution that 250 μ L are added in plate B per hole；Plate B blockades 1 h
After clean, per hole be added concatermer polyclonal antibody and concentration gradient each 70 μ L of polypeptide, and with PBS be control, 37 DEG C incubate
Educate 1 h；Plate A is cleaned 3 times with PBST, and from taking the reaction solution of 100 μ L antibody and polypeptide to be added in plate A in plate B, 37 DEG C are incubated
It is cleaned after 1 h, 100 μ L OPD developing solutions is added per hole, after 37 DEG C are protected from light 15 min of incubation, be added 50 μ L, 2 M's per hole
H2SO4Reaction is terminated, surveys light absorption value with microplate reader immediately after；The inhibiting rate that various concentration peptide is calculated according to light absorption value, according to suppression
Power of the rate height reflection concatermer polyclonal antibody processed to different epitopes peptide and blocking peptide recognition capability；
（4）Using cow's milk beta lactoglobulin as antigen, beta lactoglobulin polyclonal antibody is obtained by conventional method；
（5）By step（3）Beta lactoglobulin polyclonal antibody is purified；The beta lactoglobulin specificity that purifying is obtained again
Polyclonal antibody presses 1 with long-chain biological element:20 molar ratio mixing, 2 h of ice bath, then extra biotin is removed with desalting column；
（6）It takes the Platinum Nanoparticles solution 10 that 1 mL grain sizes are about 20 nm, 000 × g room temperatures to centrifuge 15 min, removes supernatant, add 990
Platinum Nanoparticles precipitation is resuspended in the PBS that μ L pH are 6.5, adds the biotinylated beta lactoglobulin of 10 a concentration of 1mg/mL of μ L
Polyclonal antibody, it is 1 to make antibody and Platinum Nanoparticles mass ratio:100, overnight in 4 DEG C of couplings；Then 10,000 × g room temperatures centrifugation 15
Min removes the supernatant containing unmarked antibody, and 1 mL is added and preserves the Platinum Nanoparticles probe that precipitation labelled antibody is resuspended in liquid, preserves liquid
Containing 1% BSA, 0.1 % Triton X-100, the PBS of 5% sucrose, pH 6.5；
（7）It is capture antibody with concatermer polyclonal antibody, the Platinum Nanoparticles probe of labelled antibody is detection antibody, beta lactoglobulin
To detect antigen, capture antibody and detection antibody best effort concentration are determined using chessboard method；100 μ are added in ELISA Plate per hole
The diluted capture antibody of L carbonate, overnight, PBST is washed three times 4 DEG C of coatings, 3 min and detains dry every time, 250 μ L are added per hole
2% gelatin solution, 37 DEG C are blockaded 0.5 h；After cleaning, the beta lactoglobulin of 100 μ L known concentration gradients of addition per hole, 37
DEG C be incubated 2 h；After cleaning, 100 μ L Platinum Nanoparticles probes, 37 DEG C of 1 h of incubation are added per hole；After cleaning, 100 μ L are added per hole
The Streptavidin of horseradish peroxidase-labeled, 37 DEG C of 0.5 h of incubation；After cleaning, 100 μ L TMB solution are added per hole,
After 37 DEG C are protected from light 15 min of incubation, the H of 50 μ L, 2 M is added per hole2SO4Reaction is terminated, then surveys light absorption value with microplate reader,
Beta lactoglobulin standard curve is drawn according to the relationship of light absorption value and antigen concentration；
（8）The food of required detection is subjected to leach protein, fat/precipitation is gone in centrifugation, prepares detection sample；It is coated with according to the above method
After capturing antibody, closing and cleaning, the 100 pretreated samples of μ L are added per hole, after being incubated cleaning, then press step（7）Successively plus
Enter to detect antibody, the Streptavidin of horseradish peroxidase-labeled, TMB solution, through H2SO4After terminating reaction, examined with microplate reader
Light absorption value is surveyed, light absorption value is brought into the content that standard curve calculates beta lactoglobulin and its sensitization residue in test sample.
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