CN104931663A - Immune colloidal gold method capable of quickly detecting milk allergen - Google Patents
Immune colloidal gold method capable of quickly detecting milk allergen Download PDFInfo
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- CN104931663A CN104931663A CN201510336210.2A CN201510336210A CN104931663A CN 104931663 A CN104931663 A CN 104931663A CN 201510336210 A CN201510336210 A CN 201510336210A CN 104931663 A CN104931663 A CN 104931663A
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Abstract
The invention discloses an immune colloidal gold method capable of quickly detecting a milk allergen. According to the method, two milk resistance beta LG monoclonal antibodies of the high potency with the known epitope are used, double-antibody sandwich colloidal gold testing paper slips are prepared, and the main allergen beta LG of milk can be detected quickly, simply and conveniently. According to the immune colloidal gold method, the detection sensitivity is about 0.2 ng.mL-1, the consumed time is short (about 1-5 min), and quick detection can be achieved; meanwhile, operation is easy, no requirement for professional skills of operators exists, and the method can be widely applied to detecting the milk allergen.
Description
Technical field
The present invention relates to technical field of immunoassay, particularly relate to a kind of Colloidal Gold of quick detection detecting milk allergen.
Background technology
What FAO/WHO assert causes in eight of human foods allergy large based foods, and cow's milk and goods thereof are exactly one of them.In the food labelling method of America and Europe, also specify cow's milk is the food hypersenstivity ultimate constituent that must indicate.Beta lactoglobulin (β-lactoglobulin, β LG) is the main allergen in cow's milk, and the detection method of milk allergen β LG is at present mainly for the DNA fragmentation of specific allergy crude protein in cow's milk or coding anaphylactogen.Take DNA fragmentation as the means of the detection detecting target, rely on PCR (PCR) to increase specific DNA fragmentation, detect the DNA of allergen gene in food to remain, this technology is as the detection target of food hypersenstivity material instead of detect allergen protein itself using the DNA of coding allergen protein.The current Application comparison of these class methods is extensively qualitative PCR and real-time fluorescence quantitative PCR (Real-time PCR).Real-time PCR can utilize specific probe to improve the specificity detected.At present, round pcr is for the detection of different food products anaphylactogen.But diet diversity due in food caused by allergen protein, is not mainly the gene itself that albumen is corresponding, PCR occurs that positive findings can not illustrate in food containing corresponding allergen protein.Therefore, be not desirable detection method by the method detected based on allergen gene, apply less in reality detects.
Detect specific allergen method of protein in food mainly based on the immunoreactivity of albumen and specific antibody.The specific antibody preparing high-titer is the key that allergen protein detects.It is exactly the principle of the specific bond utilizing antigen and antibody in the immune detection of milk allergen.At present, topmost immunologic detection method is exactly ELISA method (Enzyme-linked immunosorbent assay, ELISA).There are two kinds of semiquantitative ELISA method: be double antibody sandwich method (Double antibody sandwich method ELISA respectively, and competition law (Competitive ELISA S-ELISA), C-ELISA), the wherein sensitive height of double antibody sandwich method is also conventional.The method of protein detection of ELISA mainly detects cow's milk total protein in food or cow's milk main allergen (β LG, CNs, BSA), and the detection limit (LOD) of S-ELISA is generally be about 2.5ppm, and the LOD of C-ELISA is generally about 1ppm.Detection time about 2h ~ 4h of ELISA.The anti-β LG polyclonal antibody of purifying has detected the β LG of low dosage in baby milk powder and human milk for double antibody sandwich method, its detection limit can reach 0.002 mgmL
-1.At present, based on the double antibody sandwich method of anti-β LG monoclonal antibody also for detecting the β LG of natural or sex change, cow's milk and the goods thereof of sex change after thermal treatment can be detected.Anti-casein polyclonal antibody and monoclonal antibody, also for detection caseic in cow's milk, are mainly used in the immunoreactivity evaluating hydrolysis baby milk powder albumen.In recent years, double antibody sandwich method is also for detecting not containing the milk allergen in the food of milk elements, and as whether contained casein fraction in fruit juice, biscuit and chocolate, its detection limit can reach 5 mg kg
-1.But the method for ELISA is consuming time, effort; Need the equipment of specialty, and testing staff need could operate through certain professional training.
Except ELISA method, electrophoresis (SDS-PAGE), mass spectroscopy (LC/MS/MS), Western blot (Western Blotting) is also for the detection of hydrolyzed bovine Ruzhong milk allergen.But compared with traditional ELISA method, the sensitivity of these methods is not high, the Allergic skin test of low dosage is not gone out or cost too high.
Research based on anti-β LG polyclone and monoclonal antibody enzyme chain immune detection cow's milk main allergen β LG cow's milk also studies have reported that.Although enzyme chain immunologic detection method has high specific and highly sensitive advantage, these methods, for the detection of food allergen, need longer time and larger workload, also need certain experiment condition and professional and technical personnel to operate.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of Colloidal Gold of quick detection detecting milk allergen, be intended to solve existing detection method and take time and effort, sensitivity is low, and needs professional's just operable problem.
Technical scheme of the present invention is as follows:
A Colloidal Gold for quick detection detecting milk allergen, wherein, comprises step:
A, using cow's milk β LG as antigen, immune animal, obtain hybridoma;
B, by hybridoma, preparation monoclonal antibody;
C, monoclonal antibody to be identified, and obtain the epitope information of monoclonal antibody;
D, the monoclonal antibody choosing the different epi-positions of two strains are anti-and detect monoclonal antibody and prepare colloidal gold test paper slip respectively as Sheet, described colloidal gold test paper slip comprises water-absorption fiber, nitrocellulose filter and absorbent filter three part, wherein, water-absorption fiber part sprays the Sheet clonal antibody-colloidal gold conjugate of anti-cow's milk β LG, nitrocellulose filter sprays the detection monoclonal antibody of anti-cow's milk β LG; React under being put in room temperature, obtain testing result.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, described steps A specifically comprises: 4-6 BALB/c mouse in age in week, as immune animal, through screening and the clone of immunity, Fusion of Cells, hybridoma, obtains hybridoma cell clone strain.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, is cloned hybridoma wells by limiting dilution assay.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, described step B specifically comprises: get BALB/c mouse, and intraperitoneal injection paraffin oil injected hybridoma after 1 ~ 2 week; After 7-9 days, collect ascites, centrifugal, draw supernatant, then antibody purification is carried out to the supernatant collected, obtain the monoclonal antibody of anti-cow's milk β LG.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, described step C also comprises: to the subclass of the purity of monoclonal antibody and Western blotting, monoclonal antibody, monoclonal antibody tire and the intercrossing of monoclonal antibody is identified.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, described monoclonal antibody tire for: measure the tiring of monoclonal antibody with indirect ELISA method, simultaneously using NS-1 Fusion of Cells cell ascites as negative control, using P/N value > 2.1 monoclonal antibody greatest dilution tiring as monoclonal antibody.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, in described step C, improvement on synthesis is passed through in described qualification, then by the polypeptide of synthesis and monoclonal antibody specific bond, ELISA and Immuno dot-blotting is adopted to detect the epitope information of monoclonal antibody.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, in described step D, the preparation process of described monoclonal antibody-colloidal gold conjugate specifically comprises:
After being boiled by gold chloride, add trisodium citrate aqueous solution, continue to boil 10 ~ 20min; After cooling, add K
2cO
3adjust pH to 8.0 ~ 8.5; Add the anti-also rapid stirring of Sheet of anti-milk allergen β LG; Add OVA and rapid stirring, add NaCl solution, centrifugal, abandon precipitation, supernatant is centrifugal again; Abandon supernatant, obtain monoclonal antibody-colloidal gold conjugate.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, in described step D, the preparation process of described colloidal gold test paper slip specifically comprises: colloidal gold test paper slip comprises water-absorption fiber, nitrocellulose filter and absorbent filter three part; Wherein, water-absorption fiber part sprays the Sheet clonal antibody-colloidal gold conjugate of anti-cow's milk β LG, detection monoclonal antibody nitrocellulose filter spraying anti-cow's milk β LG with sheep anti-mouse igg bag by the lines becoming two 0.8 ~ 1.2mm wide, air-dry rear use is closed containing the PBS of OVA, glue on the support successively after drying, be cut into the bar of 0.2 cm × 8 cm.
The Colloidal Gold of described quick detection detecting milk allergen, wherein, in described step D, the testing result of cow's milk comprises: test-strips cellulose membrane to occur two red line are for positive findings, only occur that a red line is negative findings, losing efficacy appears then being considered as detecting in redfree lines.
Beneficial effect: the present invention utilizes the monoclonal antibody of the anti-cow's milk β LG of the high-titer of the known epi-position of preparation, then by the colloidal gold test paper slip of double-antibody sandwich of preparation, can be fast and convenient detect cow's milk major allergen protein β LG.
Accompanying drawing explanation
Fig. 1 is the test result schematic diagram of embodiment 1 colloidal gold test strip.
Embodiment
The invention provides a kind of Colloidal Gold of quick detection detecting milk allergen, for making object of the present invention, technical scheme and effect clearly, clearly, the present invention is described in more detail below.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The invention provides a kind of Colloidal Gold of quick detection detecting milk allergen, it comprises step:
A, using cow's milk β LG as antigen, immune animal, obtain hybridoma;
B, by hybridoma, preparation monoclonal antibody;
C, monoclonal antibody to be identified, and obtain the epitope information of monoclonal antibody;
D, the monoclonal antibody choosing the different epi-positions of two strains are anti-and detect monoclonal antibody and prepare colloidal gold test paper slip respectively as Sheet, described colloidal gold test paper slip comprises water-absorption fiber, nitrocellulose filter and absorbent filter three part, wherein, water-absorption fiber part sprays the Sheet clonal antibody-colloidal gold conjugate of anti-cow's milk β LG, nitrocellulose filter sprays the detection monoclonal antibody of anti-cow's milk β LG; React under being put in room temperature, obtain testing result.
The present invention utilizes the monoclonal antibody of the anti-cow's milk β LG of the high-titer of the known epi-position of preparation, then by the colloidal gold test paper slip of double-antibody sandwich of preparation, can be fast and convenient detect cow's milk major allergen protein β LG.Immune colloid gold of the present invention detects the method for cow's milk main allergen β LG, and detection sensitivity is about 0.2ngmL
-1.Compared to additive method, immune colloid gold of the present invention detects maximum advantage and is the used time few (about 1 ~ 5min), can detect fast.Simultaneously simple to operate, to the requirement of operating personnel without professional aspect, can the detection of widespread use and milk allergen.
Below by a specific embodiment, technique scheme of the present invention is described in detail.
Embodiment 1
1, animal immune
Getting 10 4-6 BALB/c mouse in age in week, is 0.5 mgmL by 1 mL concentration
-1β LG albumen and isopyknic Freund's complete adjuvant of natural cow's milk fully mix and after emulsification, in mouse back subcutaneous point 3 injections, every mouse immune dosage is 30 μ g; After 14th day, the antigen of equivalent freund 's incomplete adjuvant emulsification at mouse subcutaneous point of multi-point injection, dosage is constant; After 21st day, carry out third time routine immunization, after each immunity, get blood at mouse tail, detect the titre producing antibody with indirect elisa method; (carry out fusion first 3 days with mouse myeloma NS-1 cell) after 30 days and inject native antigen for the last time through mouse tail vein again, dosage reduces by half, booster immunization 1 time.
2, the foundation of indirect ELISA method
Using natural cow's milk β LG as envelope antigen, by square formation method (longitudinally with antigen coated, by 10,5.0,2.5,1.25,0.625,0.313,0.156,0.078,0.039,0.020 μ gmL
-1concentration bag quilt, 1:1000 pressed by horizontal antiserum, 1:2000,1:4000,1:8000,1:16000,1:32000 multiple dilutions) set up indirect ELISA method, with immunity before Normal Mouse Serum for negative control.Survey the absorbance (OD) at wavelength 450nm place by microplate reader, get male/female value (P/N value) maximum time antigen, antibody dilution is as the best effort concentration of antigen, antibody.Be greater than 2.1 times of negative serum value with the light absorption value of P/N value >2.1(and monoclonal antibody supernatant during detection) judge that testing result is as the positive, the light absorption value of P/N value <2.1(and monoclonal antibody supernatant is less than 2.1 times of negative serum value) judge that testing result is as feminine gender.
3, the screening of Fusion of Cells, hybridoma and cloning.
1), Fusion of Cells
(1) preparation of NS-1 myeloma cell
In Fusion of Cells first 7 days, take out the NS-1 myeloma cell in liquid nitrogen, recover; Cultured cell in 24 porocyte culture plates, cultivation temperature is 37 DEG C, CO
2concentration is 5%, and be that 15% hyclone DMEM nutrient solution carries out Secondary Culture by concentration, 12 h change liquid once, and 24 h go down to posterity once, and within 5-7 days, adjustment cell density is that 105-106/mL is good, merges when exponential phase.Add appropriate serum-free DMEM nutrient solution and replace nutrient solution in former bottle, and cell is blown down gently, be transferred in the centrifuge tube of 50 mL, centrifugal l 000 rpm, 5 min, wash 3 times, abandon supernatant, make cell precipitation resuspended with serum-free medium.
(2) preparation of splenocyte
The BALB/c mouse of getting immunity extracts eyeball blood sampling execution, collects blood and does negative control sera, mouse is soaked in 75% alcohol 5 min and carries out disinfection; The tweezers that transducer set is newly sterilized and scissors, cut off mouse peritoneum, exposes spleen; Press from both sides out spleen, with the fat on scissors stripping spleen and connective tissue; Spleen is rinsed with serum-free medium, spleen is transferred to sterile glass double dish (nutrient solution is housed), spleen is placed on 100 object copper mesh, with pressure spleen device, splenocyte is extruded gently, repeatedly rinse copper mesh with serum-free medium, splenocyte suspension is drawn onto the centrifuge tube of 50 mL; Centrifugal 1000 rpm, 10 min, wash 2 cells, abandon supernatant, get cell precipitation and count.
(3) preparation of feeder cells
Get non-immune BALB/c mouse to put to death, mouse is soaked in 75% alcohol 5 min and carries out disinfection; Fixing mouse of having sterilized is on dissection plate, belly, with cotton ball soaked in alcohol wiping belly, clamps lower abdomen skin with tweezers upward, with scissors mouse part skin cut off and expose mouse peritoneum and on peritonaeum, cut an osculum, in mouse peritoneal, inject 3 mL serum-free mediums with aseptic straw, draw 3 times, nutrient solution is drawn onto in 50 mL centrifuge tubes, centrifugal 1 000rpm, l0 min, abandons supernatant, the resuspended rear counting of cell.Suspend with HAT nutrient culture media afterwards, cell is stored in 37 DEG C, 5% CO
2in incubator.
(4) Fusion of Cells
Immune spleen cell about 1 × 10 will be got out
8individual with NS-1 myeloma cell about 2 × 10
7individually be mixed in 50 mL centrifuge tubes, centrifugal 1000rpm, 5min; Abandon supernatant, flick with finger and to make at the bottom of pipe that cell is loose to be evenly distributed at the bottom of pipe, 40 DEG C of water-bath preheatings; Slowly instill PEG solution 0.5 mL that 37 DEG C of concentration are 50% in lmin, limit edged stirs; DMEM(37 DEG C is added with 10 mL suction pipes) fast after minimal medium 10 mL(is first slow, within the 1st minute, dropwise add lmL, within the 2nd minute, add 1mL, within 3-4 minute, add 3 mL, after 5 minutes, add remaining 5 mL), 37 DEG C leave standstill 10 min, after centrifugal 500 rpm, 5min, abandon supernatant; Add HAT nutrient solution 5 mL, cell is dispelled city gently, and it suspends and mixes; Cell suspension after merging is added in 96 well culture plates, every hole 0.2mL, in 37 DEG C, concentration 5% CO
2cultivate in incubator.
(5) cultivation of hybridoma
The growing state of first 3 days observation of cell after merging, within the 5th day, 1 HAT nutrient culture media is added in the every hole of culture plate, within 8-10 days, changes HT nutrient solution 100 μ L; Continue the growing state of observation of cell, when fused cell group grows to 1/4 of hole floorage, sucking-off supernatant carries out antibody test.
2), positive hybridoma cell screening and clone
(1) antibody in indirect ELISA method detection fusion cell conditioned medium
Successful cell conditioned medium is merged in sucking-off, using natural cow's milk β LG albumen as envelope antigen, with the indirect ELISA method set up, cell conditioned medium liquid is detected, with Normal Mouse Serum before immunity for negative control, be greater than 2.1 times of negative serum value with the light absorption value of P/N value >2.1(and monoclonal antibody supernatant during detection) judge that testing result is as the positive, the light absorption value of P/N value <2.1(and monoclonal antibody supernatant is less than 2.1 times of negative serum value) judge that testing result is as feminine gender.
(2) clone of positive hybridoma cell
By limiting dilution assay, subclone is carried out to fixed positive hybridoma cell hole.The hybridoma that will carry out cloning is blown down gently from plate, by complete medium, cell is carried out dilution for every milliliter containing 5,10,50 cells, be dispensed into by cell suspension in 96 well culture plates, every hole 100 μ L, the cell number in every hole is respectively 0.5,1 and 5; Cultivation the 4th day, half amount changed liquid once; Observe the growing state of each porocyte afterwards every day; 10-15 days, when cell grows to about 1/4 hole floorage, carries out full dose and changes liquid, and secondary daily indirect elisa method detects cell conditioned medium liquid; According to testing result, select there is specific antibody, antibody titer is high, the good and hybridoma wells of single clonal growth of form, then carries out subclone, clones 4 times; After 4 times, the hybridoma in positive hole is moved to 24 porocyte culture plates.
4, the preparation of anti-cow's milk β LG monoclonal antibody
Adjustment hybridoma concentration is 2 × 10
5individual/mL, is inoculated in the culture flask of 80 mL, adds complete culture solution, at 37 DEG C, and 5% CO
2condition under cultivate.Treat that cytoactive reaches optimum condition, centrifugal 1000 rpm, 15min, abandon supernatant, and the full nutrient solution that cannots be used up washes 3 times, cell quantity is adjusted to about 5 × 10
6individual/mL; Get BALB/c mouse, intraperitoneal injection 0.5 mL paraffin oil, injects the hybridoma of 1mL for 7 days afterwards.About 7-9 days, when mouse web portion obviously swells, collect ascites, centrifugal 4 000rpm, 5 min, precipitate at the bottom of removing upper-layer fat and pipe, draw supernatant.With reference to HiTrap protein A affinity chromatography instructions, antibody purification is carried out to the supernatant collected, namely obtain the monoclonal antibody of anti-cow's milk β LG.
5, the specificity identification of anti-cow's milk β LG monoclonal antibody
1), the purity detecting of monoclonal antibody and Western blotting qualification
What have employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot antagonism β LG monoclonal antibody carries out purity detecting and qualification.First β LG is carried out SDS-PAGE electrophoresis (concentrated glue 5%, separation gel 12%).Protein on gel is gone to (300mA, 60min, 4 DEG C) on nitrocellulose filter by protein electrophorese transfer instrument electricity.The TBST solution added containing 3% OVA is closed, and 4 DEG C are spent the night.After shaking wash 3 times with TBST, film is cut to 8 parts, respectively room temperature effect 1 h, the antibody adding mark HRP sheep anti-mouse igg is two to resist, and room temperature effect 1 h, after TBST flushing membrane, DAB develops the color.
2), the qualification of Subclass of antibody
Carry out subgroup identification with Subclass of antibody kit to the monoclonal antibody obtained, experimental procedure is with reference to instructions.
3), the detection of antibody titer
Antibody titer is measured, simultaneously using NS-1 Fusion of Cells cell ascites as negative control, using P/N value > 2.1 antibody greatest dilution tiring as monoclonal antibody with the indirect ELISA method set up.Bag is by natural cow's milk β LG 100 ng/ hole, and 4 DEG C are spent the night, wash plate, add confining liquid 200 μ L, closes 2 h, washs 3 times for 37 DEG C.Respectively with the anti-β LG monoclonal antibody after purifying for primary antibodie, negative control is done with mice serum before immunity, monoclonal antibody two-fold dilution (being diluted to 1:1024000 from 1:1000) is added 100 μ L/ holes, 37 DEG C of incubation 1 h, wash plate, sheep anti-mouse igg-the HRP adding dilution is two anti-100 μ L/ holes, and 37 DEG C of incubation 45 min, wash plate; Add 37 DEG C of lucifuges to develop the color 10 min; Mass concentration 12.5% H is added after colour developing
2sO
450 μ L/hole cessation reactions, measure light absorption value (OD value) at wavelength 450 nm place.The dilutability of antibody is tiring of antibody.
4), monoclonal antibody intercrossing detects
Adopt the indirect elisa method set up, join in hole by β LG and other cow's milk major allergen protein (CNs, BSA and α LA) and cow's milk crude protein by 100 ng albumen/holes, 4 DEG C of bags are spent the night, and add 200 μ L/ hole confining liquids and close, in 37 DEG C of incubation 2 h, wash plate; Add monoclonal antibody 100 μ L/hole respectively, with the normal serum of mouse before immunity for negative control, in 37 DEG C of incubation 1 h, wash plate; Add the sheep anti-mouse igg-HRP 100 μ L/ hole of dilution, 37 DEG C of incubation 45 min, wash plate; Add TMB 37 DEG C of lucifuges to develop the color 10 min; Mass concentration 12.5% H is added after colour developing
2sO
450 μ L/hole cessation reactions, wavelength 450nm place measures light absorption value (OD value).
Adopt the indirect elisa method set up, cow's milk is is slightly guided and supported (peanut, egg, fish, shrimp, cashew nut, wheat, soybean) with the albumen of other Major Foods to be joined in hole by 100 ng albumen/holes, and 4 DEG C of bags are spent the night, and adds confining liquid 200 μ L/ hole and closes, in 37 DEG C of incubation 2h, wash plate; Add monoclonal antibody 100 μ L/hole, with the normal serum of mouse before immunity for negative control, in 37 DEG C of incubation 1 h, wash plate; Add the sheep anti-mouse igg-HRP 100 μ L/ hole of dilution, 37 DEG C of incubation 45 min, wash plate; Add TMB 37 DEG C of lucifuges to develop the color 10 min; Mass concentration 12.5% H is added after colour developing
2sO
450 μ L/hole cessation reactions, wavelength 450nm place measures light absorption value (OD value).
6, the Tricine-SDS-PAGE electroresis appraisal of Main Antigenic peptide chain
The formula of Tricine glue is as shown in table 1.Four sections of polypeptide of synthesis are carried out Tricine-SDS-PAGE electrophoresis, qualification molecular size range and purity.
Table 1, Tricine glue are filled a prescription
1), the ELISA of polypeptide fragment and monoclonal antibody specific bond detects
The polypeptide getting above-mentioned synthesis is buffered liquid with ELISA bag respectively and is diluted to 10 μ gmL
-1, add 100 μ L/ holes, using CNs, β LG albumen as negative, positive control, 4 DEG C of bags are spent the night.After discarding albumen coating buffer, wash plate.Add 200 μ L/ hole confining liquids, 37 DEG C of closed 2h, wash plate.Adding concentration is respectively 1 μ gmL
-1different monoclonal antibodies 100 μ L/ hole, educate warm 1h for 37 DEG C.Wash plate, add HRP and mark sheep anti-mouse igg 100 μ L/ hole, hatch 45 min, wash plate for 37 DEG C.Add TMB 37 DEG C of lucifuges to develop the color 10 min, adding stop buffer mass concentration after colour developing is 12.5% H
2sO
450 μ L/ hole cessation reactions, survey the light absorption value of 450nm.
2), the Immuno dot-blotting of polypeptide fragment and monoclonal antibody specific bond detects
The polypeptide getting above-mentioned synthesis is diluted to 20 μ gmL with PBS respectively
-1, using CNs, β LG albumen as negative, positive control, dripping 100 μ L to nitrocellulose filter, after naturally drying, close 2h with confining liquid, after the rinsing of TBST cleansing solution, is 1 μ gmL with concentration respectively
-1monoclonal antibody educate warm 1h at 37 DEG C.Marking goat anti-mouse igg with adding HRP after the rinsing of TBST cleansing solution, educating warm 45min for 37 DEG C.After cleansing solution rinsing, DAB develops the color.
7, monoclonal antibody biotin labeling
Respectively the 1H8 of known antigens epi-position, 1G5 and 4C3 are mixed lucifuge in proportion with the biotin (NSH-Biotin) of activation and react 30min, remove by the ultra filtration membrane post of 50kDa the NSH-Biotin having neither part nor lot in mark.
8, the two monoclonal antibody sandwich immunoassay colloidal gold method of cow's milk main allergen β LG is detected
1) preparation of, collaurum-anti-cow's milk total protein antibody conjugates
After gold chloride (mass concentration is 0. 01%) 100 DEG C is boiled, add trisodium citrate aqueous solution (mass concentration is 1%) (ratio adding 1.5 mL trisodium citrate aqueous solutions in every 100 mL gold chlorides adds), continue to boil 15 min.After cooling, add K
2cO
3(0.2molL
-1) adjust pH to 8.2; The best Sheet adding anti-milk allergen β LG resists 1 mg and rapid stirring 15min.Add OVA 200mg, rapid stirring 5 min, adds NaCl(mass concentration 10%) solution makes NaCl ultimate density be 1 %, and centrifugal 10 min of 2000 rpm, abandon precipitation, and supernatant is again with centrifugal 1 h of 10000 rpm; Abandon supernatant, the sediment obtained is the Sheet clonal antibody-colloidal gold conjugate of the anti-milk allergen β LG of purifying.
2), the preparation of immune chromatograph testing strip
Colloidal gold test strip comprises water-absorption fiber, nitrocellulose filter and absorbent filter three part.Water-absorption fiber part sprays the Sheet clonal antibody-colloidal gold conjugate of anti-cow's milk β LG, the use 100 μ gmL on nitrocellulose filter
-1anti-milk allergen β LG(PBS dilute) detection monoclonal antibody (being sprayed on detection line 1) and sheep anti-mouse igg (diluting with PBS) (being sprayed on control line 2) bag by the lines becoming two treaty 1 mm wide, air-dry rear use is closed containing the PBS of OVA.Glue on the support successively after drying, be cut into the bar of 0.2cm × 8 cm.Result judges: reaction is at room temperature carried out, test-strips cellulose membrane to occur two red line are for positive findings, if test-strips in Fig. 14 is positive findings, only occur that a red line is (near absorbent filter end, control line) be negative findings, if test-strips in Fig. 13 is negative findings, if redfree lines occur, be considered as detecting and lost efficacy.
In sum, the Colloidal Gold of a kind of quick detection detecting milk allergen provided by the invention, utilize the monoclonal antibody of the anti-cow's milk β LG of the high-titer of the known epi-position of two strains, by preparing the colloidal gold strip of double-antibody sandwich, fast and convenient detection cow's milk main allergen β LG can be realized.Colloidal Gold detection sensitivity of the present invention is about 0.2ngmL
-1, the used time short (about 1 ~ 5min), and detect fast.Simultaneously simple to operate, to the requirement of operating personnel without professional aspect, can the detection of widespread use and milk allergen.
Should be understood that, application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (10)
1. detect a Colloidal Gold for detecting milk allergen fast, it is characterized in that, comprise step:
A, using cow's milk β LG as antigen, immune animal, obtain hybridoma;
B, by hybridoma, preparation monoclonal antibody;
C, monoclonal antibody to be identified, and obtain the epitope information of monoclonal antibody;
D, the monoclonal antibody choosing the different epi-positions of two strains are anti-and detect monoclonal antibody and prepare colloidal gold test paper slip respectively as Sheet, described colloidal gold test paper slip comprises water-absorption fiber, nitrocellulose filter and absorbent filter three part, wherein, water-absorption fiber part sprays the Sheet clonal antibody-colloidal gold conjugate of anti-cow's milk β LG, nitrocellulose filter sprays the detection monoclonal antibody of anti-cow's milk β LG; React under being put in room temperature, obtain testing result.
2. the Colloidal Gold of quick detection detecting milk allergen according to claim 1, it is characterized in that, described steps A specifically comprises: 4-6 BALB/c mouse in age in week, as immune animal, through screening and the clone of immunity, Fusion of Cells, hybridoma, obtains hybridoma cell clone strain.
3. the Colloidal Gold of quick detection detecting milk allergen according to claim 2, be is characterized in that, cloned by limiting dilution assay to hybridoma wells.
4. the Colloidal Gold of quick detection detecting milk allergen according to claim 3, it is characterized in that, described step B specifically comprises: get BALB/c mouse, and intraperitoneal injection paraffin oil injected hybridoma after 1 ~ 2 week; After 7-9 days, collect ascites, centrifugal, draw supernatant, then antibody purification is carried out to the supernatant collected, obtain the monoclonal antibody of anti-cow's milk β LG.
5. the Colloidal Gold of quick detection detecting milk allergen according to claim 1, it is characterized in that, described step C also comprises: to the subclass of the purity of monoclonal antibody and Western blotting, monoclonal antibody, monoclonal antibody tire and the intercrossing of monoclonal antibody is identified.
6. the Colloidal Gold of quick detection detecting milk allergen according to claim 5, it is characterized in that, described monoclonal antibody tire for: measure the tiring of monoclonal antibody with indirect ELISA method, simultaneously using NS-1 Fusion of Cells cell ascites as negative control, using P/N value > 2.1 monoclonal antibody greatest dilution tiring as monoclonal antibody.
7. the Colloidal Gold of quick detection detecting milk allergen according to claim 1, it is characterized in that, in described step C, improvement on synthesis is passed through in described qualification, then by the polypeptide of synthesis and monoclonal antibody specific bond, ELISA and Immuno dot-blotting is adopted to detect the epitope information of monoclonal antibody.
8. the Colloidal Gold of quick detection detecting milk allergen according to claim 1, is characterized in that, in described step D, the preparation process of described monoclonal antibody-colloidal gold conjugate specifically comprises:
After being boiled by gold chloride, add trisodium citrate aqueous solution, continue to boil 10 ~ 20min; After cooling, add K
2cO
3adjust pH to 8.0 ~ 8.5; Add the anti-also rapid stirring of Sheet of anti-milk allergen β LG; Add OVA and rapid stirring, add NaCl solution, centrifugal, abandon precipitation, supernatant is centrifugal again; Abandon supernatant, obtain monoclonal antibody-colloidal gold conjugate.
9. the Colloidal Gold of quick detection detecting milk allergen according to claim 8, it is characterized in that, in described step D, the preparation process of described colloidal gold test paper slip specifically comprises: colloidal gold test paper slip comprises water-absorption fiber, nitrocellulose filter and absorbent filter three part; Wherein, water-absorption fiber part sprays the Sheet clonal antibody-colloidal gold conjugate of anti-cow's milk β LG, detection monoclonal antibody nitrocellulose filter spraying anti-cow's milk β LG with sheep anti-mouse igg bag by the lines becoming two 0.8 ~ 1.2mm wide, air-dry rear use is closed containing the PBS of OVA, glue on the support successively after drying, be cut into the bar of 0.2 cm × 8 cm.
10. the Colloidal Gold of quick detection detecting milk allergen according to claim 9, it is characterized in that, in described step D, the testing result of cow's milk comprises: test-strips cellulose membrane to occur two red line are for positive findings, only occur that a red line is negative findings, losing efficacy appears then being considered as detecting in redfree lines.
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