CN107918018A - A kind of method of the near field light wave targeting sensor detection shellfish allergens based on antibody technique - Google Patents
A kind of method of the near field light wave targeting sensor detection shellfish allergens based on antibody technique Download PDFInfo
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Abstract
The invention discloses a kind of method of the near field light wave targeting sensor detection shellfish allergens based on antibody technique, it is coupled with Cy5.5 fluorescent dyes and anaphylactogen monoclonal antibody, arginine kinase antigen coat optical fiber, sample to be tested first carries out pre-reaction with labelled antibody, then mixed liquor is injected into sample cell, in fibre-optical probe insertion sample cell, the envelope antigen on optical fiber is set to be combined with unreacted labelled antibody, the targeting sensor collection of near field light wave and fluorescence intensity change are recycled, so as to detect the content of anaphylactogen in sample liquid.The present invention is easy to operate, result is accurate, biologic specificity is strong, can realize the quantitative detection to trace arginine kinase in aquatic products and its product, and the development for promoting aquatic product anaphylactogen sign system is of great significance.
Description
Technical field
The invention belongs to food analysis technical field and Allergic skin test technical field, and in particular to one kind is based on antibody skill
The method of the near field light wave targeting sensor detection shell-fish main allergen of art.
Background technology
Fishery product protein rich content, delicious flavour.The consumption figure gradually risen every year with it, aquatic products are in people
In occupation of the status to become more and more important in daily life.But as one kind in eight big main allergic food, Species of Crustacea
Easily cause the allergic reaction of specific crowd.According to statistics, in Asian countries, about 40% adult and children can be to crusts
Class produces allergic reaction.
At present, the country such as the U.S., European Union and New Zealand is required to carry out anaphylactogen sign to allergy food, to remind consumption
Person, avoids eating by mistake.Therefore, the detection of allergen content be carry out the production of allergy food, assessment and indicate work basis and
Top priority.Arginine kinase (Arginine Kinase, AK) and tropomyosin (Tropomyosin, TM) are widely present in
It is two kinds of main anaphylactogens in shell-fish in the shell-fish such as shrimp, crab.
At present, shellfish allergens detection method mainly has the technologies such as ELISA, PCR, RT-PCR.There is inspection in these technologies
The problems such as surveying long time, sensitivity and accuracy not high (false positive easily occur).In order to protect consumer, Susceptible population's rule are helped
Wind sheltering danger, is established to shellfish allergens arginine kinase (Arginine Kinase, AK) and tropomyosin
Efficient, sensitive, the accurate shellfish allergens detection technique of (Tropomyosin, TM) applies valency in China with important
Value.
The content of the invention
The present invention provides a kind of method of the near field light wave targeting sensor detection shellfish allergens based on antibody technique,
To improve the detection sensitivity of arginine kinase (AK) or tropomyosin (TM) and accuracy.
A kind of method of the near field light wave targeting sensor detection shellfish allergens based on antibody technique, including following step
Suddenly：
(1) fibre-optical probe is put into allergen solution after surface hydroxylation and surface silanization processing successively and soaked,
Allergy Proantigen is coated with connection, takes out and is soaked in after pure water rinsing in BSA solution to close non-specific adsorption point；
(2) Cy5.5 fluorochrome labels monoclonal antibody corresponding with anaphylactogen is used, the monoclonal antibody after must marking is molten
Liquid；The anaphylactogen is shellfish allergens arginine kinase, and corresponding monoclonal antibody is anti-AK monoclonal antibodies；Or
For shellfish allergens tropomyosin, corresponding antibody is anti-TM monoclonal antibodies；
(3) allergen solution of various concentrations is configured, monoclonal antibody solution after being marked respectively with step (2) gained etc.
Volume mixture is uniform, carries out pre-reaction, and the mixed liquor after pre-reaction adds sample cell, and sample introduction is reacted after a certain period of time, using step
Suddenly the fibre-optical probe detection after (1) processing, compares for the ease of data between different batches and different optical fiber, usually data is carried out
Normalized, formula are as follows：
And simulated to obtain standard curve using the Logistic models of 4 parameters.Its formula is：
In two formulas, y is signal strength, and x is antigen concentration, and A1, A2 are respectively the upper and lower asymptote of curve, and x0 is curve
Flex point (503nhibiting concentration), liquid concentration draw standard curve as abscissa；P is the slope of curve at flex point.(4) allergy will be contained
Former sample to be tested mixes in equal volume with the antibody-solutions handled through step (2), is added after pre-reaction in sample cell, sample introduction reaction
The light probe after step (1) processing is inserted into after a certain period of time, is detected, will collected using the method for same step (3)
Fluorescence signal value bring into the standard curve, the concentration that anaphylactogen is corresponded in sample to be tested is calculated.
The present invention is coupled with Cy5.5 fluorescent dyes and anaphylactogen monoclonal antibody, arginine kinase antigen or former flesh
Immunoglobulin antigen is coated with optical fiber, and sample to be tested first carries out pre-reaction with labelled antibody, mixed liquor then is injected sample cell, optical fiber
In probe insertion sample cell, the envelope antigen on optical fiber is combined with unreacted labelled antibody, recycle near field light wave
Sensor collection and fluorescence intensity change are targeted, so as to detect the content of anaphylactogen in sample liquid.
In step (1), fibre-optical probe first is soaked with piraha solution, makes detecting head surface hydroxylating；Then toluene solution is used
Optical fiber is soaked with glutaraldehyde solution, makes detecting head surface silanization
Preferably, when anaphylactogen is shell class anaphylactogen arginine kinase, the concentration of allergen solution is 1 in step (1)
~2mg/mL；When anaphylactogen is shellfish allergens tropomyosin, in step (1) concentration of allergen solution for 1.5~
3mg/mL。
Preferably, the method that monoclonal antibody is corresponded to Cy5.5 fluorochrome labels is as follows：
The method that monoclonal antibody is corresponded to Cy5.5 fluorochrome labels is as follows：
Taking corresponding with anaphylactogen monoclonal antibody solution, (concentration is 1~2mg/mL when anaphylactogen is arginine kinase, mistake
Quick concentration when being tropomyosin originally is 1.5~3mg/mL) mixed with PBS buffer, then with NaCl solution and NaHCO3It is molten
Liquid dialysis monoclonal antibody solution, filters monoclonal antibody solution, by Cy5.5 solution (10mg/ with 0.22 μm of syringe filtering head
ML) by proportioning (fluorescent dye and mass ratio 20:1) add in monoclonal solution, lucifuge is slowly stirred, and antibody is filled with Cy5.5
Divide and combine, then the monoclonal antibody with NaCl solution and PBS+0.01% sodium azide solutions lucifuge dialysis Cy5.5 marks is molten
Liquid, removes small-molecule substance and unreacted Cy5.5 fluorescent dyes, finally filters Cy5.5 marks with 0.22 μm of syringe filtering head
The monoclonal antibody solution of note to obtain the final product.
Preferably, when anaphylactogen is shell class anaphylactogen arginine kinase, the concentration of monoclonal antibody solution in step (2)
For 10~15 μ g/mL；When anaphylactogen is shellfish allergens tropomyosin, monoclonal antibody solution is dense in step (2)
Spend for 5~15 μ g/mL.
Preferably, when anaphylactogen is shell class anaphylactogen arginine kinase, during pre-reaction in step (2) and step (3)
Between be 5~15min；When anaphylactogen is shellfish allergens tropomyosin, during pre-reaction in step (2) and step (3)
Between be 3~10min.
Preferably, when anaphylactogen is shell class anaphylactogen arginine kinase, the sample introduction reaction in step (2) and step (3)
Time is 10~20min；When anaphylactogen is shellfish allergens tropomyosin, the sample introduction in step (2) and step (3) is anti-
It is 5~15min between seasonable.Preferably, the fibre-optical probe is popped one's head in for silica fibre.
Preferably, the body of the anaphylactogen standard items that detect or the sample to be tested containing anaphylactogen is used in step (3) and (4)
Product is 100~200 μ L.
Sensor sheet is as the prior art in the present invention, it is preferred to use list-multi-module optical fiber coupler, effectively raises and be
The signal-to-noise ratio of system.The fluorescence signal of probe is incorporated into since sensor can only be collected, the fluorescence in reaction solution can be avoided to contaminate
Material produces interference, improves sensitivity and specificity, it is possible to prevente effectively from the false positive that ELISA is produced.In addition, the method have compared with
Good specificity, suitable for the detection demand of the shellfish allergens the aquatic product of complicated component.
The method for drafting of standard curve is as follows：
By the fibre-optical probe installation of the envelope antigen prepared on a sensor, the standard anaphylactogen for preparing various concentrations is molten
Liquid (0,0.1,0.5,1,5,10,50,100 μ g/mL), then resists allergen solution and the Cy5.5 of the equivalent monoclonal marked
Liquid solution is uniformly mixed carry out pre-reaction, mixed liquor then is added sample cell, after sample introduction reaction a period of time, with based on antibody
The near field light wave targeting sensor detection signal value of technology, to optical fiber regenerate and clear using SDS solution and PBS buffer
Wash.Test data is normalized, and standard curve is obtained using Logistic modelings.
A kind of preferred detection method, when anaphylactogen is shellfish allergens arginine kinase, includes the following steps：
(1) preparation of envelope antigen fibre-optical probe
First with piraha solution immersion fibre-optical probe, make detecting head surface hydroxylating；Then it is molten with toluene solution and glutaraldehyde
Liquid soaks optical fiber, makes detecting head surface silanization；The fibre-optical probe handled well is put into AK solution and is soaked, to connect envelope antigen
AK；Taking-up ultrapure water, is then soaked in BSA solution to close non-specific adsorption point.
(2) Cy5.5 fluorochrome labels anti-AK monoclonal antibodies (anti-AK-MAb)
Anti-AK-MAb is taken to be mixed with PBS buffer, then with NaCl solution and NaHCO3Solution dialysis anti-AK-
Mab solution, filters anti-AK-Mab solution with 0.22 μm of syringe filtering head, Cy5.5 solution is added by a certain percentage
Anti-AK-Mab solution, lucifuge are slowly stirred, and antibody is fully combined with Cy5.5, then with NaCl solution and in PBS+
The anti-AK-MAb solution of 0.01% sodium azide solution lucifuge dialysis Cy5.5 marks, removes small-molecule substance and unreacted
Cy5.5 fluorescent dyes, finally filter the anti-AK-MAb solution of Cy5.5 marks with 0.22 μm of syringe filtering head.
(3) drafting of arginine kinase standard sample standard curve
By the installation of the fibre-optical probe of the envelope antigen prepared on a sensor, prepare various concentrations AK solution (0,
0.1st, 0.5,1,5,10,50,100 μ g/mL), the anti-AK-MAb for then marking AK solution with the Cy5.5 of equivalent respectively is molten
Liquid, which is uniformly mixed, carries out 5~15min of pre-reaction, mixed liquor then is added sample cell, after sample introduction reacts 10~20min, with step
Suddenly the fibre-optical probe after (1) processing, the near field light wave targeting sensor detection signal value based on antibody technique, uses SDS solution
Optical fiber is regenerated and cleaned with PBS buffer, test data is normalized, and uses Logistic pattern dies
Plan obtains standard curve.
(4) detection of sample to be tested
By the sample to be tested of shellfish allergens arginine kinase and the anti-AK- through Cy5.5 marks obtained by step (2)
MAb solution mixes in equal volume, is added after 5~15min of pre-reaction in sample cell, and sample introduction uses step (1) after reacting 10~20min
Fibre-optical probe after processing is detected, and the fluorescence signal value collected is brought into the standard curve, is calculated to be measured
The concentration of arginine kinase AK in sample.
Another preferable technical solution, when anaphylactogen is shell-fish shellfish allergens tropomyosin, detection method
It is as follows：
(1) preparation of envelope antigen fibre-optical probe
First with piraha solution immersion fibre-optical probe, make detecting head surface hydroxylating；Then it is molten with toluene solution and glutaraldehyde
Liquid soaks optical fiber, makes detecting head surface silanization；The fibre-optical probe handled well is put into TM solution and is soaked, to connect envelope antigen
TM；Taking-up ultrapure water, is then soaked in BSA solution to close non-specific adsorption point.
(2) Cy5.5 fluorochrome labels anti-TM monoclonal antibodies (anti-TM-MAb)
Anti-TM-MAb is taken to be mixed with PBS buffer, then with NaCl solution and NaHCO3Solution dialysis anti-TM-
Mab solution, filters anti-TM-Mab solution with 0.22 μm of syringe filtering head, Cy5.5 solution is added by a certain percentage
Anti-TM-Mab solution, lucifuge are slowly stirred, and antibody is fully combined with Cy5.5, then with NaCl solution and in PBS+
The anti-TM-MAb solution of 0.01% sodium azide solution lucifuge dialysis Cy5.5 marks, removes small-molecule substance and unreacted
Cy5.5 fluorescent dyes, finally filter the anti-TM-MAb solution of Cy5.5 marks with 0.22 μm of syringe filtering head.
(3) detection of tropomyosin standard sample and the drafting of standard curve
By the installation of the fibre-optical probe of the envelope antigen prepared on a sensor, prepare various concentrations TM solution (0,
0.1st, 0.5,1,5,10,50,100 μ g/mL), then TM solution and the Cy5.5 of equivalent the anti-TM-MAb solution marked are mixed
Close and uniformly carry out 3~10min of pre-reaction, mixed liquor is then added into sample cell, after sample introduction reacts 5~15min, using step
(1) fibre-optical probe after handling, targets sensor detection signal value, using SDS solution and PBS buffer to light near field light wave
Fibre is regenerated and cleaned.Test data is normalized, and standard curve is obtained using Logistic modelings.
(4) sample to be tested is mixed with the anti-TM-MAb solution evens through Cy5.5 marks obtained by step (2), pre-reaction
Being added after 3~10min in sample cell, sample introduction is detected after reacting 5~15min using the fibre-optical probe after step (1) processing,
The fluorescence signal value collected is brought into the standard curve, the concentration of MT in sample to be tested is calculated.
It is further preferred that when anaphylactogen is shell class anaphylactogen arginine kinase, it is pre- in step (2) and step (3)
Reaction time is 6min；The sample introduction reaction time is 10min；When anaphylactogen is shellfish allergens tropomyosin, step (2) and
Pre-reaction time in step (3) is 4min, and the sample introduction reaction time in step (2) and step (3) is 6min.
The technical solution adopted in the present invention is：Envelope antigen arginine kinase or tropomyosin and optical fiber is covalently even
Connection forms optical fiber probe, and antibody used is the AK monoclonal antibodies or MT monoclonal antibodies with Cy5.5 fluorochrome labels, is coated with
Antigen can be specifically bound with determined antigen with labelled antibody.By the sample containing AK or MT and correspondence markings antibody into
Row pre-reaction, then injects reaction tank by mixed liquor, makes unreacted mark in the envelope antigen and mixed liquor on optical fiber probe
Antibody is combined, then by inputting near field light wave into sensor, so that the antibody of excitation fiber pan coating antigen binding
Cy5.5 fluorescent markers produce fluorescence signal, and the collection of signal is carried out by sensor.According to detection various concentrations AK standard items
Or the fluorescence intensity change of TM standard items can establish standard curve, further according to the fluorescence intensity change and standard curve of sample to be tested
AK contents in sample to be tested or MT contents are measured.
This method establishes a kind of near field light wave targeting sensor on-line checking shellfish allergens based on antibody technique
The method of arginine kinase or shellfish allergens tropomyosin.This method is based near field light wave targeting sensor and combines indirectly
Immuno analytical method, has the advantages such as high specific, high sensitivity, rapid reaction, favorable reproducibility.It is contaminated with Cy5.5 fluorescence
Material mark arginine kinase monoclonal antibody, while optical fiber is coated with AK or MT, then by test analyte AK or MT and necessarily
The fluorescent labeled antibody of amount is reacted certain time in advance, then mixed liquor is passed through reaction tank so that unreacted fluorescent marker resists
Body is combined with the envelope antigen modified in chip surface, the fluorescence combined in the near field light wave excitation fiber that sensing system produces
Labelled antibody produces fluorescence, and the AK in sample liquid or MT are detected by the intensity for measuring fluorescence signal.
Compared with other existing detection methods, when the present invention has high sensitivity, high specificity, easy to operate and detection
Between it is short the advantages of, for promote aquatic product anaphylactogen sign the perfect of system have great importance.
Brief description of the drawings
Fig. 1 is SDS-PAGE sample analysis result in embodiment 1；
Fig. 2 is arginine kinase-Standardization curve for fluorescence intensity；
Fig. 3 is arginine kinase specific detection result.
Fig. 4 is SDS-PAGE sample analysis result in embodiment 7；
Fig. 5 is tropomyosin-Standardization curve for fluorescence intensity；
Fig. 6 is tropomyosin specific detection result；
Fig. 7 is present invention detection equipment schematic diagram used.
Reference numeral is as follows shown in Fig. 7：
1- signal generator 2- lock-in amplifier 3- computers
4- sample 5- waste liquid 6- sample cells
7- connectors 8- is mono--multi-module optical fiber coupler 9- optical filters
10- photodiode 11- laser-fiber coupler 12- lasers
Embodiment
The raw materials used present invention is commercial goods.
The preparation of 1 sample of embodiment
The preparation of sample：2g Penaeus Vannmei muscle is taken, with liquid nitrogen grinding into powder, then using the extraction examination of triumphant base holoprotein
Agent box extracts albumen immersion liquid.Comprise the following steps that：(1) 1 μ L protease is separately added into the 1mL Lysis Buffer of precooling
Inhibitor, 10 μ L inhibitors of phosphatases and 5 μ L100mM PMSF, mix postposition preserve on ice several minutes it is stand-by；(2) one is taken newly
Fresh Penaeus Vannmei, removes shrimp head, tail, shell and gutstring.Shrimp muscle is cut with a knife into pureed, is placed in mortar, liquid nitrogen is added and grinds
Mill, until powdered.(3) 0.1g shrimp meds quickly are weighed to be placed in 1.5mL precooling centrifuge tubes, adds the above-mentioned mixed liquors of 1mL, mixed
4 DEG C of standing 2h afterwards；(4) 10000r/min, 4 DEG C of centrifugation 5min, takes supernatant, and packing is stored in -80 DEG C, should avoid freezing repeatedly
Melt.Using SDS-PAGE electrophoretic analysis sample liquids, the results are shown in Figure 1.
The coating of 2 optical fiber of embodiment and AK antigens
First use piraha solution (H2SO4:H2O2=3:1) fibre-optical probe 30min is soaked, ultra-pure water fully cleans, and nitrogen is blown
It is dry, make detecting head surface hydroxylating；Then fibre-optical probe 1h is soaked with 2% (v/v) toluene solution, toluene fully cleans, and nitrogen is blown
It is dry, fibre-optical probe 2h is next soaked at 37 DEG C with 5% (v/v) glutaraldehyde solution, toluene fully cleans, and 120 DEG C of baking ovens dry
It is dry, make detecting head surface silanization；The fibre-optical probe handled well is put into 1mg/mLAK solution, 4 DEG C of soaked overnights, to connect bag
By antigen A K；With ultrapure water fibre-optical probe, it is subsequently placed in 4mg/mL BSA solution and soaks 2h to close non-specific suction
Attachment.The optical fiber being coated with can be preserved into the several months at 4 DEG C.
3 Cy5.5 fluorochrome label anti-AK monoclonal antibodies (anti-AK-MAb) of embodiment
0.15M NaCl solutions room temperature dialysis anti-AK-MAb solution 4h, change liquid, 4 DEG C of dialysed overnights, then use 0.1M
NaHCO3(pH 8.3) solution room temperature dialysis 4h, then filters anti-AK-MAb solution with 0.22 μm of syringe filtering head
(1.5mg/mL), presses 20 in antibody-solutions:The ratio of 1 (v/v) adds Cy5.5 solution (10mg/mL), and lucifuge, is slowly stirred
40min, makes antibody fully be combined with Cy5.5, then with the anti-AK-MAb of NaCl solution room temperature lucifuge dialysis Cy5.5 marks
Solution 4h, to remove the Cy5.5 on unmarked, changes liquid, 4 DEG C of lucifuge dialysed overnights are then molten in PBS+0.01% sodium azide
The anti-AK-MAb 4h of room temperature lucifuge dialysis Cy5.5 marks in liquid, change liquid, 4 DEG C of lucifuge dialysed overnights, finally with 0.22 μm of note
The anti-AK-MAb solution of emitter filtering head filtering Cy5.5 marks.Labelled antibody should be stored in -20 DEG C.
The drafting of 4 arginine kinase standard curve of embodiment
The AK solution of various concentrations (0,0.1,0.5,1,5,10,50,100 μ g/mL) is prepared with 10mMPBS (PH 7.4),
The anti-AK-MAb solution (12 μ g/mL) that 100 μ LAK standard solutions are marked with 100 μ LCy5.5 mixes, and reacts 6min, so
Mixed liquor is added into sample cell afterwards, sample introduction, after reacting 10min, is detected with the near field light wave targeting sensor based on antibody technique.
Then optical fiber is regenerated and cleaned using 0.5%SDS solution (pH 1.9) and 10mM PBS (PH 7.4).By test data
It is normalized, and standard curve is obtained using Logistic modelings, as shown in Figure 2.As a result in 0.1~100 μ g/
Linear relationship is good in the range of mL, regression equation y=0.2283x+0.2416, and minimum detectability is 0.03 μ g/mL, quantitative
It is limited to 0.09 μ g/mL.
5 specificity experiments of embodiment
The method of shellfish allergens AK is detected to south with the near field light wave targeting sensor based on antibody technique of foundation
The foreign protein mixed liquor of penaeus vannamei carries out specific cross experiment, while blank control.As shown in Figure 3, the results showed that the party
Method specificity is stronger, only reacts to AK, and does not have cross reaction to other protein in Penaeus Vannmei.
Embodiment 6 manually adds the detection of sample
Take the foreign protein mixed liquor of 1mL Penaeus Vannmeis, be separately added into different AK, make AK concentration for 5,10,20,30,40,
50 μ g/mL, each 6 Duplicate Samples of concentration, carry out sample detection and determination of recovery rates.Testing result is as shown in table 1,5~100
In the range of the addition of μ g/mL concentration, the rate of recovery shows that this method can be used for the detection of actual sample 89%~111%.
Table 1 manually adds sample detection result (n=6)
The preparation of 7 sample of embodiment
The preparation of sample：2g Chinese prawn muscle is taken, with liquid nitrogen grinding into powder, then using triumphant base holoprotein extracts reagent
Box extracts albumen immersion liquid.Comprise the following steps that：(1) suppression of 1 μ L protease is separately added into the 1mL Lysis Buffer of precooling
Preparation, 10 μ L inhibitors of phosphatases and 5 μ L 100mM PMSF, mix postposition preserve on ice several minutes it is stand-by；(2) one is taken newly
Fresh Chinese prawn, removes shrimp head, tail, shell and gutstring.Shrimp muscle is cut with a knife into pureed, is placed in mortar, adds liquid nitrogen grinding,
Until powdered.(3) 0.1g shrimp meds quickly are weighed to be placed in 1.5mL precooling centrifuge tubes, the above-mentioned mixed liquors of 1mL is added, 4 after mixing
DEG C stand 2h；(4) 10000r/min, 4 DEG C of centrifugation 5min, takes supernatant, and packing is stored in -80 DEG C, should avoid multigelation.Adopt
With SDS-PAGE electrophoretic analysis sample liquids, the results are shown in Figure 4.
The coating of 8 optical fiber of embodiment and TM antigens
First use piraha solution (H2SO4:H2O2=3:1) fibre-optical probe 30min is soaked, ultra-pure water fully cleans, and nitrogen is blown
It is dry, make detecting head surface hydroxylating；Then fibre-optical probe 1h is soaked with 2% (v/v) toluene solution, toluene fully cleans, and nitrogen is blown
It is dry, fibre-optical probe 2h is next soaked at 37 DEG C with 5% (v/v) glutaraldehyde solution, toluene fully cleans, and 120 DEG C of baking ovens dry
It is dry, make detecting head surface silanization；The fibre-optical probe handled well is put into 1.5mg/mL TM solution, 4 DEG C of soaked overnights, with even
Meet envelope antigen TM；With ultrapure water fibre-optical probe, it is non-specific to close to be subsequently placed in immersion 2h in 4mg/mL BSA solution
Property absorption point.The optical fiber being coated with can be preserved into the several months at 4 DEG C.
9 Cy5.5 fluorochrome label anti-TM monoclonal antibodies (anti-TM-MAb) of embodiment
0.15M NaCl solutions room temperature dialysis anti-TM-MAb solution 4h, change liquid, 4 DEG C of dialysed overnights, then use 0.1M
NaHCO3(pH 8.3) solution room temperature dialysis 4h, then filters anti-TM-MAb solution (1mg/ with 0.22 μm of syringe filtering head
ML), 20 are pressed in antibody-solutions:The ratio of 1 (v/v) adds Cy5.5 solution (10mg/mL), and lucifuge, is slowly stirred 40min, makes
Antibody is fully combined with Cy5.5, the anti-TM-MAb solution 4h then marked with NaCl solution room temperature lucifuge dialysis Cy5.5, with
The Cy5.5 on unmarked is removed, changes liquid, 4 DEG C of lucifuge dialysed overnights, then room temperature is kept away in PBS+0.01% sodium azide solutions
The anti-TM-MAb 4h of light dialysis Cy5.5 marks, change liquid, 4 DEG C of lucifuge dialysed overnights, finally with 0.22 μm of syringe filtering head
Filter the anti-TM-MAb solution of Cy5.5 marks.Labelled antibody should be stored in -20 DEG C.
The drafting of 10 tropomyosin standard curve of embodiment
The TM solution of various concentrations (0,0.1,0.5,1,5,10,50,100 μ g/mL) is prepared with 10mMPBS (PH 7.4),
50 μ LTM standard solutions are mixed (9 μ g/mL) with the 50 μ L Cy5.5 anti-TM-MAb solution marked, react 4min, then
Mixed liquor is added into sample cell, sample introduction, after reacting 12min, is detected near field light wave targeting sensor.Then 0.5%SDS is used
Solution (pH 1.9) and 10mM PBS (PH 7.4) are regenerated and cleaned to optical fiber.Test data is normalized,
And standard curve is obtained using Logistic modelings, as shown in Figure 5.As a result it is linear in the range of 0.1~100 μ g/mL
Relation is good, regression equation y=0.2386x+0.2651, and minimum detectability is 0.026 μ g/mL, is quantitatively limited to 0.08 μ g/
mL。
11 specificity experiments of embodiment
With the method centering of the near field light wave targeting sensor detection shellfish allergens TM based on antibody technique of foundation
The foreign protein mixed liquor of state prawn carries out specific cross experiment, while blank control.As shown in Figure 6, the results showed that this method
Specificity is stronger, only reacts to TM, and does not have cross reaction to other protein in Chinese prawn.
Embodiment 12 manually adds the detection of sample
The foreign protein mixed liquor of 1mL Chinese prawns is taken, is separately added into different TM, it is 5,10,20,30,40,50 to make TM concentration
μ g/mL, each 6 Duplicate Samples of concentration, carry out sample detection and recovery of standard addition measure.Testing result is as shown in table 1,5~
In the range of the addition of 100 μ g/mL concentration, the rate of recovery shows that this method can be used for the detection of actual sample 88%~106%.
Table 2 manually adds sample detection result (n=6)
The foregoing is merely the specific implementation case of patent of the present invention, but the technical characteristic of patent of the present invention is not limited to
This, any those skilled in the relevant art in the field of the invention, all cover in the special of the present invention by the change or modification made
Among sharp scope.
Claims (7)
- A kind of 1. method of the near field light wave targeting sensor detection shellfish allergens based on antibody technique, it is characterised in that Include the following steps：(1) fibre-optical probe is put into allergen solution after surface hydroxylation and surface silanization processing successively and soaked, with even Coating allergy Proantigen is connect, takes out and is soaked in after pure water rinsing in bovine serum albumin solution to close non-specific adsorption Point；(2) Cy5.5 fluorochrome labels monoclonal antibody corresponding with anaphylactogen, the monoclonal antibody solution after must marking are used； The anaphylactogen is shellfish allergens arginine kinase, and corresponding monoclonal antibody is anti-AK monoclonal antibodies；Or it is first Shell class anaphylactogen tropomyosin, corresponding antibody are anti-TM monoclonal antibodies；(3) allergen solution of various concentrations is configured, it is isometric with the monoclonal antibody solution after step (2) gained mark respectively It is uniformly mixed, carries out pre-reaction, the mixed liquor after pre-reaction adds sample cell, and sample introduction is reacted after a certain period of time, by step (1) place Fibre-optical probe installation after reason is inserted into sample cell on a sensor, gathers the fluorescence signal value excited by near field light wave, will survey Examination data are normalized, and obtain standard curve using Logistic modelings；(4) sample to be tested containing anaphylactogen is mixed in equal volume with the antibody-solutions handled through step (2), sample is added after pre-reaction In product pond, sample introduction reaction be inserted into after a certain period of time through step (1) processing after light probe, using same step (3) method into Row detection, the fluorescence signal value collected is brought into the standard curve, is calculated in sample to be tested and corresponds to anaphylactogen Concentration.
- 2. method according to claim 1, it is characterised in that when anaphylactogen is shell class anaphylactogen arginine kinase, step (1) The concentration of middle allergen solution is 1~2mg/mL；When anaphylactogen is shellfish allergens tropomyosin, allergy in step (1) The concentration of original solution is 1.5~3mg/mL.
- 3. method according to claim 1, it is characterised in that the side of monoclonal antibody is corresponded to Cy5.5 fluorochrome labels Method is as follows：Monoclonal antibody corresponding with anaphylactogen is taken to be mixed with PBS buffer, then with NaCl solution and NaHCO3Solution dialysis is single Clonal antibody solution, monoclonal antibody solution is filtered with 0.22 μm of syringe filtering head, and Cy5.5 solution is added Dan Ke by proportioning In grand solution, lucifuge is slowly stirred, and antibody is fully combined with Cy5.5, then with NaCl solution and PBS+0.01% Azides The monoclonal antibody solution of sodium solution lucifuge dialysis Cy5.5 marks, removes small-molecule substance and unreacted Cy5.5 fluorescence dye Material, finally filters the monoclonal antibody solution of Cy5.5 marks with 0.22 μm of syringe filtering head to obtain the final product.
- 4. method according to claim 1, it is characterised in that when anaphylactogen is shell class anaphylactogen arginine kinase, step (2) concentration of monoclonal antibody solution is 10~15 μ g/mL in；When anaphylactogen is shellfish allergens tropomyosin, step Suddenly the concentration of monoclonal antibody solution is 5~15 μ g/mL in (2).
- 5. method according to claim 1, it is characterised in that when anaphylactogen is shell class anaphylactogen arginine kinase, step (2) pre-reaction time and in step (3) is 5~15min；When anaphylactogen is shellfish allergens tropomyosin, step (2) pre-reaction time and in step (3) is 3~10min.
- 6. method according to claim 1, it is characterised in that when anaphylactogen is shell class anaphylactogen arginine kinase, step (2) the sample introduction reaction time and in step (3) is 10~20min；When anaphylactogen is shellfish allergens tropomyosin, step Suddenly the sample introduction reaction time in (2) and step (3) is 5~15min.
- 7. method according to claim 1, it is characterised in that the fibre-optical probe is popped one's head in for silica fibre.
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| CN108866060A (en) * | 2018-06-12 | 2018-11-23 | 浙江工商大学 | A kind of aptamer, kit and detection method specifically binding shell-fish arginine kinase |
| CN110456059A (en) * | 2019-07-01 | 2019-11-15 | 浙江工商大学 | A kind of detection card of shellfish allergens and its application |
| CN110501506A (en) * | 2018-07-05 | 2019-11-26 | 东莞东阳光医疗智能器件研发有限公司 | A kind of biosensor and its application |
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| CN110865060A (en) * | 2019-11-29 | 2020-03-06 | 中国人民大学 | Method for detecting glycocholic acid by one-step method |
| CN110951696A (en) * | 2019-12-19 | 2020-04-03 | 浙江工商大学 | Hybridoma cell strain and anti-shrimp arginine kinase monoclonal antibody secreted by same |
| CN111073860A (en) * | 2019-12-19 | 2020-04-28 | 浙江工商大学 | Application of anti-shrimp arginine kinase monoclonal antibody in anti-allergic medicine and detection |
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| CN110501506A (en) * | 2018-07-05 | 2019-11-26 | 东莞东阳光医疗智能器件研发有限公司 | A kind of biosensor and its application |
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| CN110736833A (en) * | 2019-11-13 | 2020-01-31 | 浙江工商大学 | Crustacean arginine kinase detection kit based on coffee ring effect and detection method thereof |
| CN110736833B (en) * | 2019-11-13 | 2022-10-14 | 浙江工商大学 | Detection kit and detection method for crustacean arginine kinase based on coffee ring effect |
| CN110865060A (en) * | 2019-11-29 | 2020-03-06 | 中国人民大学 | Method for detecting glycocholic acid by one-step method |
| CN110951696A (en) * | 2019-12-19 | 2020-04-03 | 浙江工商大学 | Hybridoma cell strain and anti-shrimp arginine kinase monoclonal antibody secreted by same |
| CN111073860A (en) * | 2019-12-19 | 2020-04-28 | 浙江工商大学 | Application of anti-shrimp arginine kinase monoclonal antibody in anti-allergic medicine and detection |
| CN110951696B (en) * | 2019-12-19 | 2021-08-10 | 浙江工商大学 | Hybridoma cell strain and anti-shrimp arginine kinase monoclonal antibody secreted by same |
| CN111073860B (en) * | 2019-12-19 | 2021-08-24 | 浙江工商大学 | Application of anti-shrimp arginine kinase monoclonal antibody in anti-allergic medicine and detection |
| CN113702344A (en) * | 2021-08-30 | 2021-11-26 | 北京协同创新研究院 | Method for rapidly detecting imidacloprid in water |
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