CN103558381A - Immunochromatographic test paper for detecting human immunodeficiency virus antibodies and preparation method thereof - Google Patents
Immunochromatographic test paper for detecting human immunodeficiency virus antibodies and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an immunochromatographic test paper for detecting human immunodeficiency virus antibodies and a preparation method thereof, belonging to the field of reagents for detecting human immunodeficiency virus antibodies. The immunochromatographic test paper comprises a sample pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected, wherein the sample pad contains a superparamagnetic composite particle labeled recombinant protein A; the nitrocellulose membrane is coated with a detection line and a quality control line which are mutually separate; the detection line contains a human immunodeficiency virus recombinant antigen; the quality control line contains an anti-recombinant protein A antibody which can be specifically combined with the recombinant protein A; and the human immunodeficiency virus is human immunodeficiency virus Type 1+2. The immunochromatographic test paper has the advantages of high sensitivity and high specificity, is quick and convenient, and can implement objective determination.
Description
Technical field
The detection technique field that the invention belongs to mankind antibody of AIDS virus, is specifically related to immune chromatography test paper of a kind of mankind of detection antibody of AIDS virus and preparation method thereof.
Background technology
Acquired immune deficiency syndrome (AIDS), be acquired immunodeficiency syndrome (Acquired Immunodeficiency Syndrome, AIDS), its cause of disease is human immunodeficiency virus (Human Immunodeficiency Virus, HIV), also claims AIDS virus, this virus is destroyed the immunocompetence of human body, cause immune system to lose resistibility, and cause various diseases and cancer to be able to survive in human body, finally suffer from acquired immune deficiency syndrome (AIDS).At present, acquired immune deficiency syndrome (AIDS) has not only become the public health problem of serious threat China people ' s health, and has had influence on economic development and social stability.The detection of human immunodeficiency virus (HIV) 1+2 type antibody has great importance for the diagnosis of acquired immune deficiency syndrome (AIDS) and discriminating, EPDML investigation, blood donor's screening, prognosis judgement and the investigation of result for the treatment of and the screening of medicine.
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 flow measurement immunochromatography, on its film surface, detection zone, observe approximately 10% signaling molecule intensity only, and with the superparamagnetism composite particle material that serves as a mark, the all magnetic signal molecules in the film 3-D solid structure of detection zone can be detected, can greatly improve sensitivity, and the magnetic signal detector of available correspondence reaches quantitative measurement.Therefore, superparamagnetism composite particle is the material receiving publicity in LFIAs in recent years.
Yet at present the biological detection of report adopts chemical method first to prepare after the magnetic nano-particle of organic phase as coprecipitation with magnetic nano-composite particle more, then adopts silicon (SiO
2) or the macromolecular material such as polystyrene, polyacrylic acid, gelatin 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 obtaining 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 much in 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 cannot 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
The immune chromatography test paper that the object of this invention is to provide a kind of mankind of detection antibody of AIDS virus.
Another object of the present invention is to provide the preparation method of above-mentioned immune chromatography test paper.
The technical solution used in the present invention is to detect the immune chromatography test paper of mankind antibody of AIDS virus, comprises the sample pad, nitrocellulose membrane and the adsorptive pads that connect successively, and described sample pad contains superparamagnetism composite particle mark recombinant protein A; Described nitrocellulose membrane is coated with detection line and the nature controlling line being separated from each other, and described detection line contains mankind's AIDS virus recombinant antigen, described nature controlling line contain can with the anti-recombinant protein A antibody of described recombinant protein A specific bond; Described mankind's AIDS virus behaviour AIDS-like disease virus 1+2 type.Wherein, adsorptive pads provides power for sample chromatography on test paper.
As preferably, described superparamagnetism composite particle mark recombinant protein A is that recombinant protein A and superparamagnetism composite particle are formed to condensate with peptide bond covalent bond.
As preferably, the preparation method of described superparamagnetism composite particle mark recombinant protein A comprises the following steps:
The preparation of a, activation magnetic particle: by ratio of weight and the number of copies, take 2～3 parts of superparamagnetism composite particles, EDC0.5～1.5 part and NHS1～3 part, mix, react 20～40 minutes at 35 ℃～40 ℃, with damping fluid washing, obtain activation magnetic particle subsequently;
B, the reactant liquor preparation that contains magnetic particle after coupling: by ratio of weight and the number of copies, take 0.5～1 part of 2～3 parts of described activation magnetic particles, 0.1～0.2 part of recombinant protein A and borate buffer solution, mix, at 35 ℃～40 ℃, react 3～4 hours, must contain the reactant liquor of magnetic particle after coupling;
C, contain the reactant liquor of magnetic particle after coupling described in getting, add the BSA solution that accounts for whole described reactant liquor volume 0.5%～1.5%, mix, at 35 ℃～40 ℃, react 20～40 minutes, after the sealing subsequently reaction being obtained, magnetic particle washs, suspends, and obtains superparamagnetism composite particle mark recombinant protein A finished product.
Further, described superparamagnetism composite particle is superparamagnetism Fe
3o
4nano particle.
Due to superparamagnetism composite particle will be by the sample pad swimming of immune chromatography test paper the test section to nitrocellulose membrane, realize specific bond and the labeled reactant of Ag-Ab, during its particle diameter > 300nm, the swimming time on immune chromatography test paper is longer, and developing time is slow; When coupling, be easier to assemble, and easily produce non-specific responding.If particle diameter is too little, magnetic intensity is often inadequate again, and swimming is not.Therefore the particle diameter of described superparamagnetism composite particle is 60nm～300nm.Preferably, the particle diameter of described superparamagnetism composite particle is 80nm～200nm.Particularly preferred, the particle diameter of described superparamagnetism composite particle is 80nm.Described in it, the deviation of superparamagnetism composite particle particle diameter is between 10%～30%.Preferably, the deviation of described superparamagnetism composite particle particle diameter is between 10%～20%.Particularly preferred, the deviation of described superparamagnetism composite particle particle diameter is 15%.
The magnetic saturation intensity of superparamagnetism composite particle and external magnetic field response speed thereof have directly determined the height of detection sensitivity and accuracy thereof, the magnetic saturation intensity of superparamagnetism composite particle prepared by classic method is all less than 30emu/g conventionally, and corresponding external magnetic field response speed was over 100 seconds.For improving sensitivity and the accuracy thereof of immune chromatography test paper of the present invention, the magnetic saturation intensity of described superparamagnetism composite particle is 30emu/g～80emu/g, and corresponding external magnetic field response speed is 20 seconds～100 seconds.Preferably, the magnetic saturation intensity of described superparamagnetism composite particle is 35emu/g～70emu/g, and corresponding external magnetic field response speed is 20 seconds～50 seconds.Particularly preferred, the magnetic saturation intensity of described superparamagnetism composite particle is 40emu/g, and corresponding external magnetic field response speed is 20 seconds.
For for improving the detection performance of mankind's AIDS virus 1+2 type antibody, described recombinant protein A and superparamagnetism composite particle are being formed before condensate with peptide bond covalent bond, this superparamagnetism composite particle can be activated, make superparamagnetism composite particle surface with the functional group being easy to recombinant protein A coupling.As preferably, described functional group is a kind of in carboxyl or amino; The content of described functional group is 50～500 μ mol/g.Preferably, functional group is carboxyl, and the content of carboxyl is 50～300 μ mol/g.Particularly preferred, the content of carboxyl is 80 μ mol/g.
Further, the concentration that contains mankind's AIDS virus recombinant antigen in described detection line is 0.5～2mg/ml; The concentration that contains anti-recombinant protein A antibody in described nature controlling line is 0.5～2mg/ml.
Further, described mankind's AIDS virus recombinant antigen behaviour AIDS-like disease virus 1+2 type genetic engineering recombinant antigen; Described mankind's AIDS virus 1+2 type genetic engineering recombinant antigen is a kind of in gp36, gp41 or gp120.
A kind of preparation method who detects the immune chromatography test paper of mankind antibody of AIDS virus, comprise the following steps: sample pad, nitrocellulose membrane and adsorptive pads are connected successively, by containing mankind's AIDS virus recombinant antigen and containing, can be sprayed at respectively with the anti-recombinant protein A antibody of described recombinant protein A specific bond the zones of different at nitrocellulose membrane two ends, corresponding detection line and the nature controlling line of forming, obtains finished product.
The preparation method of sample pad is as follows: the damping fluid that all-glass paper is placed in to 37 ℃ soaks 1 hour, then takes out and dries, standby.Again superparamagnetism composite particle mark recombinant protein A is adopted to 50 times of diluted, be then sprayed onto on standby all-glass paper, obtain sample pad finished product.Damping fluid in this preparation method and dilution are same solution, in every 2ml TritonX100, add 10g BSA, 50g sucrose and 950ml concentration and be 0.02M, pH and be 7.4 PBS damping fluid and be mixed to get damping fluid, adjust PH to 7.4, and constant volume makes to 1000ml.
When preparing immune chromatography test paper, before sample pad, nitrocellulose membrane and adsorptive pads are connected successively, sample pad and the nitrocellulose membrane that is coated with detection line and nature controlling line can be dried.
The present invention relates to the application of immune chromatography test paper in preparation mankind AIDS virus 1+2 type antibody test test paper.Sample to be detected specifically can be serum, blood plasma etc.
Know-why of the present invention is:
HIV1+2 type antibody test reagent is relevant to the immuno-chromatographic assay technology of superparamagnetism composite particle mark, 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 immune chromatography test paper of the present invention can detect mankind's AIDS virus (HIV) 1+2 type antibody, a kind of method that has been to adopt flow measurement immunochromatography based on superparamagnetism composite particle mark to detect, be about to mankind's AIDS virus (HIV) 1+2 type recombinant antigen and anti-recombinant protein A antibody and be sprayed at respectively detection line and nature controlling line place, in sample pad, spray the recombinant protein A of superparamagnetism composite particle mark.Principle based on flow measurement immunochromatography, add after testing sample, mankind's AIDS virus (HIV) 1+2 type antibody in sample is combined mankind AIDS virus (HIV) the 1+2 type recombinant antigen of rear chromatography to detection line place spraying with the recombinant protein A of magnetic mark, at detection line place, form envelope antigen-antibody-magnetic mark recombinant protein A immune complex, unnecessary magnetic mark recombinant protein A forms magnetic mark immune complex at nature controlling line place and anti-recombinant protein A antibody.With magnetic test paper interpretoscope, measure the magnetic strength intensity of detection line place superparamagnetism microballoon, by comparing and determine its positive or negative result with the threshold value of setting, nature controlling line measurement result is marked in the Quality Control as this assay method.
Its concrete technical step comprises:
(1) preparation of superparamagnetism composite particle mark recombinant protein A
Adopt applicable nanometer superparamagnetism composite particle, activate after its surperficial carboxyl, adopt the mode of chemical coupling that recombinant protein A orientation is connected to this superparamagnetism composite particle surface.
(2) test section detection line and nature controlling line place antigen/antibody is coated
Adopt special spray film instrument, in the detection line place of test section spraying mankind's AIDS virus (HIV) 1+2 type recombinant antigen, in nature controlling line place, spray anti-recombinant protein A antibody.
(3) sample pad place label probe is coated
Adopt spraying instrument, in the recombinant protein A of sample pad specific location spraying superparamagnetism microballoon mark.
(4) assembled formation of immune chromatography test paper
According to the structural representation (see figure 1) requirement of immune chromatography test paper, in the middle of plastic support backboard, paste cellulose nitrate (NC) film as test section, in the detection line end of NC film, paste sample pad, Quality Control line end is pasted adsorptive pads.Paste in the above transparent protective film.Adopt special test paper cutting machine, monoblock immune chromatography test paper is divided to the paper slip that is cut to certain broadband, with the special aluminium foil bag that drying agent is housed, pack.
(5) formation of Ag-Ab magnetic mark immune complex
Well place in the immune chromatography test paper of above-mentioned assembled formation adds testing sample, mankind's AIDS virus (HIV) 1+2 type antibody in sample is combined mankind AIDS virus (HIV) the 1+2 type recombinant antigen of rear chromatography to detection line place spraying with the recombinant protein A of magnetic mark, at detection line place, form envelope antigen-antibody-magnetic mark recombinant protein A immune complex, unnecessary magnetic mark recombinant protein A is the magnetic mark immune complex with the formation of anti-recombinant protein A antibody at nature controlling line place.
(6) magnetic mark immune complex magnetic field intensity detects
With magnetic test paper interpretoscope, measure the magnetic field intensity of detection line place superparamagnetism microballoon, by comparing and determine its positive or negative result with the threshold value of setting, nature controlling 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 that Fig. 2 is shown in by the water-solubility nanocrystalline TEM photo that MP-2(poly hexadecanol ester (PMAH) is modified), the preparation method who adopts is by the oil-soluble Fe making with chemical method
3o
4be dissolved in and in organic reagent, obtain solution A, amphiphilic oligomer is 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 organic solvent volatilization, carry out centrifuging, after the product of centrifuging is dried, get final product to obtain water miscible superparamagnetism composite particle.The saturated magnetic intensity of magnetic compound particles for preparing by this method is high, magnetic response fast, 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.
In the present invention, magnetic mark immune complex refers to add and detects after sample, through immune combination, mankind's AIDS virus (HIV) 1+2 type envelope antigen-antibody-magnetic mark recombinant protein A immune complex forming at detection line place, and the anti-recombinant protein A antibody-magnetic mark recombinant protein A immune complex forming at nature controlling line place.
In the present invention, the magnetic field intensity of magnetic mark immune complex, the quantity that refers to the combination magnetic bead under detection line and nature controlling line place are detained respectively with the superparamagnetic resonance detector MAR of U.S. Quantum Dot, measure after resulting numerical value.By immunochromatography repercussion study, find, through the normal sample of large quantitative determination different people serum, blood plasma etc., can determine the mensuration average of variant normal sample, using that this determines the positive or negative result of detection line detection sample as critical value (cutoff).Nature controlling line measurement result is marked in the Quality Control as this assay method.
Of the present invention experimental results show that, by to superparamagnetism composite particle, the research of mankind's AIDS virus (HIV) 1+2 type antibody and mankind's AIDS virus (HIV) 1+2 type recombinant antigen molecular characterization, by prepared by multiple superparamagnetism composite particle, the optimization of coated 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 immunochromatography reaction, reach the objective detection to mankind's AIDS virus (HIV) 1+2 type antibody, realized the quick and Sensitive Determination to mankind's AIDS virus (HIV) 1+2 type antibody.
In the present invention, BSA is bovine serum albumin(BSA); EDC is 1-ethyl-(3-dimethylaminopropyl) carbodiimide; NHS is N-maloyl imines.
In the present invention, recombinant protein A and can be commercially available common reagent with the anti-recombinant protein A antibody of recombinant protein A specific bond.
Beneficial effect of the present invention is: highly sensitive, high specificity, quick, easy, can realize the mensuration that objectifies.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing in the following describes is only one of them embodiment of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the structural representation of immune chromatography test paper of the present invention;
Fig. 2 is superparamagnetism composite particle Electronic Speculum figure.
In figure: 1-sample pad, 2-nitrocellulose membrane, 3-adsorptive pads, 4-detection line, 5-nature controlling line, 6-backboard.
Embodiment
For making those skilled in the art understand in detail production technology of the present invention and technique effect, with concrete production instance, further introduce application of the present invention and technique effect below.
Embodiment mono-:
(1) preparation of superparamagnetism composite particle mark recombinant protein A
Employing particle diameter is that 100nm, particle diameter deviation are 15%, magnetic saturation intensity is 40emu/g, and corresponding external magnetic field response speed is that 20 seconds, surperficial carboxyl-content are the superparamagnetism Fe of 80 μ mol/g
3o
4nano particle.
Concrete grammar is: the above-mentioned superparamagnetism Fe that gets 2.5mg
3o
4nano particle is that 0.1mol, pH are 4.7 MES damping fluid washing by concentration, and after the magnet stand separation and concentration with 0.4T, resuspended with 1 milliliter of above-mentioned MES damping fluid, then add the EDC of 0.96mg and the NHS of 2.17mg, mix, in temperature of reaction, being to react 0.5 hour under the condition of 37 ℃, is that 50mmol, pH are 8.5 borate buffer solution washing by concentration subsequently, obtains 2.5mg activation magnetic particle.
Get 1.5mg recombinant protein A and above-mentioned gained activation magnetic particle, being placed in concentration and being 50mmol, pH and be 8.5 borate buffer solution fully mixes, at 37 ℃, react 3.5 hours, allow albumen and magnetic particle form stable peptide bond covalent bond, must contain the reactant liquor of magnetic particle after coupling.Adding the final concentration that accounts for whole this reactant liquor volume 1% is 5%(percentage by weight) BSA solution, mix, residual activity amino sites is sealed, at 37 ℃, react 0.5 hour.After having reacted, by concentration, be the magnetic particle obtaining after the PBS damping fluid washing sealing that is 7.4 of 0.02 mole, pH, suspend, obtain superparamagnetism composite particle mark recombinant protein A finished product, be placed in 4 ℃ of preservations stand-by.
Wherein, being prepared as of borate buffer solution: take 1.9g Na
2b
4o
710H
2o is dissolved in 100ml pure water, adjusts pH to 8.5, obtains.Being prepared as of PBS damping fluid: take 2.3 grams of Na
2hPO
4, 0.524 gram of NaH
2pO
4h
2o, 8.77 grams of NaCl are dissolved in 1L pure water, adjust pH to 7.4, obtain.
(2) preparation of immune chromatography test paper
Shown in Fig. 1, it is that 0.02mol, pH, in 7.4 PB damping fluid, mix that the anti-recombinant protein A antibody of 250ul of getting concentration and be 4mg/ml joins 750ul concentration, and obtaining concentration is the anti-recombinant protein A antibody of 1mg/ml.It is that 0.02mol, pH, in 7.4 PB damping fluid, mix that the 250ul gp36 that gets concentration again and be 4mg/ml joins 750ul concentration, obtains the gp36 that concentration is 1mg/ml.Select the BioJet shower nozzle in the XYZ3050 spray film system of BioDot the anti-recombinant protein A antibody after preparing to be sprayed onto to the position of nature controlling line 5 on nitrocellulose filter 2, the gp36 solution preparing is sprayed onto to the position of detection line 4, in relative humidity is the drying plant below 10%, carries out dehumidifier dried for standby after 4 hours.
With the 0.02mol PBS(pH=7.4 containing 0.2%TritonX100,1%BSA, 1% sucrose) solution soaks all-glass paper 1 hour, the temperature of soaking is 37 ℃, in same dehumidifier condition, carry out dehumidifier after 4 hours, with the damping fluid of above-mentioned immersion glass fibre, by 50 times of weight, dilute after the recombinant protein A of superparamagnetism composite particle marks, adopt AirJet shower nozzle in the XYZ3050 spray film system of BioDot this dilution magnetic mark compound to be sprayed into preparation on the glass fibre element film of above-mentioned processing and form sample pad 1, in same dehumidifier condition, be dried.In 100,000 grades of cleanings and dry workshop, above-mentioned dried sample pad 1, nitrocellulose filter 2 and adsorptive pads 3 are got up by backboard 6 bondings successively; in sample pad 1, arrange after well; again at the surperficial bonding protective film of sample pad 1, nitrocellulose filter 2 and adsorptive pads 3; then the CM4000 cutting system that adopts BioDot is the width of 5mm/ bar by the Paperboard cutting posting; pack into detect and use intermediate plate stand-by, obtain detecting the immune chromatography test paper of mankind's AIDS virus 1+2 type antibody.
Embodiment bis-:
(1) preparation of superparamagnetism composite particle mark recombinant protein A
Employing particle diameter is that 60nm, particle diameter deviation are 10%, magnetic saturation intensity is 80emu/g, and corresponding external magnetic field response speed is that 100 seconds, surperficial carboxyl-content are the superparamagnetism Fe of 50 μ mol/g
3o
4nano particle.
Concrete grammar is: the above-mentioned superparamagnetism Fe that gets 2.5mg
3o
4nano particle is that 0.1mol, pH are 4.7 MES damping fluid washing by concentration, and after the magnet stand separation and concentration with 0.4T, resuspended with 1 milliliter of above-mentioned MES damping fluid, then add the EDC of 0.96mg and the NHS of 2.17mg, mix, in temperature of reaction, being to react 40 minutes under the condition of 35 ℃, is that 50mmol, pH are 8.5 borate buffer solution washing by concentration subsequently, obtains the activation magnetic particle of 2.5mg.
Get 1.5mg recombinant protein A and above-mentioned gained activation magnetic particle, being placed in concentration and being 50mmol, pH and be 8.5 borate buffer solution fully mixes, at 40 ℃, react 3 hours, allow albumen and magnetic particle form stable peptide bond covalent bond, must contain the reactant liquor of magnetic particle after coupling.Adding the final concentration that accounts for whole this reactant liquor volume 1.5% is 5%(percentage by weight) BSA solution, mix, residual activity amino sites is sealed, at 38 ℃, react 20 minutes.After having reacted, by concentration, be magnetic particle after the PBS damping fluid washing sealing that is 7.4 of 0.02 mole, pH, suspend, obtain superparamagnetism composite particle mark recombinant protein A finished product, be placed in 4 ℃ of preservations stand-by.
Wherein, being prepared as of borate buffer solution: take 1.9g Na
2b
4o
710H
2o is dissolved in 100ml pure water, adjusts pH to 8.5, obtains.Being prepared as of PBS damping fluid: take 2.3 grams of Na
2hPO
4, 0.524 gram of NaH
2pO
4h
2o, 8.77 grams of NaCl are dissolved in 1L pure water, adjust pH to 7.4, obtain.
(2) preparation of immune chromatography test paper
Shown in Fig. 1, employing concentration is the PB damping fluid of 0.02mol, pH=7.4, will resist recombinant protein A antibody to be formulated as concentration 1.5mg/ml (manner of formulation is as embodiment mono-); The concentration of gp41 is formulated as to concentration 1.5mg/ml (manner of formulation is as embodiment mono-); Select the BioJet shower nozzle in the XYZ3050 spray film system of BioDot the anti-recombinant protein A antibody after preparing to be sprayed onto to the position of nature controlling line 5 on nitrocellulose filter 2, the gp41 preparing is sprayed onto to the position of detection line 4, in relative humidity is the drying plant below 10%, carries out dehumidifier dried for standby after 4 hours.With the PBS solution of 0.02mol, pH=7.4 containing 0.2%TritonX100,1%BSA, 1% sucrose, soak all-glass paper 1 hour, the temperature of soaking is 37 ℃, in same dehumidifier condition, carry out dehumidifier after 4 hours, the damping fluid of processing with above-mentioned glass fibre is pressed after the recombinant protein A of 50 times of dilution superparamagnetism composite particle marks, adopt AirJet shower nozzle in the XYZ3050 spray film system of BioDot this dilution magnetic mark compound to be sprayed into preparation on the glass fibre element film of above-mentioned processing and form sample pad 1, in same dehumidifier condition, be dried.In 100,000 grades of cleanings and dry workshop, above-mentioned dried sample pad 1, nitrocellulose filter 2 and adsorptive pads 3 are got up by backboard 6 bondings successively; can be at the surperficial bonding protective film of sample pad 1, nitrocellulose filter 2 and adsorptive pads 3; then the CM4000 cutting system that adopts BioDot is the width of 5mm/ bar by the Paperboard cutting posting; pack into detect and use intermediate plate stand-by, obtain detecting the immune chromatography test paper of mankind's AIDS virus 1+2 type antibody.
Embodiment tri-:
(1) preparation of superparamagnetism composite particle mark recombinant protein A
Employing particle diameter is that 300nm, particle diameter deviation are 20%, magnetic saturation intensity is 30emu/g, and corresponding external magnetic field response speed is that 100 seconds, surperficial carboxyl-content are the superparamagnetism Fe of 300 μ mol/g
3o
4nano particle.
Concrete grammar is: the above-mentioned superparamagnetism Fe that gets 2.5mg
3o
4nano particle is that 0.1mol, pH are 4.7 MES damping fluid washing by concentration, and after the magnet stand separation and concentration with 0.4T, resuspended with 1 milliliter of above-mentioned MES damping fluid, then add the EDC of 0.96mg and the NHS of 2.17mg, mix, in temperature of reaction, being to react 20 minutes under the condition of 37 ℃, is that 50mmol, pH are 8.5 borate buffer solution washing by concentration subsequently, obtains the activation magnetic particle of 2.5mg.
Get 1.5mg recombinant protein A and above-mentioned gained activation magnetic particle, being placed in concentration and being 50mmol, pH and be 8.5 borate buffer solution fully mixes, at 35 ℃, react 4 hours, allow albumen and magnetic particle form stable peptide bond covalent bond, must contain the reactant liquor of magnetic particle after coupling.Adding the final concentration that accounts for whole this reactant liquor volume 0.5% is 5%(percentage by weight) BSA solution, mix, residual activity amino sites is sealed, at 36 ℃, react 20 minutes.After having reacted, by concentration, be magnetic particle after the PBS damping fluid washing sealing that is 7.4 of 0.02 mole, pH, suspend, obtain superparamagnetism composite particle mark recombinant protein A finished product, be placed in 4 ℃ of preservations stand-by.
Wherein, being prepared as of borate buffer solution: take 1.9g Na
2b
4o
710H
2o is dissolved in 100ml pure water, adjusts pH to 8.5, obtains.Being prepared as of PBS damping fluid: take 2.3 grams of Na
2hPO
4, 0.524 gram of NaH
2pO
4h
2o, 8.77 grams of NaCl are dissolved in 1L pure water, adjust pH to 7.4, obtain.
(2) preparation of immune chromatography test paper
Shown in Fig. 1, employing concentration is the PB damping fluid of 0.02M, pH=7.4, will resist recombinant protein A antibody to be formulated as concentration 2mg/ml (manner of formulation is as embodiment mono-); The concentration of gp120 is formulated as to concentration 2mg/ml (manner of formulation is as embodiment mono-); Select the BioJet shower nozzle in the XYZ3050 spray film system of BioDot the anti-recombinant protein A antibody after preparing to be sprayed onto to the position of nature controlling line 5 on nitrocellulose filter 2, the gp120 preparing is sprayed onto to the position of detection line 4, in relative humidity is the drying plant below 10%, carries out dehumidifier dried for standby after 4 hours.With the 0.02M PBS(pH=7.4 containing 0.2%TritonX100,1%BSA, 1% sucrose) solution soaks all-glass paper 1 hour, the temperature of soaking is 37 ℃, in same dehumidifier condition, carry out dehumidifier after 4 hours, the damping fluid of processing with above-mentioned glass fibre is pressed after the recombinant protein A of 50 times of dilution superparamagnetism composite particle marks, adopt AirJet shower nozzle in the XYZ3050 spray film system of BioDot this dilution magnetic mark compound to be sprayed into preparation on the glass fibre element film of above-mentioned processing and form sample pad 1, in same dehumidifier condition, be dried.In 100,000 grades of cleanings and dry workshop, above-mentioned dried sample pad 1, nitrocellulose filter 2 and adsorptive pads 3 are got up by backboard 6 bondings successively; can be at the surperficial bonding protective film of sample pad 1, nitrocellulose filter 2 and adsorptive pads 3; then the CM4000 cutting system that adopts BioDot is the width of 5mm/ bar by the Paperboard cutting posting; pack into detect and use intermediate plate stand-by, obtain detecting the immune chromatography test paper of mankind's AIDS virus 1+2 type antibody.
For the effect of checking immune chromatography test paper of the present invention, spy analyzes the performance of this immune chromatography test paper.Test as follows:
1, main material:
1.1 mankind's AIDS virus (HIV) 1+2 type antibody immune chromatography test paper are obtained by embodiment mono-;
1.2 mankind's AIDS virus (HIV) 1+2 type antibody National references (ELISA): provided by Nat'l Pharmaceutical & Biological Products Control Institute;
1.3 clinical serum: select altogether clinical sample 1064 examples, wherein 390 parts, HIV-1 the infected's sample of HIV antibody positive (sample preserved of middle inspection comprise 187 parts of HIV-1 antibody and nucleic acid all positive, 1 part of nucleic acid positive but the negative HIV-I of HIV antibody infects window phase sample, wherein 153 duplicate samples are successfully completed Genotyping, B hypotype accounts for 33.33%, BC recombinant type accounts for 57.52%, AE recombinant type accounts for 9.15%), the HIV-1 of HIV negative antibody infects 1 part, window phase sample, from Nat'l Pharmaceutical & Biological Products Control Institute and PLA's acquired immune deficiency syndrome (AIDS), detects Confirmatory laboratory; 673 parts of HIV negative samples, wherein each 100 parts, HBV and HCV the infected's sample are from 302 hospitals of PLA, and 473 parts of HIV negative samples detect Confirmatory laboratory from Nat'l Pharmaceutical & Biological Products Control Institute and PLA's acquired immune deficiency syndrome (AIDS).
2, detection method: first detected sample is recovered to room temperature before detection.Mankind's AIDS virus (HIV) 1+2 type antibody immune chromatography test paper is taken out and well is lain on experiment table top outwardly.Use accurate pipettor to get the filling opening front end aperture that detected sample 50 μ l vertically slowly splash into test strips, along filling opening rear end aperture, vertically slowly splash into 100 μ l washing fluids immediately.In incubated at room, after 20 minutes, with magnetic test paper analyser, carry out test judgement result.Its washing fluid is 7.4PBS damping fluid for containing 0.02M, the pH of 2mlTween20.
3, testing result:
3.1 mankind's AIDS virus (HIV) 1+2 type antibody National references (ELISA) detect: National reference serum dish is detected, and result is consistent with expection, by national Specification (in Table 1).
Table 1:HIV1+2 type antibody National reference (ELISA) testing result
| Test item | Quantity | Standard code | Testing result |
| HIV(+) | 20 | 20/20 | 20/20 |
| HIV(-) | 20 | ≥18/20 | 20/20 |
| |
6 | ≥3/6 | 3/6 |
| |
2 | 10/10+/+ | 10/10+ /+, testing result is consistent |
3.2 clinical serum detect: clinical detection the results are shown in Table 2:
Table 2: clinical serum testing result
The positive HIV-1 the infected's sample of 390 parts of HIV antibody is through this detection paper, and result is all positive; 1 part of negative HIV-1 of HIV antibody infects window phase sample through this detection paper, and result is negative.Therefore the susceptibility of this test paper is 99.74%.In 673 parts of HIV negative antibody samples, 663 these detection paper of duplicate samples are negative, and 10 these detection paper of duplicate samples are positive.This 10 duplicate samples, from many HIV antibody diagnosing reagent marks of Nat'l Pharmaceutical & Biological Products Control Institute ，Jing China, is HIV negative antibody.Thus, there are 10 parts of false positives in this test paper, and specificity is 98.51%.This test paper sample consistent with Confirmation reagent result is 1054 parts altogether, and overall coincidence rate is 99.06%.
Finally it should be noted that, above embodiment is the unrestricted technical scheme of the present invention in order to explanation only, although the present invention is had been described in detail with reference to above-described embodiment, those skilled in the art are to be understood that, still can modify or be equal to replacement the present invention, and not departing from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in claim scope of the present invention.
Claims (10)
1. detect the immune chromatography test paper of mankind antibody of AIDS virus, it is characterized in that: comprise the sample pad (1), nitrocellulose membrane (2) and the adsorptive pads (3) that connect successively, described sample pad (1) contains superparamagnetism composite particle mark recombinant protein A; Described nitrocellulose membrane (2) is coated with detection line (4) and the nature controlling line (5) being separated from each other, described detection line (4) contains mankind's AIDS virus recombinant antigen, described nature controlling line (5) contain can with the anti-recombinant protein A antibody of described recombinant protein A specific bond; Described mankind's AIDS virus behaviour AIDS-like disease virus 1+2 type.
2. the immune chromatography test paper of detection mankind antibody of AIDS virus according to claim 1, is characterized in that: described superparamagnetism composite particle mark recombinant protein A is that recombinant protein A and superparamagnetism composite particle are formed to condensate with peptide bond covalent bond.
3. the immune chromatography test paper of detection according to claim 2 mankind antibody of AIDS virus, is characterized in that: the preparation method of described superparamagnetism composite particle mark recombinant protein A comprises the following steps:
The preparation of a, activation magnetic particle: by ratio of weight and the number of copies, take 2～3 parts of superparamagnetism composite particles, EDC0.5～1.5 part and NHS1～3 part, mix, react 20～40 minutes at 35 ℃～40 ℃, with damping fluid washing, obtain activation magnetic particle subsequently;
B, the reactant liquor preparation that contains magnetic particle after coupling: by ratio of weight and the number of copies, take 0.5～1 part of 2～3 parts of described activation magnetic particles, 0.1～0.2 part of recombinant protein A and borate buffer solution, mix, at 35 ℃～40 ℃, react 3～4 hours, must contain the reactant liquor of magnetic particle after coupling;
C, contain the reactant liquor of magnetic particle after coupling described in getting, add the BSA solution that accounts for whole described reactant liquor volume 0.5%～1.5%, mix, at 35 ℃～40 ℃, react 20～40 minutes, after the sealing subsequently reaction being obtained, magnetic particle washs, suspends, and obtains superparamagnetism composite particle mark recombinant protein A finished product.
4. the immune chromatography test paper of detection mankind antibody of AIDS virus according to claim 3, is characterized in that: described superparamagnetism composite particle is superparamagnetism Fe
3o
4nano particle; The particle diameter of described superparamagnetism composite particle is 60nm～300nm, and the deviation of this particle diameter is between 10%～30%; The magnetic saturation intensity of described superparamagnetism composite particle is 30emu/g～80emu/g, and corresponding external magnetic field response speed is 20 seconds～100 seconds.
5. the immune chromatography test paper of detection according to claim 2 mankind antibody of AIDS virus, it is characterized in that: described recombinant protein A and superparamagnetism composite particle are being formed before condensate with peptide bond covalent bond, also comprise this superparamagnetism composite particle is activated, make superparamagnetism composite particle surface with functional group.
6. the immune chromatography test paper of detection according to claim 5 mankind antibody of AIDS virus, is characterized in that: described functional group is a kind of in carboxyl or amino; The content of described functional group is 50～500 μ mol/g.
7. the immune chromatography test paper of detection mankind antibody of AIDS virus according to claim 1, is characterized in that: the concentration that contains mankind's AIDS virus recombinant antigen in described detection line (4) is 0.5～2mg/ml; The concentration that contains anti-recombinant protein A antibody in described nature controlling line (5) is 0.5～2mg/ml.
8. the immune chromatography test paper of detection mankind antibody of AIDS virus according to claim 1, is characterized in that: described mankind's AIDS virus recombinant antigen behaviour AIDS-like disease virus 1+2 type genetic engineering recombinant antigen; Described mankind's AIDS virus 1+2 type genetic engineering recombinant antigen is a kind of in gp36, gp41 or gp120.
9. the preparation method of the immune chromatography test paper of the detection mankind antibody of AIDS virus based on described in any one in claim 1-8, it is characterized in that: comprise the following steps: sample pad (1), nitrocellulose membrane (2) and adsorptive pads (3) are connected successively, by containing mankind's AIDS virus recombinant antigen and containing, can be sprayed at respectively with the anti-recombinant protein A antibody of described recombinant protein A specific bond the zones of different at nitrocellulose membrane (2) two ends, corresponding detection line (4) and the nature controlling line (5) of forming, obtains finished product.
10. the application of immune chromatography test paper in preparation mankind AIDS virus 1+2 type antibody test test paper according to claim 1.
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