CN110196330A - Immunity colloidal gold test paper strip and its application of pseudo- cow's milk are mixed in a kind of detection bactrian camel milk - Google Patents
Immunity colloidal gold test paper strip and its application of pseudo- cow's milk are mixed in a kind of detection bactrian camel milk Download PDFInfo
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- CN110196330A CN110196330A CN201910223473.0A CN201910223473A CN110196330A CN 110196330 A CN110196330 A CN 110196330A CN 201910223473 A CN201910223473 A CN 201910223473A CN 110196330 A CN110196330 A CN 110196330A
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- 239000012723 sample buffer Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
Abstract
The present invention discloses a kind of immunity colloidal gold test paper strip and its application for detecting and mixing pseudo- cow's milk in bactrian camel milk, by preparing bactrian camel milk ɑ-lactoalbumin gold labeling antibody, bactrian camel milk αs1Casein gold labeling antibody, cow's milk beta lactoglobulin gold labeling antibody and cow's milk κ-casein gold labeling antibody, and the test strips of detection whey are prepared respectively and detect the test strips II of Caseinum componemt, obtain a kind of immunity colloidal gold test paper strip for detecting and mixing pseudo- cow's milk in bactrian camel milk, the test strips can monitor ox source and the camel source protein of milk and milk products simultaneously, mix pseudo- monitoring field with wide applicability and Development volue in bactrian camel milk.
Description
Technical field
The invention mainly relates to the technical fields of immune detection, specifically, the present invention relates in a kind of detection bactrian camel milk
Mix the technical field of the colloidal gold detection of pseudo- cow's milk.
Background technique
With economic development, the enhancing of people's health care consciousness, camel dairy products are progressed into daily life, camel
Newborn rich in nutrition content, immunoglobulin, lactoferrin, insulin and insulin-like growth factor in protein active ingredients
Content is higher, and bactrian camel milk ɑ-lactoalbumin structure is more easily absorbed compared to cow's milk ɑ-lactoalbumin, and bactrian camel milk is free of
Main allergic source beta lactoglobulin in cow's milk, is more suitable for the consumption by infants to Milk allergy.Oligosaccharide, insatiable hunger in bactrian camel milk
It is special with the compositional models such as fatty acid, minerals, have the characteristics that easy to digest.In recent years, domestic and foreign scholars are raw to bactrian camel milk
The research of reason function gradually increases, and bactrian camel milk is improving immunity, reducing blood sugar in diabetic patients and blood lipid level, treatment tumour
Etc. have auxiliary therapeutic action.Camel aquaculture is slower in the presence of developing, and camel breeding stock is lower, and camel lactation condition is severe
It carves, camel milk production is lower for the cow's milk of industry development maturation.Due to the nutritive value and physiological function of bactrian camel milk
The characteristics of special, low output, the commercially available price of bactrian camel milk is generally higher than cow's milk.Due to the difference of price, then may go out in market
Cow's milk is now mixed into the phenomenon in bactrian camel milk, therefore quickly and precisely detection camel Methods for detection of cows is necessary
's.Currently, existing Adulteration detection technology is mostly for the detection method for mixing cow's milk in sheep cream, buffalo's milk, in white horse with a black mane both at home and abroad
The detection method that cow's milk is mixed in hunchbacked cream is still rare.
Currently, the detection method that can be applied to mix cow's milk in bactrian camel milk mainly has: first is that electrophoresis detection technology: although
This method is easy to operate, but its sensitivity is low, and is not suitable for the detection of a large amount of samples.Second is that liquid-phase chromatographic analysis: this method spirit
Sensitivity is high, and sample size needed for detecting is few, is suitble to a large amount of sample detections, but this method needs expensive instrument and skilled instrument behaviour
Make personnel and complicated for operation, unsuitable field test.Third is that nucleic acid differences are analyzed: this method sensitivity is higher, but inhereditary material
Extraction process is complex.Fourth is that immunology detection: immunology detection is the method for the specific reaction based on Ag-Ab,
More application is enzyme-linked immunosorbent assay and colloidal gold immunochromatographimethod technology in the technology of detection, and reaction result can lead to
It crosses monitoring reaction and whether has the depth of color change and color change to distinguish in detectable substance whether there is corresponding antigen or anti-
Body can qualitatively or quantitatively detect the target substance in object to be checked, and in existing detection method, be mostly using precision instrument into
Row detection does not occur also mixing cow's milk using in quick, convenient and fast colloidal gold immuno-chromatography test paper strip technology detection bactrian camel milk
Method.
Colloidal gold immunochromatographimethod technology (Gold ImmunochromatographicAssay, GICA) is to make colloidal gold
The immunochromatographic techniques for being marker in conjunction with antibody.Immunochromatographic techniques are then to utilize microporous barrier using cellulose membrane as carrier
Capillarity migrate liquid on film, by a kind of solid phase labelling of antigen and antibody specific combination iodine process
Immune Fast Detection Technique.Have the characteristics that high sensitivity, high specificity, stability are high, easy to operate, easy to carry, is not required to
Complicated instrument and equipment and technical professional are wanted, analysis result is easy to judge, can directly detect by an unaided eye in the short time
To testing result, it is widely used in the on-site tests analysis such as rapid disease diagnosis, food inspection and environmental monitoring.
Summary of the invention
For it is current it is domestic for bactrian camel milk Adulteration detection there is no specific standard, to bactrian camel milk and its Related product
Detection, the country is more common in for nutritional ingredient in bactrian camel milk and the detection of medicament residue etc., and pseudo- inspection is mixed for bactrian camel milk
Less technical problem is surveyed, the present invention is intended to provide immune colloid gold test paper and its application of pseudo- cow's milk are mixed in detection bactrian camel milk,
By preparing bactrian camel milk ɑ-lactoalbumin gold labeling antibody, bactrian camel milk αs1Casein gold labeling antibody, cow's milk beta lactoglobulin gold mark are anti-
Body and cow's milk κ-casein gold labeling antibody, and the test strips I and detection casein group of detection whey are prepared respectively
The test strips II divided, obtain a kind of immune colloid gold test paper for detecting and mixing pseudo- cow's milk in bactrian camel milk, which can monitor simultaneously
The ox source of milk and milk products and hunchbacked source protein mix pseudo- monitoring field with wide applicability and Development volue in bactrian camel milk.
What the invention is realized by the following technical scheme:
The present invention, which is provided in a kind of detection bactrian camel milk, mixes the immune colloid gold test paper of pseudo- cow's milk, including is assembled into detection whey
The test strips I of protein ingredient and the test strips II of detection casein fraction, test strips I include using cow's milk beta lactoglobulin gold
Labeling antibody and the gold-labelled pad of bactrian camel milk α-lactalbumin gold labeling antibody mixed solution spraying use 0.5~1.5mg/mL cow's milk β-
The T of lactoglobulin1Liquid draws T1Line and the T for using 0.5~1.5mg/mL bactrian camel milk α-lactalbumin2Liquid draws T2Line and make
NC film, sample pad and the water absorption pad that C line is drawn with the C liquid of 0.5~1.5mg/mL sheep anti-mouse igg or rabbit anti-mouse igg, carry out group
Dress；Test strips II include using cow's milk κ-casein gold labeling antibody and bactrian camel milk αs1The spraying of casein gold labeling antibody mixed solution
Gold-labelled pad, use 0.5~1.5mg/mL cow's milk κ-casein T1Liquid draws T1Line and use 0.5~1.5mg/mL bactrian camel milk
αs1The T of casein2Liquid draws T2The C liquid of line and use 0.5~1.5mg/mL sheep anti-mouse igg or rabbit anti-mouse igg draws C line
NC film, sample pad and water absorption pad, assembled.
In the present invention, it is described it is a kind of detection bactrian camel milk in mix pseudo- cow's milk immune colloid gold test paper specific preparation process
Are as follows:
(1) preparation of gold-labelled pad: glass fibre element film being submerged into glass fibre element film confining liquid and is closed, and 37 DEG C
1~2h is dried, then cow's milk beta lactoglobulin gold labeling antibody and bactrian camel milk α-lactalbumin gold labeling antibody mixed solution or cow's milk
κ-casein gold labeling antibody and bactrian camel milk αs1Casein gold labeling antibody mixed solution is sprayed at the glass fibre element film closed
On, it is transferred quickly to be freeze-dried in freeze drier, production becomes gold-labelled pad；
(2) preparation of sample pad and water absorption pad: sample pad is submerged into confining liquid for glass fibre element film and is closed, and 37 DEG C
Drying for standby, water absorption pad are the preferable absorbent filter material of water absorbing properties；
(3) coating of NC film: by C liquid, the T of preparation1Liquid and T2Liquid is crossed on NC film respectively, from left to right sequentially successively
For T1Liquid, T2Liquid and C liquid set 37 DEG C of dry 20~40min, and confining liquid closing is submerged into after dry, is placed in 37 DEG C of insulating boxs and does
It is dry spare；
(4) assembling of test strips: the sample pad prepared, gold-labelled pad, NC film and water absorption pad are cut to of same size
Rectangle, and carry out overlapping assembling, be assembled into the test strips I and detection casein fraction of detection whey protein fraction respectively
Test strips II.
In the present invention, the overlapping assembling, overlapped sequential is that sample pad right end is laminated on gold-labelled pad left end from left to right,
Gold-labelled pad right end is laminated on NC film left end, and water absorption pad left end is laminated on the right end of NC film, and stack laminate section mutually overlapping 1~
3cm。
In the present invention, the confining liquid is the 0.01mol/L phosphate buffer containing 2~3% bovine serum albumin(BSA)s.
In the present invention, the T1Liquid, with 0.01mol/L phosphate buffer by cow's milk beta lactoglobulin or cow's milk κ-casein
It is diluted to the solution of 0.5~1.5mg/mL.
In the present invention, the T2Liquid, with 0.01mol/L phosphate buffer by bactrian camel milk α-lactalbumin or bactrian camel milk αs1-
Casein is diluted to the solution of 0.5~1.5mg/mL.
In the present invention, the C liquid, with 0.01mo/L phosphate buffer by sheep anti-mouse igg or rabbit anti-mouse igg monoclonal antibody
It is diluted to the solution of 0.5~1.5mg/mL.
In the present invention, the test strips I is detection whey protein fraction, and test strips I includes: using cow's milk beta lactoglobulin
Gold labeling antibody and the gold-labelled pad of bactrian camel milk α-lactalbumin gold labeling antibody mixed solution spraying use 0.5~1.5mg/mL cow's milk
The T of beta lactoglobulin1Liquid draws T1Line and the T for using 0.5~1.5mg/mL bactrian camel milk α-lactalbumin2Liquid draws T2Line and
NC film, sample pad and the water absorption pad of C line are drawn using the C liquid of 0.5~1.5mg/mL sheep anti-mouse igg or rabbit anti-mouse igg, are carried out
Assembling.
In the present invention, the test strips II are detection casein fraction, and test strips II include: using cow's milk κ-casein gold
Labeling antibody and bactrian camel milk αs1The gold-labelled pad of casein gold labeling antibody mixed solution spraying uses 0.5~1.5mg/mL cow's milk κ-junket
The T of albumen1Liquid draws T1Line and use 0.5~1.5mg/mL bactrian camel milk αs1The T of casein2Liquid draws T2Line and use 0.5
The C liquid of~1.5mg/mL sheep anti-mouse igg or rabbit anti-mouse igg draws NC film, sample pad and the water absorption pad of C line, is assembled.
In the present invention, the cow's milk beta lactoglobulin gold labeling antibody, bactrian camel milk α-lactalbumin gold labeling antibody, cow's milk κ-junket
Albumen gold labeling antibody, bactrian camel milk αs1The preparation process of casein gold labeling antibody are as follows:
(1) reduction of sodium citrate method prepare AuNPs: gold chloride is dissolved in distilled water, make its final concentration of 0.1%~
0.15%, it is heated to boiling, 0.75~1.5mL1% citric acid three sodium solution is taken to be added in the conical flask of above-mentioned boiling, it is mixed rapidly
It closes uniformly, is kept for 100 DEG C, 10~20min, after solution is cooled to room temperature, with distilled water make-up solution to original volume；
(2) preparation of gold labeling antibody: by the cow's milk beta lactoglobulin monoclonal antibody of 18~36 μ g or cow's milk β-milk-globule egg
White polyclonal antibody or cow's milk κ-casein monoclonal antibody or cow's milk κ-casein polyclonal antibody or bactrian camel milk α-milky white egg
White monoclonal antibody or bactrian camel milk α-lactalbumin polyclonal antibody or bactrian camel milk αs1Casein monoclonal antibody or bactrian camel milk
αs1Casein polyclonal antibody is separately added into colloidal gold solution, adjusts the pH to 7.6~8.5 of the AuNPs solution of tape label,
It mixes and stands 2~5min；
(3) it the Quality Identification of AuNPs: using the character and aggregation situation of scanning electron microscope observation AuNPs, and uses
400~700nm ultraviolet specrophotometer detects the AuNPs solution prepared, records the maximum as a result, AuNPs solution
There are linear relationships with AuNPs average grain diameter for absorption peak wavelength, can be according to the average grain diameter of regression equation calculation AuNPs: y=
0.4271x+514.56, in formula: y is colloidal gold solution maximum absorption peak, and x is gold particle average grain diameter；
(4) activity identification of gold labeling antibody: taking secondary antibody point sample and to carry out position mark on nitrocellulose membrane, by nitric acid fibre
It ties up film and places drying in 37 DEG C of drying boxes, then take gold labeling antibody to stand in the position point sample of secondary antibody, rinsed with distilled water rapidly,
The gold labeling antibody that do not react with secondary antibody is rinsed out.
In the present invention, the secondary antibody is sheep anti-mouse igg or rabbit anti-mouse igg.
In the present invention, the cow's milk beta lactoglobulin, cow's milk κ-casein, bactrian camel milk α-lactalbumin, bactrian camel milk αs1Junket
The monoclonal antibody preparation process of albumen are as follows:
The female Balb/c mouse of about 7 week old is chosen as immune animal, immunogene is cow's milk beta lactoglobulin, cow's milk κ-
Casein, bactrian camel milk α-lactalbumin and bactrian camel milk αs1Casein, immunogene and Freund's complete adjuvant or incomplete Freund's adjuvant
It is immunized after ultrasound mixes well emulsification, subcutaneous multiple spot and the immune mouse of intraperitoneal injection；Serum is separated to survey using indirect elisa method
Determine potency, when serum titer reaches 1:64000 or more, response value highest mouse tail vein and abdominal cavity are injected respectively without adjuvant
100 μ g immunogenes carry out last booster immunization；Spleen is taken to prepare the fusion of lymphocyte inducing cell, using indirect elisa method
Screening with sheep, horse, donkey, camel or cow's milk no cross reaction and with cow's milk beta lactoglobulin or cow's milk κ-casein or bactrian camel milk α-cream
Albumin or bactrian camel milk αs1The higher positive colony further expansion culture of casein response value, injection expand thin after cultivating
Born of the same parents purify into ascites is induced in Mice Body, obtain monoclonal antibody, are lyophilized spare.
In the present invention, the cow's milk beta lactoglobulin, cow's milk κ-casein, bactrian camel milk α-lactalbumin, bactrian camel milk αs1Junket
The Anti-TNF-α production procedure of albumen are as follows: screen healthy New Zealand male White Rabbit, with ear edge vein exploitating blood, centrifugation obtains yin
Property serum is spare；Respectively with the standby cow's milk beta lactoglobulin of Sigma corporation, cow's milk κ-casein and homemade bactrian camel milk α-
Lactoalbumin, bactrian camel milk αs1White Rabbit is immunized in casein mixing Freund's complete adjuvant or incomplete Freund's adjuvant, with indirect
Elisa technique detects beta lactoglobulin polyclonal antibody, α-lactalbumin polyclonal antibody, αs1Casein polyclonal antibody, κ-
The potency of casein polyclonal antibody, each protein antibodies potency put to death rabbit with Culling heart blood when being at least up to 1:20000 or more
Method collect serum, purifying, obtain polyclonal antibody.
In the present invention, the homemade bactrian camel milk α-lactalbumin, bactrian camel milk αs1The extracting method of casein, including it is following
Technical step:
(1) ammonium sulfate graded precipitation slightly proposes bactrian camel milk α-lactalbumin: extracting degreasing bactrian camel milk, adjusts pH to 4.3~4.8,
3000~12000r/min be centrifuged 10~60min, repeated multiple times centrifugation, supernatant by ammonium sulfate saturation degree 70%~100% into
It is spare by desalination freeze-drying to obtain precipitating for row fractional precipitation；
(2) calcium precipitation coarse extraction bactrian camel milk αs1Casein: pH is adjusted to 10~12, is added by extracting degreasing bactrian camel milk
Calcium ion generates precipitating, and pH to 6~8,3000~8000r/min is adjusted to be centrifuged 10~30min, and precipitating is taken to be dissolved in 3.3mol/L urine
Element adjusts pH to 4~5,3000~8000r/min to be centrifuged 10~30min, obtains spare after precipitating is dried；
(3) ultrafiltration centrifugation crude separation bactrian camel milk α-lactalbumin or bactrian camel milk αs1Casein: extracting degreasing bactrian camel milk adjusts pH
To 4.3~4.8,3000~12000r/min is centrifuged 10~60min, and repeated multiple times centrifugation obtains supernatant and precipitating, supernatant
It is centrifuged 20~30min using 5000~10000r/min of super filter tube, isolating protein of the molecular weight lower than 20kDa is bactrian camel milk
α-lactalbumin is centrifuged 20~30min using 5000~10000r/min of super filter tube after precipitating dissolution, isolates molecular weight and be lower than
The protein of 30kDa is bactrian camel milk αs1Casein.
In addition, the present invention provides a kind of application for detecting and mixing the immune colloid gold test paper of pseudo- cow's milk in bactrian camel milk, it is specific to wrap
Include following steps:
(1) sample treatment: liquid milk sample can be used without pre-treatment, milk powder and cheese sample through dissolution；
(2) it detects: the sample pad part of immune colloid gold test paper is immersed in measuring samples solution, liquid is waited to diffuse to
It is taken out after water absorption pad, stands the appearance situation of observation T line and C line after 10~15min；
(3) testing result: C line goes out to represent result effective in test strips；T in test strips1Line and T2Line do not go out represent to
Contain corresponding antigens in sample product；T in test strips1Line does not occur and T2Line occurs, and illustrates to contain cow's milk protein in measuring samples,
Without containing camel lactoprotein；T in test strips1Line occurs and T2Line does not occur, illustrates containing camel lactoprotein in measuring samples, no
Contain cow's milk protein；T in test strips1Line and T2Line does not occur, illustrates to contain cow's milk and bactrian camel milk egg in measuring samples simultaneously
It is white；T in test strips1Line and T2Line occurs, and illustrates to try in sample product without cow's milk and camel lactoprotein；C line does not go out in paper slip
It is existing, it is invalid to represent result.
By implement technical solution of the present invention, can achieve it is following the utility model has the advantages that
(1) present invention, which is provided in a kind of detection bactrian camel milk, mixes immune colloid gold test paper and its application of pseudo- cow's milk, based on exempting from
The detection method of epidemiology, by preparing bactrian camel milk ɑ-lactoalbumin gold labeling antibody, bactrian camel milk αs1Casein gold labeling antibody, cow's milk
Beta lactoglobulin gold labeling antibody and cow's milk κ-casein gold labeling antibody, and the test strips of detection whey are prepared respectively
I and the test strips II for detecting Caseinum componemt obtain a kind of immune colloid gold test paper for detecting and mixing pseudo- cow's milk in bactrian camel milk, the examination
Paper slip can monitor ox source and the camel source protein of milk and milk products simultaneously, mix pseudo- monitoring field with extensive practical in bactrian camel milk
Property and Development volue.
(2) present invention provides a kind of immune colloid gold test paper and its application for detecting and mixing pseudo- cow's milk in bactrian camel milk, can be fast
Speed easily carries out mixing pseudo- cow's milk detection in bactrian camel milk, and lowest detection is limited to mix pseudo- 5% cow's milk in bactrian camel milk, can satisfy
Daily production and processing, the needs quickly detected.
Detailed description of the invention
Fig. 1 is shown as bactrian camel milk compared with cow's milk Discrepancy proteome and bactrian camel milk characteristic protein purity check figure；In figure, A
For cow's milk, figure, A1 are cow's milk whey compared with bactrian camel milk whey differential protein, and A2 is bactrian camel milk whey, and A3 is self-control bactrian camel milk α-
Lactoalbumin；B is cow's milk figure compared with camel milk casein differential protein, and B1 is bovine casein, and B2 is camel milk casein,
B3 is self-control bactrian camel milk αs1Casein, B4 are commercialization cow's milk κ-casein.
Fig. 2 is shown as cow's milk beta lactoglobulin antibody titer figure.
Fig. 3 is shown as cow's milk κ-casein antibody titer figure.
Fig. 4 is shown as bactrian camel milk α-lactalbumin antibody titer figure.
Fig. 5 is shown as bactrian camel milk αs1Casein monoclonal antibody figure.
Fig. 6 is shown as cow's milk beta lactoglobulin polyclonal antibody potency figure.
Fig. 7 is shown as bactrian camel milk α-lactalbumin polyclonal antibody potency figure.
Fig. 8 is shown as cow's milk κ-casein polyclonal antibody potency figure.
Fig. 9 is shown as bactrian camel milk αs1Casein polyclonal antibody potency figure.
Figure 10 is shown as AuNPs electron-microscope scanning figure.
Figure 11 is shown as AuNPs solution ultraviolet spectrogram.
Figure 12 is shown as the activity identification figure of gold labeling antibody；In figure, A is hunchbacked α-lactalbumin, and B is cow's milk β-milk-globule egg
White, C is cow's milk κ-casein, and D is bactrian camel milk αs1Casein.
Figure 13 is shown as test strips assembling schematic diagram.
Figure 14 is shown as test strips result judgement figure.
Specific embodiment
In the following, illustrating the present invention for embodiment, still, the present invention is not limited to following embodiments.
The main agents of use: glass fibre element film is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd., NC film (cellulose nitrate
Plain film) it is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd.；Female Balb/c mouse is purchased from Zhenjiang biology Co., Ltd；Cow's milk β-cream
Globulin, cow's milk κ-casein are purchased from SIGMA company, and sheep anti-mouse igg is public purchased from SIGMA purchased from SIGMA company, rabbit anti-mouse igg
Department, Freund's complete adjuvant are purchased from PIERCE company；Incomplete Freund's adjuvant is purchased from SIGMA company；Chromogenic substrate pNPP is purchased from KPL
Company；RPMI basal medium, L-Glutamine, penicillin+streptomysin be dual anti-, HAuCl4, HAT, HT, DMSO, ox blood are pure
Albumen, skimmed milk power, Tween-20, trisodium citrate are to analyze pure, Quick StartTM Bradford 1x Dye
Reagent is purchased from Bole's life medical product (Shanghai) Co., Ltd., Quick StartTM Bovine Gamma Globulin
Standard Set is raw purchased from Bole purchased from Bole's life medical product (Shanghai) Co., Ltd., Native Sample Buffer
Order medical product (Shanghai) Co., Ltd..
The main instrument and equipment used: BioTek full-automatic enzyme-linked immunoassay instrument, the desk-top low-temperature and high-speed of Eppendorf
Centrifuge, the full-automatic protein purification instrument of AKTA pure, AL104 electronic balance, BRANSON Ultrasound Instrument, BIO-RAD electrophoresis apparatus.
The reagent and equipment that the present invention uses can be bought or be customized by public channel.
What all material, reagent and the instrument selected in the present invention were all well known in the art, but reality of the invention is not limited
It applies, other some reagents well known in the art and equipment are applied both to the implementation of following implementation of the present invention.
The polyacrylamide gel electrophoresis (SDS-PAGE) that is used in the present invention, the artificial board-washing of PBST, reduction of sodium citrate
Method prepares gold nanoparticle (AuNPs) and uses Dot-immunogold filtration (DIGFA) all for those of ordinary skill in the art
Known technological means, the specific embodiment that following example provides are not intended to limit implementation of the invention.
Embodiment one: SDS-PAGE compares cow's milk and bactrian camel milk albumen composition
Bactrian camel milk whey, cow's milk whey, camel milk casein, bovine casein are through polyacrylamide gel electrophoresis (SDS-
PAGE), recorded using gel imager after Coomassie gel and decoloration as a result, referring to figure 1, finding camel
Whey lacks beta lactoglobulin relative to bovine whey, and α-lactalbumin protein content is significantly higher than bovine whey in camel whey, because
This selects cow's milk beta lactoglobulin and bactrian camel milk α-lactalbumin to carry out the preparation of polyclonal antibody；Relative to cow's milk, camel
κ-casein content is less in milk casein, αs1Casein molecule amount is larger, therefore selects cow's milk κ casein and bactrian camel milk
αs1Casein carries out the preparation of polyclonal antibody or monoclonal antibody.
Embodiment two: bactrian camel milk α-lactalbumin and αs1The extraction of casein
(1) ammonium sulfate graded precipitation slightly proposes bactrian camel milk α-lactalbumin
Extracting degreasing bactrian camel milk, adjust pH to 4.3~4.8,3000~12000r/min be centrifuged 10~60min, it is repeated multiple times from
The heart, supernatant by ammonium sulfate saturation degree 70%~100% carry out fractional precipitation, obtain precipitating be lyophilized through desalination it is spare.
(2) calcium precipitation coarse extraction bactrian camel milk αs1Casein
PH is adjusted to 10~12 by extracting degreasing bactrian camel milk, and calcium ion is added and generates precipitating, adjusts pH to 6~8,3000~
8000r/min is centrifuged 10~30min, and precipitating is taken to be dissolved in 3.3mol/L urea, adjusts pH to 4~5,3000~8000r/min centrifugation
10~30min is obtained spare after precipitating is dried.
(3) ultrafiltration centrifugation crude separation bactrian camel milk α-lactalbumin or bactrian camel milk αs1Casein
Extracting degreasing bactrian camel milk, adjust pH to 4.3~4.8,3000~12000r/min be centrifuged 10~60min, it is repeated multiple times from
The heart, obtains supernatant and precipitating, and supernatant is centrifuged 20~30min using 5000~10000r/min of super filter tube, isolates molecule
Amount lower than 20kDa protein be bactrian camel milk α-lactalbumin, precipitating dissolution after using 5000~10000r/min of super filter tube from
20~30min of the heart, isolating protein of the molecular weight lower than 30kDa is bactrian camel milk αs1Casein.
(4) SDS-PAGE detects bactrian camel milk α-lactalbumin and bactrian camel milk αs1Casein purity
It is derived from bactrian camel milk α-lactalbumin processed and αs1Casein carries out the purity that SDS-PAGE examines α-lactalbumin,
Referring to figure 1, A3 is self-control bactrian camel milk α-lactalbumin, and B3 is self-control bactrian camel milk αs1Casein finds two kinds of protein
Purity is higher, can be used for preparing polyclonal antibody or monoclonal antibody.
Embodiment three: cow's milk beta lactoglobulin, cow's milk κ-casein, bactrian camel milk α-lactalbumin, bactrian camel milk αs1Casein
Monoclonal antibody preparation
(1) cow's milk beta lactoglobulin, cow's milk κ-casein, bactrian camel milk α-lactalbumin, bactrian camel milk αs1The Dan Ke of casein
Grand Antibody preparation:
The female Balb/c mouse of about 7 week old is chosen as immune animal, immunogene is cow's milk beta lactoglobulin, cow's milk κ-
Casein, bactrian camel milk α-lactalbumin and bactrian camel milk αs1Casein, immunogene and Freund's complete adjuvant (initial immunity) or Freund
Freund's incomplete adjuvant (booster immunization) is immune after ultrasound mixes well emulsification, subcutaneous multiple spot and the immune mouse of intraperitoneal injection.Separation
Serum measures potency using indirect elisa method, when serum titer reaches 1:64000 or more, to response value highest mouse tail vein
It injects the 100 μ g immunogenes without adjuvant respectively with abdominal cavity and carries out last booster immunization.Spleen is taken to prepare lymphocyte induction thin
Born of the same parents fusion, using indirect elisa method screening with sheep, horse, donkey, camel or cow's milk no cross reaction and with cow's milk beta lactoglobulin or
Cow's milk κ-casein or bactrian camel milk α-lactalbumin or bactrian camel milk αs1The higher positive colony further expansion of casein response value
Culture, cell purifies into ascites is induced in Mice Body, obtains monoclonal antibody, be lyophilized spare after injection expands culture.
(2) cow's milk beta lactoglobulin, cow's milk κ-casein, bactrian camel milk α-lactalbumin, bactrian camel milk αs1The Dan Ke of casein
Grand antibody titer detection:
With 2 μ g/mL cow's milk beta lactoglobulins or cow's milk κ-casein or bactrian camel milk α-lactalbumin or bactrian camel milk αs1Junket egg
White coated elisa plate, 50 holes μ l/, 4 DEG C of overnight or 37 DEG C of 2~4h of coating.Coating buffer of inclining adds after washing 3~5 times with PBST
Enter 0.4% fishskin gelatin to close, 200 holes μ L/, 37 DEG C, 1~3h.Confining liquid is discarded, after being added PBST washing 3~5 times, is added
The doubling dilution antibody purification since 1000 times (mother liquid concentration 1mg/ml), blank control PBS, 50~100 holes μ l/, 37
DEG C be incubated for 1~3h.After the artificial board-washing of PBST 3 times, the goat anti-mouse IgG two of the diluted alkali phosphatase enzyme mark of 1:5000 is added
It is anti-, 50 holes μ l/, 37 DEG C of 1~3h of incubation.After the artificial board-washing of PBST 5 times, chromogenic substrate pNPP, 50 holes μ l/, 37 DEG C of colour developings are added
0.5~1h.Measure the absorbance under wavelength 405nm.
Test result: result is referring to shown in attached drawing 2 to attached drawing 5, cow's milk beta lactoglobulin, cow's milk κ-junket egg as the result is shown
White, bactrian camel milk α-lactalbumin, bactrian camel milk αs1The potency of casein monoclonal antibody reaches 1:16000 or more, potency difference
For 1:16000,1:16000,1:64000,1:16000.
Example IV: α-lactalbumin, beta lactoglobulin, αs1The preparation of casein, κ-casein polyclonal antibody
1, cow's milk beta lactoglobulin polyclonal antibody, bactrian camel milk α-lactalbumin polyclonal antibody, bactrian camel milk αs1Casein
Polyclonal antibody, the preparation of cow's milk κ-casein polyclonal antibody
This experiment is respectively with the standby cow's milk beta lactoglobulin of Sigma corporation, cow's milk κ-casein and homemade camel
Newborn α-lactalbumin, bactrian camel milk αs1Casein is immunizing antigen, mixes Freund's complete adjuvant (initial immunity) or Freund is incomplete
White Rabbit is immunized in adjuvant (booster immunization), detects polyclonal antibody potency with indirect ELISA technology, each protein antibodies potency is at least
Rabbit is put to death when reaching 1:20000 or more effectively and collects serum in the method for Culling heart blood, and purifying obtains polyclonal antibody.
2, indirect elisa method detects cow's milk beta lactoglobulin, bactrian camel milk α-lactalbumin, cow's milk κ-casein, bactrian camel milk
αs1Casein polyclonal antibody potency
Indirect elisa method detects cow's milk beta lactoglobulin, bactrian camel milk α-lactalbumin, cow's milk κ-casein, bactrian camel milk αs1-
Casein polyclonal antibody potency experimentation is as follows: primary antibody is serum to be checked, and serum: dilution gradient dilution multiple proportions is 1:
16000 to 1:3276800, prepared serum is as negative control before being immunized, and PBST is as blank control.
(1) be coated with: into polystyrene micropore plate every hole be added 100 μ L 2 μ g/mL cow's milk beta lactoglobulin dilutions or
2 μ g/mL bactrian camel milk α-lactalbumin dilutions or 2 μ g/mL bactrian camel milk αs1Casein dilution or 2 μ g/mL cow's milk κ-casein
Dilution, 4 DEG C of overnight coatings or 37 DEG C of 2~4h of coating；
(2) wash: after polystyrene micropore plate restores room temperature, 200~300 μ L cleaning solutions are added in every hole, discard residual
Liquid repeats 2~5 times；
(3) close: 100~200 μ L confining liquids are added in every hole, close 1~3h under the conditions of 37 DEG C；
(4) add primary antibody: being washed by step (2), every hole adds cow's milk beta lactoglobulin polyclonal antibody or bactrian camel milk α-milky white
Protein polyclone antibody or bactrian camel milk αs1Casein polyclonal antibody or cow's milk κ-casein polyclonal antibody or negative serum
100~200 μ L, 37 DEG C of 1~3h of incubation；
(5) add ELIAS secondary antibody: after step (2) washing, with diluted secondary antibody, counting by volume, secondary antibody: dilution
=1:10000 is set on ice for use, and every hole adds secondary antibody 100~200 the μ L, 37 DEG C of 1~3h of incubation after gradient dilution；
(6) develop the color: after step (2) washing, 100 μ L of TMB developing solution is added in every hole, and room temperature is protected from light developing time and is
10min；
(7) terminate reaction: 50~100 μ L terminate liquids are added in every hole, terminate reaction, measure the absorbance under 450nm wavelength
Value.It is the positive, the highest dilution times of antibody serum with P (serum to be tested)/N (serum rather) > 2, P (serum to be tested) > 0.2
Count polyclonal antibody potency thus.
3, test result
(1) cow's milk beta lactoglobulin polyclonal antibody potency
Gradient dilution: 1:16000,1:32000,1:64000,1 is carried out to cow's milk beta lactoglobulin polyclonal antibody:
128000,1:256000,1:512000,1:1024000,1:204800,1:409600,1:819200,1:1638400 and 1:
3276800。
The light absorption value of P/N value close to 2.5 corresponding polyclonal antibody dilutions is polyclonal antibody potency.β-milk-globule egg
P/N value corresponding to white polyclonal antibody dilution 1:819200 is 2.72, it is thus determined that beta lactoglobulin polyclonal antibody potency
For 1:819200, it is shown that the results are shown in attached figure 6.
(2) bactrian camel milk α-lactalbumin polyclonal antibody potency
Bactrian camel milk α-lactalbumin polyclonal antibody dilution selects 1:128000,1:256000,1:512000,1:
1024000,1:204800,1:409600,1:819200 and 1:1638400.
P/N value corresponding to bactrian camel milk α-lactalbumin polyclonal antibody dilution 1:1638400 is 2.74, it is thus determined that hunchbacked
α-lactalbumin polyclonal antibody potency is 1:1638400, and it is shown that the results are shown in attached figure 7.
(3) cow's milk κ-casein polyclonal antibody potency
Cow's milk κ-casein polyclonal antibody dilution selects 1:64000,1:128000,1:256000,1:512000,1:
1024000,1:204800,1:409600,1:819200 and 1:1638400.
P/N value corresponding to cow's milk κ-casein polyclonal antibody dilution 1:819200 is 2.82, it is thus determined that cow's milk κ-
Casein polyclonal antibody potency is 1:819200, and it is shown that the results are shown in attached figure 8.
(4) bactrian camel milk αs1Casein polyclonal antibody potency
Bactrian camel milk αs1Casein polyclonal antibody dilution selects 1:64000,1:128000,1:256000,1:
512000,1:1024000,1:204800,1:409600,1:819200 and 1:1638400.
Bactrian camel milk αs1P/N value corresponding to casein polyclonal antibody dilution 1:204800 is 2.68, it is thus determined that camel
Newborn αs1Casein polyclonal antibody potency is 1:204800, and it is shown that the results are shown in attached figure 9.
Embodiment five: the preparation of gold labeling antibody
(1) reduction of sodium citrate method prepares gold nanoparticle (AuNPs)
Gold chloride is dissolved in distilled water, make its final concentration of 0.1%~0.15%, be heated to boiling, take 0.75~
1.5mL1% citric acid three sodium solution is added in the conical flask of above-mentioned boiling, is uniformly mixed rapidly, 100 DEG C of holding, 10~
20min, after solution is cooled to room temperature, with distilled water make-up solution to original volume.
(2) preparation of gold labeling antibody
By the cow's milk beta lactoglobulin of 18~36 μ g or cow's milk κ-casein or bactrian camel milk α-lactalbumin or bactrian camel milk αs1-
Casein monoclonal antibody is separately added into colloidal gold solution, adjusts the pH to 7.6~8.5 of the AuNPs solution of tape label, mixing
And stand 2~5min.
(3) Quality Identification of AuNPs
Using the character and aggregation situation of scanning electron microscope observation AuNPs, and use ultraviolet specrophotometer (400
~700nm) the AuNPs solution prepared is detected, record maximum absorption band wavelength and AuNPs as a result, AuNPs solution
There are linear relationships for average grain diameter, can be according to the average grain diameter of regression equation calculation AuNPs: y=0.4271x+514.56, formula
In: y is colloidal gold solution maximum absorption peak, and x is gold particle average grain diameter.
For scanning result referring to shown in attached drawing 10, the colloidal gold shape of preparation is more uniform, without a large amount of aggregations, referring to attached drawing 11
Shown, the AuNPs ultraviolet spectra waveform of preparation is relatively smooth, and no trailing phenomenon, absorption peak narrower width illustrates AuNPs evenness
Preferably, maximum absorption peak is 526nm, can show that average grain diameter is 26.785nm by regression equation.
(4) activity identification of gold labeling antibody
The activity of gold labeling antibody is identified using Dot-immunogold filtration (DIGFA).Take secondary antibody (sheep anti mouse
IgG or rabbit anti-mouse igg) on cellulose nitrate (NC) film point sample and position mark is carried out, NC film is placed in 37 DEG C of drying boxes and is done
It is dry, it then takes gold labeling antibody to stand in the position point sample of secondary antibody, is rinsed with distilled water rapidly, the gold mark not reacted with secondary antibody is anti-
Body rinses out.
There is apparent punctation to show that gold labeling antibody is reacted with secondary antibody on NC film, it is active to be positive,
Redfree spot then indicates that antibody is inactive for feminine gender.As a result referring to shown in attached drawing 12, gold labeling antibody on NC film can be recognized
There is pink spot on point sample position, shows that there is prepared immune colloid gold particle bioactivity can tie with secondary antibody
Close reaction.
Embodiment six: the preparation of immune colloid gold test paper
(1) preparation of gold-labelled pad
Glass fibre element film is submerged into confining liquid (the 0.01mol/L phosphoric acid buffer containing 2~3% bovine serum albumin(BSA)s
Liquid) in closed, 37 DEG C of dry 1~2h, then cow's milk beta lactoglobulin gold labeling antibody and bactrian camel milk α-lactalbumin gold mark
Antibody mixed solution or cow's milk κ-casein gold labeling antibody and bactrian camel milk αs1Casein gold labeling antibody mixed solution, which is sprayed at, have been sealed
It on the glass fibre element film closed, is transferred quickly to be freeze-dried in freeze drier, production becomes gold-labelled pad.
(2) preparation of sample pad and water absorption pad
Sample pad is that glass fibre element film is submerged into confining liquid (the 0.01mol/L phosphorus containing 2~3% bovine serum albumin(BSA)s
Acid buffer) it is closed, 37 DEG C of drying for standby.Water absorption pad is the preferable absorbent filter material of water absorbing properties.
(3) coating of nitrocellulose filter (NC film)
Cow's milk beta lactoglobulin or cow's milk κ-casein are diluted to 0.5~1.5mg/ with 0.01mol/L phosphate buffer
The solution of mL is as T1Liquid, bactrian camel milk α-lactalbumin or bactrian camel milk αs1The solution that casein is diluted to 0.5~1.5mg/mL is made
For T2Liquid, sheep anti-mouse igg or rabbit anti-mouse igg monoclonal antibody are diluted to the solution of 0.5~1.5mg/mL as C liquid.It will preparation
C liquid, T1Liquid and T2Liquid is crossed on NC film respectively, is sequentially followed successively by T from left to right1Liquid, T2Liquid and C liquid set 37 DEG C of dryings 20
~40min is submerged into confining liquid closing, is placed in drying for standby in 37 DEG C of insulating boxs after dry.
(4) assembling of test paper
The sample pad prepared, gold-labelled pad, NC film and water absorption pad are cut to rectangle of same size, and press attached drawing
Overlapping assembling is carried out shown in 13, overlapped sequential is that sample pad right end is laminated on gold-labelled pad left end from left to right, gold-labelled pad right end
It is laminated on NC film left end, water absorption pad left end is laminated on the right end of NC film, mutually overlapping 1~3cm between each section.Wherein make
The gold-labelled pad and T sprayed with cow's milk beta lactoglobulin gold labeling antibody and bactrian camel milk α-lactalbumin gold labeling antibody mixed solution1Line
For cow's milk beta lactoglobulin, T2Line is that the NC film of bactrian camel milk α-lactalbumin carries out the test paper for being assembled into detection whey protein fraction
I；Use cow's milk κ-casein gold labeling antibody and bactrian camel milk αs1The gold-labelled pad and T of casein gold labeling antibody mixed solution spraying1
Line is cow's milk κ-casein, T2Line is bactrian camel milk αs1The NC film of casein carries out the test strips for being assembled into detection casein fraction
Ⅱ。
Embodiment seven: pseudo- bactrian camel milk is mixed in immune colloid gold test paper detection
In experimental basis by embodiment one to embodiment five, the present invention further provides immune colloid gold test paper detections
It mixes pseudo- bactrian camel milk and mixes fake method, i.e., the present invention further provides the immune colloid gold test papers for mixing pseudo- cow's milk in a kind of detection bactrian camel milk
Application, specifically includes the following steps:
(1) sample treatment: liquid milk sample can be used without pre-treatment, milk powder and cheese sample through dissolution；
(2) it detects: the sample pad part of immune colloid gold test paper is immersed in measuring samples solution, liquid is waited to diffuse to
It is taken out after water absorption pad, stands the appearance situation of observation T line and C line after 10~15min；
(3) testing result: referring to shown in attached drawing 14, C line goes out to represent result effective in test paper schematic diagram, T1Line and T2Line
Do not go out to represent and contains corresponding antigens in measuring samples.A, C line occurs in several results of B, C, D, and it is effective to represent result, wherein
The T of A result1Line does not occur and T2Line occurs, and illustrates to contain cow's milk protein in measuring samples, does not contain camel lactoprotein；B result
Middle T1Line occurs and T2Line does not occur, illustrates not containing cow's milk protein containing camel lactoprotein in measuring samples；T in C result1Line
And T2Line does not occur, illustrates to contain cow's milk and camel lactoprotein in measuring samples simultaneously；T in D result1Line and T2Line occurs,
Illustrate in sample product without cow's milk and camel lactoprotein.E, C line does not occur in several results of F, G, H, and it is invalid to represent result.
Embodiment eight: pseudo- bactrian camel milk application is mixed in immune colloid gold test paper detection
Cow's milk is mixed by volume in bactrian camel milk sample, the ratio for mixing cow's milk is respectively 100%, 50%, 30%,
20%, 10%, 5%, 1%, 0.1%, 0%, and distilled water is set as blank control, the step of according to embodiment six, it will exempt from
The sample pad part of epidemic disease colloid gold test paper is soaked respectively into sample liquids, is taken out after waiting liquid to diffuse to water absorption pad, and standing 10~
The appearance situation of T line and C line is observed after 15min, test result is as shown in table 1.
Table 1: pseudo- different proportion cow's milk experimental result is mixed in immunity colloidal gold test paper strip detection bactrian camel milk
| Sample additive ratio | C line | T1Line | T2Line | As a result judge |
| 100% | + | - | + | There are cow's milk, no bactrian camel milk |
| 50% | + | - | - | Cow's milk is detected in bactrian camel milk |
| 30% | + | - | - | Cow's milk is detected in bactrian camel milk |
| 20% | + | - | - | Cow's milk is detected in bactrian camel milk |
| 10% | + | - | - | Cow's milk is detected in bactrian camel milk |
| 5% | + | - | - | Cow's milk is detected in bactrian camel milk |
| 1% | + | + | - | Cow's milk is not detected in bactrian camel milk |
| 0.5% | + | + | - | Cow's milk is not detected in bactrian camel milk |
| 0% | + | + | - | There are bactrian camel milk, no cow's milk |
| Blank control | + | + | + | Bactrian camel milk cow's milk is not detected |
Note: "+" indicates occur, and "-" expression does not occur
Pass through test result shown in table 1, it can be seen that the immune colloid gold test paper prepared through the invention can be fast
Speed easily carries out mixing pseudo- cow's milk detection in bactrian camel milk, and lowest detection is limited to mix pseudo- 5% cow's milk in bactrian camel milk, can satisfy
Daily production and processing, the needs quickly detected.
As described above, the present invention can be realized preferably, the above embodiments are only to preferred implementation side of the invention
Formula is described, and is not intended to limit the scope of the present invention, and without departing from the spirit of the design of the present invention, this field is general
The various changes and improvement that logical technical staff makes technical solution of the present invention, should all fall into present invention determine that protection scope
It is interior.
Claims (10)
1. mixing the immune colloid gold test paper of pseudo- cow's milk, the examination including being assembled into detection whey protein fraction in a kind of detection bactrian camel milk
The test strips II of paper slip and detection casein fraction.
2. mixing the immune colloid gold test paper of pseudo- cow's milk in a kind of detection bactrian camel milk as described in claim 1, which is characterized in that institute
Test strips are stated as detection whey protein fraction, test strips include: using cow's milk beta lactoglobulin gold labeling antibody and bactrian camel milk α-
The gold-labelled pad of lactoalbumin gold labeling antibody mixed solution spraying uses the T of 0.5 ~ 1.5mg/mL cow's milk beta lactoglobulin1Liquid is drawn
T1Line and the T for using 0.5 ~ 1.5mg/mL bactrian camel milk α-lactalbumin2Liquid draws T2Line and use 0.5 ~ 1.5mg/mL sheep anti mouse
The C liquid of IgG or rabbit anti-mouse igg draws NC film, sample pad and the water absorption pad of C line, is assembled.
3. mixing the immune colloid gold test paper of pseudo- cow's milk in a kind of detection bactrian camel milk as described in claim 1, which is characterized in that institute
Test strips II are stated as detection casein fraction, test strips II include: using cow's milk κ-casein gold labeling antibody and bactrian camel milk αs1Junket
The gold-labelled pad of albumen gold labeling antibody mixed solution spraying uses 0.5 ~ 1.5mg/mL cow's milk κ-casein T1Liquid draws T1Line and
Use 0.5 ~ 1.5mg/mL bactrian camel milk αs1The T of casein2Liquid draws T2Line and using 0.5 ~ 1.5mg/mL sheep anti-mouse igg or
The C liquid of rabbit anti-mouse igg draws NC film, sample pad and the water absorption pad of C line, is assembled.
4. mixing the preparation method of the immune colloid gold test paper of pseudo- cow's milk in a kind of detection bactrian camel milk, which is characterized in that described immune
The preparation detailed process of colloid gold test paper are as follows:
(1) preparation of gold-labelled pad: glass fibre element film being submerged into confining liquid and is closed, 37 DEG C of 1 ~ 2 h of drying, then
Cow's milk beta lactoglobulin gold labeling antibody and bactrian camel milk α-lactalbumin gold labeling antibody mixed solution or cow's milk κ-casein gold mark are anti-
Body and bactrian camel milk αs1-Casein gold labeling antibody mixed solution is sprayed on the glass fibre element film closed, and is transferred quickly to
It is freeze-dried in freeze drier, production becomes gold-labelled pad；
(2) preparation of sample pad and water absorption pad: sample pad is submerged into confining liquid for glass fibre element film and is closed, and 37 DEG C dry
Dry spare, water absorption pad is the preferable absorbent filter material of water absorbing properties；
(3) coating of NC film: by C liquid, the T of preparation1Liquid and T2Liquid is crossed on NC film respectively, is sequentially followed successively by T from left to right1
Liquid, T2Liquid and C liquid set 37 DEG C of dry 20 ~ 40min, and confining liquid closing is submerged into after dry, are placed in 37 DEG C of insulating boxs dry
It is spare；
(4) sample pad prepared, gold-labelled pad, NC film and water absorption pad the assembling of test strips: are cut to length of same size
It is rectangular, and overlapping assembling is carried out, it is assembled into the test strips of detection whey protein fraction and the test paper of detection casein fraction respectively
Item II；
(5) overlapping described in step (4) assembles, and overlapped sequential is that sample pad right end is laminated on gold-labelled pad left end from left to right,
Gold-labelled pad right end is laminated on NC film left end, and water absorption pad left end is laminated on the right end of NC film, and stack laminate section mutually overlapping 1 ~
3cm。
5. the preparation method of the immune colloid gold test paper of pseudo- cow's milk is mixed in a kind of detection bactrian camel milk as claimed in claim 4,
It is characterized in that, the T1Cow's milk beta lactoglobulin or cow's milk κ-casein are diluted to by liquid with 0.01mol/L phosphate buffer
The solution of 0.5 ~ 1.5mg/mL.
6. the preparation method of the immune colloid gold test paper of pseudo- cow's milk is mixed in a kind of detection bactrian camel milk as claimed in claim 4,
It is characterized in that, the T2Liquid, with 0.01mol/L phosphate buffer by bactrian camel milk α-lactalbumin or bactrian camel milk αs1Casein is dilute
It is interpreted as the solution of 0.5 ~ 1.5mg/mL.
7. the preparation method of the immune colloid gold test paper of pseudo- cow's milk is mixed in a kind of detection bactrian camel milk as claimed in claim 4,
It is characterized in that, the C liquid, is diluted to sheep anti-mouse igg or rabbit anti-mouse igg monoclonal antibody with 0.01mol/L phosphate buffer
The solution of 0.5 ~ 1.5mg/mL.
8. the preparation method of the immune colloid gold test paper of pseudo- cow's milk is mixed in a kind of detection bactrian camel milk as claimed in claim 4,
It is characterized in that, the cow's milk beta lactoglobulin gold labeling antibody, bactrian camel milk α-lactalbumin gold labeling antibody, cow's milk κ-casein gold mark
Antibody, bactrian camel milk αs1The preparation of casein gold labeling antibody specifically includes the following steps:
(1) reduction of sodium citrate method prepare AuNPs: gold chloride is dissolved in distilled water, make its final concentration of 0.1% ~ 0.15%, add
Heat takes 0.75 ~ 1.5mL, 1% citric acid three sodium solution to be added in the conical flask of above-mentioned boiling to boiling, and is uniformly mixed rapidly, protects
100 DEG C, 10 ~ 20 min are held, after solution is cooled to room temperature, with distilled water make-up solution to original volume；
(2) preparation of gold labeling antibody: the cow's milk beta lactoglobulin monoclonal antibody of 18 ~ 36 μ g or cow's milk beta lactoglobulin is more
Clonal antibody or cow's milk κ-casein monoclonal antibody or cow's milk κ-casein polyclonal antibody or bactrian camel milk α-lactalbumin list
Clonal antibody or bactrian camel milk α-lactalbumin polyclonal antibody or bactrian camel milk αs1Casein monoclonal antibody or bactrian camel milk αs1Junket
Protein polyclone antibody is separately added into colloidal gold solution, adjusts the pH to 7.6 ~ 8.5 of the AuNPs solution of tape label, mixing is simultaneously
Stand 2 ~ 5min；
(3) Quality Identification of AuNPs: using the character and aggregation situation of scanning electron microscope observation AuNPs, and 400 are used
~ 700 nm ultraviolet specrophotometers detect the AuNPs solution prepared, record the absorption maximum as a result, AuNPs solution
Spike is long, and there are linear relationships with AuNPs average grain diameter, can be according to the average grain diameter of regression equation calculation AuNPs: y=
0.4271x+514.56, in formula: y is colloidal gold solution maximum absorption peak, and x is gold particle average grain diameter；
(4) activity identification of gold labeling antibody: taking sheep anti-mouse igg or rabbit anti-mouse igg as secondary antibody, secondary antibody point on nitrocellulose membrane
Sample simultaneously carries out position mark, and nitrocellulose membrane is placed drying in 37 DEG C of drying boxes, then takes gold labeling antibody in the position of secondary antibody
Point sample is stood, and is rinsed with distilled water rapidly, the gold labeling antibody that do not react with secondary antibody is rinsed out.
9. the preparation method of the immune colloid gold test paper of pseudo- cow's milk is mixed in a kind of detection bactrian camel milk as claimed in claim 8,
It is characterized in that, the cow's milk beta lactoglobulin, cow's milk κ-casein, bactrian camel milk α-lactalbumin, bactrian camel milk αs1Casein Dan Ke
The preparation of grand antibody specifically includes: choosing the female Balb/c mouse of about 7 week old as immune animal, immunogene is cow's milk β-cream
Globulin, cow's milk κ-casein, bactrian camel milk α-lactalbumin and bactrian camel milk αs1Casein, immunogene and Freund's complete adjuvant or
Incomplete Freund's adjuvant is immune after ultrasound mixes well emulsification, subcutaneous multiple spot and the immune mouse of intraperitoneal injection；Separation serum is adopted
Potency is measured with indirect elisa method, when serum titer reaches 1:64000 or more, to response value highest mouse tail vein and abdominal cavity
100 μ g immunogenes of the injection without adjuvant carry out last booster immunization respectively；It takes spleen to prepare lymphocyte inducing cell to melt
Close, using indirect elisa method screening with sheep, horse, donkey, camel or cow's milk no cross reaction and with cow's milk beta lactoglobulin or cow's milk
κ-casein or bactrian camel milk α-lactalbumin or bactrian camel milk αs1The higher positive colony further expansion training of casein response value
It supports, injection expands the cell after culture into ascites is induced in Mice Body, purifies, obtains monoclonal antibody, is lyophilized spare.
10. such as the application for the immune colloid gold test paper for mixing pseudo- cow's milk in a kind of detection bactrian camel milk in any one of claim 1 or 4, tool
Body the following steps are included:
(1) sample treatment: liquid milk sample can be used without pre-treatment, milk powder and cheese sample through dissolution；
(2) it detects: the sample pad part of immune colloid gold test paper is immersed in measuring samples solution, liquid is waited to diffuse to water suction
It is taken out after pad, stands the appearance situation of observation T line and C line after 10 ~ 15min；
(3) testing result: C line goes out to represent result effective in test strips；T in test strips1Line and T2Line does not go out to represent sample to be checked
Contain corresponding antigens in product；T in test strips1Line does not occur and T2Line occurs, and illustrates to contain cow's milk protein in measuring samples, be free of
There is camel lactoprotein；T in test strips1Line occurs and T2Line does not occur, illustrates not containing in measuring samples containing camel lactoprotein
Cow's milk protein；T in test strips1Line and T2Line does not occur, illustrates to contain cow's milk and camel lactoprotein in measuring samples simultaneously；Examination
T in paper slip1Line and T2Line occurs, and illustrates to try in sample product without cow's milk and camel lactoprotein；C line does not occur in paper slip, represents
As a result invalid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910223473.0A CN110196330A (en) | 2019-03-22 | 2019-03-22 | Immunity colloidal gold test paper strip and its application of pseudo- cow's milk are mixed in a kind of detection bactrian camel milk |
Applications Claiming Priority (1)
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| CN113092787A (en) * | 2021-04-09 | 2021-07-09 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Preparation method and application of sheep milk protein and goat milk protein duplex detection card |
| CN113311155A (en) * | 2021-04-09 | 2021-08-27 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Preparation method and application of cow milk protein and buffalo milk protein duplex detection card |
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| CN111704667A (en) * | 2020-08-20 | 2020-09-25 | 北京金智准科技有限公司 | Bovine and goat milk casein monoclonal antibody, detection kit and application |
| CN113092787A (en) * | 2021-04-09 | 2021-07-09 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Preparation method and application of sheep milk protein and goat milk protein duplex detection card |
| CN113311155A (en) * | 2021-04-09 | 2021-08-27 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Preparation method and application of cow milk protein and buffalo milk protein duplex detection card |
| CN113092787B (en) * | 2021-04-09 | 2022-07-19 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Preparation method and application of sheep milk protein and goat milk protein duplex detection card |
| CN113311155B (en) * | 2021-04-09 | 2022-11-29 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Preparation method and application of cow milk protein and buffalo milk protein dual-detection card |
| CN114716531A (en) * | 2022-05-17 | 2022-07-08 | 中国农业科学院农业质量标准与检测技术研究所 | Casein polypeptide, polypeptide antigen, antibody, test strip and application thereof |
| CN114716531B (en) * | 2022-05-17 | 2023-11-03 | 中国农业科学院农业质量标准与检测技术研究所 | Casein polypeptide, polypeptide antigen, antibody, test strip and application thereof |
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