CN108717122A - It is a kind of detection copper ion immunofluorescence chromatograph test strip and its application - Google Patents
It is a kind of detection copper ion immunofluorescence chromatograph test strip and its application Download PDFInfo
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Abstract
The present invention provides a kind of immunofluorescence chromatograph test strip of detection copper ion and its applications in detecting sample copper ion concentration, belong to copper ion detection technique field；The immunofluorescence chromatograph test strip includes overlapped fiberglass packing, nitrocellulose membrane and blotting paper successively；Copper ion monoclonal antibody-fluorescent microsphere compound is coated on the fiberglass packing；The nitrocellulose filter sets gradually the nature controlling line of the detection line and coating secondary antibody of coating copper ion detection comlete antigen；The immunofluorescence chromatograph test strip is easy to use, high sensitivity；Detection sensitivity (IC50) it is 3.6ng/ml, detection range (IC20‑IC80) it is 0.4-32.3ng/ml, detection limit (IC10) it is 0.2ng/ml.
Description
Technical field
The invention belongs to heavy metal technical field of immunoassay more particularly to a kind of immunofluorescence chromatographies of detection copper ion
Test strips and its application.
Background technology
Heavy metal is widely distributed on earth, will not cause damages under normal circumstances to the living environment of the mankind.But with
The fast development of the explosion in city and industry, plurality of heavy metal with abnormal speed enter our life because of human activity
Environment, these heavy metals and its compound have teratogenesis, carcinogenesis more, and are all accumulative toxicants, pass through drinking water or food
The modes such as object chain enrichment seriously endanger the health of the mankind and animal.
Include for the heavy metal of environmental pollution most serious at present：Lead, cadmium, mercury, chromium, arsenic (being known as " five poisonous creatures: scorpion, viper, centipede, house lizard, toad "), except this it
Outside, further include the relatively weak nickel of toxicity, zinc, copper, cobalt, tin, vanadium etc..Although the toxicity of heavy metal copper is relatively weak, because
It also more and more causes the concern of people in industry, agriculture and animal husbandry extensive use, the harm of Cu-W ore deposit.Human body is taken the photograph
A series of lesions can be caused by entering excessive copper, and acute copper poisoning can cause gastrointestinal tract mucosa irritation, and such as nausea is retched, abdomen
It rushes down or even hemolytic anemia, renal failure, shock, stupor or death.Chronic intake copper is excessively high, can cause respiratory system disease
Disease, copper powder dirt can cause pharyngalgia, cough, coughs up phlegm and suppressed with gas uncomfortable in chest, or even pneumoconiosis occur etc..In addition, mantoquita has certain sterilization
Effect, so be also widely used in feed, but it is difficult to be metabolized that the copper of high dose, which enters the internal of domestic animal, as biology is tired
Product effect causes Livestock poisoned dead, can also directly threaten human health by fat stock meat products.Therefore, reinforce food
It is extremely urgent with the inspection detection of the heavy metal copper of environmental sanitation, it is significant.
Traditional heavy metal detection method mostly uses physical/chemical method, such as atomic absorption spectrography (AAS) (AAS), atomic fluorescence
Spectroscopic methodology (AFS), inductively coupled plasma-atomic emission spectrometry method (ICP) etc., it is with a high credibility although its testing result is accurate,
Large-scale instrument and equipment and professional is needed to operate, analysis time is long, expensive, there is certain time place limitation, it is difficult to
Adapt to paroxysmal Environment Pollution Event or extensive environmental quality screening detection.The detection method cost of existing heavy metal copper
It is high, efficiency is low, poor accuracy.
Invention content
In view of this, the purpose of the present invention is to provide a kind of detection copper ions at low cost, efficient, accuracy rate is high
Immunofluorescence chromatograph test strip and its application.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：A kind of immunofluorescence of detection copper ion
Chromatograph test strip, including overlapped successively fiberglass packing, nitrocellulose membrane and blotting paper；The fiberglass packing surface spraying
There is copper ion monoclonal antibody-fluorescent microsphere compound；It is complete that the nitrocellulose filter sets gradually coating copper ion detection
The nature controlling line of the detection line and coating secondary antibody of antigen；Copper ion monoclonal antibody-fluorescent microsphere the compound is by copper ion list
Clonal antibody is obtained with fluorescent microsphere coupling；Copper ion detection comlete antigen includes copper ion, carrier protein and by institute
State the chelating agent of copper ion and carrier protein couplet.
Preferably, a concentration of 0.03~0.05 μ g/ μ l of the copper ion monoclonal antibody-fluorescent microsphere compound；Institute
The quantity for spray for stating copper ion monoclonal antibody-fluorescent microsphere compound on fiberglass packing is 3~5 μ l/cm.
Preferably, the copper ion monoclonal antibody is generated by hybridoma cell strain 4F12 secretions, the hybridoma
The deposit number of strain 4F12 is CCTCC NO：C201670.
Preferably, the spraying concentration of the copper ion detection comlete antigen is 0.3~0.6 μ g/ μ l；The nitrocellulose
The quantity for spray of copper ion detection comlete antigen is 0.1~0.2 μ l/mm on film.
Preferably, the chelating agent in the copper ion detection comlete antigen is bifunctional chelating agent p-SCN-Bn-NOTA；Institute
It is chicken ovalbumin OVA to state carrier protein.
The present invention also provides application of the immunofluorescence chromatograph test strip in detecting sample copper ion concentration.
Preferably, include the following steps：1) measuring samples are added drop-wise on glass fibre membrane and are chromatographed；2) described in reading
Fluorescence signal the value Bt and Bc of Immunofluorescence test paper strip detection line, nature controlling line after chromatography；3) by the fluorescence signal value Bt/Bc
Scheduled standard curve equation of linear regression is brought into as B values calculates copper ion concentration in acquisition measuring samples；It is described scheduled
The independent variable of standard curve equation of linear regression be copper ion concentration log10 logarithms, dependent variable be shine inhibiting rate, it is described because
Variable is calculated by Formulas I, (B0-B)/B0× 100% Formulas I, B0It is glimmering when to be not added with measuring samples or be not added with standard sample
Optical signal value Bt0/Bc0, B is fluorescence signal value Bt/Bc when adding measuring samples.
Preferably, the standard curve equation of linear regression is prepared by the following：
With the competitor standard items Cu-NOTA replacement steps 1 of gradient concentration) in measuring samples, according to step 1)~2)
Method obtain competitor standard items fluorescence signal value Bt/Bc, with the fluorescence signal value Bt of 0ng/ml competitor standard items0/
Bc0For B0, the luminous inhibiting rate of gradient concentration competitor standard items is calculated, using the inhibiting rate that shines as ordinate, with Cu2+It is dense
Degree log10 logarithms are abscissa, draw standard curve, obtain standard curve equation of linear regression.
Preferably, the dripping quantity of measuring samples described in step 1) is 60~90 μ l；The chromatography time be 12~
18min。
Preferably, the sample includes Chinese herbal medicine sample, pedotheque and water sample.
Beneficial effects of the present invention：Immunofluorescence chromatograph test strip provided by the invention includes overlapped glass fibre successively
Pad, nitrocellulose membrane and blotting paper；Copper ion monoclonal antibody-fluorescent microsphere compound is coated on the fiberglass packing；
The nitrocellulose filter sets gradually the nature controlling line of the detection line and coating secondary antibody of coating copper ion detection comlete antigen.It is described
Copper ion detects comlete antigen by the copper ion and carrier protein couplet of small molecule, and copper ion is enable to be examined by immune method
It surveys, copper ion concentration is quantitative determined using the linear relationship of luminous inhibiting rate and copper ion concentration log10 logarithms；The present invention is to send out
Xanthophyll cycle rate is ordinate, with Cu2+Concentration log10 logarithms are abscissa, draw standard curve, detect copper ion in measuring samples
Concentration, can realize the rapidly and efficiently detection of copper ion concentration；The immunofluorescence chromatograph test strip, it is easy to use, it is sensitive
Degree is high；Detection sensitivity (IC50) it is 3.6ng/ml, detection range (IC20-IC80) it is 0.4-32.3ng/ml, detection limit (IC10)
For 0.2ng/ml.Meet GB5749《Standards for drinking water quality》Drinking Water, GB3838《Surface water environment quality mark
It is accurate》The testing requirements of the minimum Limited Doses 0.01mg/L of copper in the class water of I class~V.
Description of the drawings
Fig. 1 is the structure chart of the immunofluorescence chromatograph test strip in embodiment 1；
Fig. 2 is the SDS-PAGE qualification figures that copper ion detects comlete antigen；
Fig. 3 is the standard curve that BCA methods measure that copper ion detects comlete antigen albumen concentration；
Fig. 4 is the result of immunofluorescence chromatograph test strip detection card detection various concentration competitor standard items in embodiment 2
Photo；
Fig. 5 is that immunofluorescence chromatograph test strip detects card fluorescence signal curve graph in embodiment 2；
Fig. 6 is that immunofluorescence chromatograph test strip method measures copper ion concentration standard curve in embodiment 2.Biological deposits explanation
Hybridoma cell strain 4F12 is preserved in China typical culture collection center, preservation address：China, Wuhan, Wuhan
University；The preservation time：On June 8th, 2016, deposit number are CCTCC NO：C201670.
Specific implementation mode
The present invention provides a kind of immunofluorescence chromatograph test strips of detection copper ion, as shown in Figure 1, including overlapping successively
Fiberglass packing (6), nitrocellulose membrane (3) and blotting paper (2)；It is anti-that copper ion monoclonal is coated on the fiberglass packing
Body-fluorescent microsphere compound；The nitrocellulose filter set gradually coating copper ion detection comlete antigen detection line (4) and
It is coated with the nature controlling line (5) of secondary antibody；Wherein arrow direction is chromatography direction；Copper ion monoclonal antibody-the fluorescent microsphere
Compound is obtained by copper ion monoclonal antibody and fluorescent microsphere coupling；Copper ion detection comlete antigen include copper ion,
Carrier protein and by the chelating agent of the copper ion and carrier protein couplet.
In the present invention, the fiberglass packing is as sample pad；The fiberglass packing uses available glass fiber pad
?.Copper ion monoclonal antibody-fluorescent microsphere compound is sprayed on fiberglass packing of the present invention；It is described be coated with copper from
The fiberglass packing of sub- monoclonal antibody-fluorescent microsphere compound is prepared by following steps：By the copper ion Dan Ke
Grand antibody-fluorescent microsphere compound is dried after being sprayed on fiberglass packing.The copper ion monoclonal in the present invention
The spraying concentration of antibody-fluorescent microsphere compound is 0.03~0.05 μ g/ μ l；More preferably 0.04 μ g/ μ l；The copper ion list
The quantity for spray of clonal antibody-fluorescent microsphere compound is preferably 3~5 μ l/cm, more preferably 4 μ l/cm.The spraying is preferred
It is carried out using spraying instrument；The present invention is preferably coated with copper ion monoclonal antibody-fluorescent microsphere after spraying by described
The fiberglass packing of compound is dried；The temperature of the drying is preferably 45~55 DEG C, more preferably 50 DEG C；The drying
Time be preferably 12~for 24 hours, more preferably 16~20h.In the present invention, the copper ion monoclonal antibody-fluorescent microsphere
Compound is by obtaining the copper ion monoclonal antibody and fluorescent microsphere coupling.The coupling is preferably adopted in the present invention
With EDC-NHS mediated methods, the carboxyl on EDC and NHS activation fluorescent microspheres surface, the carboxyl on the fluorescent microsphere surface after activation are utilized
It is compound that amido bond acquisition copper ion monoclonal antibody-fluorescent microsphere is formed with the amino condensation coupling in copper ion monoclonal antibody
Object.The fluorescent microsphere is commercial goods in the present invention；The fluorescent microsphere commodity exist with liquid condition.Have in the present invention
In body implementation process, ultrasonic disperse fluorescent microsphere obtains fluorescent microsphere dispersion liquid first；Then by the fluorescent microsphere dispersion liquid
It mixes and is activated with EDC and NHS, obtain activation fluorescent microsphere dispersion liquid；The activation fluorescent microsphere dispersion liquid is centrifuged, is obtained
To activation microballoon；By the activation microballoon and phosphate buffer solution mixing ultrasonic disperse, activation microballoon buffer system is obtained；By institute
It states activation microballoon buffer system and mixes coupling with copper ion monoclonal antibody, it is multiple to obtain copper ion monoclonal antibody-fluorescent microsphere
Close object.
In the present invention, the general power of the ultrasonic disperse is preferably 20w, the work(of selection 10~15% when described ultrasonic
The mode of rate, the ultrasound is preferably intermittent ultrasound；Specially 3~5s of ultrasound, 3~5s of interval.The present invention is obtaining fluorescence
The fluorescent microsphere dispersion liquid is mixed with EDC and NHS after microballoon dispersion liquid and is activated, activation fluorescent microsphere dispersion is obtained
Liquid.The dosage of the EDC and NHS is preferably more excellent using each 4~6mg of EDC and NHS per 1ml fluorescent microspheres in the present invention
Choosing is 5mg.In the present invention, the time of the activation is preferably 12~18min, more preferably 15min.
After obtaining activation fluorescent microsphere dispersion liquid, the centrifugation activation fluorescent microsphere dispersion liquid obtains activating micro- the present invention
Ball.In the present invention, the rotating speed of the centrifugation is preferably 10000rpm~14000rpm；More preferably 12000rpm；It is described from
The time of the heart is preferably 8~12min, more preferably 10min.The present invention after obtaining and activating microballoon, by the activation microballoon and
Phosphate buffer solution mixing ultrasonic disperse obtains activation microballoon buffer system；The concentration of heretofore described phosphate buffer is excellent
Be selected as 0.01mol/L, the dosage of the phosphate buffer with the mixed total volume meter of solid phase components, after preferably making mixing
Activation microballoon buffer system volume it is consistent with the volume of fluorescent microsphere dispersion liquid；Herein the conditional parameter of ultrasonic disperse with it is upper
The condition for stating ultrasonic disperse is consistent with parameter, and details are not described herein；The present invention is clear with cleaning activator after the ultrasonic disperse
It washes 1~3 time；In the present invention, the cleaning activator is preferably trehalose containing 300mM, the phosphorus of the 0.01Mol/L of 1% albumin
Acid buffer, PH7.2.；The effect of the cleaning activator is to wash away unbonded EDC and NHS.
The present invention mixes the fluorescent microsphere with copper ion monoclonal antibody after the fluorescent microsphere for cleaning the activation
Coupling obtains copper ion monoclonal antibody-fluorescent microsphere compound.The copper ion monoclonal antibody and fluorescence in the present invention
The mass volume ratio of microballoon is preferably (0.3~0.5) mg：1ml.It is heretofore described be coupled at be protected from light under the conditions of carry out, it is described
The time of coupling is preferably 1.5~3h, more preferably 2~2.5h.
The present invention carries out closing and obtains copper ion monoclonal antibody-fluorescent microsphere compound after the coupling.In this hair
In bright, the closing is preferably 1% BSA solution with confining liquid, and the present invention carries out low temperature ultrasonic point after confining liquid is added
It dissipates, the temperature of the low temperature ultrasonic dispersion is preferably 4~10 DEG C, and the general power of the low temperature ultrasonic dispersion is preferably 20w, institute
The power of selection 10~15% when stating low temperature ultrasonic dispersion, the low temperature ultrasonic dispersion are preferably intermittent ultrasound；It is specially super
3~5s of sound, 3~5s of interval.The closed time described in the present invention is preferably 0.8~1.2h, more preferably 1h；The closing
Preferably carried out under the conditions of being protected from light.Preferably supernatant is removed in centrifugation to the present invention after the closing, collect solid phase components be copper from
Sub- monoclonal antibody-fluorescent microsphere compound.
Copper ion monoclonal antibody-fluorescent microsphere the compound is preferably uniformly mixed guarantor with protection liquid in the present invention
It is stored in 4 DEG C.In the present invention, the protection liquid is preferably containing point 1% (quality) BSA, the phosphoric acid of 0.05% (quality) tween
Buffer solution；Heretofore described uniformly mixed method is preferably low temperature ultrasonic dispersion, with the above-mentioned low temperature ultrasonic referred to point
Scattered parameter is consistent, and details are not described herein.
The copper ion monoclonal antibody is generated by hybridoma cell strain 4F12 secretions in the present invention, and the hybridoma is thin
The deposit number of born of the same parents' strain 4F12 is CCTCC NO：C201670.The hybridoma cell strain 4F12 secretions production copper in the present invention
The method that the method for ion monoclonal antibody produces monoclonal antibody using the hybridoma cell strain secretion of this field routine,
Without other particular/special requirements.In specific implementation process of the present invention, the cell strain 4F12 intraperitoneal injection of mice is taken out after 6~8 days
Ascites is taken, ascites is isolated and purified and obtains copper ion monoclonal antibody.
Immunofluorescence chromatograph test strip of the present invention further includes nitrocellulose filter, on the nitrocellulose filter according to
The nature controlling line of the detection line and coating secondary antibody of secondary setting coating copper ion detection comlete antigen.In the present invention, the nitric acid is fine
The spraying concentration of copper ion detection comlete antigen is preferably 0.3~0.6 μ g/ μ l in the plain film detection line of dimension, and more preferably 0.4
~0.5 μ g/ μ l.The quantity for spray of heretofore described copper ion detection comlete antigen is preferably 0.1~0.2 μ l/mm, more preferably
0.15μl/mm.In the present invention, the spraying concentration of secondary antibody is preferably 0.3~0.6 μ g/ μ on the nitrocellulose filter nature controlling line
L, more preferably 0.4~0.5 μ g/ μ l；The quantity for spray of the secondary antibody is preferably 0.1~0.2 μ l/mm, more preferably 0.15 μ l/
mm.Heretofore described spraying is preferably carried out using spraying instrument；The present invention is preferred after detection line and Quality Control wire spraying
Nitrocellulose filter is dried；The temperature of the drying is preferably 45~55 DEG C, more preferably 50 DEG C；The drying
Time is preferably 24~72h, more preferably 36~60h.
In the present invention, the secondary antibody is preferably commercially available secondary antibody product, more preferably rabbit igg.In the present invention, institute
It includes copper ion, carrier protein and by the chelating agent of the copper ion and carrier protein couplet to state copper ion detection comlete antigen
The chelating agent in the copper ion detection comlete antigen is preferably bifunctional chelating agent p-SCN-Bn-NOTA in the present invention；Institute
It is preferably chicken ovalbumin OVA to state carrier protein；Heretofore described bifunctional chelating agent p-SCN-Bn-NOTA and carrier protein
It is all made of commercial goods.Heretofore described copper ion detection comlete antigen is prepared by the following method acquisition：C1) by chela
Mixture solution is added dropwise to chelating in carrier protein solution and obtains chelating protein solution dropwise；C2) copper ion solution is added dropwise dropwise
It is in light blue to chelating in the chelating protein solution to reaction solution, and obtains copper ion with muddiness and detect comlete antigen.
In the present invention, the p-SCN-Bn-NOTA powder is preferably dissolved in DMSO by the chelating agent solution
It obtains, the concentration of the p-SCN-Bn-NOTA solution is preferably 25mg/ml；The OVA protein solutions are preferably by OVA
Protein powder is dissolved in HEPES buffer solution and obtains, and the concentration of the HEPES buffer solution is preferably 0.1M, and pH value is preferred
It is 8.9；The concentration of the OVA protein solutions is preferably 10mg/ml；In the present invention, the molar ratio of the NOTA and OVA is excellent
Choosing is (68~72):1, more preferably 70:1；The chelating agent solution is added dropwise in OVA liquid the present invention, 0~4 DEG C
2~4h of stirring chelating, 4 DEG C obtain chelating protein solution overnight.
Bifunctional chelating agent of the present invention includes two different structural units, when functional group, it can be with biology
Macromolecular (such as albumen) is connected in the form of covalent bond；Second is that metal chelating groups, make metal ion in three azo-cycle ligand holes
Be combined with it solidly, and introduce metal ion far from large biological molecule, to ensure that its bioactivity is unaffected.p-SCN-
Bn-NOTA is one kind minimum in polyaminopolycarboxylic group class annular chelating agent, be on the basis of Isosorbide-5-Nitrae, 7- 7-triazacyclononanes,
Three carboxyls are connected on three nitrogen-atoms, are also connected with characteristic functional group on NOTA heterocycles in addition --- end carries SCN
The benzyl of (thiocyanate) ion, by being covalently keyed the coupling that can be realized with OVA protein moleculars.
In the present invention, the copper ion solution is preferably by CuSO4·5H2O powder is dissolved in ultra-pure water and prepares,
The concentration of the copper ion solution is preferably 0.8~1.2mg/ml, more preferably 1.0mg/ml.In the present invention, described
Copper ion is preferably (75~80) with NOTA molar ratios:(68~72), more preferably 1.1:1.It in the present invention will be described
Copper ion solution is added dropwise in chelating protein solution, and 0~4 DEG C of stirring 25~35min of chelating, reaction solution is in light blue, and companion
There are muddiness, chelating to terminate.
The present invention is preferably surpassed obtained chelatropic reaction product using 30kD after copper ion is reacted with chelating protein chelates
Chimney filter ultrafiltration removes the NOTA and copper ion not being coupled, obtains copper ion and detects comlete antigen.The copper that the present invention obtains
Copper ion and carrier protein OVA coupling ratios are (18~22) in ion detection comlete antigen:1.In the present invention, the ultrafiltration turns
Speed is preferably 4000~6000g, more preferably 5000g；The time of the ultrafiltration is preferably 8~12min, more preferably
For 10min；The number of the ultrafiltration is preferably 3~8 times, more preferably 5 times.
The present invention combines after obtaining copper ion detection comlete antigen preferably through SDS-PAGE, ICP-AES, BCA method
Identify whether the copper ion detection comlete antigen is coupled successfully and calculates its coupling ratio.The copper ion in the present invention
Copper ion in detection comlete antigen and measuring samples competes copper ion monoclonal antibody jointly, inhibit copper in measuring samples from
Son is combined with copper ion monoclonal antibody, and inhibiting rate is calculated according to the size of fluorescence signal value.
In the present invention, the immunofluorescence chromatograph test strip further includes preferably PVC backboards (1), the PVC backboards
(1) it is used as supporter, overlapped that fiberglass packing, nitrocellulose filter and blotting paper stick on PVC backboards by described successively.
In the present invention, the immunofluorescence chromatograph test strip further includes preferably detection card shell；The detection card is outer
Shell is preferably one kind in PCV, PC, PE, ABS and PS.In heretofore described detection card shell face glass fibre membrane
There are wells for the heart, facilitate the addition of measuring samples.
The present invention also provides application of the immunofluorescence chromatograph test strip in detecting sample copper ion concentration.
Application of the present invention specifically includes following steps：1) measuring samples are added drop-wise on glass fibre membrane and carry out layer
Analysis；2) read the Immunofluorescence test paper strip detection line after the chromatography, nature controlling line fluorescence signal value Bt and Bc；It 3) will be described glimmering
Optical signal value Bt/Bc brings scheduled standard curve equation of linear regression into as B and calculates copper ion concentration in acquisition measuring samples；
The independent variable of the scheduled standard curve equation of linear regression is the log10 logarithms of copper ion concentration, and dependent variable is the suppression that shines
Rate processed, the dependent variable are calculated by Formulas I, (B0-B)/B0× 100% Formulas I, B0To be not added with measuring samples or being not added with mark
Fluorescence signal value Bt when quasi- sample0/Bc0, B is fluorescence signal value Bt/Bc when adding measuring samples.
In specific implementation process of the present invention, the sample-adding amount of each test strips is preferably 60~90 μ l, more preferably
For 75 μ l.The measuring samples are added dropwise to sample application zone by the present invention according to above-mentioned sample-adding amount, upper after 15min machine-readable to take fluorescence signal
B.The reading fluorescence signal value B preferably (is inspired confidence in Guangzhou ten thousand using winged II generation immunofluorescence instrument of surveying in the present invention
Biotechnology Ltd.).
The present invention brings scheduled standard curve linear regression into after obtaining fluorescence signal value B, by the fluorescence signal value B
Equation calculation obtains copper ion concentration in measuring samples.Heretofore described scheduled standard curve equation of linear regression is y=
31.58X+32.346, wherein x are the inhibiting rate that shines, and y is the copper ion concentration in measuring samples, x=(B0-B)/B0×
100%, B0To be not added with the fluorescence signal value that measuring samples are.
In the present invention, the standard curve equation of linear regression is preferably prepared by the following：It is dense with different gradients
The competitor standard items Cu-NOTA of degree replaces the measuring samples in step 2) described in above-mentioned technical proposal, according to above-mentioned technical side
The application method detection of immunofluorescence chromatograph test strip described in case obtains fluorescence signal value B, with 0ng/ml competitor standard items
Fluorescence signal value is B0, calculates the luminous inhibiting rate x of different gradient concentration competitor standard items, x=(B0-B)/B0 × 100%,
Using the inhibiting rate that shines as ordinate, with Cu2+Concentration log10 logarithms are abscissa, draw standard curve, and it is linear to obtain standard curve
Regression equation.
In the present invention, the concentration of the competitor standard items Cu-NOTA of the different gradient concentrations includes preferably 0ng/
Ml, 0.8ng/ml, 1.6ng/ml, 3.12ng/ml, 6.25ng/ml, 12.5ng/ml, 25ng/ml, 50ng/ml and 100ng/ml.
The method of the invention prepares the R of scheduled standard curve equation of linear regression2=0.995, detection is sensitive
Spend (IC50) it is 3.6ng/ml, detection range (IC20-IC80) it is 0.4-32.3ng/ml, detection limit (IC10) it is 0.2ng/ml.It can
Meet GB5749《Standards for drinking water quality》Drinking Water, GB3838《Water environment quality standard》The class of I class~V
The testing requirements of the minimum Limited Doses 0.01mg/L of copper in water.
The present invention provides application of the immunofluorescence chromatograph test strip in detecting sample copper ion concentration.In this hair
In bright, the sample includes preferably Chinese herbal medicine sample, pedotheque and water sample.In the present invention, the sample is excellent before detection
Being coupled with chelating agent for choosing obtains measuring samples.In specific implementation process of the present invention, when the sample be Chinese herbal medicine sample or
When pedotheque, preferably by Chinese herbal medicine sample or pedotheque micro-wave digestion co-precipitation method, after redissolution again with chelating agent
NOTA is coupled to prepare sample to be tested.In the present invention, the micro-wave digestion preferably uses microwave dissolver to carry out；It is described micro-
The purpose of wave resolution is will to fix treatments of the sample as liquid.In the present invention, the resolution program of the condition of the micro-wave digestion is excellent
It is selected as：130 DEG C of 10min that climb, 130 DEG C of holding 3min；160 DEG C of 3min that climb, 160 DEG C of holding 5min；180 DEG C of 3min that climb,
180 DEG C of holding 40min；It is cooling；Heretofore described solid sample is preferably cleared up in advance before resolution；The pre- resolution is excellent
It is selected as draught cupboard overnight or 130 DEG C of sour instrument is caught up with to preheat 30min.In the present invention, the purpose of the precipitation method is disappeared by adjusting
The pH of solution sample liquid makes copper ion precipitate, to efficiently separate copper ion and most metal ion.In the present invention, described heavy
The condition in shallow lake is preferably：By after resolution Chinese herbal medicine sample and pedotheque be settled to 10ml and 25ml respectively, take 5ml fixed respectively
Hold sample liquid, pH value of solution is adjusted to neutrality 6.7~7.2 by 1.0~2.0mol/LNaOH, and 0.2~0.4ml 1 ‰ one is added
Quantitative flocculant (polyacrylamide, PAM), 1500~2500rpm centrifuges 5~10min after standing 15~20min, goes to take back completely
Collection precipitation, then passes through the dilute HNO of 1.0~2.0mol/L3Slowly redissolution precipitation is used to prepare the haptens with reactionogenicity and waits for
Sample.In the present invention, the concrete operations being coupled with chelating agent after the sample micro-wave digestion co-precipitation redissolves：It will resolution
Chinese herbal medicine sample afterwards is settled to 10ml, takes 5ml that mono- 1~2 μ l mixings of the NOTA liquid reaction 15min of 5 μ g/ μ l are added；Or it will resolution
Pedotheque is settled to 25ml afterwards, takes 5ml that mono- 2~4 μ l mixings of the NOTA liquid reaction 15min of 5 μ g/ μ l are added.
When the sample is water sample, including river water, pond water, farmland water, surface water；By the water sample filter after again with
Chelating agent NOTA couplings are to prepare sample to be tested；The filtering is preferred first to use filter paper to filter, and then uses membrane filtration；Institute
The aperture for stating filter membrane is preferably 0.22um.In the present invention, the concrete operations side of the filtered water sample and chelating agent coupling
Method：It takes 5ml to cross film liquid and mono- 1~2 μ l mixings of the NOTA liquid reaction 15min of 2 μ g/ μ l is added.
With reference to embodiment to it is provided by the invention it is a kind of detection copper ion immunofluorescence chromatograph test strip and its answer
With being described in detail, but they cannot be interpreted as limiting the scope of the present invention.
The preparation of 1 immunofluorescence chromatograph test strip of embodiment
Copper ion detects the preparation and identification of comlete antigen
Cu ions and carrier protein OVA are coupled by bifunctional chelating agent p-SCN-Bn-NOTA
PH8.9 is configured, OVA protein powders are dissolved in HEPES buffer solution, obtain a concentration of by 0.1M HEPES buffer solutions
The OVA protein solutions of 10mg/ml；NOTA powder is dissolved in DMSO solution, the NOTA solution of a concentration of 25mg/ml is obtained；It will
NOTA liquid is added dropwise in OVA protein solutions, and stirring chelating 3h in ice chest, 4 DEG C are made NOTA-OVA liquid (NOTA and OVA overnight
Molar ratio about 70:1)；
By CuSO4·5H2O powder is dissolved in ultra-pure water, obtains the copper ion solution of a concentration of 1.0mg/ml；By Cu2+Solution
(Cu in NOTA-OVA liquid is added dropwise2+With NOTA molar ratios about 1.1:1), stirring chelating 30min in ice chest, reaction solution are in pale blue
Color, and with muddiness, obtain Cu-NOTA-OVA comlete antigens, and 4 DEG C of preservations.Then use 30kD super filter tube ultrafiltration that Cu- is made
NOTA-OVA comlete antigens, ultra-filtration conditions：Centrifuge speed 5000g, centrifuges 10min, and ultrafiltration 5 times removes the p- not being coupled
SCN-Bn-NOTA and Cu ions obtain copper ion and detect comlete antigen.
Combine whether identification comlete antigen is coupled successfully and calculates its coupling by SDS-PAGE, ICP-AES, BCA method
Ratio.
The results are shown in Figure 2 by SDS-PAGE, heavy metal copper comlete antigen Cu-NOTA-OVA bands and carrier protein OVA items
Band, which is compared, has apparent hysteresis, the comlete antigen molecular weight of synthesis to show that Cu-NOTA is successfully coupled knot than carrier protein bigger
OVA is closed, detection antigen is successfully prepared.
Flame Atomic Absorption Spectrometry instrumental method measures a concentration of 0.1107mg/ml of Cu in comlete antigen Cu-NOTA-OVA, BCA methods
Standard curve as shown in figure 3, measure a concentration of 3.962mg/ml of OVA albumen in comlete antigen Cu-NOTA-OVA, calculate complete
Holoantigen Cu ions and carrier protein OVA coupling ratios are about 20:1, it meets the requirements.
The preparation and identification of copper ion monoclonal antibody
Recover freeze can stably excreting preventing from heavy metal copper monoclonal antibody cell strain 4F12, the cell strain was in 2016
It was preserved in Wuhan University on June 8, deposit number is CCTCC NO：C201670, and expand culture, by the good sun of growth conditions
Property cell is with 5 × 105The Balb/c mouse for 10 week old for having broken Freund's incomplete adjuvant in advance are only injected intraperitoneally in cells/, are inoculated with 7 days
Afterwards daily observation mouse survival state, when mouse web portion obvious tumefaction, in time with aseptic syringe needle take out ascites, 3000rpm from
Heart 15min takes -20 DEG C of packing of supernatant to freeze.By 4 DEG C of slow mechanism dissolveds of ascitic type antibody of collection, 1200rpm centrifugations 30min is gone
It except impurity, is slowly stirred under the conditions of 0 DEG C and the 4 DEG C saturated ammonium sulfate solution that are pre-chilled isometric with it is added dropwise, 4 DEG C after 30min
Precipitates overnight.Mixed liquor is shifted, 10000rpm centrifugations 20min abandons supernatant at 4 DEG C, takes precipitation to redissolve and prepares re-suspension liquid, 4 DEG C of dialysis
It 3 times, each 6h, is diluted after the completion with combination buffer and 0.45um filter membranes is used to filter, then pass through ProteinG affinity columns
Antibody purification.The 4 DEG C of ultrafiltration of 30kD super filter tubes of the antibody of purified pool, it is pure using antibody after PAGE gel purification Identification
Degree, the albumen concentration of BCA kit measurement purified antibodies, and antibody purification is measured by indirect ELISA and chessboard method respectively
Potency and affinity.B4 potency 6 × 105Times, affinity of antibody constant K=1.51 × 1010L/mol。
The preparation of copper ion monoclonal antibody-fluorescent microsphere compound
Pass through EDC-NHS mediated methods (0.3-0.5mg antibody is added in 1ml fluorescent microspheres) coupling fluorescent microsphere and monoclonal
Antibody protein, to prepare fluorescent microsphere antibody complex.Ultrasonic disperse fluorescent microsphere, ultrasonic disperse general power is 20W, when ultrasonic
Select 10%-15% power, ultrasonic 3-5s, interval 3-5s；Then the EDC liquid configured and NHS liquid are sequentially added into ultrasound point
In the fluorescent microsphere liquid dissipated, mixing and room temperature uniform activation 15min are overturned rapidly；Centrifugation (12000rpm, 10min) is gone
Clearly, be added reaction solution (phosphate buffer solution of 0.01mol/L, such as prepare fluorescent microsphere antibody complex total volume be 1ml, from
Polishing reaction solution is to 1ml after the heart) ultrasonic disperse mixing afterwards, cleaning activator (cleaning unbonded on EDC and NHS) 2 times
Afterwards, copper ion monoclonal antibody is added, room temperature is protected from light 2~2.5h of mixing and is coupled；Supernatant is removed in centrifugation, and 1%BSA closings are added
Liquid, after low temperature ultrasonic dispersion (power is same as above, and temperature is 4~10 DEG C), room temperature is protected from light mixing 1h and is closed；Supernatant is removed in centrifugation,
Preservation liquid is added, and (it is that 0.01mol/L contains 1%BSA to preserve liquid, and the phosphate buffer of 0.05% tween preserves the addition body of liquid
Product is same as above, polishing 1ml) low temperature ultrasonic disperses afterwards and 4 DEG C are kept in dark place, that is, complete the system of fluorescent microsphere antibody complex
It is standby.
The spraying of fiberglass packing and nitrocellulose filter
After coating detection antigen is diluted to best effort concentration (0.45 μ g/ μ l) with rabbit igg, by its even application (spray
Film instrument sprays, and spraying rate is 0.15 μ l/mm) on suitable for NC films, 50 DEG C of freeze-day with constant temperature 40h, to prepare T lines antigen and C lines
Antibody.After fluorescent microsphere antibody complex is diluted to best effort concentration (0.04 μ g/ μ l), by its even application (spray pad instrument
Spraying, spraying rate are 4 μ l/cm) on suitable glass fiber mat, 50 DEG C of freeze-day with constant temperature 20h.Later by dry glass fibre
Pad, NC films, blotting paper are bonded successively, and cutting machine cuts into 4mm wide test strips, and is assembled into ELISA test strip card, drying at room temperature
It is kept in dark place.
Embodiment 2
With the performance evaluation of the copper ion concentration in the immunofluorescence chromatograph test strip detection sample in embodiment 1
The preparation of standard curve：By graded series concentration (0,0.8,1.6,3.12,6.25,12.5,25,50,100ng/
Ml) competitor standard items Cu-NOTA is added dropwise to sample application zone by every card 75ul sample-adding amount, upper after 15min machine-readable to take fluorescence signal.
Each detection acquires mean value using 10 parallel groups, calculates standard curve and inhibiting rate (sensitivity).By 0ng/ml standard sample wells
Fluorescence signal T/C values be set to B0, the T/C values in other holes are B, to detect inhibiting rate (B0-B)/B0× 100% is ordinate, with
Cu2+Concentration log10 logarithms are abscissa, draw standard suppression curve.
Photo such as Fig. 4 after copper ion is detected in the immunofluorescence chromatograph test strip product standard curve preparation process
Shown, its visible graded of ELISA test strip card T lines fluorescent dye signal naked eyes, C lines fluorescent dye signal naked eyes are invisible,
Quantitative fluorescence signal value can be read by immunofluorescence instrument.Fig. 5 is the fluorescence signal curve graph read, standard curve
The results are shown in Figure 6, and standard curve equation of linear regression is y=31.58X+32.346, R2=0.995, detection sensitivity
(IC50) it is 3.6ng/ml, detection range (IC20-IC80) it is 0.4-32.3ng/ml, detection limit (IC10) it is 0.2ng/ml.It can expire
Sufficient GB5749《Standards for drinking water quality》Drinking Water, GB3838《Water environment quality standard》The class water of I class~V
The testing requirements of the middle minimum Limited Doses 0.01mg/L of copper.
Veracity and precision is evaluated：
3 batch ELISA test strip cards of production and assembly are selected in the range of linearity of immunofluorescence chromatograph test strip method
The titer of basic, normal, high 3 kinds of various concentrations, each concentration point are repeated 10 times, and are measured fluorescence signal using the above method, are passed through
Standard curve calculates detectable concentration.Immunofluorescence chromatograph test strip method batch is interior, differences between batches are shown in Tables 1 and 2：Wherein, it is returned in criticizing
Yield is between 91.3%~117.8%, and RSD is below 15%；Between 91.3%~119.8%, RSD's rate of recovery exists between batch
10% hereinafter, meet the testing requirements of general heavy-metal residual analysis.
Difference test (n=10) in 1 immunofluorescence chromatograph test strip method of table batch
2 immunofluorescence chromatograph test strip method differences between batches of table test (n=3)
Recovery test is added in sample liquid：
Add competitor standard items respectively in PB buffer solutions, CB buffer solutions, special Sample dilution, configuration is basic, normal, high
The detection liquid of 3 kinds of various concentrations measures fluorescence signal using the above method.In the present invention, the special Sample dilution is
300mM trehaloses, the phosphate buffer of the 0.01Mol/L of 1% bovine serum albumin(BSA), PH7.2.PB, CB buffer solution and test paper
Sample-specific dilution addition recovery test the results are shown in Table 3, table 4 and table 5：By comparing each buffer solution system detection value,
The rate of recovery and relative standard deviation are it can be found that different buffer systems will produce significantly fluorimetric analysis process accuracy
It influences, and sample-specific dilution is compared with the Stability and veracity higher of other buffer solution detected values.
3 immunofluorescence chromatograph test strip method PB buffer solutions of table add recovery test (n=3)
4 immunofluorescence chromatograph test strip method CB buffer solutions of table add recovery test (n=3)
The special Sample dilution addition recovery test (n=3) of 5 immunofluorescence chromatograph test strip method of table
Evaluation on specificity
Each metallic element standard suppression curve sensitivity (IC is found out by immunofluorescence chromatograph test strip method50Value), pass through
Formula Cross Reactivity=[IC50(Cu)/IC50(other metal ions)] × 100% intersection for acquiring each metallic element
Reactivity.
As shown in table 6, except Mg elements, Zn elements respectively have copper monoclonal antibody 14.2%, 5.3% friendship in this method
It pitches outside reactivity, the cross reacting rate of remaining common metal element is respectively less than or approximation 1%, and specificity is overall good, meets
General testing requirements.
6 immunofluorescence chromatograph test strip method specific test of table
Estimation of stability：
A collection of ELISA test strip card is prepared according to established immunofluorescence chromatograph test strip method, guarantor is protected from light in 37 DEG C of dryings
Several weeks are deposited, are taken out respectively when 1 week, 2 weeks, 3 weeks, 4 weeks, the titer of basic, normal, high 3 kinds of various concentrations are detected, using above-mentioned side
Method measures fluorescence signal, and detectable concentration is calculated by standard curve.Immunofluorescence chromatograph test strip method stability test result is shown in
Table 7.
7 immunofluorescence chromatograph test strip method stability test result of table
Embodiment 3
Utilize immunofluorescence chromatograph test strip described in embodiment 1 detection Chinese herbal medicine sample, pedotheque and Copper in Water Samples
Ion concentration
1, the micro-wave digestion rate of recovery of Chinese herbal medicine, pedotheque
The condition precise sample 0.2000g of micro-wave digestion, is added nitric acid 8ml, and 130 DEG C of sour instrument is stayed overnight or caught up with to draught cupboard
Preheating 30min clears up in advance；Selection resolution program (130 DEG C of 10min that climb, 130 DEG C of holding 3min；160 DEG C of 3min that climb, 160 DEG C
Keep 5min；180 DEG C of 3min that climb, 180 DEG C of holding 40min；It is cooling)；In the present invention after the sample micro-wave digestion with chela
The concrete operations of mixture coupling：10ml volumetric flask constant volumes are used after Chinese herbal medicine Specimen eliminating, take 5ml digestion solutions that 5 μ g/ μ l are added mono-
1~2 μ l mixings of NOTA liquid react 15min；25ml volumetric flask constant volumes are used after pedotheque resolution, take 5ml digestion solutions that 5 μ g/ μ are added
Mono- 2~4 μ l mixings of NOTA liquid of l react 15min.While sample micro-wave digestion, by standard substance together with blank control into
Row resolution is cleared up back using standard substance after graphite oven atomic absorption detection resolution and the Cu contents in blank control, calculating
Yield, it is whether up to standard with assessment resolution efficiency.
Plant standard substance is national standard reference substance GBW 07602 (GSV-1) bush branch and leaf, copper content standard value
For：5.2μg/g；Reference material of soil is national standard reference substance GBW07404 (GSS-4) soil constituent analytical standard substance,
Copper content standard value is：40.0μg/g；Blank control is nitric acid AR, copper highest content：≤ 0.00001%, standard substance and sky
The results are shown in Table 8 for Cu assays after white control resolution：
Cu assay results after 8 standard substance of table and blank control micro-wave digestion
The practical weighing 0.2000g of standard substance, 25ml is settled to after catching up with acid, so standard substance Cu contents after micro-wave digestion
It calculates：
(1) plant：(39.705-2.757) × 0.025 ÷ 0.2=4.62 μ g/g；
(2) soil：(346.5-2.757) × 0.025 ÷ 0.2=42.97 μ g/g.
The resolution rate of recovery calculates：
(1) plant：(4.62/5.2) × 100%=88.8%；
(2) soil：(42.97/40.0) × 100%=107.4%.
Resolution is qualified.
Chinese herbal medicine and water sample sample wait for the preparation and detection of test sample
The measurement of copper content in Chinese herbal medicine sample
Chinese herbal medicine sample drying is ground rear micro-wave digestion co-precipitation to redissolve, then is coupled with excessive chelating agent NOTA
To prepare sample to be tested.By above-mentioned chemiluminescence indirect competitive and ICP-AES instrumental methods simultaneously to Cu contents in sample
It is measured, the results are shown in Table 9
9 Chinese herbal medicine sample heavy metal copper assay result (n=3) of table
* immunofluorescence chromatograph test strip method detected value is result (the correction letter based on above 13 samples after coefficient correction
Number is y=1.7877x+1.8287)
Immunofluorescence chromatograph test strip method and ICP-AES instrumental method testing results using GraphPad Prism and SPSS into
Row correlation statistics are analyzed, and Pearson correlations are respectively 0.877, and testing result is horizontal significantly correlated 0.05, can apply
The detection of copper content in general (Chinese herbal medicine) plant.
The measurement of copper content in water sample sample
0.22um filters after the water samples sample filter paper such as river water, pond water, farmland water, (Metal Processing Factory) surface water are filtered
Film is crossed, then with chelating agent NOTA couplings to prepare sample to be tested.Pass through above-mentioned immunofluorescence chromatograph test strip and ICP-AES instrument
Device method is simultaneously measured Cu contents in sample, as a result as shown in table 11.
11 water sample sample heavy metal copper assay result of table
* immunofluorescence chromatograph test strip method detected value is result (the correction letter based on above 15 samples after coefficient correction
Number is y=1.6982x+5.9604)
Immunofluorescence chromatograph test strip method and ICP-AES instrumental method testing results using GraphPad Prism and SPSS into
Row correlation statistics are analyzed, and Pearson correlations are respectively 0.929, and testing result is horizontal significantly correlated 0.05, can apply
In the detection of general Copper in Water Samples content.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of immunofluorescence chromatograph test strip of detection copper ion, which is characterized in that including overlapped successively fiberglass packing,
Nitrocellulose membrane and blotting paper；The fiberglass packing surface spraying has copper ion monoclonal antibody-fluorescent microsphere compound；Institute
State the nature controlling line that nitrocellulose filter sets gradually the detection line and coating secondary antibody of coating copper ion detection comlete antigen；
Copper ion monoclonal antibody-fluorescent microsphere the compound is obtained by copper ion monoclonal antibody and fluorescent microsphere coupling；
Copper ion detection comlete antigen includes copper ion, carrier protein and by the copper ion and carrier protein couplet
Chelating agent.
2. immunofluorescence chromatograph test strip according to claim 1, which is characterized in that the copper ion monoclonal antibody-
A concentration of 0.03~0.05 μ g/ μ l of fluorescent microsphere compound；Copper ion monoclonal antibody-fluorescence is micro- on the fiberglass packing
The quantity for spray of ball compound is 3~5 μ l/cm.
3. immunofluorescence chromatograph test strip according to claim 1 or 2, which is characterized in that the copper ion monoclonal is anti-
Body is generated by hybridoma cell strain 4F12 secretions, and the deposit number of the hybridoma cell strain 4F12 is CCTCCNO：C201670.
4. immunofluorescence chromatograph test strip according to claim 1, which is characterized in that the copper ion detects comlete antigen
A concentration of 0.3~0.6 μ g/ μ l；The quantity for spray of copper ion detection comlete antigen is 0.1~0.2 μ on the nitrocellulose filter
l/mm。
5. immunofluorescence chromatograph test strip according to claim 1 or 4, which is characterized in that the copper ion detection is complete
Chelating agent in antigen is bifunctional chelating agent p-SCN-Bn-NOTA；The carrier protein is chicken ovalbumin OVA.
6. application of the immunofluorescence chromatograph test strip described in Claims 1 to 5 any one in detecting sample copper ion concentration.
7. the application described in claim 6, which is characterized in that include the following steps：
1) measuring samples are added drop-wise on glass fibre membrane and are chromatographed；
2) read the Immunofluorescence test paper strip detection line after the chromatography, nature controlling line fluorescence signal value Bt and Bc；
3) it brings the fluorescence signal value Bt/Bc into scheduled standard linear regression equation calculations as B values and obtains measuring samples
Middle copper ion concentration；
The independent variable of the scheduled standard linear regression equation is the log10 logarithms of copper ion concentration, and dependent variable is the suppression that shines
Rate processed, the dependent variable are calculated by Formulas I, (B0-B)/B0× 100% Formulas I, B0To be not added with the fluorescence signal of measuring samples
Value Bt0/Bc0, B is fluorescence signal value Bt/Bc when adding measuring samples.
8. application according to claim 7, which is characterized in that the standard linear regression equation obtains by the following method
?：
With the competitor standard items Cu-NOTA replacement steps 1 of gradient concentration) in measuring samples, according to step 1)~2) side
Method obtains the fluorescence signal value Bt/Bc of competitor standard items, with the fluorescence signal value Bt of 0ng/ml competitor standard items0/Bc0For
B0, the luminous inhibiting rate of gradient concentration competitor standard items is calculated, using the luminous inhibiting rate as ordinate, with Cu2+It is dense
Degree log10 logarithms are abscissa, draw standard curve, obtain standard linear regression equation.
9. application according to claim 7, which is characterized in that the dripping quantity of measuring samples described in step 1) is 60~90
μl；The chromatography time is 12~18min.
10. application according to claim 7, which is characterized in that the sample includes Chinese herbal medicine sample, pedotheque and water
Sample.
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Application publication date: 20181030 |