CN103983776A - Colloidal gold immunochromatography test strip for simultaneously detecting viral antigens and antibodies - Google Patents
Colloidal gold immunochromatography test strip for simultaneously detecting viral antigens and antibodies Download PDFInfo
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- CN103983776A CN103983776A CN201410228165.4A CN201410228165A CN103983776A CN 103983776 A CN103983776 A CN 103983776A CN 201410228165 A CN201410228165 A CN 201410228165A CN 103983776 A CN103983776 A CN 103983776A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention relates to a colloidal gold immunochromatography test strip for simultaneously detecting viral antigens and antibodies. The test strip comprises a first glue strip and a second glue strip which are arranged in parallel, wherein the first glue strip comprises a first sample pad, a first conjugate pad, a first nitrocellulose membrane and first absorbent paper, which are sequentially lapped with one another, the first conjugate pad is coated with colloidal gold marked by the viral antigens, the first nitrocellulose membrane is coated with viral antibodies and antibodies, the viral antibodies are taken as a first detection line, and the antibodies are used for resisting the viral antigens on the colloidal gold and are taken as a quality control line; the second glue strip comprises a second sample pad, a second conjugate pad, a second nitrocellulose membrane and second absorbent paper, which are sequentially lapped with one another, the second conjugate pad is coated with the colloidal gold marked by the viral antigens, the second nitrocellulose membrane is coated with anti-human IgG antibodies and anti-human IgM antibodies, the anti-human IgG antibodies are taken as a second detection line, and the anti-human IgM antibodies are taken as a third detection line. The colloidal gold immunochromatography test strip can be used for simultaneously detecting the viral antigens as well as the IgG antibodies and IgM antibodies of the viral antigens.
Description
Technical field
The invention belongs to immune detection analysis technical field, relate to a kind of colloidal gold immuno-chromatography test paper strip that simultaneously detects viral antigen and specific immunoglobulin M (IgM) and immunoglobulin G (IgG).
Background technology
Influenza (abbreviation influenza) is the ARI that influenza virus causes, is also the disease that a kind of infectiousness is strong, velocity of propagation is fast.Its mainly by the airborne spittle, interpersonal contact or with the contact transmission of contaminated article.Typical clinical symptoms is: anxious high heat, whole body pain, the remarkable weak and slight respiratory symptom of rising.Generally, autumn and winter season is its high-incidence season, and caused complication and the phenomena of mortality are very serious.This disease is to cause influenza by influenza virus, Tobamovirus orthomyxoviridae family, diameter 80～120nm is spherical or thread.Influenza virus can be divided into first (A), second (B), third (C) three types, and antigenic variation often occurs influenza A virus, and infectiousness is large, propagates rapidly, very easily occurs popular on a large scale.H1N1 is a kind of influenza A virus namely, and this disease has self limiting, but infant, the elderly with have the severe complications such as the easy Complicating Pneumonia In Patients of patient of cardiopulmonary underlying diseases and cause death.Correct this pathogen of diagnosis is of great significance rapid healing tool.
Influenza A virus can be divided into 1-16 kind hypotype according to the difference of influenza virus hemagglutinin albumen (HA), according to the difference of viral neuraminic acid zymoprotein (NA), can be divided into 1-9 kind hypotype, HA different subtype can be combined to form different influenza viruses mutually from the different subtype of NA.
Immunoglobulin M (IgM) and immunoglobulin G (IgG) are most important two kinds of antibody in the mankind or animal body.As shown in Figure 1, after organism infection influenza virus, originally do not have enough time to cause the immune response of body, therefore, now viral antigen can only be detected.Afterwards, cause the immune response of body, stimulate body to produce antibody IgM and IgG antibody.As shown in Figure 2, after organism infection influenza virus, what occur the earliest in vivo is the IgM antibody of influenza virus, occurs afterwards the specific IgG antibodies of influenza virus again.Therefore, by detecting body inner virus antigen, and the existence of specific IgM antibody and IgG antibody whether, can diagnose the immune response state of body infected by influenza.If viral antigen can only be detected, illustrate that body just infects several days, if detect the IgM antibody of virus-specific in serum, prove that this body has the generation of recent influenza, but because the IgM half life period is shorter, the detection feminine gender of IgM can not prove that body is not subject to the infection of influenza, also needs to detect the IgG antibody that the half life period is long, content is the highest, to clarify a diagnosis.And viral antigen exists a mxm. in body, concentration also can change over time, can have situation about can't detect.Therefore, by detect influenza antigen and specific IgM antibody and IgG antibody simultaneously, can judge accurately the immunological response state of this body, thereby according to testing result, provide testing result accurately, finally give certain clinical treatment.Regrettably, do not detect the test strips of influenza antigen and specific IgM antibody and IgG antibody at present simultaneously.
Summary of the invention
The object of the invention is to, a kind of colloidal gold immuno-chromatography test paper strip that simultaneously detects viral antigen and antibody is provided.Described colloidal gold immuno-chromatography test paper strip can not only detect viral antigen and specific IgM antibodies and IgG antibody in one-time detection simultaneously, and can avoid undetected, can greatly shorten detection time and reduce testing cost, reaches the object of Site Detection.
For realizing object of the present invention, the invention provides following technical scheme:
Detect a colloidal gold immuno-chromatography test paper strip for viral antigen and antibody simultaneously, comprise the first adhesive tape and the second adhesive tape that are set up in parallel; Described the first adhesive tape comprises the first sample pad, the first bond pad, the first nitrocellulose filter and first thieving paper of overlap joint in turn, on wherein said the first bond pad, be coated with the collaurum of antiviral antibody mark, on described the first nitrocellulose filter, be coated with antiviral antibody as the first detection line the antibody that is coated with antiviral antibody on anti-collaurum as nature controlling line; Described the second adhesive tape comprises the second sample pad, the second bond pad, the second nitrocellulose filter and second thieving paper of overlap joint in turn, on wherein said the second bond pad, be coated with the collaurum of viral antigen mark, on described the second nitrocellulose filter, be coated with anti-human IgG antibody as the second detection line and be coated with anti-human IgM antibody as the 3rd detection line.
As the preferred technical solution of the present invention, on described collaurum, antiviral antibody is mouse antibody, and the described antibody as nature controlling line is sheep anti-mouse igg.Mouse antibody is modal antibody formation in current commercialization antibody, wide material sources, and cost is lower, and specificity and good stability.Described antiviral antibody can be polyclonal antibody or monoclonal antibody, the method of preparing described polyclonal antibody or monoclonal antibody is well known to a person skilled in the art, such as can be with corresponding virus immunity animal, as mouse, purifying polyclonal antibody from animal blood serum, but polyclonal antibody is compared monoclonal antibody, may there is the problem that specificity is not strong, likely have the generation of false positive results.Specific to the present invention, although described antiviral antibody can adopt polyclonal antibody, but monoclonal antibody has more obvious advantage, therefore be of the present invention preferred, the method of preparing described monoclonal antibody is such as can be with corresponding virus immunity animal, as mouse, get animal spleen B cell and murine myeloma cell and merge formation hybridoma, therefrom obtain the monoclonal antibody that specificity is stronger.
As the preferred technical solution of the present invention, described anti-human IgG antibody is rabbit anti-human igg's antibody or goat anti-human igg antibody, is preferably rabbit anti-human igg's antibody.
As the preferred technical solution of the present invention, described anti-human IgM antibody is the anti-human IgM antibody of rabbit or goat-anti human IgM antibody, is preferably the anti-human IgM antibody of rabbit.
As the preferred technical solution of the present invention, described virus is influenza A virus, influenza B virus, adenovirus or parainfluenza virus, is preferably influenza A virus, influenza B virus or adenovirus.
As the preferred technical solution of the present invention, the particle diameter of described collaurum is 20～30nm.Particle size range has excellent dispersed and mobility in fluid at the collaurum of 20～30nm, does not have the generation of the phenomenons such as reunion, is conducive in the present invention the immune response between antibody and antigen and with the movement under capillary action.
As the preferred technical solution of the present invention, described sample pad is glass fibre membrane or dacron film, is preferably glass fibre membrane.
As the preferred technical solution of the present invention, described bond pad is glass fibre membrane.
As the preferred technical solution of the present invention, described colloidal gold immuno-chromatography test paper strip also comprises base plate, and described the first adhesive tape and the second adhesive tape are all arranged on described base plate.
Preferably, described base plate is polyvinyl chloride plastic sheet.
As the preferred technical solution of the present invention, described colloidal gold immuno-chromatography test paper strip also comprises and getting stuck, in getting stuck described in described the first adhesive tape and the second adhesive tape are all placed in.
Preferably, described in have sample application zone and colour developing district on getting stuck, described sample application zone is corresponding with described the first sample pad and the second sample pad, described colour developing district is corresponding with described the first nitrocellulose filter and the second nitrocellulose filter.
Preferably, the quantity of described sample application zone is 2, corresponding with described the first sample pad and the second sample pad respectively.
Preferably, the quantity in described colour developing district is 2, corresponding with described the first nitrocellulose filter and the second nitrocellulose filter respectively.
Colloidal gold immuno-chromatography test paper strip of the present invention is measured viral antigen and specific IgM and IgG antibody according to immunocapture method principle and double antibody sandwich method principle simultaneously, by disposable operation, can joint-detection go out the viral antigens such as influenza and specific IgM and IgG antibody.This colloidal gold immuno-chromatography test paper strip has the advantages such as high specificity, cheap and reading result be quick and directly perceived, does not need special instruments and equipment, does not also need professional's operation, and the overall coincidence rate of testing result is higher, is suitable for Site Detection.And detect when importantly realizing antigen and antibody, avoid undetected, play double insurance effect, thereby obtain more relevant informations of organism infection influenza virus, there is important clinical meaning.
Accompanying drawing explanation
After Fig. 1 is organism infection influenza virus, the concentration of body inner virus antigen and total antibody thereof is situation over time;
After Fig. 2 is organism infection influenza virus, body endoantigen/antibody is situation over time;
Fig. 3 is the schematic diagram that the present invention detects the colloidal gold immuno-chromatography test paper strip of viral antigen and antibody simultaneously, wherein, and 1: influenza antigen sample application zone; 2: Serum Antibody Detection sample application zone; 3: antigen detects observation window; 4: antibody test observation window; 5: the first detection lines (T1 line); 6: nature controlling line (C line); 8: the three detection lines (T3 line) of 7: the second detection lines (T2 line);
Fig. 4 is viral antigen and the antibody test schematic diagram in the present invention;
Fig. 5 is viral antigen positive test symbol schematic diagram of the present invention;
Fig. 6 is viral antigen/IgM antibody positive testing result schematic diagram of the present invention;
Fig. 7 is viral antigen/IgM/IgG antibody positive testing result schematic diagram of the present invention;
Fig. 8 is viral antigen/IgG antibody positive testing result schematic diagram of the present invention;
Fig. 9 is virus IgM antibody positive test symbol schematic diagram of the present invention;
Figure 10 is virus IgM/IgG antibody positive testing result schematic diagram of the present invention;
Figure 11 is virus IgG antibody positive test symbol schematic diagram of the present invention;
Figure 12 is the present invention's virus negative result schematic diagram;
Figure 13 is the present invention's virus invalid detection result schematic diagram;
Figure 14 is transmission electron microscope (TEM) photo of gold nano grain in the embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that following examples are only the preferred embodiments of the present invention, so that understand better the present invention, thereby should not be considered as limiting scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method; Experiment material used, if no special instructions, is and is purchased available from routine biochemistry chemical reagent work.
The present invention can detect the colloidal gold immuno-chromatography test paper strip of influenza antigen and specific IgM thereof, IgG antibody simultaneously can prepare (with reference to figure 2) by the following method:
On Polyvinylchloride (PVC) offset plate, one end is sticked sample pad, bond pad (two), nitrocellulose filter, thieving paper in turn mutually overlap joint, then the material of PVC offset plate and attaching is cut into two test strips (width is as 4mm), and in packing into and getting stuck, on getting stuck, have two sample application zone and two colour developing districts's (viewing area).
Wherein, described sample pad is glass fibre membrane; Described bond pad comprises: be coated with the first bond pad of the collaurum of Antibody of Influenza mark, be coated with the second bond pad of the collaurum of influenza antigen mark; Described nitrocellulose filter comprises: the first nitrocellulose filter and the second nitrocellulose filter, on described the first nitrocellulose filter, be coated with: the coated antibody that T1 line is influenza virus, C line is sheep anti-mouse igg antibody, on described the second nitrocellulose filter, be coated with: T2 line is the anti-human IgG antibody of rabbit, T3 line is the anti-human IgM antibody of rabbit.
As shown in Figure 4, the present invention can detect the detection principle of the colloidal gold immuno-chromatography test paper strip of influenza antigen and specific IgM thereof, IgG antibody simultaneously, specific as follows:
In sample application zone (sample pad), add after detected sample, by capillary action, detect in the region of viral antigen: in sample, the bond of Ag-Ab-collaurum moves to the direction of thieving paper one end.If the antigen that contains this influenza virus in detected sample, sample moves to T1 line while being the coated line of coated antibody of influenza virus, the bond of Ag-Ab-collaurum is hunted down, and generates the bond of antibody-Ag-Ab-collaurum at T1 line place, therefore shows a red stripes; Sample continue to flow, and when flowing to C line and being sheep anti-mouse igg antibody, there will be a red stripes, proves the validity of this test strips.If there is not viral antigen in the actual sample detecting, can not show red stripes at T1 line place.In the region of detecting serum antibody, while adding serum to be detected, if the IgG in sample contains the IgG antibody for influenza antigen, when sample is the coated line of anti-human IgG antibody by T2 line, IgG antibody in sample is combined with anti-human IgG antibody, the bond of IgG-antigen-collaurum is hunted down, and at T2 line, shows a red stripes; Sample continues to move to T3 place while being the anti-human coated line of IgM, and the IgM antibody in sample is combined with anti-human IgM antibody, and the bond of IgM-antigen-collaurum is hunted down, in a red stripes of T3 line demonstration.If during not for the IgG of this influenza virus and IgM antibody, can not show red stripes at T2 and T3 line place in the actual sample detecting.If only have the colour developing of C line, all negative, prove that this body does not infect influenza.As long as C line does not develop the color, prove that this test strips is invalid, need to again detect actual sample.Various testing result schematic diagram are as shown in Fig. 5-13, and table 1 has gathered the explanation of various testing results.
Table 1 testing result statistics ("+" colour developing, "-" do not develop the color)
The preparation and determination methods of colloidal gold immuno-chromatography test paper strip of the present invention is described below by embodiment.Be to be understood that these embodiment are only exemplary, should not limit the scope of the invention.
Reagent used and the source of instrument and equipment in embodiment: the antigen of influenza A virus, influenza B virus and adenovirus and antibody thereof are purchased from Beijing Mo Zhidong Bioisystech Co., Ltd; Rabbit/goat-anti human IgM antibody and rabbit/goat anti-human igg antibody are purchased from Beijing Bo Aosen Bioisystech Co., Ltd; Nitrocellulose filter is purchased from Merck Mi Libo; Three-dimensional planar is drawn film instrument, three-dimensional planar gold spraying instrument purchased from Jin Biao bio tech ltd, Shanghai; Freeze drier is purchased from Beijing Bo Yikang experimental apparatus company limited; Cutting cutter is purchased from Jin Biao bio tech ltd, Shanghai; PVC offset plate, thieving paper, glass fibre membrane and get stuck purchased from Jin Biao bio tech ltd, Shanghai.
Embodiment 1: influenza A virus detects
The detection of influenza A virus antigen:
(1) get 200 μ g Flu-A labelled antibodies and be placed in bag filter to the 5mM Tris-HCl 24h that dialyses, in this process, every 2h, change water one time, after having dialysed, antibody is taken out and to be placed in centrifuge tube, add ultrapure water to 2mL, discard precipitated impurities after centrifugal.
(2) prepare golden labeling antibody bond pad: adopt sodium citrate reduction gold chloride legal system for collaurum, glass apparatus used, in advance with the washing lotion immersion of spending the night, is then rinsed well with deionized water.In beaker, add 1000mL ultrapure water, then add 1% the sodium citrate solution of 10mL, be heated to after boiling, then add the chlorauric acid solution of 10mL1%, boil after 15min to cooling, be placed in 4 ℃ of preservations.Particle diameter is approximately 20～30nm (as Figure 14), gets 20mL colloidal gold solution and is placed in beaker, adds the K of 200 μ L0.01M
2cO
3the pH of solution regulator solution, stir, then add antibody, stir 20min left and right, then the BSA solution that adds 2mL10%, carry out centrifugal (40min, 10000rpm), then abandoning supernatant, add again the solution (buffer solution of the pH8.6 that contains 1mM) of 1% BSA to continue centrifugal, abandoning supernatant, sediment is recovered, the composition that recovers liquid is: the NaCl of 50mM, 1% BSA, 0.5% sucrose and 0.5% casein-sodium, be coated in above 200 square centimeters of big or small glass fibre membranes,-45 ℃ freezing after, as bond pad, after freeze-drying, room temperature keeps in Dark Place standby.
(3) by the coated antibody of sheep anti-mouse igg and influenza A virus, as nature controlling line (C) and the first p-wire (T1), be coated on nitrocellulose filter respectively, then nitrocellulose filter, thieving paper, gold mark bond pad and sample pad are attached on PVC base plate successively, and assemble.Carry out the detection of first stream viral antigen, the detection sensitivity of viral antigen is 30ppb.
The detection of influenza A virus antibody:
(1) get 200 μ g influenza A virus antigens and be placed in bag filter to the 5mM Tris-HCl 24h that dialyses, in this process, every 2h, change water one time, after having dialysed, antigen is taken out and to be placed in centrifuge tube, add ultrapure water to 2mL, discard precipitated impurities after centrifugal.
(2) preparation gold mark antigen bond pad: adopt sodium citrate reduction gold chloride legal system for collaurum, glass apparatus used, in advance with the washing lotion immersion of spending the night, is then rinsed well with deionized water.To in beaker, add 1000mL tri-distilled water, then add 1% the sodium citrate solution of 10mL, be heated to after boiling, then add the chlorauric acid solution of 10mL1%, boil after 15min to cooling, be placed in 4 ℃ of preservations.Particle diameter is approximately 20～30nm, gets 20mL colloidal gold solution and is placed in beaker, adds the K of 200 μ L0.01M
2cO
3the pH of solution regulator solution, stir, then add antigen, stir 20min left and right, then the BSA solution that adds 2mL10%, carry out centrifugal (40min, 10000rpm), then abandoning supernatant, add again the solution (buffer solution of the pH8.6 that contains 1mM) of 1% BSA to continue centrifugal, abandoning supernatant, sediment is recovered, the composition that recovers liquid is: the NaCl of 50mM, 1% BSA, 0.5% sucrose and 0.5% casein-sodium, be coated in above 200 square centimeters of big or small glass fibre membranes,-45 ℃ freezing after, as bond pad, after freeze-drying, room temperature keeps in Dark Place standby.
(3) rabbit anti-human igg's antibody and the anti-human IgM antibody of rabbit are coated on nitrocellulose filter as T2 and T3 line respectively, then nitrocellulose filter, thieving paper, gold mark bond pad and sample pad are attached on PVC base plate successively, and assemble.Carry out the detection of influenza A virus antibody.Get the clinical first stream patient's who has made a definite diagnosis clarification serum sample.
The testing result of influenza A virus antigen and antibody is as shown in table 2, will in institute's testing result and table 1, compare, and judges patient's Infection Status.
The testing result of table 2 Flu-A actual sample
| T1 line | T2 line | T3 line | C line | Read testing result number |
| + | - | - | + | 2 |
| + | - | + | + | 3 |
| + | + | + | + | 6 |
| + | + | - | + | 4 |
| - | - | + | + | 5 |
| - | + | + | + | 4 |
| - | + | - | + | 4 |
| - | - | - | + | 0 |
| -/+ | -/+ | -/+ | - | 0 |
Embodiment 2: influenza B virus detects
The detection of influenza B virus antigen:
(1) get 200 μ g influenza B labelled antibodies and be placed in bag filter to the 5mM Tris-HCl 24h that dialyses, in this process, every 2h, change water one time, after having dialysed, antibody is taken out and to be placed in centrifuge tube, add ultrapure water to 2mL, discard precipitated impurities after centrifugal.
(2) prepare golden labeling antibody bond pad: adopt sodium citrate reduction gold chloride legal system for collaurum, glass apparatus used, in advance with the washing lotion immersion of spending the night, is then rinsed well with deionized water.To in beaker, add 1000mL ultrapure water, then add 1% the sodium citrate solution of 10mL, be heated to after boiling, then add the chlorauric acid solution of 10mL1%, boil after 15min to cooling, be placed in 4 ℃ of preservations.Particle diameter is approximately 20～30nm, gets 20mL colloidal gold solution and is placed in beaker, adds the K of 200 μ L0.01M
2cO
3the pH of solution regulator solution, stir, then add antibody, stir 20min left and right, then the BSA solution that adds 2mL10%, carry out centrifugal (40min, 10000rpm), then abandoning supernatant, add again the solution (buffer solution of the pH8.6 that contains 1mM) of 1% BSA to continue centrifugal, abandoning supernatant, sediment is recovered, the composition that recovers liquid is: the NaCl of 50mM, 1% BSA, 0.5% sucrose and 0.5% casein-sodium, be coated in above 200 square centimeters of big or small glass fibre membranes,-45 ℃ freezing after, as bond pad, after freeze-drying, room temperature keeps in Dark Place standby.
(3) by the coated antibody of sheep anti-mouse igg and influenza B virus, as nature controlling line (C) and the first p-wire (T1), be coated on nitrocellulose filter respectively, then nitrocellulose filter, thieving paper, gold mark bond pad and sample pad are attached on PVC base plate successively, and assemble.Carry out the detection of influenza B virus antigen, the detection sensitivity of viral antigen is 15ppb.
The detection of influenza B virus antibody:
(1) get 200 μ g influenza B virus antigens and be placed in bag filter to the 5mM Tris-HCl 24h that dialyses, in this process, every 2h, change water one time, after having dialysed, antigen is taken out and to be placed in centrifuge tube, add ultrapure water to 2mL, discard precipitated impurities after centrifugal.
(2) preparation gold mark antigen bond pad: adopt sodium citrate reduction gold chloride legal system for collaurum, glass apparatus used, in advance with the washing lotion immersion of spending the night, is then rinsed well with deionized water.To in beaker, add 1000mL tri-distilled water, then add 1% the sodium citrate solution of 10mL, be heated to after boiling, then add the chlorauric acid solution of 10mL1%, boil after 15min to cooling, be placed in 4 ℃ of preservations.Particle diameter is approximately 20～30nm, gets 20mL colloidal gold solution and is placed in beaker, adds the K of 200 μ L0.01M
2cO
3the pH of solution regulator solution, stir, then add centrifugal good antigen, stir 20min left and right, then the BSA solution that adds 2mL10%, carry out centrifugal (40min, 10000rpm), then abandoning supernatant, add again the solution (buffer solution of the pH8.6 that contains 1mM) of 1% BSA to continue centrifugal, abandoning supernatant, sediment is recovered, the composition that recovers liquid is: the NaCl of 50mM, 1% BSA, 0.5% sucrose and 0.5% casein-sodium, be coated in above 200 square centimeters of big or small glass fibre membranes,-45 ℃ freezing after, as bond pad, after freeze-drying, room temperature keeps in Dark Place standby.
(3) rabbit anti-human igg's antibody and the anti-human IgM antibody of rabbit are coated on nitrocellulose filter as T2 and T3 line respectively, then nitrocellulose filter, thieving paper, gold mark bond pad and sample pad are attached on PVC base plate successively, and assemble.Carry out the detection of second stream antiviral antibody.The clarification serum sample of getting the clinical infection influenza B virus patient who has made a definite diagnosis, the testing result of influenza B virus antigen and antibody is as shown in table 3, will in institute's testing result and table 1, compare, and judges patient's Infection Status.
The detection of table 3 influenza B actual sample
| T1 line | T2 line | T3 line | C line | Read testing result number |
| + | - | - | + | 10 |
| + | - | + | + | 2 |
| + | + | + | + | 3 |
| + | + | - | + | 5 |
| - | - | + | + | 4 |
| - | + | + | + | 5 |
| - | + | - | + | 3 |
| - | - | - | + | 0 |
| -/+ | -/+ | -/+ | - | 0 |
Embodiment 3: the detection of adenovirus
The detection of Adenovirus Antigen:
(1) labelled antibody of getting 200 μ g adenovirus is placed in bag filter to the 5mM Tris-HCl 24h that dialyses, and in this process, every 2h, changes water one time, after having dialysed, antibody is taken out and to be placed in centrifuge tube, add ultrapure water to 2mL, discard precipitated impurities after centrifugal.
(2) prepare golden labeling antibody bond pad: adopt sodium citrate reduction gold chloride legal system for collaurum, glass apparatus used, in advance with the washing lotion immersion of spending the night, is then rinsed well with deionized water.To in beaker, add 1000mL ultrapure water, then add 1% the sodium citrate solution of 10mL, be heated to after boiling, then add the chlorauric acid solution of 10mL1%, boil after 15min to cooling, be placed in 4 ℃ of preservations.Particle diameter is approximately 20～30nm, gets 20mL colloidal gold solution and is placed in beaker, adds the K of 200 μ L0.01M
2cO
3the pH of solution regulator solution, stir, then add antibody, stir 20min left and right, then the BSA solution that adds 2mL10%, carry out centrifugal (40min, 10000rpm), then abandoning supernatant, add again the solution (buffer solution of the pH8.6 that contains 1mM) of 1% BSA to continue centrifugal, abandoning supernatant, sediment is recovered, the composition that recovers liquid is: the NaCl of 50mM, 1% BSA, 0.5% sucrose and 0.5% casein-sodium, be coated in above 200 square centimeters of big or small glass fibre membranes,-45 ℃ freezing after, as bond pad, after freeze-drying, room temperature keeps in Dark Place standby.
(3) by the coated antibody of sheep anti-mouse igg and adenovirus, as nature controlling line (C) and the first p-wire (T1), be coated on nitrocellulose filter respectively, then nitrocellulose filter, thieving paper, gold mark bond pad and sample pad are attached on PVC base plate successively, and assemble.Carry out the detection of Adenovirus Antigen, the detection sensitivity of viral antigen is 30ppb.
The detection of adenovirus antibody:
(1) get 200 μ g Adenovirus Antigens and be placed in bag filter to the 5mM Tris-HCl 24h that dialyses, in this process, every 2h, change water one time, after having dialysed, antigen is taken out and is placed in centrifuge tube, add ultrapure water to 2mL, discard precipitated impurities after centrifugal.
(2) preparation gold mark antigen bond pad: adopt sodium citrate reduction gold chloride legal system for collaurum, glass apparatus used, in advance with the washing lotion immersion of spending the night, is then rinsed well.To in beaker, add 1000mL tri-distilled water, then add 1% the sodium citrate solution of 10mL, be heated to after boiling, then add the chlorauric acid solution of 10mL1%, boil after 15min to cooling, be placed in 4 ℃ of preservations.Particle diameter is approximately 20～30nm, gets 20mL colloidal gold solution and is placed in beaker, adds the K of 200 μ L0.01M
2cO
3the pH of solution regulator solution, stir, then add antigen, stir 20min left and right, then the BSA solution that adds 2mL10%, carry out centrifugal (40min, 10000rpm), then abandoning supernatant, add again the solution (buffer solution of the pH8.6 that contains 1mM) of 1% BSA to continue centrifugal, abandoning supernatant, sediment is recovered, the composition that recovers liquid is: the NaCl of 50mM, 1% BSA, 0.5% sucrose and 0.5% casein-sodium, be coated in above 200 square centimeters of big or small glass fibre membranes,-45 ℃ freezing after, as bond pad, after freeze-drying, room temperature keeps in Dark Place standby.
(3) rabbit anti-human igg's antibody and the anti-human IgM antibody of rabbit are coated on nitrocellulose filter as T2 and T3 line respectively, then nitrocellulose filter, thieving paper, gold mark bond pad and sample pad are attached on PVC base plate successively, and assemble.Carry out the detection of adenovirus antibody.The clarification serum sample of getting the clinical infection adenovirus patient who has made a definite diagnosis, the testing result of Adenovirus Antigen and antibody is as shown in table 4, will in institute's testing result and table 1, compare, and judges patient's Infection Status.
The testing result of table 4 adenovirus actual sample
| T1 line | T2 line | T3 line | C line | Read testing result number |
| + | - | - | + | 4 |
| + | - | + | + | 2 |
| + | + | + | + | 5 |
| + | + | - | + | 3 |
| - | - | + | + | 5 |
| - | + | + | + | 3 |
| - | + | - | + | 5 |
| - | - | - | + | 0 |
| -/+ | -/+ | -/+ | - | 0 |
Applicant's statement, the present invention illustrates detailed features of the present invention and detailed method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and detailed method, do not mean that the present invention must rely on above-mentioned detailed features and detailed method could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention is selected the selection of the equivalence replacement of component and the interpolation of auxiliary element, concrete mode etc., within all dropping on protection scope of the present invention and open scope to the present invention.
Claims (10)
1. detect a colloidal gold immuno-chromatography test paper strip for viral antigen and antibody simultaneously, comprise the first adhesive tape and the second adhesive tape that are set up in parallel; Described the first adhesive tape comprises the first sample pad, the first bond pad, the first nitrocellulose filter and first thieving paper of overlap joint in turn, on wherein said the first bond pad, be coated with the collaurum of antiviral antibody mark, on described the first nitrocellulose filter, be coated with antiviral antibody as the first detection line the antibody that is coated with antiviral antibody on anti-collaurum as nature controlling line; Described the second adhesive tape comprises the second sample pad, the second bond pad, the second nitrocellulose filter and second thieving paper of overlap joint in turn, on wherein said the second bond pad, be coated with the collaurum of viral antigen mark, on described the second nitrocellulose filter, be coated with anti-human IgG antibody as the second detection line and be coated with anti-human IgM antibody as the 3rd detection line.
2. colloidal gold immuno-chromatography test paper strip according to claim 1, is characterized in that, on described collaurum, antiviral antibody is mouse antibody, and the described antibody as nature controlling line is sheep anti-mouse igg.
3. colloidal gold immuno-chromatography test paper strip according to claim 1 and 2, is characterized in that, described anti-human IgG antibody is rabbit anti-human igg's antibody or goat anti-human igg antibody, is preferably rabbit anti-human igg's antibody.
4. according to the colloidal gold immuno-chromatography test paper strip described in claim 1-3 any one, it is characterized in that, described anti-human IgM antibody is the anti-human IgM antibody of rabbit or goat-anti human IgM antibody, is preferably the anti-human IgM antibody of rabbit.
5. according to the colloidal gold immuno-chromatography test paper strip described in claim 1-4 any one, it is characterized in that, described virus is influenza A virus, influenza B virus, adenovirus or parainfluenza virus, is preferably influenza A virus, influenza B virus or adenovirus.
6. according to the colloidal gold immuno-chromatography test paper strip described in claim 1-5 any one, it is characterized in that, the particle diameter of described collaurum is 20～30nm.
7. according to the colloidal gold immuno-chromatography test paper strip described in claim 1-6 any one, it is characterized in that, described sample pad is glass fibre membrane or dacron film, is preferably glass fibre membrane.
8. according to the colloidal gold immuno-chromatography test paper strip described in claim 1-7 any one, it is characterized in that, described bond pad is glass fibre membrane.
9. according to the colloidal gold immuno-chromatography test paper strip described in claim 1-8 any one, it is characterized in that, described colloidal gold immuno-chromatography test paper strip also comprises base plate, and described the first adhesive tape and the second adhesive tape are all arranged on described base plate;
Preferably, described base plate is polyvinyl chloride plastic sheet.
10. according to the colloidal gold immuno-chromatography test paper strip described in claim 1-9 any one, it is characterized in that, described colloidal gold immuno-chromatography test paper strip also comprises and getting stuck, in getting stuck described in described the first adhesive tape and the second adhesive tape are all placed in;
Preferably, described in have sample application zone and colour developing district on getting stuck, described sample application zone is corresponding with described the first sample pad and the second sample pad, described colour developing district is corresponding with described the first nitrocellulose filter and the second nitrocellulose filter;
Preferably, the quantity of described sample application zone is 2, corresponding with described the first sample pad and the second sample pad respectively;
Preferably, the quantity in described colour developing district is 2, corresponding with described the first nitrocellulose filter and the second nitrocellulose filter respectively.
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Application publication date: 20140813 |