CN102253224B - Immunochromatographic test paper for detecting diethylstilbestrol and preparation method thereof - Google Patents
Immunochromatographic test paper for detecting diethylstilbestrol and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an immunochromatographic test paper for detecting diethylstilbestrol and a preparation method thereof. The immunochromatographic test paper for detecting diethylstilbestrol provided in the invention comprises a sample pad containing superparamagnetic composite particle marked antibodies to diethylstilbestrol, a cellulose nitrate membrane connected with one end of the pad and a water absorbing pad connected with another 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 diethylstilbestrol antigen, and the quality control line contains antiantibodies capable of specific binding to the antibodies to diethylstilbestrol. Experiments in the invention indicate that the test paper provided in the invention has the characteristics of high sensitivity, strong specificity, rapidness, simpleness and capacity of realizing objective detection in detecting diethylstilbestrol.
Description
Technical field
The present invention relates to the detection reagent of diethylstilbestrol residue, be specifically related to a kind of immune chromatography test paper that detects diethylstilbestrol and preparation method thereof.
Background technology
Diethylstilbestrol (Diethylstilbestrol, DES) be a kind of artificial synthetic nonsteroidal estrogen, because it can promote albumen synthetic, accelerate weightening finish and bone calcification, and the minimizing food consumption, be used as the promoting animal growth agent and be applied to the aspects such as Production of Livestock and Poultry and aquaculture.But it is a kind of carcinogen that many scientific experimentations have confirmed diethylstilbestrol, larger to the health hazard of humans and animals, and therefore, many countries have been defined in livestock and poultry and the aquaculture and have forbidden diethylstilbestrol.China is aquatic products production, consumption and big export country, and diethylstilbestrol is that the medicine in recent years aquatic products mainly monitored is one of residual.At present field screening detects with detecting the multiplex diethylstilbestrol colloid 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 that grows 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, is subjected to the restriction of its sensitivity etc., can only be used for qualitative detection.
The superparamagnetism composite particle has good magnetism characteristic, because it is subjected 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 are used for the detection of flow measurement immunochromatography, observe about 10% signaling molecule intensity on its detection zone film surface only, and with the superparamagnetism composite particle material that serves as a mark, then can detect all the magnetic signal molecules in the detection zone film 3-D solid structure, can greatly improve sensitivity, and the magnetic signal detector of available correspondence reaches quantitative measurement, and therefore, the superparamagnetism composite particle is the material that receives publicity in LFIAs in recent years.
Yet, after the biological detection of report adopts chemical method such as coprecipitation to prepare first the magnetic nano-particle of organic phase with magnetic nano-composite particle more at present, adopt again silicon (SiO
2) or the macromolecular material such as polystyrene, polyacrylic acid, gelatin the stabilization coating decoration is carried out on its surface, to obtain stable, water miscible magnetic label material.But these preparation method of modifying are very complicated comparatively often, the superparamagnetism composite particle that obtains can not satisfy the requirement of LFIAs simultaneously at aspects such as size, biocompatibility, saturated magnetic intensity, external magnetic field response speed, stability and labeling effciencies: 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 diethylstilbestrol.
The immune chromatography test paper of detection diethylstilbestrol provided by the invention comprises sample pad, the reacting pad that is connected in described sample pad one end that contains superparamagnetism composite particle mark diethylstilbestrol 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 that is separated from each other, and described detection line contains diethylstilbestrol antigen, described nature controlling line contain can with the antiantibody of described diethylstilbestrol antibody specific bond.
Described reacting pad is nitrocellulose filter;
The condensate that the peptide bond covalent bond that described superparamagnetism composite particle mark diethylstilbestrol antibody is diethylstilbestrol antibody and superparamagnetism composite particle forms;
Described diethylstilbestrol antibody is diethylstilbestrol monoclonal antibody or diethylstilbestrol polyclonal antibody, described diethylstilbestrol antibody be specially the diethylstilbestrol monoclonal antibody (Yunnan University monoclonal antibody engineering center, DES-002);
Described diethylstilbestrol antigen is the conjugate of diethylstilbestrol haptens and carrier protein; When little molecular antigen material and carrier protein couplet, the haptenic average number that connects 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 again the extension in site, diethylstilbestrol haptens space 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.General mol ratio by adjusting haptens and carrier, reaction environment pH value, temperature, ionic strength etc. are controlled coupling ratio.
Described carrier protein and the haptenic coupling ratio of described diethylstilbestrol are 1: 7～1: 9, are suitable for the extension in the coated and site, diethylstilbestrol haptens space of antigen, and described carrier protein and the haptenic coupling ratio of described diethylstilbestrol are specially 1: 9;
The antiantibody of described and described diethylstilbestrol antibody specific bond is sheep anti-mouse igg antibody;
The concentration of described diethylstilbestrol antigen and described sheep anti-mouse igg antibody is 1mg/ml.
Described superparamagnetism composite particle mark diethylstilbestrol antibody is prepared as follows:
1) with 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 be 4.7 2-(N-morpholine) ethyl sulfonic acid (MES) damping fluid (take by weighing 1.066g MES, 0.45g NaCl is dissolved in the 50ml pure water, transfer pH to 4.7) mixing, reaction obtains activating rear magnetic particle;
2) with every 2.5mg step 1) to obtain activating rear magnetic particle, 0.15mg diethylstilbestrol antibody and 0.8ml concentration be that 50mM pH is 8.5 borate buffer solution mixing, reaction, obtains containing the reactant liquor of magnetic particle after the coupling;
3) to step 2) add the BSA mixing in the reactant liquor that obtains and obtain mixed liquor, reaction, obtain containing the reactant liquor of magnetic particle after the sealing;
The concentration of described BSA in described mixed liquor is 5% (quality percentage composition),
Described diethylstilbestrol antigen is prepared as follows:
A) get first every 26.8mg diethylstilbestrol (DES), 20mg succinic anhydride and 5ml pyridine mixing, reaction obtains reactant liquor a, removes pyridine among the described reactant liquor a again, obtains the DES intermediate; Add again the described DES intermediate of 5mlDMF (DMF) dissolving 82mg, obtain the DES midbody solution;
B) with every 5ml steps A) the DES midbody solution that obtains and the positive amine of 26.2 μ l tributyls is mixed, obtains reactant liquor b, adds 1.5 μ l isobutyl chlorocarbonate mixings, reaction, the DES midbody solution after obtaining activating again in described reactant liquor b;
C) get every 100mg carrier protein and be dissolved in 10ml concentration 0.1mol/L, pH obtains the carrier protein BAS in 8.5 the sodium borate aqueous solution;
D) with every 5ml step B) DES midbody solution and 10ml step C obtain after the activation that obtains carrier protein BAS mixing, reaction, obtain reactant liquor c.
Among the preparation method of described superparamagnetism composite particle mark diethylstilbestrol 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.
Among the preparation method of described diethylstilbestrol antigen:
Steps A) in, described temperature of reaction is 45 ℃, and the described reaction time is 15h;
Step B) in, described mixing temperature is 4 ℃, and described incorporation time is 1h; Described temperature of reaction is 25 ℃, and the described reaction time is 1h;
Step D) in, described temperature of reaction is 4 ℃, and the described reaction time is 12h.
Among the preparation method of described superparamagnetism composite particle mark diethylstilbestrol antibody:
In described step 3) after, also comprise magnetic particle after the described sealing that contains in the reactant liquor that seals rear magnetic particle is washed, suspends, obtain the step of superparamagnetism composite particle mark diethylstilbestrol antibody, it is 7.4 PBS damping fluid that described cleansing solution and suspending liquid are 0.02M pH.
Among the preparation method of described diethylstilbestrol antigen,
Among the preparation method of described diethylstilbestrol antigen,
At described step D) after also comprise step D) the reactant liquor c that obtains is through dialysis, purifying, freeze-drying, obtains the step of diethylstilbestrol antigen.
The dislysate that described dialysis is adopted is that concentration is that 0.02mol/L, pH are 7.4 PBS damping fluid, and dialysis time is 48h, and every 6h changes a dislysate.
The purification column that described purifying adopts is Sephadex G-25.
Described diethylstilbestrol haptens is diethylstilbestrol;
Described carrier protein is BSA;
Because diethylstilbestrol 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 could reaching by means of the character of surface of carrier protein after the coupling good combination with the NC film.Can be used as the seralbumin that various animals are arranged of carrier protein, such as bovine serum albumin(BSA) (Bovine Serum 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, in the situation that contains organic solvent (such as pyridine, DMF etc.), all can carry out coupling with haptens, and after coupling, still keep solvable state, the splendid selection as carrier protein, so the present invention selects BSA as coupling protein.
Described superparamagnetism composite particle is Fe
3O
4Nano particle;
Because this superparamagnetism composite particle will be realized by the sample pad swimming in the above-mentioned reaction plate specific bond and the labeled reactant of Ag-Ab to the test section in the middle of the reaction plate, its particle diameter too large (＞300nm) the swimming time on test paper long, developing time is slow; When coupling, be easier to assemble, and produce non-specific responding easily; The too little then 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%, preferred 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 the superparamagnetism composite particle of classic method preparation usually all＜30emu/g, the external magnetic field response speed is then 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;
Detect for being used for the diethylstilbestrol residue, superparamagnetism composite particle surface needs with the group that is easy to the diethylstilbestrol 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, namely with behind EDC and the NHS activation superparamagnetism composite particle, close reaction and finish coupling reaction with antibody generation carboxylic again.Before the condensate that forms with the peptide bond covalent bond with diethylstilbestrol antibody and superparamagnetism composite particle, comprise that also with 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 that detects, and usually, high specificity, the antibody that affinity is high can improve the accuracy of detection significantly.Research finds that for improving sensitivity, described diethylstilbestrol antibody affinity costant is 10
6～10
8M
-1Described diethylstilbestrol antibody affinity costant is specially 10
7～10
8M
-1Described diethylstilbestrol antibody affinity costant especially is preferably 10
8M
-1
Another object of the present invention provides a kind of method for preparing the immune chromatography test paper that detects diethylstilbestrol.
Method provided by the invention comprises the steps:
I, prepare sample pad and contain detection line and the reacting pad of nature controlling line respectively;
The reacting pad that contains detection line and nature controlling line and adsorptive pads that II, sample pad, step I that step I is obtained obtain paste on the backboard successively, obtain detecting the immune chromatography test paper of diethylstilbestrol;
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 diethylstilbestrol antigen and described and diethylstilbestrol antibody specific bond is sprayed on respectively reacting pad, form detection line and nature controlling line, obtain containing the reacting pad of detection line and nature controlling line;
Described sample pad is prepared as follows: described superparamagnetism composite particle mark diethylstilbestrol antibody is sprayed onto on the all-glass paper, obtains sample pad.
In described sample pad preparation method: go forward in that described superparamagnetism composite particle mark diethylstilbestrol antibody is sprayed onto 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 diethylstilbestrol antibody:
The described all-glass paper of described pre-service is that described all-glass paper was soaked in damping fluid 1 hour;
Described damping fluid is prepared as follows: be that 0.02M, pH are that 7.4 PBS damping fluid is mixed to get damping fluid with every 2mlTritonX100,10g BSA, 50g sucrose and 950ml concentration, transfer 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 diethylstilbestrol antibody is that described extension rate is 50 times with described superparamagnetism composite particle mark diethylstilbestrol antibody dilution.Described dilution is damping fluid used in the pre-service all-glass paper.
Described reacting pad is nitrocellulose filter.
Behind 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 drying steps I obtains and step I obtain.
The application in the residual diethylstilbestrol in test sample of described test paper also is the scope of protection of the invention, and described sample is specially animal tissue's sample, animal urine, feed, honey or milk sample, and described sample especially is specially pig urine.
Know-why of the present invention:
Diethylstilbestrol detection reagent of the present invention is relevant with the immuno-chromatographic assay technology of superparamagnetism composite particle mark, to adopt the superparamagnetism composite particle material that serves as a mark, carry out the class methods that fast immune chromatographic is measured, 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 diethylstilbestrol, be to adopt a kind of method that detects based on the flow measurement immunochromatography of superparamagnetism composite particle mark, being about to p-wire (T line) and nature controlling line (C line) that diethylstilbestrol antigen and dynamics be sprayed at respectively the test section that is positioned in the middle of the reaction plate (be that NC film consist of by nitrocellulose filter) locates, the anti-diethylstilbestrol antibody of spraying superparamagnetism composite particle mark on the sample pad of reaction plate lower end, the reaction plate upper end then 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 diethylstilbestrol antigenic competition of the diethylstilbestrol in the sample and T line place spraying is combined magnetic mark diethylstilbestrol antibody, form Ag-Ab binary magnetic mark immune complex at T line place, unnecessary magnetic mark diethylstilbestrol antibody is the magnetic mark immune complex with anti-mouse IgG formation at C line place then.Measure the magnetic strength intensity of T line place superparamagnetism microballoon with magnetic test paper interpretoscope, by with the threshold ratio of setting to determining its positive or negative result, C line measurement result is then marked in the Quality Control as this assay method.
Its concrete technical step comprises:
(1) preparation of superparamagnetism composite particle label probe
The nanometer superparamagnetism composite particle that adopt to be fit to, activate its surperficial carboxyl after, adopt the mode of chemical coupling that the diethylstilbestrol 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 diethylstilbestrol antigen, in C line place spraying dynamics.
(3) sample pad place label probe is coated
Adopt special spraying instrument, in the anti-diethylstilbestrol 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, in the middle of the plastic support backboard, paste cellulose nitrate (NC) film as the test section, 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, divide the paper slip that is cut to certain broadband with the monoblock reaction plate, pack 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 diethylstilbestrol antigenic competition of the diethylstilbestrol in the sample and T line place spraying is combined magnetic mark diethylstilbestrol antibody, form Ag-Ab binary magnetic mark immune complex at T line place, unnecessary magnetic mark diethylstilbestrol antibody is the magnetic mark immune complex with anti-mouse IgG formation at C line place then.
(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 with the threshold ratio of setting to determining its positive or negative result, C line measurement result is then marked in the Quality Control as this assay method.
The superparamagnetism composite particle that the present invention adopts is available from Shenzhen's TELUS Science and Technology Ltd., catalog number is MP-2 (the water-solubility nanocrystalline TEM photo of poly hexadecanol ester (PMAH) modification is seen Fig. 2), and the preparation method who adopts is the oil-soluble Fe that will make with chemical method
3O
4Be dissolved in and obtain solution A in the organic reagent, be dissolved in amphiphilic oligomer in 3 distilled water and regulate pH be 8～10 solution B, under the normal temperature solution B is injected solution A, mixed liquor fully stirs and makes the organic solvent volatilization, carry out centrifuging, get final product to get water miscible superparamagnetism composite particle after the product drying with centrifuging.High with the saturated magnetic intensity of the standby magnetic particle that obtains of this legal system, magnetic response is fast, the magnetic bead size uniform, monodispersity is good, stability is strong, it is fast to spring up the time, can satisfy well the testing requirement of LFIAs.
Described Ag-Ab magnetic mark immune complex, after referring to add test sample, through the competition combination, the diethylstilbestrol antigen that forms at T line place-magnetic mark diethylstilbestrol antibody immune complex, and anti-mouse IgG two antiantibodys that form at C line place-magnetic mark diethylstilbestrol antibody immune complex.
The magnetic field intensity of described magnetic mark immune complex refers to resulting numerical value after the measuring with the superparamagnetic resonance detector MAR of U.S. Quantum Dot in conjunction with the quantity of magnetic bead under T line and C line place are detained respectively.By optimizing the condition of competitive reaction, research is found, normal sample through large quantitative determination separate sources such as urine sample, blood and tissue sample extract etc., can determine the mensuration average of the normal sample in variant source, determine that as critical value (cutoff) the T line detects the positive or negative result of sample with this.C line measurement result is then marked in the Quality Control as this assay method.
The chemical name of diethylstilbestrol be (E)-4,4 '-(1,2-diethyl-1,2-ethenylidene) biphenol.
Of the present invention experimental results show that, by the research to superparamagnetism composite particle, diethylstilbestrol antigen and diethylstilbestrol antibody molecular characterization, by the optimization to the preparation of multiple superparamagnetism composite particle, coating and finishing condition, superparamagnetism composite particle and the specific antibody selecting to be fit 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 diethylstilbestrol left drug, realized the quick and Sensitive Determination to the diethylstilbestrol left drug.Have following advantage: highly sensitive, high specificity, quick, easy, can realize the mensuration that objectifies.
Description of drawings
Fig. 1 is magnetic test paper structure synoptic diagram
Fig. 2 is water-soluble superparamagnetism composite particle Electronic Speculum figure
Fig. 3 is diethylstilbestrol magnetic detection paper value and concentration standard curve figure
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The preparation of embodiment 1, diethylstilbestrol residue magnetic mark quick detection test paper
(1) preparation of superparamagnetism composite particle mark diethylstilbestrol antibody
The employing particle diameter is that 100nm, magnetic saturation intensity are 40emu/g, and corresponding external magnetic field response speed is that 20 seconds, surperficial carboxyl-content are superparamagnetism composite particle (the superparamagnetism Fe of 80 μ mol/g
3O
4Nano particle) (available from Shenzhen's TELUS Science and Technology Ltd., catalog number is MP-2) mark diethylstilbestrol antibody.
Concrete grammar is:
1) get the above-mentioned magnetic particle of 2.5mg with the MES damping fluid of 0.1M (take by weighing 1.066g MES, 0.45g NaCl is dissolved in the 50ml pure water, accent pH to 4.7) behind washing and the magnet stand separation and concentration with 0.4T, be that 0.1M, pH value are that 4.7 MES damping fluid is resuspended with 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 ℃, the reaction half an hour after, obtain activating rear magnetic particle;
2) with the borate buffer solution washing of 50mM pH=8.5, (the gloomy bio tech ltd in river, Guangzhou, JS-23-0004, diethylstilbestrol antibody tire 10 to get 0.15mg diethylstilbestrol monoclonal antibody
6, the diethylstilbestrol antibody affinity costant is 10
8M
-1) and 2.5mg activation after the magnetic particle borate buffer solution that is mixed into 0.8ml 50mM pH=8.5 (take by weighing 1.9g Na
2B
4O
7.10H
2O is dissolved in the 100ml pure water, transfers pH to 8.5) in abundant mixing.The lower reaction of room temperature (25 ℃) 3.5 hours allows antibody and magnetic particle form stable peptide bond covalent bond, obtains containing the reactant liquor of magnetic particle after the coupling;
3) after reaction finishes, to step 2) to add final concentration in the reactant liquor that obtains be that the BSA (Sigma-aldrich, 85041C) of 5% (quality percentage composition) seals the residual activity amino sites, reaction was carried out 0.5 hour under 37 ℃.After finishing, (take by weighing 2.3g Na with the 0.02M PBS damping fluid of pH=7.4
2HPO
4, 0.524gNaH
2PO
4.H
2O, 8.77g NaCL are dissolved in the 1L pure water, transfer pH to 7.4) washing, resuspended rear 4 ℃ of preservations are stand-by, obtain superparamagnetism composite particle mark diethylstilbestrol antibody.
(2) diethylstilbestrol antigen is synthetic
The diethylstilbestrol haptens is that (DES, a day Chemical Engineering Technology research institute is opened to diethylstilbestrol by the permanent unit in Beijing, 30216CDCT-C12607000).
The synthetic employing of DES-BSA antigen is converted into intermediate with the DES molecule first, carries out coupling with BSA again.Concrete steps are as follows:
(1) gets 26.8mg diethylstilbestrol (DES), 20mg succinic anhydride and 5ml pyridine mixing, at 45 ℃ of lower stirring reaction 15h; Obtain reactant liquor a, remove pyridine among the reactant liquor a by Rotary Evaporators, obtain the DES intermediate after drying up remaining pyridine with nitrogen; Add 5mlDMF (DMF) dissolving DES intermediate, obtain the DES midbody solution;
(2) 5mlDES midbody solution, the positive amine of 26.2 μ l (about 0.1mmol) tributyl (making the bundle acid binding agent) are mixed 15min under 4 ℃, obtain reactant liquor b; In reactant liquor b, add 1.5 μ l (about 0.1mmol) isobutyl chlorocarbonate at 25 ℃ of lower stir-activating 1h, the DES midbody solution that obtains activating again;
(3) get 100mg BSA and be dissolved in 10ml concentration 0.1mol/L, pH obtains the BSA BAS in 8.5 the dobell's solution;
(4) with 5ml step 3) the good DES midbody solution of activation that obtains slowly dropwise adds 10ml step 4 by separating funnel under ice bath) in the described BSA BAS that obtains, stirring reaction spends the night, and (temperature is 4 ℃, time is 12h), obtain mixed liquor d;
(5) be that 0.01mol/L, pH are 4 ℃ of dialysis 2 days in 7.4 the PBS solution with mixed liquor d in concentration, every 6h changes a dislysate, remove unreacted little molecule, collect the dialysis product, the product of will dialysing is again crossed post (GE Healthcare with Sephadex G-25,17-0034-01) purifying makes DES-BSA.Products obtained therefrom freeze dryer freeze-drying in-20 ℃ of preservations, obtains diethylstilbestrol antigen.
(3) preparation of diethylstilbestrol magnetic mark quick detection test paper
Adopt 0.02M PBS (pH=7.4) damping fluid, with sheep anti-mouse igg antibody (the rich eugenic thing in Changsha Science and Technology Ltd., ABGAM-0500) and the concentration of above-mentioned (two) diethylstilbestrol antigen of obtaining all be formulated as concentration 1mg/ml, select BioJet shower nozzle in the XYZ3050 spray film system of BioDot sheep anti-mouse igg antibody to be sprayed onto control line (the Control Line of nitrocellulose filter (NC film), the C line) position, the diethylstilbestrol antigen that above-mentioned (two) are obtained is sprayed onto detection line (Test Line, the T line) position is that dehumidifier dried for standby after 4 hours is carried out in drying plant below 10% in relative humidity.Soaked all-glass paper 1 hour with 0.02M PBS (pH=7.4) solution that contains 2%TritonX100,1%BSA, 1% sucrose, the temperature of soaking is 37 ℃, carry out dehumidifier after 4 hours in same dehumidifier condition, behind the diethylstilbestrol antibody of above-mentioned film processing damping fluid by 50 times of dilution paramagnetism composite particle marks, adopt AirJet shower nozzle in the XYZ3050 spray film system of BioDot this dilution magnetic labeling antibody to be sprayed into preparation forms sample pad on the glass fibre element film of above-mentioned processing, carry out drying in same dehumidifier condition.In 100,000 grades of cleanings and dry workshop above-mentioned drying good NC film, magnetic pad, thieving paper, backboard and diaphragm by shown in Figure 1 arrange in pairs or groups assemble after; adopting the CM4000 cutting system of BioDot is the width of 5mm/ bar with the Paperboard cutting that posts; packing into, it is stand-by to detect with intermediate plate, obtains detecting the immune chromatography test paper of diethylstilbestrol.
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, diethylstilbestrol residue
1, the mensuration of sensitivity
(a day Chemical Engineering Technology research institute is opened, catalog number by the permanent unit in Beijing: 30216CDCT-C12607000), be formulated as the standard inventory solution of 100ug/ml with methyl alcohol to take by weighing an amount of diethylstilbestrol.With the PBS (pH=7.4) of 0.02M dilution be formulated as 0,0.005,0.01,0.05,0.1,1,5, the standard solution of 10ug/L, add respectively in the immune chromatography test paper of the detection diethylstilbestrol that is obtained by embodiment 1, and employing superparamagnetic resonance detector MAR (MagnaBioSciences, 8094-101-01﹠amp; 8094-101-02) read.Detecting step: first detected sample is recovered room temperature (25 ℃) before the detection, get the application of sample end that detected sample 100 μ l vertically slowly splash into the magnetic test strips with accurate pipettor, then splash into the 50ul washing fluid (0.02M, pH 7.4, PBS), test with MAR after 20 minutes.
Its testing result is as shown in table 1 below.Detected value when detected value was with 0ug/L when we can find that diethylstilbestrol concentration is 0.005ug/L from the testing result data has and intersects, and detected value and 0ug/L value can distinguish fully when concentration is 0.01ug/L, and concentration curve R
2=0.9865, better linear, illustrate that the detection sensitivity of test paper can reach 0.01ug/L.
Diethylstilbestrol magnetic detection paper value and concentration curve are as shown in Figure 3.
The magnetic detection paper value of table 1 diethylstilbestrol variable concentrations sample
2, the mensuration of cross reaction
Select dienestrol (Sigma-aldrich, 46190-100MG), hexestrol (Sigma-aldrich, 46320-100MG-R), glucosiduronic acid diethylstilbestrol (Sigma-aldrich, D8647), (a day Chemical Engineering Technology research institute is opened to estriol by the permanent unit in Beijing, 13783NIC-100934) with hexin estradiol (Sigma-aldrich, E4876-100MG) 5 kinds of medicines, be made into respectively series concentration, use the immune chromatography test paper by the detection diethylstilbestrol of embodiment 1 to detect.Calculate the IC50 that respectively competes thing, calculate respectively the cross reacting rate of these 5 kinds of medicines and DES magnetic test paper with following formula.Computing formula is: cross reacting rate (%)=[IC50 (CAP)/IC50 (medicine to be measured)] * 100.
Mensuration and result of calculation are as shown in table 2.The result shows that diethylstilbestrol magnetic test paper has certain intersection to dienestrol, hexestrol, glucosiduronic acid diethylstilbestrol, and all the other 2 kinds of medicine cross reacting rates are all less than 0.1%.
The cross reaction of table 2 diethylstilbestrol magnetic test paper and other medicines
3, the mensuration of accuracy and the recovery
3.1 urine sample:
The urine of clarification can be directly used in detection, if cloudy urine needs first centrifugal (4000g) 10min, gets supernatant and detects.
3.2 the assay method of accuracy
60 parts of pig urine 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 Britain's ELISA of Randox company kit of the detection diethylstilbestrol that is obtained by embodiment 1 to detect simultaneously 60 duplicate samples.
Measure the magnetic field intensity of T line place superparamagnetism microballoon with magnetic test paper interpretoscope, by with the threshold ratio of setting to determining its positive or negative result, C line measurement result is then marked in the Quality Control as this assay method.
Threshold value less than 615 positive, threshold value greater than 615 negative.
3.3 the mensuration of accuracy
The testing result of the immune chromatography test paper of the detection diethylstilbestrol that is obtained by embodiment 1 is that to be numbered the sample of 1-55 negative, and its threshold value is respectively 645.5,641.3,655.6,651.7,655.8,631.2,634.6,643.2,640.7,662.3,633.2,630.6,649.8,652.4,646.7,644.8,636.9,638.6,653.3,641.1,647.5,646.6,657.8,640.2,643.3,649.3,636.5,631.7,638.4,636.3,644.9,652.7,647.2,641.5,642.2,632.8,629.6,658.4,661.5,646.9,648.8,641.9,639.7,638.4,632.1,629.6,637.3,631.8,644.9,647.2,643.5,633.4,648.2,645.3,640.8.
The sample that is numbered 56-60 is positive, and its threshold value is respectively 471.4,355.7,260.5,204.8,153.6.
The testing result of the Britain ELISA of Randox 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 were mixed, the diethylstilbestrol titer (0.1,0.5,1,2,5ug/L) of variable concentrations was 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 diethylstilbestrol adds in the pig urine is 83.8%～95.6%, average recovery rate 90.82%, and the coefficient of variation 4%～13.65%, average coefficient of variation 9.28%, accuracy is better.
Table 3 determination of recovery rates
Claims (12)
1. an immune chromatography test paper that detects diethylstilbestrol comprises sample pad, the reacting pad that is connected in described sample pad one end that contains superparamagnetism composite particle mark diethylstilbestrol 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 that is separated from each other, and described detection line contains diethylstilbestrol antigen, described nature controlling line contain can with the antiantibody of described diethylstilbestrol antibody specific bond;
The condensate that the peptide bond covalent bond that described superparamagnetism composite particle mark diethylstilbestrol antibody is diethylstilbestrol antibody and superparamagnetism composite particle forms; Described diethylstilbestrol antibody is diethylstilbestrol monoclonal antibody or diethylstilbestrol polyclonal antibody;
Described superparamagnetism composite particle is Fe
3O
4Nano particle;
The particle diameter of described superparamagnetism composite particle is 60～300nm;
The magnetic saturation intensity of described superparamagnetism composite particle is 30～80emu/g, and corresponding external magnetic field response speed is 20～100 seconds; The carboxyl-content on described superparamagnetism composite particle surface is 50～500 μ mol/g;
Described diethylstilbestrol antibody affinity costant is 10
6～10
8M
-1
Described diethylstilbestrol antigen is the conjugate of diethylstilbestrol haptens and carrier protein;
The haptenic coupling ratio of described carrier protein and diethylstilbestrol is 1:8～1:10, and described diethylstilbestrol haptens is diethylstilbestrol; Described carrier protein is BSA.
2. test paper according to claim 1 is characterized in that:
Described reacting pad is nitrocellulose filter;
Described diethylstilbestrol antibody is the diethylstilbestrol monoclonal antibody;
The haptenic coupling ratio of described carrier protein and diethylstilbestrol is 1:10;
The antiantibody of described and described diethylstilbestrol antibody specific bond is sheep anti-mouse igg antibody;
The concentration of described diethylstilbestrol antigen and described sheep anti-mouse igg antibody is 1mg/ml.
3. test paper according to claim 1 and 2 is characterized in that:
Described superparamagnetism composite particle mark diethylstilbestrol antibody is prepared as follows:
1) be that 0.1M, pH value are 4.7 2-(N-morpholine) ethyl sulfonic acid damping fluid mixing with every 2.5mg superparamagnetism composite particle, 0.96mg1-ethyl-(3-dimethylaminopropyl) carbodiimide, 1.15mg N-maloyl imines and 1ml concentration, reaction obtains activating rear magnetic particle;
2) every 2.5mg step 1) being obtained activating rear magnetic particle, 0.15mg diethylstilbestrol antibody and 0.8ml concentration is that 50mM pH is 8.5 borate buffer solution mixing, reaction, obtains containing the reactant liquor of magnetic particle after the coupling;
3) to step 2) add the BSA mixing in the reactant liquor that obtains and obtain mixed liquor, reaction, obtain containing the reactant liquor of magnetic particle after the sealing;
The concentration of described BSA in described mixed liquor is 5% quality percentage composition;
Described diethylstilbestrol antigen is prepared as follows:
A) get first every 26.8mg diethylstilbestrol, 20mg succinic anhydride and 5ml pyridine mixing, reaction obtains reactant liquor a, removes pyridine among the described reactant liquor a again, obtains the diethylstilbestrol intermediate; Add 5mlN, the described diethylstilbestrol intermediate of dinethylformamide dissolving 82mg obtains the diethylstilbestrol midbody solution again;
B) with every 5ml steps A) the diethylstilbestrol midbody solution that obtains and the positive amine of 26.2 μ l tributyls is mixed, obtains reactant liquor b, adds 1.5 μ l isobutyl chlorocarbonate mixings, reaction, the diethylstilbestrol midbody solution after obtaining activating again in described reactant liquor b;
C) get every 100mg carrier protein and be dissolved in 10ml concentration 0.1mol/L, pH obtains the carrier protein BAS in 8.5 the sodium borate aqueous solution;
D) with every 5ml step B) diethylstilbestrol midbody solution and 10ml step C obtain after the activation that obtains carrier protein BAS mixing, reaction, obtain reactant liquor c; Reactant liquor c obtains diethylstilbestrol antigen through dialysis, purifying, freeze-drying.
4. test paper according to claim 3 is characterized in that:
Among the preparation method of described superparamagnetism composite particle mark diethylstilbestrol antibody:
In the step 1), temperature of reaction is 37 ° of C, and the reaction time is 0.5h;
Step 2) in, temperature of reaction is 25 ° of C, and the reaction time is 3.5h;
In the step 3), temperature of reaction is 37 ° of C, and the reaction time is 0.5h.
Among the preparation method of described diethylstilbestrol antigen:
Steps A) in, described temperature of reaction is 45 ℃, and the described reaction time is 15h;
Step B) in, described mixing temperature is 4 ℃, and described incorporation time is 1h; Described temperature of reaction is 25 ° of C, and the described reaction time is 1h;
Step D) in, described temperature of reaction is 4 ℃, and the described reaction time is 12h.
5. test paper according to claim 4 is characterized in that:
Among the preparation method of described superparamagnetism composite particle mark diethylstilbestrol antibody:
After described step 3), also comprise magnetic particle after the described sealing that contains in the reactant liquor that seals rear magnetic particle is washed, suspends, obtain the step of superparamagnetism composite particle mark diethylstilbestrol antibody, it is that 0.02M pH is 7.4 PBS damping fluid that described cleansing solution and suspending liquid are concentration.
6. test paper according to claim 5, it is characterized in that: the particle diameter of described superparamagnetism composite particle is for being 80～200nm;
The magnetic saturation intensity of described superparamagnetism composite particle is 35～70emu/g, and corresponding external magnetic field response speed is 20～50 seconds;
The carboxyl-content on described superparamagnetism composite particle surface is specially 50～300 μ mol/g;
Described diethylstilbestrol antibody affinity costant is specially 10
7～10
8M
-1
7. test paper according to claim 6, it is characterized in that: the particle diameter of described superparamagnetism composite particle is that described particle diameter 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 diethylstilbestrol antibody affinity costant especially is preferably 10
8M
-1
8. a method for preparing such as the immune chromatography test paper of the described detection diethylstilbestrol of one of claim 1-7 comprises the steps:
I, prepare sample pad and contain detection line and the reacting pad of nature controlling line respectively;
II, the sample pad that the step I is obtained, the reacting pad that contains detection line and nature controlling line and the adsorptive pads that the step I obtains paste on the backboard successively, obtain detecting the immune chromatography test paper of diethylstilbestrol;
The described reacting pad that contains detection line and nature controlling line is prepared as follows: the two ends zones of different that the described and antiantibody diethylstilbestrol antibody specific bond in the test paper among the described diethylstilbestrol antigen in the test paper among the claim 1-7 and the claim 1-7 is sprayed on respectively reacting pad, form detection line and nature controlling line, obtain containing the reacting pad of detection line and nature controlling line;
Described sample pad is prepared as follows: the described superparamagnetism composite particle mark diethylstilbestrol antibody in the test paper among the claim 1-7 is sprayed onto on the all-glass paper, obtains sample pad.
9. method according to claim 8 is characterized in that:
In described sample pad preparation method: go forward in that described superparamagnetism composite particle mark diethylstilbestrol antibody is sprayed onto 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 diethylstilbestrol antibody:
The described all-glass paper of described pre-service is that described all-glass paper was soaked in damping fluid 1 hour;
Described damping fluid is prepared as follows: be that 0.02M, pH are that 7.4 PBS damping fluid is mixed to get damping fluid with every 2mlTritonX100,10g BSA, 50g sucrose and 950ml concentration, transfer 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 ° of C;
The described superparamagnetism composite particle of described pre-service mark diethylstilbestrol antibody is that described extension rate is 50 times with described superparamagnetism composite particle mark diethylstilbestrol antibody dilution;
Described reacting pad is nitrocellulose filter.
10. it is characterized in that according to claim 8 or 9 described methods:
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.
11. the application in the residual diethylstilbestrol in test sample of the arbitrary described test paper of claim 1-7, described sample is specially animal tissue's sample, animal urine, feed, honey or milk sample.
12. application according to claim 11 is characterized in that: described sample is pig urine.
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