CN102353773B - Immune chromatographic test paper for detecting melamine and preparation method thereof - Google Patents
Immune chromatographic test paper for detecting melamine and preparation method thereof Download PDFInfo
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- CN102353773B CN102353773B CN201110157278.6A CN201110157278A CN102353773B CN 102353773 B CN102353773 B CN 102353773B CN 201110157278 A CN201110157278 A CN 201110157278A CN 102353773 B CN102353773 B CN 102353773B
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Abstract
The invention discloses an immune chromatographic test paper for detecting melamine and a preparation method thereof. The immune chromatographic test paper for detecting melamine comprises a sample pad containing superparamagnetic composite particle labeled melamine antibody, a cellulose nitrate membrane connected with one end of the sample pad, and an absorbent pad connected with the other end of the cellulose nitrate membrane, wherein, the cellulose nitrate membrane is coated with a detection line and a quality control line which are mutually separated, the detection line contains a melamine antigen, and the quality control line contains an antiantibody which can be combined with the melamine antibody specifically. Experiments indicate that the immunochromatography test paper for detecting melamine has the advantages of high sensitivity, strong specificity, convenience and rapidness, and available objective determination.
Description
Technical field
The present invention relates to the detection reagent of melamine residual thing, be specifically related to a kind of immune chromatography test paper that detects melamine and preparation method thereof.
Background technology
Melamine (Melamine), be commonly called as melamine, extract of protein, is a kind of triazines nitrogen heterocyclic ring organic compound, is used as industrial chemicals.Melamine is harmful to health, is not useable for food processing or food additives.On October 8th, 2008, the Ministry of Public Health, the Ministry of Industry and Information Technology, the Ministry of Agriculture, State Administration for Industry and Commerce and State Administration for Quality Supervision and Inspection and Quarantine combine the issue bulletin, and formulate the temporary control and education value of melamine in breast and dairy products: the Limited Doses that the Limited Doses of baby formula melamine in dry milk is melamine in 1.0mg/kg liquid milk (comprising raw milk), milk powder, other prescription emulsifiable powder is 2.5mg/kg (L).In other food containing breast more than 15%, the Limited Doses of melamine is 2.5mg/kg (L), higher than the product of above Limited Doses, must not sell without exception.At present field screening is detected with detecting the available melamine colloidal gold test paper of reagent, but can not finely meet the demands because its sensitivity is low, therefore need develop can be at the scene more accurate and detection method fast.
Sidestream immune chromatography detection technique (LFIAs) based on the Ag-Ab immunological response is the emerging technology grown up early 1990s, because of its characteristic fast and easily, is suitable for very much on-the-spot fast monitored.But such technology adopts collaurum as the signal labeled molecule more at present, be subject to the restriction of its sensitivity etc., can only be for qualitative detection.
The superparamagnetism composite particle has good magnetism characteristic, because it is subject to background interference little, is particularly suitable for not containing the detection of the biological sample of magnetisable material.When collaurum and fluorescent tag molecule detect for the flow measurement immunochromatography, observe approximately 10% signaling molecule intensity on its detection zone film surface only, and with the superparamagnetism composite particle material that serves as a mark, the all magnetic signal molecules in detection zone film 3-D solid structure can be detected, can greatly improve sensitivity, and the magnetic signal detector of available correspondence reaches quantitative measurement, therefore, the superparamagnetism composite particle is the material received publicity in LFIAs in recent years.
Yet at present the biological detection of report adopts chemical method as after coprecipitation first prepares the magnetic nano-particle of organic phase with magnetic nano-composite particle more, then adopts silicon (SiO
2) or the macromolecular material such as polystyrene, polyacrylic acid, gelatin the stabilization coating decoration is carried out in its surface, to obtain stable, water miscible magnetic label material.But these prepare often very complicated comparatively of method of modifying, the superparamagnetism composite particle obtained can not meet the requirement of LFIAs at aspects such as size, biocompatibility, saturated magnetic intensity, external magnetic field response speed, stability and labeling effciencies simultaneously: its size is many between 200～300nm, because magnetic bead particles is bigger than normal, the swimming time on test paper is slower, and developing time is longer; And too little particle can't provide enough magnetic resonance signals; Also have in addition the problems such as biocompatibility is unstable, the easy polymerization of magnetic bead; These deficiencies have limited the application of superparamagnetism composite particle in LFIAs.
Summary of the invention
An object of the present invention is to provide a kind of immune chromatography test paper that detects melamine.
The immune chromatography test paper of detection melamine provided by the invention, comprise sample pad, the reacting pad that is connected in described sample pad one end that contains superparamagnetism composite particle mark melamine antibody, the adsorptive pads that is connected in the described reacting pad other end; Described reacting pad is coated with detection line and the nature controlling line be separated from each other, and described detection line contains melamine antigen, described nature controlling line contain can with the antiantibody of described melamine antibody specific bond.
Described reacting pad is nitrocellulose filter;
Described superparamagnetism composite particle mark melamine antibody is by the condensate formed with the peptide bond covalent bond of melamine antibody and superparamagnetism composite particle;
Described melamine antibody is melamine monoclonal antibody or melamine polyclonal antibody, described melamine antibody be specially the melamine monoclonal antibody (your bio tech ltd of Zhengzhou Kinsey, C010110);
The conjugate that described melamine antigen is melamine hapten and carrier protein; When little molecular antigen material and carrier protein couplet, the haptenic average number connected on each carrier protein molecule be called coupling ratio or in conjunction with than.For reaching the good combination of carrier protein and NC film, can farthest guarantee the extension in site, melamine hapten space again simultaneously, it is very important selecting suitable coupling ratio.The size of coupling ratio feeds intake with activity, steric hindrance, the reaction of haptens functional group, and when reaction conditions is relevant.The general mol ratio by adjusting haptens and carrier, reaction environment pH value, temperature, ionic strength etc. are controlled coupling ratio.
The coupling ratio of described carrier protein and described melamine hapten is 1: 10～1: 12, is suitable for the extension in the coated and site, melamine hapten space of antigen, and the coupling ratio of described carrier protein and described melamine hapten is specially 1: 12;
The antiantibody of described and described melamine antibody specific bond is sheep anti-mouse igg antibody;
The concentration of described melamine antigen and described sheep anti-mouse igg antibody is 1mg/ml.
Described superparamagnetism composite particle mark melamine antibody is prepared as follows:
1) by every 2.5mg superparamagnetism composite particle, 0.96mg1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), 1.15mg N-maloyl imines (NHS) and 1ml concentration, be 0.1M, pH value 2-(N-morpholine) ethyl sulfonic acid (MES) damping fluid that is 4.7 (take 1.066g MES, 0.45g NaCl is dissolved in the 50ml pure water, adjust pH to 4.7) mix, reaction, obtain activating rear magnetic particle;
2) by every 2.5mg step 1) to obtain activating rear magnetic particle, 0.15mg melamine antibody and 0.8ml concentration be that the borate buffer solution that 50mM pH is 8.5 mixes, reacts, and obtains containing the reactant liquor of magnetic particle after coupling;
3) to step 2) add BSA to mix in the reactant liquor that obtains to obtain mixed liquor, reaction, obtain containing the reactant liquor of magnetic particle after sealing;
The concentration of described BSA in described mixed liquor is 5% (quality percentage composition).
Described melamine antigen is prepared as follows:
The synthetic employing of melamine-BSA first is converted into intermediate by melamine molecule, then carries out coupling with BSA.Concrete steps are:
A) first get every 63mg melamine, 50mg succinic anhydride and 5ml pyridine and mix, reaction, obtain reactant liquor a, then remove pyridine in described reactant liquor a, obtains the melamine intermediate; Add again 5ml DMF (DMF) to dissolve the described melamine intermediate of 82mg, obtain the melamine midbody solution;
B) by every 5ml steps A) the melamine midbody solution and the 20.6mg DCC (N that obtain, the N-dicyclohexyl carbodiimide) mixed, obtain reactant liquor b, in described reactant liquor b, add 28.6mg NHS (N-maloyl imines) to mix, react, the melamine midbody solution after being activated again;
C) get every 100mg carrier protein and be dissolved in 10ml concentration 0.1mol/L, in the sodium borate aqueous solution that pH is 8.5, obtain the carrier protein BAS;
D) by every 5ml step B) melamine midbody solution and 10ml step C obtain after the activation that obtains carrier protein BAS mixes, reacts, and obtains reactant liquor c.
In the preparation method of described superparamagnetism composite particle mark melamine antibody:
Step 1) in, temperature of reaction is 37 ℃, and the reaction time is 0.5h;
Step 2) in, temperature of reaction is 25 ℃, and the reaction time is 3.5h;
Step 3) in, temperature of reaction is 37 ℃, and the reaction time is 0.5h.
In the preparation method of described melamine antigen:
Steps A) in, described temperature of reaction is 25 ℃, and the described reaction time is 6h;
Step B) in, described mixing temperature is 25 ℃, and described incorporation time is 15min, and described reaction is first at 25 ℃, to react 1h, then reacts 1.5h under 4 ℃;
Step D) in, described temperature of reaction is 4 ℃, and the described reaction time is 3.5h.
In the preparation method of described superparamagnetism composite particle mark melamine antibody:
In described step 3) after, also comprise magnetic particle after the sealing in the described reactant liquor that contains the rear magnetic particle of sealing is washed, suspends, obtain the step of superparamagnetism composite particle mark melamine antibody, described cleansing solution and suspending liquid are the PBS damping fluid that 0.02M pH is 7.4.
In the preparation method of described melamine antigen,
At described step D) after also comprise step D) the reactant liquor c that obtains dialysis, freeze-drying, obtain melamine antigen;
Described dislysate is that concentration is the PBS damping fluid that 0.02mol/L, pH are 7.4, and dialysis time is 24h, and every 6h changes a dislysate.
Described melamine hapten is melamine;
Described carrier protein is BSA;
Because melamine is small-molecule substance, its molecular surface characteristic is unfavorable for reaction zone being the direct combination of NC film, itself and carrier protein need to be carried out after coupling reaching by means of the character of surface of carrier protein the good combination with the NC film.Can be used as the seralbumin that various animals are arranged of carrier protein, as bovine serum albumin(BSA) (BovineSerum Albumin, BSA), human serum albumins (Human Serum Albumin, HSA), also has keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin, KLH), gamma globulin of thyroglobulin, albumin rabbit serum (RSA), ovalbumin (Ovalbumin, OVA), fibrinogen or rabbit and chicken etc.Research is found, the BSA physicochemical property is stable, lysine content is high, free amino group is many, larger solubleness is all arranged under different pH and ionic strength, all can carry out coupling with haptens in the situation that contain organic solvent (as pyridine, DMF etc.), and still keep solvable state after coupling, the splendid selection as carrier protein, therefore the present invention selects BSA as coupling protein.
Described superparamagnetism composite particle is Fe
3o
4nano particle;
Because this superparamagnetism composite particle will the sample pad swimming in above-mentioned reaction plate be realized specific bond and the labeled reactant of Ag-Ab to the test section in the middle of reaction plate, too large (>300nm) swimming time on test paper of its particle diameter is long, and developing time is slow; Be easier to assemble when coupling, and easily produce non-specific responding; The too little magnetic intensity of particle diameter is often inadequate again.The particle diameter of described superparamagnetism composite particle is 60～300nm, and described particle diameter is specially 80～200nm, and described particle diameter especially is preferably 100nm; The deviation of its particle diameter (CV) between 10～30%, preferably between 10～20%, preferably 15%.
The saturated magnetic intensity of superparamagnetism composite particle and external magnetic field response speed thereof have directly determined the height of detection sensitivity and accuracy thereof, the saturated magnetic intensity of superparamagnetism composite particle prepared by classic method usually all<30emu/g, the external magnetic field response speed is at 100～200 seconds.For improving its sensitivity and accuracy, the magnetic saturation intensity of described superparamagnetism composite particle is 30～80emu/g, corresponding external magnetic field response speed is 20～100 seconds, the magnetic saturation intensity of described superparamagnetism composite particle is specially 35～70emu/g, corresponding external magnetic field response speed is 20～50 seconds, the magnetic saturation intensity of described superparamagnetism composite particle especially is preferably 40emu/g, and corresponding external magnetic field response speed is 20 seconds;
For surveying for the melamine residual quality testing, superparamagnetism composite particle surface need be with the group be easy to the melamine antibody coupling, these groups can be carboxyl, amino groups, the group of optimizing is the surface functional group with carboxyl, usually adopt chemical method to connect antibody, with after EDC and NHS activation superparamagnetism composite particle, then close and react and complete coupling reaction with antibody generation carboxylic.Before the condensate formed with the peptide bond covalent bond by melamine antibody and superparamagnetism composite particle, also comprise that by superparamagnetism composite particle surface functional group be the activation of carboxyl.The carboxyl-content difference on superparamagnetism composite particle surface can have influence on the sensitivity of detection, for improving sensitivity, described functional group is specially carboxyl, the content of described carboxyl is 50～500 μ mol/g, the content of described carboxyl is specially 50～300 μ mol/g, and the content of described carboxyl especially is preferably 80 μ mol/g;
In immune detection, the performance index of antibody are most important for the accuracy detected, and typically, high specificity, the antibody that affinity is high, can improve the accuracy of detection significantly.Research is found, is raising sensitivity, and described melamine antibody affinity costant is 10
6～10
8m
-1; Described melamine antibody affinity costant is specially 10
7～10
8m
-1; Described melamine antibody affinity costant especially is preferably 10
8m
-1.
Another object of the present invention is to provide a kind of method for preparing the immune chromatography test paper that detects melamine.
Method provided by the invention comprises the steps:
I, prepare sample pad respectively and contain detection line and the reacting pad of nature controlling line;
The reacting pad that contains detection line and nature controlling line and adsorptive pads that II, the sample pad that step I is obtained, step I obtain paste on backboard successively, obtain detecting the immune chromatography test paper of melamine;
The described reacting pad that contains detection line and nature controlling line is prepared as follows: the two ends zones of different that the antiantibody of described melamine antigen and described and melamine antibody specific bond is sprayed on respectively to reacting pad, form detection line and nature controlling line, obtain the reacting pad that contains detection line and nature controlling line;
Described sample pad is prepared as follows: described superparamagnetism composite particle mark melamine antibody is sprayed onto on all-glass paper, obtains sample pad.
In described sample pad preparation method: go forward described superparamagnetism composite particle mark melamine antibody is sprayed onto to described all-glass paper, also comprise the step of the described all-glass paper of pre-service and the described superparamagnetism composite particle of pre-service mark melamine antibody:
The described all-glass paper of described pre-service is that described all-glass paper is soaked 1 hour in damping fluid,
Described damping fluid is prepared as follows: be that the PBS damping fluid that 0.02M, pH are 7.4 is mixed to get damping fluid by every 2mlTritonX100,10g BSA, 50g sucrose and 950ml concentration, adjust PH to 7.4, and constant volume be to 1000ml.
The time of described immersion is 1 hour, and the temperature of immersion is 37 ℃;
The described superparamagnetism composite particle of described pre-service mark melamine antibody is that described extension rate is 50 times by described superparamagnetism composite particle mark melamine antibody dilution.Described dilution is damping fluid used in the pre-service all-glass paper.
Described reacting pad is nitrocellulose filter.
After step I, before Step II, also comprise the step of the nitrocellulose filter that contains detection line and nature controlling line that sample pad that drying steps I obtains and step I obtain.
The application in residual melamine in detecting sample of described test paper is also the scope of protection of the invention, and described sample is specially milk powder or milk.
Know-why of the present invention:
It is relevant to the immuno-chromatographic assay technology of superparamagnetism composite particle mark that melamine of the present invention detects reagent, to adopt the superparamagnetism composite particle material that serves as a mark, carry out class methods of fast immune chromatographic mensuration, this Technology Integration the research of the association areas such as magnetic Nano material chemosynthesis, labelling technique, flow measurement immunochromatography technique.
Why the present invention can detect melamine, a kind of method that has been to adopt flow measurement immunochromatography based on superparamagnetism composite particle mark to detect, being about to melamine antigen and dynamics is sprayed at respectively the p-wire (T line) and the nature controlling line (C line) that are positioned at the test section (being that the NC film forms by nitrocellulose filter) in the middle of reaction plate and locates, the anti-melamine antibody of spraying superparamagnetism composite particle mark on the sample pad of reaction plate lower end, the reaction plate upper end is connected with adsorptive pads, and the formation of whole reaction plate as shown in Figure 1.Principle based on the flow measurement immunochromatography, after adding testing sample, the melamine antigen competition of the melamine in sample and the spraying of T line place is in conjunction with magnetic mark melamine antibody, form Ag-Ab binary magnetic mark immune complex at T line place, unnecessary magnetic mark melamine antibody is the magnetic mark immune complex with anti-mouse IgG formation at C line place.Measure the magnetic strength intensity of T line place superparamagnetism microballoon with magnetic test paper interpretoscope, by the threshold value with setting, compare and determine its positive or negative result, C line measurement result is marked in the Quality Control as this assay method.
Its concrete technical step comprises:
(1) preparation of superparamagnetism composite particle label probe
Adopt applicable nanometer superparamagnetism composite particle, after activating its surperficial carboxyl, adopt the mode of chemical coupling that the melamine antibody orientation is connected to this superparamagnetism composite particle surface.
(2) test section T line and C line place antigen/antibody is coated
Adopt special spray film instrument, in the T of test section line place spraying melamine antigen, in C line place spraying dynamics.
(3) sample pad place label probe is coated
Adopt special spraying instrument, in the anti-melamine antibody of sample pad specific location spraying superparamagnetism microballoon mark.
(4) assembled formation of reaction plate
According to structural drawing (the seeing Fig. 1) requirement of reaction plate, paste cellulose nitrate (NC) film as test section in the middle of the plastic support backboard, paste sample pad in the T of NC film line end, the C line end is pasted adsorptive pads.Paste in the above transparent protective film.Adopt special test paper cutting machine, the monoblock reaction plate is divided to the paper slip that is cut to certain broadband, packed with the special aluminium foil bag that drying agent is housed.
(5) formation of Ag-Ab magnetic mark immune complex
Well place in the reaction plate of above-mentioned assembled formation adds testing sample, the melamine antigen competition of the melamine in sample and the spraying of T line place is in conjunction with magnetic mark melamine antibody, form Ag-Ab binary magnetic mark immune complex at T line place, unnecessary magnetic mark melamine antibody is the magnetic mark immune complex with anti-mouse IgG formation at C line place.
(6) magnetic mark immune complex magnetic field intensity detects
Measure the magnetic field intensity of T line place superparamagnetism microballoon with magnetic test paper interpretoscope, by the threshold value with setting, compare and determine its positive or negative result, C line measurement result is marked in the Quality Control as this assay method.
The superparamagnetism composite particle that the present invention adopts is purchased from Shenzhen TELUS Science and Technology Ltd., catalog number is MP-2 (Fig. 2 is shown in by the water-solubility nanocrystalline TEM photo of poly hexadecanol ester (PMAH) modification), and the preparation method who adopts is the oil-soluble Fe will made with chemical method
3o
4be dissolved in organic reagent and obtain solution A, by amphiphilic oligomer, be dissolved in 3 distilled water and regulate pH be 8～10 solution B, under normal temperature, solution B is injected to solution A, mixed liquor fully stirs and makes the organic solvent volatilization, carry out centrifuging, will get final product to obtain water miscible superparamagnetism composite particle after the product drying of centrifuging.High with the standby saturated magnetic intensity of magnetic particle obtained of this legal system, magnetic response fast, the magnetic bead size uniform, monodispersity is good, stability is strong, it is fast to spring up the time, can meet well the testing requirement of LFIAs.
Described Ag-Ab magnetic mark immune complex, after referring to and adding and detect sample, through the competition combination, the melamine antigen formed at T line place-magnetic mark melamine antibody immune complex, and anti-mouse IgG bis-antiantibodys that form at C line place-magnetic mark melamine antibody immune complex.
The magnetic field intensity of described magnetic mark immune complex, refer to the quantity in conjunction with magnetic bead under T line and C line place are detained respectively with the superparamagnetic resonance detector MAR of U.S. Quantum Dot, measure after resulting numerical value.By optimizing the condition of competitive reaction, research is found, normal sample through large quantitative determination separate sources as urine sample, blood and tissue sample extract etc., can determine the mensuration average of the normal sample in variant source, using that this determines that as critical value (cutoff) the T line detects the positive or negative result of sample.C line measurement result is marked in the Quality Control as this assay method.
The chemical name of melamine is 2,4,6-triamido-1,3,5-triazines.
Of the present invention experimental results show that, by the research to superparamagnetism composite particle, melamine antigen and melamine antibody molecular characterization, by the optimization to the preparation of multiple superparamagnetism composite particle, coating and finishing condition, select applicable superparamagnetism composite particle and specific antibody to carry out directed covalent chemical coupling, obtain functional magnetic mark probe, and by optimizing the various conditions of competitive immunization reaction, reach the objective detection to the melamine residual medicine, realized the quick and Sensitive Determination to the melamine residual medicine.There is following advantage: highly sensitive, high specificity, quick, easy, can realize the mensuration that objectifies.
The accompanying drawing explanation
Fig. 1 is magnetic test paper structure schematic diagram
Fig. 2 is water-soluble superparamagnetism composite particle Electronic Speculum figure
Fig. 3 is melamine magnetic detection paper value and concentration standard curve figure
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The preparation of embodiment 1, melamine residual thing magnetic mark quick detection test paper
(1) preparation of superparamagnetism composite particle mark melamine antibody
The employing particle diameter is that 100nm, magnetic saturation intensity are 40emu/g, and corresponding external magnetic field response speed is superparamagnetism composite particle (the superparamagnetism Fe that 20 seconds, surperficial carboxyl-content are 80 μ mol/g
3o
4nano particle) (purchased from Shenzhen TELUS Science and Technology Ltd., catalog number is MP-2) mark melamine antibody.
Concrete grammar is:
1) get the MES damping fluid of 0.1M for the above-mentioned magnetic particle of 2.5mg (take 1.066g MES, 0.45g NaCl is dissolved in the 50ml pure water, adjust pH to 4.7) wash and use the magnet stand separation and concentration of 0.4T after, be that 0.1M, pH value are 4.7 the MES damping fluid is resuspended by 1ml concentration, add the 1-ethyl of 0.96mg (final concentration is 5mM)-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.15mg (final concentration is 10mM) N-maloyl imines (NHS) in wherein.Temperature of reaction is 37 ℃, after reacting half an hour, obtains activating rear magnetic particle;
2), with the borate buffer solution washing of 50mM pH=8.5, (melamine antibody tires 10 for Zhengzhou Kinsey that bio tech ltd, C010110 to get 0.15mg melamine monoclonal antibody
6, the melamine antibody affinity costant is 10
8m
-1the borate buffer solution that is mixed into 0.8ml 50mMpH=8.5 with magnetic particle after the 2.5mg activation (takes 1.9gNa
2b
4o
7.10H
2o is dissolved in the 100ml pure water, adjusts pH to 8.5) in fully mix.The lower reaction of room temperature (25 ℃) 3.5 hours, allow antibody and magnetic particle form stable peptide bond covalent bond, obtains containing the reactant liquor of magnetic particle after coupling;
3) after reaction finishes, to step 2) add the BSA (Sigma-aldrich that final concentration is 5% (quality percentage composition) in the reactant liquor that obtains, 85041C) the residual activity amino sites is sealed, reaction is carried out 0.5 hour under 37 ℃, obtains the reactant liquor that contains the rear magnetic particle of sealing; After completing, with the 0.02M PBS damping fluid of pH=7.4, (take 2.3g Na
2hPO
4, 0.524g NaH
2pO
4.H
2o, 8.77g NaCL are dissolved in the 1L pure water, adjust pH to 7.4) washing, resuspended rear 4 ℃ of preservations are stand-by, obtain superparamagnetism composite particle mark melamine antibody.
(2) melamine antigen is synthetic
Melamine hapten is that (a day Chemical Engineering Technology research institute is opened to melamine by the permanent unit in Beijing, 29921CDCT-C14861400).
The synthetic employing of melamine-BSA first is converted into intermediate by melamine molecule, then carries out coupling with BSA.
Concrete steps are:
1) get 63mg (0.5mmol) melamine in vial, add 50mg succinic anhydride and 5ml pyridine to make it to dissolve fully, at the lower stirring reaction 6h of room temperature (25 ℃), remove pyridine by Rotary Evaporators, obtain the melamine intermediate after drying up remaining pyridine with nitrogen; Get the melamine intermediate 82mg (about 0.2mmol) prepared and be dissolved in 5ml DMF and make it abundant dissolving, obtain the melamine midbody solution;
2) by 5ml step 1) the melamine midbody solution that obtains adds 20.6mg DCC (N, the N-dicyclohexyl carbodiimide) (about 0.1mmol) is at the lower activation of room temperature (25 ℃) 15min, add NHS (N-maloyl imines) 28.6mg (about 0.2mmol) at room temperature to react 1h, proceed to afterwards under 4 ℃ and react 1.5h, the melamine midbody solution after being activated;
3) get 100mg BSA and be dissolved in 10ml 0.1mol/L, in the dobell's solution that pH is 8.5, obtain the BSA BAS; Under 4 ℃, preserve stand-by;
4) melamine-DMF of 5ml activation is slowly dropwise added to 4 ℃ of reaction 3.5h in 10ml BSA solution by separating funnel under ice bath;
5) product is in 0.02mol/L, and in pH=7.5PBS, dialysis is 24 hours, and every 6h changes time dislysate.Products obtained therefrom freeze dryer freeze-drying, in-20 ℃ of preservations, obtain melamine antigen.
(3) preparation of melamine magnetic mark quick detection test paper
Adopt 0.02M PBS (pH=7.4) damping fluid, by sheep anti-mouse igg antibody (Changsha rich eugenic thing Science and Technology Ltd., ABGAM-0500) and the concentration of above-mentioned (two) melamine antigen of obtaining all be formulated as concentration 1mg/ml, select the BioJet shower nozzle in the XYZ3050 spray film system of BioDot sheep anti-mouse igg antibody to be sprayed onto to control line (the Control Line of nitrocellulose filter (NC film), the C line) position, the melamine antigen that above-mentioned (two) are obtained is sprayed onto detection line (Test Line, the T line) position, in relative humidity, be that dehumidifier dried for standby after 4 hours is carried out in drying plant below 10%.Soak all-glass paper 1 hour with 0.02M PBS (pH=7.4) solution containing 2%TritonX100,1%BSA, 1% sucrose, the temperature of soaking is 37 ℃, carry out dehumidifier after 4 hours in same dehumidifier condition, after processing the melamine antibody of damping fluid by 50 times of dilution paramagnetism composite particle marks with above-mentioned film, adopt AirJet shower nozzle in the XYZ3050 spray film system of BioDot this dilution magnetic labeling antibody to be sprayed into to preparation on the glass fibre element film of above-mentioned processing and form sample pad, in same dehumidifier condition, carry out drying.In 100,000 grades of cleanings and dry workshop above-mentioned dried NC film, magnetic pad, thieving paper, backboard and diaphragm by the assembling of arranging in pairs or groups shown in Fig. 1 after; the width that the CM4000 cutting system that adopts BioDot is the 5mm/ bar by the Paperboard cutting that posts; packing into, it is stand-by to detect with intermediate plate, obtains detecting the immune chromatography test paper of melamine.
The result schematic diagram of this test paper as shown in Figure 1.
The detection of immune chromatography test paper sensitivity, cross reaction and the accuracy of embodiment 2, melamine residual thing.
1, the mensuration of sensitivity
(MEL, a day Chemical Engineering Technology research institute is opened by the permanent unit in Beijing, 29921CDCT-C14861400), dissolves the standard inventory solution that is formulated as 100mg/ml with methyl alcohol to take appropriate melamine.With the PBS (pH=7.4) of 0.02M dilution be formulated as 0,1,2,5,10,25,50,100, the standard solution of 200ug/L; add respectively in the immune chromatography test paper of the detection melamine obtained by embodiment 1; and employing superparamagnetic resonance detector MAR (Magna BioSciences, 8094-101-01& 8094-101-02) read.Detecting step: first detected sample is recovered to room temperature (25 ℃) before detection, get with accurate pipettor the application of sample end that detected sample 100 μ l vertically slowly splash into the magnetic test strips, then splash into the 50ul washing fluid (0.02M, pH 7.4, PBS), after 20 minutes, with MAR, tested.
Its testing result is as shown in table 1 below.Can find from the testing result data that detected value when when melamine concentration is 1ug/L, detected value is with 0ug/L has intersects, and when concentration is 2ug/L, detected value and 0ug/L value can distinguish fully, and concentration curve R
2=0.9884, better linear, illustrate that the detection sensitivity of test paper can reach 2ug/L.
Melamine magnetic detection paper value and concentration curve are as shown in Figure 3.
The magnetic detection paper value of table 1 melamine variable concentrations sample
2, the mensuration of cross reaction
(a day Chemical Engineering Technology research institute is opened by the permanent unit in Beijing to select cyanuric acid, 29922 CDCT-C11815000), triazine (Sigma-aldrich, T46051-1G), triazinediamine (Sigma-aldrich, S367753-1EA), be made into respectively series concentration, detected by the magnetic test strips.Calculate the IC50 that respectively competes thing, calculate respectively the cross reacting rate of these 5 kinds of medicines and MEL magnetic test paper with following formula.Computing formula is: cross reacting rate (%)=[IC50 (MEL)/IC50 (medicine to be measured)] * 100.
Mensuration and result of calculation are as shown in table 2.Result shows that melamine magnetic test paper has certain intersection to cyanuric acid, is less than 1% to all the other two kinds of crossing-over rates.
The cross reaction of table 2 melamine magnetic test paper and other medicines
The mensuration of 3 accuracys and the recovery
3.1 sample extraction:
Measure 10ml milk, add 20mL 50% acetonitrile solution, supersonic oscillations extraction 10min.Add 1ml1M hydrochloric acid, the vibration 1min, the centrifugal 10min of 4000g, get supernatant, with after 0.45 μ m membrane filtration for detection of.
3.2 the assay method of accuracy:
60 parts of milk samples to be measured are provided by Animal &. Plant Inspection and Quarantine Techn Center, Shenzhen Bureau of Impor, and known wherein 55 parts of (being numbered 1-55) negative samples, 5 parts of (being numbered 56-60) positive samples.Use immune chromatography test paper and the U.S. Beacon company Melamine Plate KIT kit of the detection melamine obtained by embodiment 1 to detect 60 duplicate samples simultaneously;
Measure the magnetic field intensity of T line place superparamagnetism microballoon with magnetic test paper interpretoscope, by the threshold value with setting, compare and determine its positive or negative result, C line measurement result is marked in the Quality Control as this assay method.
Threshold value be less than 565 positive, threshold value be greater than 565 negative.
3.3 the mensuration of accuracy
The testing result of the immune chromatography test paper of the detection melamine obtained by embodiment 1 is that to be numbered the sample of 1-55 negative, its threshold value is respectively 611.4, 581.2, 582.5, 587.8, 595.9, 612.3, 590.5, 603.4, 614.6, 605.5, 622.3, 596.5, 587.6, 615.8, 597.7, 594.9, 615.2, 626.3, 611.5, 596.7, 599.8, 628.3, 622.5, 603.6, 609.5, 617.1, 596.7, 608.5, 625.4, 618.8, 629.2, 594.4, 609.7, 592.9, 613.4, 616.8, 621.5, 605.6, 595.7, 583.3, 610.5, 589.6, 622.5, 604.4, 626.9, 602.9, 588.6, 596.5, 587.4, 616.3, 595.8, 608.4, 614.6, 586.1, 607.6.
The sample that is numbered 56-60 is positive, and its threshold value is respectively 485.7,411.6,264.8,198.4,162.7.
The testing result of the U.S. ELISA of Beacon company kit is consistent with above-mentioned detection paper result.
Illustrate that detection paper of the present invention is correct.
3.4 the assay method of the recovery
After 10 duplicate samples of the numbering 1-10 of above-mentioned detection feminine gender are mixed, the melamine titer (5,10,25,50,100ug/L) of variable concentrations is added in preparation.Add each test of sample 5 times, and calculate recovery rate.
3.5 the mensuration of the recovery
Determination of recovery rates the results are shown in Table 3, and the recovery that melamine adds in milk is 88%～114%, average recovery rate 99.12%, and the coefficient of variation 7.54%～11.31%, average coefficient of variation 9.25%, accuracy is better.
Table 3 determination of recovery rates
Claims (9)
1. an immune chromatography test paper that detects melamine, comprise sample pad, the reacting pad that is connected in described sample pad one end that contains superparamagnetism composite particle mark melamine antibody, the adsorptive pads that is connected in the described reacting pad other end; Described reacting pad is coated with detection line and the nature controlling line be separated from each other, and described detection line contains melamine antigen, described nature controlling line contain can with the antiantibody of described melamine antibody specific bond;
The condensate that the peptide bond covalent bond that described superparamagnetism composite particle mark melamine antibody is melamine antibody and superparamagnetism composite particle forms; Described melamine antibody is melamine monoclonal antibody or melamine polyclonal antibody; The conjugate that described melamine antigen is melamine hapten and carrier protein; The coupling ratio of described carrier protein and melamine hapten is 1:8～1:10;
Described melamine hapten is melamine;
Described carrier protein is BSA;
Described superparamagnetism composite particle is Fe
3o
4nano particle;
The particle diameter of described superparamagnetism composite particle is 100nm;
The magnetic saturation intensity of described superparamagnetism composite particle is 40emu/g, and corresponding external magnetic field response speed is 20 seconds; The carboxyl-content on described superparamagnetism composite particle surface is 80 μ mol/g;
Described melamine antibody affinity costant is 10
8m
-1.
2. test paper according to claim 1 is characterized in that:
Described reacting pad is nitrocellulose filter;
Described melamine antibody is the melamine monoclonal antibody;
The coupling ratio of described carrier protein and melamine hapten is 1:10;
The antiantibody of described and described melamine antibody specific bond is sheep anti-mouse igg antibody;
The concentration of described melamine antigen and described sheep anti-mouse igg antibody is 1mg/ml.
3. test paper according to claim 2 is characterized in that:
Described superparamagnetism composite particle mark melamine antibody is prepared as follows:
1) by every 2.5mg superparamagnetism composite particle, 0.96mg1-ethyl-(3-dimethylaminopropyl) carbodiimide, 1.15mg N-maloyl imines and 1ml concentration, be that 2-(N-morpholine) the ethyl sulfonic acid damping fluid that 0.1M, pH value are 4.7 mixes, reaction, obtain activating rear magnetic particle;
2) every 2.5mg step 1) being obtained activating rear magnetic particle, 0.15mg melamine antibody and 0.8ml concentration is that the borate buffer solution that 50mM pH is 8.5 mixes, reacts, and obtains containing the reactant liquor of magnetic particle after coupling;
3) to step 2) add BSA to mix in the reactant liquor that obtains to obtain mixed liquor, reaction, obtain containing the reactant liquor of magnetic particle after sealing;
The mass percentage concentration of described BSA in described mixed liquor is 5%;
Described melamine antigen is prepared as follows:
A) first get every 63mg melamine, 50mg succinic anhydride and 5ml pyridine and mix, reaction, obtain reactant liquor a, then remove pyridine in described reactant liquor a, obtains the melamine intermediate; Add again the 5ml DMF to dissolve the described melamine intermediate of 82mg, obtain the melamine midbody solution;
B) by every 5ml steps A) the melamine midbody solution and the 20.6mg N that obtain, N-bis-cyclohexyl carbodiimides are mixed, obtain reactant liquor b, then add 28.6mgN-maloyl imines to mix, react in described reactant liquor b, the melamine midbody solution after being activated;
C) get every 100mg carrier protein and be dissolved in 10ml concentration 0.1mol/L, in the sodium borate aqueous solution that pH is 8.5, obtain the carrier protein BAS;
D) by every 5ml step B) melamine midbody solution and 10ml step C after the activation that obtains) the carrier protein BAS that obtains mixes, reacts, and obtains reactant liquor c, by reactant liquor c dialysis, freeze-drying, obtains melamine antigen.
4. test paper according to claim 3 is characterized in that:
In the preparation method of described superparamagnetism composite particle mark melamine antibody:
In step 1), temperature of reaction is 37 ℃, and the reaction time is 0.5h;
Step 2) in, temperature of reaction is 25 ℃, and the reaction time is 3.5h;
In step 3), temperature of reaction is 37 ℃, and the reaction time is 0.5h;
In the preparation method of described melamine antigen:
Steps A) in, described temperature of reaction is 25 ℃, and the described reaction time is 6h;
Step B) in, described mixing temperature is 25 ℃, and described incorporation time is 15min, and described reaction is first at 25 ℃, to react 1h, then reacts 1.5h under 4 ℃;
Step D) in, described temperature of reaction is 4 ℃, and the described reaction time is 3.5h.
5. test paper according to claim 4 is characterized in that:
In the preparation method of described superparamagnetism composite particle mark melamine antibody:
After described step 3), also comprise magnetic particle after the sealing in the described reactant liquor that contains the rear magnetic particle of sealing is washed, suspends, obtain the step of superparamagnetism composite particle mark melamine antibody, it is the PBS damping fluid that 0.02M pH is 7.4 that described cleansing solution and suspending liquid are concentration.
6. a method for preparing the immune chromatography test paper that detects melamine, comprise the steps:
I, prepare sample pad respectively and contain detection line and the reacting pad of nature controlling line;
The reacting pad that contains detection line and nature controlling line and adsorptive pads that II, the sample pad that the step I is obtained, step I obtain paste on backboard successively, obtain detecting the immune chromatography test paper of melamine;
The described reacting pad that contains detection line and nature controlling line is prepared as follows: the two ends zones of different that the antiantibody of the described and melamine antibody specific bond in arbitrary described test paper in the described melamine antigen in arbitrary described test paper in claim 1-5 and claim 1-5 is sprayed on respectively to reacting pad, form detection line and nature controlling line, obtain the reacting pad that contains detection line and nature controlling line;
Described sample pad is prepared as follows: the described superparamagnetism composite particle mark melamine antibody in arbitrary described test paper in claim 1-5 is sprayed onto on all-glass paper, obtains sample pad.
7. method according to claim 6 is characterized in that:
In described sample pad preparation method: go forward described superparamagnetism composite particle mark melamine antibody is sprayed onto to described all-glass paper, also comprise the step of the described all-glass paper of pre-service and the described superparamagnetism composite particle of pre-service mark melamine antibody:
The described all-glass paper of described pre-service is that described all-glass paper is soaked 1 hour in damping fluid,
Described damping fluid is prepared as follows: be that the PBS damping fluid that 0.02M, pH are 7.4 is mixed to get damping fluid by every 2mlTritonX100,10g BSA, 50g sucrose and 950ml concentration, adjust pH to 7.4, and constant volume be to 1000ml;
The time of described immersion is 1 hour; The temperature of soaking is 37 ℃;
The described superparamagnetism composite particle of described pre-service mark melamine antibody is that described extension rate is 50 times by described superparamagnetism composite particle mark melamine antibody dilution;
Described reacting pad is nitrocellulose filter.
8. according to the described method of claim 6 or 7, it is characterized in that:
After the step I, before the step II, also comprise the step of the nitrocellulose filter that contains detection line and nature controlling line that sample pad that the drying steps I obtains and step I obtain.
9. arbitrary described test paper application in residual melamine in detecting sample in claim 1-5, described sample is milk powder or milk.
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| CN104569326A (en) * | 2013-10-23 | 2015-04-29 | 上海捷门保林迈生物工程有限公司 | Method for detecting beta-lactamase in milk and test paper |
| CN105866410A (en) * | 2016-03-25 | 2016-08-17 | 上海交通大学 | Immunochromatographic detection test strip for joint detection of NSE and CEA, and preparation method and application method thereof |
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