CN105092861A - Immunofluorescence chromatography test paper for CRP (C-reaction protein)/SAA (Serum amyloid A protein) quantitative combined detection and preparation method of immunofluorescence chromatography test paper - Google Patents
Immunofluorescence chromatography test paper for CRP (C-reaction protein)/SAA (Serum amyloid A protein) quantitative combined detection and preparation method of immunofluorescence chromatography test paper Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Abstract
The invention discloses immunofluorescence chromatography test paper for CRP (C-reaction protein)/SAA (Serum amyloid A protein) quantitative combined detection, aiming at providing a test strip capable of realizing quantitative combined detection on the content of CRP/SAA in human peripheral blood and quantitative combined detection on the content of CRP/SAA in venous blood. The immunofluorescence chromatography test paper is technically characterized by comprising a box with a cover, wherein a detection test strip and a blood diluent bottle are arranged in the box, the detection test strip comprises a bottom lining, the bottom lining is provided with a nitrocellulose membrane, one end of the nitrocellulose membrane is connected with a fluorescent microsphere marked antibody fixation pad, the fluorescent microsphere marked antibody fixation pad is connected with a sample pad, the other end of the nitrocellulose membrane is connected with an absorption pad, the fluorescent microsphere marked antibody fixation pad is coated by a CRP monoclonal antibody and an SAA monoclonal antibody, a CRP detection line coated by a CRP monoclonal antibody, an SAA detection line coated by an SAA monoclonal antibody, and a quality control line coated by a goat-anti-mouse IgG polyclonal antibody are arranged on the nitrocellulose membrane in parallel. The immunofluorescence chromatography test paper belongs to the technical field of biological medicines.
Description
Technical field
The present invention relates to a kind of fluorescent chromatographic test paper and preparation method thereof, specifically, is a kind of CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper and preparation method thereof; Belong to biomedicine technical field.
Background technology
C reactive protein (C-reaction, CRP) be a kind of acute phase protein synthesized by liver, trace is present in Healthy Human Serum, when body be infected or tissue damage time content raise rapidly, to improve along with the state of an illness or its content of recovery of institutional framework function returns to normal level.Current CRP as the sensitive indexes of systemic inflammatory reaction early detection, widespread use in the diagnosis of clinical numerous disease, treatment and prognosis.According to the concentration level of CRP in serum, blood plasma or whole blood, effectively can determine whether infection, the hazard level of disease and disease and whether be in active stage.
Serum amyloid A protein (SerumamyloidAprotein, SAA), a kind of Acute reaction protein equally, it is the sensitive indicator of a kind of inflammation or acute stage of infection, clinical meaning and CRP similar, detect the concentration level of SAA in blood, contribute to diagnosing non-specific inflammation, assessment course inflammatory activity degree, monitoring course inflammatory activity and the rear effect for the treatment of.
Research shows that the order of severity of CRP/SAA concentration level and numerous disease in blood is certain positive correlation, it is the test item of numerous disease auxiliary diagnosis, joint-detection CRP/SAA concentration level can the hazard level of more enough effective predictive diseases and morbidity thereof, and in body, CRP/SAA level rises and imply that body exists inflammation infection or disease is in active stage; In addition CRP/SAA joint-detection effectively can be differentiated, diagnose bacterium and virus infections, particularly infant infection disease is early stage, bacterium and virus are difficult to differentiate, and the change of joint-detection serum CA125 and SAA level and SAA/CRP ratio contributes to the antidiastole of children's's bacteriological infection and virus infections.CRP and SAA joint-detection can improve detection sensitivity and specificity, provides foundation more fully to antidiastole, and the two joint-detection has mutual supplement with each other's advantages and clinical value-added meaning, more meaningful than detecting separately a project.
And so, there is not the product of CRP/SAA simultaneous quantitative joint-detection in the market, detecting while the CRP/SAA concentration clinically to patient can only be detect item by item.Take time and effort on the one hand, cause the waste of reagent, consumptive material on the other hand, add the cost detecting treatment, and CRP and SAA is when body is in Infection Status, all measures to exist in body in units of mg/L, has the condition simultaneously detected.
In addition, the sample size that current most of quick detection reagent need is larger, finger tip peripheral blood cannot be applied detect, need to gather venous blood to detect, and the sample collection of blood needs professional to operate, this makes Rapid detection test strip be difficult to popularize community or the utilization of individual in diagnosis, health check-up etc.Research shows that finger peripheral blood acquisition operations is easy, does not need professional to operate, little to the harm of body, some Testing index clinically and venous blood do not have difference.Lee gallops and show that the content detecting peripheral blood and venous blood PCT, CRP does not have difference in the research finger using value of peripheral blood PCT, hs-CRP joint-detection in Children's Infectious Diseases, finger peripheral blood may be used for the content of PCT, CRP in joint-detection serum, can diagnose difference Children's Infectious Diseases.As can be seen here, invent a kind of test paper being simultaneously suitable for finger peripheral blood CRP/SAA content detection and community is spread to Rapid detection test strip or the utilization of individual in diagnosis, health check-up etc. has great importance.
Summary of the invention
For above-mentioned deficiency, the object of the invention is to provide one can quantitative CRP/SAA content in joint-detection people peripheral blood, also the CRP/SAA that can be used for CRP/SAA content in quantitative joint-detection venous blood quantitatively combines Rapid detection test strip, present invention also offers the preparation method of this test paper.
For solving the problems of the technologies described above, previous technical scheme provided by the invention is such:
A kind of CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, comprises box body with cover, is provided with test strip in box body, also establish the blood dilution liquid bottle that blood dilution liquid is housed in box body; Described test strip comprises end liner, end liner is provided with nitrocellulose filter, one end of nitrocellulose filter is connected fluorescent microsphere labelled antibody pad, and fluorescent microsphere labelled antibody pad uplink is connected to sample pad, and the other end of nitrocellulose filter is connected absorption pad; Described fluorescent microsphere labelled antibody pad is coated with CRP monoclonal antibody, SAA monoclonal antibody; Parallelly on described nitrocellulose filter be provided with a CRP detection line being coated with CRP monoclonal antibody, nature controlling line that a SAA detection line being coated with SAA monoclonal antibody and one are coated with sheep anti-mouse igg polyclonal antibody.
Further, above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, the lid of described box body is provided with well and result watch window.
Further, above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, described blood dilution liquid is the isotonic solution containing the non-blood soluble surfactants of 0.05-0.15wt%, 0.01%-0.03wt% anti-coagulants, 0.03-0.07wt% antiseptic.
Further, above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, described sample pad is Whole Blood Filtration membrane sample pad.
Further, above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, described fluorescent microsphere labelled antibody pad is enclosed with the CRP/SAA monoclonal antibody of the polystyrene latex microballoon mark of Eu3+.
After provided by the invention, a technical scheme is such:
The preparation method of above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, comprises the steps: successively
Nitrocellulose filter pasted by end liner, one end of nitrocellulose filter pastes sample pad, fluorescent microsphere labelled antibody pad successively, the other end of nitrocellulose filter is connected absorption pad and obtains test strip, the Test paper rule assembled is arranged on bottom box body in draw-in groove, covers lid and get final product;
Wherein:
1) preparation method of sample pad
Whole Blood Filtration membrane sample pad is soaked the pH7.520mmol/L boric acid-borate buffer solution process containing 2% sucrose, 0.5%PVP10000, after drying at room temperature, 150 μ L are evenly sprayed containing 1% blocking agent, 0.5%Tetronic1307 and 2%BSA aqueous solution according to every 1.5 × 30cm2, again after drying, kept dry in sealing bag;
2) preparation method of fluorescent microsphere labelled antibody pad
The fluorescent microsphere being marked with CRP monoclonal antibody, the fluorescent microsphere being marked with SAA monoclonal antibody is diluted respectively with fluorescent microsphere redissolution liquid, by two kinds of fluorescent microspheres after mark, by volume you mix than 1:1, be sprayed onto on pad, prepare fluorescent microsphere labelled antibody pad;
3) preparation of detection line and nature controlling line
With bag be buffered liquid dilute respectively can with the monoclonal antibody of determined antigen CRP specific binding, can with the monoclonal antibody of SAA specific binding, and sheep anti-mouse igg polyclonal antibody, then adopt three kinds of antibody after dilution to form CRP detection line, SAA detection line, nature controlling line at nitrocellulose filter successively line abreast.
Further, the preparation method of above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, step 3) described in the concentration of three kinds of antibody be 0.5 ~ 1.5mg/ml, consumption is 30 μ l/30cm.
Further, the preparation method of above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, described bag is buffered the PBS of 0.01 ~ 0.05MpH7.4 containing 3 ~ 5% trehaloses in liquid.
Further, the preparation method of above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, the method of described fluorescent microsphere labelled antibody is: with 10mg/mlEDC and 50mg/mlNHS to after fluorescent microsphere activation, centrifugal segregation EDC (1-(3-dimethylamino-propyl)-3-ethyl carbodiimide) and NHS (N-hydroxy-succinamide), ultrasonic resuspended with 50mMMES (ethyl sulfonic acid) 100W of pH6.0 ~ 7.0, measure according to 10 μ g ~ 50 μ g/0.1mg, CRP monoclonal antibody is added respectively to fluorescent microsphere, SAA monoclonal antibody, then after above-mentioned two kinds being added the mixing of the fluorescent microsphere after antibody, room temperature vortex stirs 1h, centrifuge washing 2 ~ 3 times, precipitate and redissolve with rear fluorescent microsphere redissolution liquid 100W ultrasonic process 30s, 4 DEG C of preservations.Further, the preparation method of above-mentioned CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, described fluorescent microsphere redissolution liquid is the pH8.010mmol/LTris-HCl damping fluid containing 2%BSA, 0.1%PEG4000,5% trehalose, 5% sucrose.Compared with prior art, technical scheme tool provided by the invention has the following advantages: 1, the present invention's application fluorescent chromatographic technology, wrap by the detection line of CRP monoclonal antibody by being provided with on test paper simultaneously, be coated with the detection line of SAA monoclonal antibody and the nature controlling line of sheep anti-mouse igg, CRP and SAA concentration in blood can be detected accurately simultaneously, and two kinds of antigens do not interfere with each other, result is read by instrument, have quick, easy, without the need to advantages such as professional's operations, can be applied in hospital, community, individual disease forecasting, diagnosis and monitoring.2, CRP detection and SAA detect and are incorporated into same test paper by technical scheme provided by the invention, not only can quantitative CRP/SAA content in joint-detection people peripheral blood, also can be used for CRP/SAA content in quantitative joint-detection venous blood, prior art can be solved and in same test paper, the problem of CRP/SAA can not be detected by simultaneous quantitative; In addition this invention also solves and use finger peripheral blood as the problem of pattern detection CRP/SAA content, simplify operation easier, promote test strip utilization in disease forecasting, diagnosis, health check-up etc. in community or individual.
Accompanying drawing explanation
Fig. 1 is CRP/SAA provided by the invention quantitative joint-detection immunofluorescence chromatographic test paper perspective view;
Fig. 2 is CRP/SAA provided by the invention quantitative joint-detection immunofluorescence chromatograph test strip structural representation.
Wherein: box body 1, blood dilution liquid bottle 2, lid 3, well 31, result watch window 32, end liner 4, nitrocellulose filter 5, CRP detection line 51, SAA detection line 52, nature controlling line 53, fluorescent microsphere labelled antibody pad 6, sample pad 7, absorption pad 8.
Embodiment
Mode below in conjunction with specific embodiment is described in further detail claim of the present invention, but do not form any limitation of the invention, the amendment of anyone limited number of time made within the scope of the claims in the present invention, still within right of the present invention.
Embodiment 1
A kind of CRP/SAA provided by the invention quantitative joint-detection immunofluorescence chromatographic test paper, consults Fig. 1, Fig. 2, comprises box body 1 with cover, and the lid 3 of described box body 1 is provided with well 31 and result watch window 32.
Be provided with test strip in box body, in box body, be also provided with the blood dilution liquid bottle 2 of the isotonic solution containing the non-blood soluble surfactants of 0.05-0.15wt%, 0.01%-0.03wt% anti-coagulants, 0.03-0.07wt% antiseptic; Described test strip comprises end liner 4, end liner 4 is provided with nitrocellulose filter 5, one end of nitrocellulose filter 5 is connected fluorescent microsphere labelled antibody pad 6, and described fluorescent microsphere labelled antibody pad 6 is enclosed with the CRP/SAA monoclonal antibody of the polystyrene latex microballoon mark of Eu3+.Fluorescent microsphere labelled antibody pad 6 uplink is connected to sample pad 7, and described sample pad 7 is Whole Blood Filtration membrane sample pad; The other end of nitrocellulose filter 5 is connected absorption pad 8, and described absorption pad 8 is strong absorptive filter paper; Described fluorescent microsphere labelled antibody pad 6 is coated with CRP monoclonal antibody, SAA monoclonal antibody; Parallelly on described nitrocellulose filter 5 be provided with a CRP detection line 51, being coated with CRP monoclonal antibody and be coated with the nature controlling line 53 that the SAA detection line 52 of SAA monoclonal antibody and are coated with sheep anti-mouse igg polyclonal antibody.
Specifically, the blood dilution liquid in described blood dilution liquid bottle 2 is the phosphate buffer composition of the 0.01MpH7.4 containing 0.1%Tween-20 and 0.05% Sodium azide; Or the composition of described blood dilution liquid is containing 0.02% sodium citrate, 0.1%tetronic1307 and 0.05%ProClin300 in the physiological saline of 0.01mol/L.
Embodiment 2
CRP/SAA provided by the invention combines the preparation method of Quantitative detection test strips:
The pre-treatment of sample pad: Whole Blood Filtration membrane sample pad is soaked the pH7.520mmol/L boric acid-borate buffer solution process containing 2% sucrose, 0.5%PVP10000, after drying at room temperature, 150 μ L are evenly sprayed containing 1% blocking agent, 0.5%Tetronic1307 and 2%BSA aqueous solution according to every 1.5 × 30cm2, again after drying, kept dry in sealing bag.
The preparation of fluorescent microsphere bond pad: the fluorescent microsphere (Eu diluting mark CRP monoclonal antibody with fluorescent microsphere redissolution liquid respectively
3+polystyrene latex microballoon, adopt the Fluoro-MaxDyedCarboxylate-ModifiedMicroparticles that ThermoScientific produces), and the fluorescent microsphere (Eu of mark SAA monoclonal antibody
3+polystyrene latex microballoon, the Fluoro-MaxDyedCarboxylate-ModifiedMicroparticles that ThermoScientific produces), by these two kinds of fluorescent microsphere mixings, be sprayed onto on pad with the three-dimensional specking instrument of spray BiodotXYZ3060, prepare immune chromatography test paper bond pad.The wherein grand antibody of CRP of every milligram of fluorescent microsphere mark and the equal 0.20mg of the grand antibody of SAA of fluorescent microsphere mark, the pH8.010mmol/LTris-HCl damping fluid containing 2%BSA, 0.1%PEG4000,5% trehalose, 5% sucrose in described fluorescence redissolution liquid;
The method of described fluorescent microsphere labelled antibody is: with 10mg/mlEDC and 50mg/mlNHS to after fluorescent microsphere activation, centrifugal segregation EDC and NHS, ultrasonic resuspended with the 50mMMES100W of pH6.0 ~ 7.0, measure according to 10 μ g ~ 50 μ g/0.1mg, CRP monoclonal antibody, SAA monoclonal antibody is added respectively to fluorescent microsphere, then after above-mentioned two kinds being added the mixing of the fluorescent microsphere after antibody, room temperature vortex stirs 1h, centrifuge washing 2 ~ 3 times, precipitate and redissolve with rear fluorescent microsphere redissolution liquid 100W ultrasonic process 30s, 4 DEG C of preservations.
The preparation of detection line and nature controlling line: with bag be buffered liquid dilution can with another strain monoclonal antibody of determined antigen CRP specific binding, can with another strain monoclonal antibody of SAA specific binding and sheep anti-mouse igg polyclonal antibody, and by three kinds of antibody after dilution on described nitrocellulose filter successively line abreast form CRP detection line, SAA detection line, nature controlling line; The concentration of three kinds of antibody is 1.0mg/ml, and consumption is 30 μ l/30cm; Containing 5% trehalose in described coating buffer, the PBS of 0.01MpH7.4;
The assembling of test-strips: paste sample pad, fluorescent microsphere bond pad, nitrocellulose filter and adsorptive pads above end liner successively;
The assembling of test card: cut into rectangular by the test-strips assembled, and being arranged in test card outer casing bottom draw-in groove, covers and gets stuck.
Sample diluting liquid: containing the phosphate buffer of the 0.01MpH7.4 of 0.1%Tween-20 and 0.05% Sodium azide.
The cardinal principle of this CRP/SAA associating Quantitative detection test strips is the association reaction based on immunology antigen-antibody.
Finger peripheral blood detection method: tear outer packaging bag, take out and detect reagent, with cotton ball soaked in alcohol to after the sterilization of blood sampling finger, use disposable blood taking needle in finger puncture rapidly, then with there being the capillary syring of exact scale quantitatively to draw 10 μ l peripheral bloods, mix in sample diluting liquid rapidly, getting 75 μ l detection samples with sample injector is added in well, use timer timing, after keeping flat reaction 10min, use the immunofluorescence Rapid reading instrument being provided with CRP/SAA and combining the fluorescent quantitation spectral detection system of Quantitative detection test paper, detect the concentration of CRP and SAA in sample fast.
Under capillarity, sample moves along test strips to absorption pad end, antigen in sample and fluorescently-labeled antibody combine on bond pad, flowing through in detection zone, coated antibody recognition also catches combination on detection zone, and the antigen of combined delay and the amount of fluorescent-labeled antibody compound are proportionate with antigen concentration.After detection line is irradiated by the exciting light of suitable wavelength, fluorescent microsphere launches certain fluorescence signal, and fluorescence signal through the process of fluorescence automatic detection analysis, and shows result on a display screen, and testing result is printed, and realizes the accurate quantification to sample.
Claims (10)
1. a CRP/SAA quantitative joint-detection immunofluorescence chromatographic test paper, comprise box body (1) with cover, test strip is provided with in box body, it is characterized in that, the blood dilution liquid bottle (2) that blood dilution liquid is housed also is established in box body, described test strip comprises end liner (4), one end linking that end liner (4) is provided with nitrocellulose filter (5) has fluorescent microsphere labelled antibody pad (6), fluorescent microsphere labelled antibody pad (6) uplink is connected to sample pad (7), the other end of nitrocellulose filter (5) is connected absorption pad (8), described fluorescent microsphere labelled antibody pad (6) is coated with CRP monoclonal antibody, SAA monoclonal antibody, the nature controlling line (53) that described nitrocellulose filter (5) is upper is parallelly provided with a CRP detection line (51) being coated with CRP monoclonal antibody, a SAA detection line (52) being coated with SAA monoclonal antibody and one are coated with sheep anti-mouse igg polyclonal antibody.
2. CRP/SAA according to claim 1 quantitative joint-detection immunofluorescence chromatographic test paper, it is characterized in that, the lid (3) of described box body (1) is provided with well (31) and result watch window (32).
3. CRP/SAA according to claim 1 quantitative joint-detection immunofluorescence chromatographic test paper, it is characterized in that, described blood dilution liquid is the isotonic solution containing the non-blood soluble surfactants of 0.05-0.15wt%, 0.01%-0.03wt% anti-coagulants, 0.02-0.1wt% antiseptic.
4. CRP/SAA according to claim 1 quantitative joint-detection immunofluorescence chromatographic test paper, is characterized in that, described sample pad (7) is Whole Blood Filtration membrane sample pad.
5. CRP/SAA according to claim 1 quantitative joint-detection immunofluorescence chromatographic test paper, is characterized in that, described fluorescent microsphere labelled antibody pad (6) is enclosed with Eu
3+polystyrene latex microballoon mark CRP/SAA monoclonal antibody.
6. the preparation method of CRP/SAA according to claim 1 quantitative joint-detection immunofluorescence chromatographic test paper, is characterized in that, comprise the steps: successively
End liner (4) is pasted nitrocellulose filter (5), one end of nitrocellulose filter (5) pastes sample pad (7), fluorescent microsphere labelled antibody pad (6) successively, the other end of nitrocellulose filter (5) is connected the obtained test strip of absorption pad (8), the test strip assembled is arranged on bottom box body in draw-in groove, covers lid (3) and get final product;
Wherein:
1) preparation method of sample pad
Whole Blood Filtration membrane sample pad is soaked the pH7.520mmol/L boric acid-borate buffer solution process containing 2% sucrose, 0.5%PVP10000, after drying at room temperature, according to every 1.5 × 30cm
2even sprinkling 150 μ L contains 1% blocking agent, 0.5%Tetronic1307 and 2%BSA aqueous solution, again after drying, and kept dry in sealing bag;
2) preparation method of fluorescent microsphere labelled antibody pad
The fluorescent microsphere being marked with CRP monoclonal antibody, the fluorescent microsphere being marked with SAA monoclonal antibody is diluted respectively with fluorescent microsphere redissolution liquid, by the 1:1 mixing by volume of two kinds of fluorescent microspheres after mark, be sprayed on pad, prepare fluorescent microsphere labelled antibody pad;
3) preparation of detection line and nature controlling line
With bag be buffered liquid dilute respectively can with the monoclonal antibody of determined antigen CRP specific binding, can with the monoclonal antibody of SAA specific binding, and sheep anti-mouse igg polyclonal antibody, then adopt three kinds of antibody after dilution to rule abreast successively on nitrocellulose filter and form CRP detection line, SAA detection line, nature controlling line.
7. the preparation method of the quantitative joint-detection immunofluorescence of the CRP/SAA according to claims 6 chromatographic test paper, is characterized in that, step 3) described in the concentration of three kinds of antibody be 0.5 ~ 1.5mg/ml, consumption is 30 μ l/30cm.
8. the preparation method of the quantitative joint-detection immunofluorescence of the CRP/SAA according to claims 6 chromatographic test paper, is characterized in that, described bag is buffered the PBS of 0.01 ~ 0.05MpH7.4 containing 3 ~ 5% trehaloses in liquid.
9. the preparation method of the quantitative joint-detection immunofluorescence of the CRP/SAA according to claims 6 chromatographic test paper, it is characterized in that, the method of described fluorescent microsphere labelled antibody is: with 10mg/mlEDC and 50mg/mlNHS to after fluorescent microsphere activation, centrifugal segregation EDC and NHS, ultrasonic resuspended with the 50mMMES100W of pH6.0 ~ 7.0, measure according to 10 μ g ~ 50 μ g/0.1mg, CRP monoclonal antibody is added respectively to fluorescent microsphere, SAA monoclonal antibody, then after above-mentioned two kinds being added the mixing of the fluorescent microsphere after antibody, room temperature vortex stirs 1h, centrifuge washing 2 ~ 3 times, precipitate and redissolve with rear fluorescent microsphere redissolution liquid 100W ultrasonic process 30s, 4 DEG C of preservations.
10. the preparation method of the quantitative joint-detection immunofluorescence of the CRP/SAA according to claims 6 chromatographic test paper, it is characterized in that, described fluorescent microsphere redissolution liquid is the pH8.010mmol/LTris-HCl damping fluid containing 2%BSA, 0.1%PEG4000,5% trehalose, 5% sucrose.
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