CN110133281A - CRP and SAA combined detection kit and preparation method thereof - Google Patents
CRP and SAA combined detection kit and preparation method thereof Download PDFInfo
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- CN110133281A CN110133281A CN201910371125.8A CN201910371125A CN110133281A CN 110133281 A CN110133281 A CN 110133281A CN 201910371125 A CN201910371125 A CN 201910371125A CN 110133281 A CN110133281 A CN 110133281A
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Abstract
The present invention provides a kind of CRP and SAA combined detection kit and preparation method thereof.The kit includes CRP and SAA combined detection test paper, the test strips include sample pad, bonding pad, analyzing film, absorption pad and bottom plate, the analyzing film is equipped with 2 detection lines (CRP, SAA detection line) and 1 nature controlling line, and sample pad, bonding pad, analyzing film and absorption pad are pasted on bottom plate and partially overlap respectively with successively overlapping；The bonding pad is coated with CRP and SAA antibody mark fluorescent microballoon.The present invention uses the kit of fluorescence immune chromatography method preparation, CRP and SAA can be detected simultaneously, clinical detection efficiency is improved, has the advantages that highly sensitive, high specific and quickly detection, the auxiliary diagnosis for diseases such as infectious diseases, tissue damage necrosis diseases provides effective means.
Description
Technical field
The present invention relates to medicine detection and technical field of immunoassay, specifically, being related to a kind of CRP and SAA joint inspection
Test agent box and preparation method thereof.
Background technique
C reactive protein (CRP) and serum amyloid A protein (SAA) are the Earlier period of inflammation indexs of infectious diseases, for thin
Bacterium infection, the diagnosis of tissue infection and assessment degree of inflammation are particularly significant.C reactive protein (CRP) is a kind of to be synthesized by liver
Acute reaction protein, after bacterium infection body, patient's body CRP is significantly raised.When CRP concentration is greater than 10mg/L Shi Jike
Energy bacterial infection, clinically needs antibiotic to treat, after rational therapy, can restore to normal level within general 3~7 days.And
Hs-CRP (super quick CRP) is a kind of, detection limit lower project sensitiveer than common CRP detection, is had been used at present
The assessment of early detection Pediatrics and coronary heart disease risk.Clinically, the joint-detection of super quick CRP and cardiac troponin is made
For the index of coronary heart disease risk and the index of prediction recurrence.
Serum amyloid protein (SAA) is the precursor substance of tissue amyloid A, is mainly generated by stem cell, phase
It is about 12000 to molecular weight, content concn is reaction infectious diseases Earlier period of inflammation index.Unlike CRP, SAA is not
It all can significantly be increased by bacterium or virus infection.In acute-phase response period, stimulated through IL-1, IL-6 and TNF, SAA
It can be increased to 100-1000 times of initial concentration, increasing degree is higher than CRP, but half-life short, only 50min or so.Pass through joint-detection
SAA and CRP is can overcome the disadvantages that just at present to the blank of virus infection detection, brings foundation for viral diagnosis.
CN201610826542.3 disclose it is a kind of detect CRP, SAA immune chromatography reagent kit and the side of making and using
Method, the kit use colloidal gold immunity chromatography, can primary sample, while detecting two indices, it is simple and fast, but by
The signal value of the limitation of colloidal gold methodology, concentration comparable sample is not easily distinguishable, and easily causes measured deviation.Still lacking at present can be with
The joint inspection kit of above two index quickly, is quantitatively accurately detected simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of CRP and SAA combined detection kits and preparation method thereof.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a kind of CRP and SAA combined detection test paper, including
Sample pad, bonding pad, analyzing film, absorption pad and bottom plate, the analyzing film are equipped with 2 detection lines and 1 nature controlling line, the sample
Product pad, bonding pad, analyzing film and absorption pad are pasted on bottom plate and partially overlap respectively with successively overlapping.
Wherein, 2 detection lines are close to bonding pad one end, and nature controlling line is close to water absorption pad one end；
2 detection lines are respectively CRP detection line, SAA detection line；The CRP detection line is coated with CRP detection antibody, institute
It states SAA detection line and is coated with SAA detection antibody；
The bonding pad is coated with CRP antibody mark fluorescent microballoon and SAA antibody mark fluorescent microballoon；
The coated antibody of detection line is respectively provided with different antigen binding sites from the coated antibody of the bonding pad.
In the present invention, the sample pad is glass fibre element film or polyester cellulose film.
The bonding pad is glass fibre element film or polyester cellulose film.
The analyzing film is nitrocellulose filter.
The material of the bottom plate is PVC.
Fluorescent microsphere used is the microballoon made from Silica-coated fluorescent material,
The fluorescent material be selected from fluorescein isothiocynate, rhodamine, tetramethylisothiocyanate rhodamine, TO, Cy3,
At least one of Cy5, phycoerythrin, quantum dot etc..
The partial size of the fluorescent microsphere be 20nm~600nm, launch wavelength be 500nm~700nm (preferably 525nm~
620nm), the use concentration of fluorescent microsphere is 0.00001mg/ml~0.1mg/ml；The surface modification of the fluorescent microsphere has official
It can roll into a ball, the functional group is selected from least one of carboxyl, amino or aldehyde radical etc..
The CRP detection antibody is the antibody 801001 of Abgree company production, and the SAA detection antibody is Medix
The antibody 2203 of Biochemica company production.
CRP antibody used in the CRP antibody mark fluorescent microballoon is the antibody 801002 of Abgree company production, institute
State the antibody 2201 that SAA antibody used in SAA antibody mark fluorescent microballoon is the production of Medix Biochemica company.
The nature controlling line is coated with secondary antibody, the secondary antibody in sheep anti mouse, goat-anti rabbit, rabbit-anti goat-anti body at least one
Kind.It is preferred that the secondary antibody is sheep anti-mouse antibody.
The CRP antibody mark fluorescent microballoon is made on CRP antibody by EDC and NHS on N-terminal amino and fluorescent microsphere
Carboxyl passes through chemistry key connection.
The SAA antibody mark fluorescent microballoon is made on SAA antibody by EDC and NHS on N-terminal amino and fluorescent microsphere
Carboxyl passes through chemistry key connection.
The length of CRP and SAA combined detection test paper of the present invention is 5~10cm, and width is 3~5mm.
Second aspect, the present invention provide a kind of CRP and SAA combined detection kit, the kit include above-mentioned CRP and
SAA combined detection test paper, optionally comprising for loading getting stuck for the test strips.
The third aspect, the present invention provide the preparation method of CRP and SAA combined detection test paper, include the following steps:
(1) microballoon activate: into 1~5ml of 1mg/ml fluorescent microsphere be added use 50mM, pH6.1MES dissolution 0.1~
10mg EDC and 10mg NHS, activate 10~60min, 7000~15000rpm be centrifuged 30min, abandon supernatant, precipitating with pH7.0~
8.0 50mM PBS is resuspended, and obtains the fluorescent microsphere solution of 1mg/ml activation；
(2) 0.2mg CRP labelled antibody the coupling of CRP antibody: is added to the fluorescent microsphere solution 2ml of 1mg/ml activation
In, room temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating is closed with the 50mM PBS solution of the BSA containing 5mg
60min is stirred and evenly mixed, and 10000rpm is centrifuged 30min, and it is slow that the precipitating of collection is stored in 20mM Tris of the pH7.8 containing 0.5%BSA
In fliud flushing, obtaining concentration is 0.1mg/ml CRP antibody mark fluorescent microspheres solution；
Wherein, the CRP labelled antibody is the antibody 801002 of Abgree company production；
(3) 0.4mg SAA labelled antibody the coupling of SAA antibody: is added to the fluorescent microsphere solution 2ml of 1mg/ml activation
In, room temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating is closed with the 50mM PBS solution containing 1%BSA
60min is stirred and evenly mixed, and 10000rpm is centrifuged 30min, and it is slow that the precipitating of collection is stored in 20mM Tris of the pH7.8 containing 0.5%BSA
In fliud flushing, obtaining concentration is 0.1mg/mlSAA antibody mark fluorescent microspheres solution；
Wherein, the SAA labelled antibody is the antibody 2201 of Medix Biochemica company production；
(4) by fluorescent microsphere solution obtained by step (2) and step (3), 1:0.1~1:10 is mixed by volume；
(5) it the preparation of bonding pad: using glass fibre element film or polyester cellulose film as bonding pad, prepared by step (4)
1 μ L/cm of fluorescent microsphere mixed liquor be sprayed on film, drying for standby；
(6) preparation of analyzing film: CRP is detected into antibody, SAA detection antibody and secondary antibody (sheep anti-mouse igg) and is diluted to 0.8-
1mg/ml is sprayed on nitrocellulose filter in point film instrument with the discharge rate of 0.8-1.2 μ L/cm, forms CRP detection line, SAA inspection
Survey line and nature controlling line, as analyzing film, drying for standby；
Wherein, the CRP detection antibody is the antibody 801001 of Abgree company production, and the SAA detection antibody is
The antibody 2203 of Medix Biochemica company production, the secondary antibody are sheep anti-mouse igg；
(7) assembling of test strips: by sample pad (glass fibre element film or polyester cellulose film), bonding pad, analyzing film and
Water absorption pad is pasted on PVC bottom plate and partially overlaps respectively with successively overlapping, cut into length be 5~10cm, width be 3~
The paper slip of 5mm is to get CRP and SAA combined detection test paper.
Preferably, activator EDC and NHS are added in step (1) Xiang Suoshu fluorescent microsphere, activates 20~30min, is centrifuged institute
It must precipitate and be resuspended with the 50mM PBS of pH7.4, the fluorescent microsphere solution activated, wherein the mass ratio of EDC and NHS is 1:2-
5, EDC and fluorescent microsphere mass ratio be 1:20-100.
In the specific embodiment of the present invention, CRP and SAA combined detection test paper the preparation method is as follows:
(1) microballoon activates: being to be added in carboxyl fluorescent microsphere 1ml that 100nm launch wavelength is 535nm to 1mg/ml partial size
With 50mM, 1mg EDC and the 10mg NHS of pH6.1MES dissolution, 30min is activated, 10000rpm is centrifuged 30min and abandons supernatant, precipitating
It is resuspended with the 50mM PBS of pH7.4, obtains the fluorescent microsphere solution of 1mg/ml activation；
(2) the activation fluorescent microsphere that 2ml concentration is 1mg/ml the coupling of CRP antibody: is added in 0.2mgCRP labelled antibody
In solution, room temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating is sealed with the 50mM PBS solution of the BSA containing 5mg
60min is closed, is stirred and evenly mixed, 10000rpm is centrifuged 30min, and the precipitating of collection is stored in 20mM Tris of the pH7.8 containing 0.5%BSA
In buffer, obtaining concentration is 0.1mg/ml CRP antibody mark fluorescent microspheres solution；
(3) the activation fluorescent microsphere that 2ml concentration is 1mg/ml the coupling of SAA antibody: is added in 0.4mgSAA labelled antibody
In solution, room temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating is sealed with the 50mM PBS solution containing 1%BSA
60min is closed, is stirred and evenly mixed, 10000rpm is centrifuged 30min, and the precipitating of collection is stored in 20mM Tris of the pH7.8 containing 0.5%BSA
In buffer, obtaining concentration is 0.1mg/ml SAA antibody mark fluorescent microspheres solution；
(4) by fluorescent microsphere solution obtained by step (2) and step (3), 1:1 is mixed by volume；
(5) preparation of bonding pad: using glass fibre membrane as bonding pad, fluorescent microsphere mixed liquor prepared by step (4),
It is sprayed on pretreated glass fibre element film with point film instrument with 0.8 μ L/cm, drying for standby；
(6) preparation of analyzing film: CRP is detected into antibody, SAA detection antibody and secondary antibody sheep anti-mouse igg and is diluted to 0.8mg/
Ml is sprayed on nitrocellulose filter with the discharge rate of 1 μ L/cm, is respectively formed CRP detection line, SAA detection line and nature controlling line, i.e.,
For analyzing film, drying for standby；
(7) sample pad, bonding pad, analyzing film and water absorption pad the assembling of test strips: are pasted onto PVC bottom plate with successively overlapping
It above and respectively partially overlaps, cutting into length is 8cm, and width is the paper slip of 4mm to get CRP and SAA combined detection test paper.
Fourth aspect, the present invention provide the preparation method of CRP and SAA combined detection kit, and above-mentioned CRP and SAA is joined
Close test strip, or the test strips that prepare according to the method described above be loaded into get stuck in get.
5th aspect, the present invention provide CRP and SAA combined detection test paper/kit joint-detection (including it is qualitative and
Quantitative detection) application in CRP and SAA.
6th aspect, the present invention provide the quantitative joint inspection method of CRP and SAA, comprising the following steps:
1. the drafting of standard curve: preparing CRP the and SAA standard solution of various concentration respectively, take 60 μ L to add to above-mentioned
Test strips are placed under fluorescence immunity analyzer after 10min by the sample pad of CRP and SAA combined detection test paper, detection T line (inspection
Survey line) and C line (nature controlling line) fluorescence intensity, is drawn respectively according to the ratio of C line and the fluorescence intensity of T line and reflects CRP and SAA
The standard curve of content；
2. pattern detection: by the sample of acquisition, 60 μ L is taken to add to the sample pad of above-mentioned CRP and SAA combined detection test paper,
Test strips are placed under fluorescence immunity analyzer after 10min, T line (detection line) is detected respectively and the fluorescence of C line (nature controlling line) is strong
Degree substitutes into above-mentioned standard curve, CRP's and SAA is dense in calculating acquisition sample according to the ratio of C line and the fluorescence intensity of T line
Degree.
Method above-mentioned, the linear detection range of CRP are 0.1-100mg/L, Monitoring lower-cut 0.1mg/L.
Method above-mentioned, the linear detection range of SAA are 0.5-250mg/L, Monitoring lower-cut 0.5mg/L.
In the present invention, above-mentioned sample can be the sample of serum, blood plasma, whole blood and peripheral blood.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) using CRP and SAA combined detection kit provided by the invention, it can be achieved that a sample detect simultaneously it is super quick
Two projects of c reactive protein and serum amyloid A protein, the sample dosage for comparing single project separate detection is few, simplifies inspection
Step is surveyed, detection time has been saved.
(2) this method can examine that specimen types are more compared to other detection methods, include serum, blood plasma, whole blood and peripheral blood
Sample, and without carrying out pre-treatment to sample.
(3) detection sensitivity of kit of the present invention is high, can detect in serum sample the c reactive protein of 0.1mg/L and
The serum amyloid A protein of 0.5mg/L；Mating fluorescence detection equipment is small and exquisite to be can be carried around, and detection process is easy to operate, detection
Quickly, 10min can be obtained testing result after blood sampling.
(4) testing principle of kit of the present invention is immunoreacted based on double antibodies sandwich.Fluorescence immune chromatography is as a kind of micro-
Analysis method has many advantages, such as easy to operate, high sensitivity, low in cost, while having the advantages that POCT quick diagnosis, is
Clinical detection provides convenient.
Detailed description of the invention
Fig. 1 is the structure of the CRP and SAA combined detection test paper prepared in the embodiment of the present invention 1.
Fig. 2 is the structure of the CRP and SAA combined detection reagent card prepared in the embodiment of the present invention 2.
In figure, 1- sample pad, 2- bonding pad, 3- analyzing film, 4- water absorption pad, 5- bottom plate, 6- detection line T1,7- detection line
T2,8- nature controlling line C, 9- well, 10- get stuck.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Fluorescent microsphere used in the following embodiment is the polystyrene microsphere containing fluorescent material, raw purchased from Nanjing micrometering
Object Science and Technology Ltd..
The preparation of 1 CRP and SAA combined detection test paper of embodiment
1, the preparation of solution
The preparation of activating solution: weighing 10.66g activator MES in beaker, purified water 1L be added, and after stirring and evenly mixing, uses
NaOH solution tune pH to 6.1,4 DEG C save backup.
It is coupled the preparation of liquid PBS: weighing the Na of 2.865g2HPO4·12H2O successively weighs 0.272g's in beaker
KH2PO4·H2O, the KCl of the NaCl and 1.983g of 7.948g are added in above-mentioned beaker, with 1L purified water constant volume, after stirring and evenly mixing,
PH to 7.4 is adjusted, 4 DEG C save backup.
2, fluorescent microsphere labelled antibody
(1) microballoon activates: being to be added in carboxyl fluorescent microsphere 1ml that 100nm launch wavelength is 535nm to 1mg/ml partial size
With 50mM, 1mg EDC and the 10mg NHS of pH6.1MES dissolution, 30min is activated, 10000rpm is centrifuged 30min and abandons supernatant, precipitating
It is resuspended with the 50mM PBS of pH7.4, obtains the fluorescent microsphere solution of 1mg/ml activation；
(2) coupling of CRP antibody: it is 1mg/ml that 2ml concentration, which is added, in 0.2mgCRP labelled antibody (Abgree 801002)
Activation fluorescent microsphere solution in, room temperature mix 2~4h, 10000rpm be centrifuged 30min, abandon supernatant, precipitate with the BSA's containing 5mg
50mM PBS solution closes 60min, stirs and evenly mixs, and 10000rpm is centrifuged 30min, and the precipitating of collection is stored in pH7.8 containing 0.5%
In the 20mM Tris buffer of BSA, obtaining concentration is 0.1mg/ml CRP antibody mark fluorescent microspheres solution；
(3) 2ml concentration the coupling of SAA antibody: is added in 0.4mgSAA labelled antibody (Medix Biochemica 2201)
For in the activation fluorescent microsphere solution of 1mg/ml, room temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating is with containing
The 50mM PBS solution of 1%BSA closes 60min, stirs and evenly mixs, and 10000rpm is centrifuged 30min, and the precipitating of collection is stored in
In 20mM Tris buffer of the pH7.8 containing 0.5%BSA, obtaining concentration is that 0.1mg/ml SAA antibody mark fluorescent microballoon is molten
Liquid；
The mixing of (4) two kinds of antibody mark fluorescent microspheres solutions: by above-mentioned (2), solution prepared in (3) by volume 1:
1 mixing, mixes well rear spare；
(5) preparation of bonding pad: using glass fibre membrane as bonding pad, fluorescent microsphere mixed liquor prepared by step (4),
It is sprayed on pretreated glass fibre element film with point film instrument with 0.8 μ L/cm, drying for standby；
3, the preparation of line containing C, T line analysis film
CRP is detected into antibody (Abgree 801001), SAA detection antibody (Medix Biochemica 2203) and secondary antibody
Sheep anti-mouse igg is diluted to 0.8mg/ml, is crossed on analyzing film with point film instrument and (is sprayed on cellulose nitrate respectively with the amount of 1 μ L/cm
On plain film): it is respectively formed CRP detection line (T1 line), SAA detection line (T2 line) and nature controlling line (C line), alternatively, being respectively formed CRP
Detection line (T2 line), SAA detection line (T1 line) and nature controlling line (C line), as analyzing film, drying for standby；
4, the assembling of test strips: sample pad (glass fibre element film), bonding pad, analyzing film and water absorption pad are successively overlapped ground
It is pasted on PVC bottom plate and partially overlaps respectively, cutting into length is 8cm, and width is that the paper slip of 4mm joins to get CRP and SAA
Close test strip.
The structure of test strips is as shown in Figure 1.Wherein, sample pad 1, sample pad ride on bonding pad 2, and bonding pad 2 rides over point
3 left side of film is analysed, water absorption pad 4 rides over 3 right side of analyzing film, and analyzing film 3 is attached on bottom plate 5, successively has CRP detection anti-on analyzing film
T1 line 6, the T2 line 7 of SAA detection antibody scribing line and the line 8 of secondary antibody sheep anti-mouse igg scribing line of body scribing line.
The preparation of 2 CRP and SAA combined detection kit of embodiment
The present embodiment provides the kits of a kind of hs-CRP and serum amyloid A protein joint-detection, include examination
Agent card.Reagent card structure is as shown in Figure 2.Wherein, 9 be well, and 10 be the card for loading test strips prepared by embodiment 1
Shell, 6,7,8 respectively correspond CRP detection T1 line, SAA detection T2 line and Quality Control C line on watch window.
The Performance Evaluation of 3 CRP and SAA combined detection kit of embodiment
1, linear assessment
Using newborn bovine serum as dilution, by hs-CRP standard items be configured to concentration be 100,50,20,
10, the standard solution of 2,0.5,0.1 and 0mg/L takes 60 μ L sample-adding to be detected, test strips is placed in fluorescence after 10min and are exempted from
Under epidemic disease analyzer, the fluorescence intensity of T line (detection line) and C line (nature controlling line) is detected, according to the ratio of T line and the fluorescence intensity of C line
Value draws the standard curve of reflection CRP content respectively, and each concentration repeats detection 3 times, is averaged as detectable concentration, with preparation
Concentration and detectable concentration do linear equation and calculate related coefficient.The result shows that linear correlation coefficient r > 0.99, curvilinear equation y
=0.9781x+0.163.The linear detection range of CRP is 0.1-100mg/L, Monitoring lower-cut 0.1mg/L.
Using newborn bovine serum as dilution, by serum amyloid A protein standard items be configured to concentration be 250,100,
50, the standard solution of 20,10,2,0.5 and 0mg/L takes 60 μ L sample-adding to be detected, test strips is placed in fluorescence after 10min
Under immunity analysis instrument, the fluorescence intensity of T line (detection line) and C line (nature controlling line) is detected, according to the fluorescence intensity of T line and C line
Ratio draws the standard curve of reflection SAA content respectively, and each concentration repeats detection 3 times, is averaged as detectable concentration, with matching
Concentration processed and detectable concentration do linear equation and calculate related coefficient.The result shows that linear correlation coefficient r > 0.99, curve side
Journey y=0.9484x+0.7498.The linear detection range of SAA is 0.5-250mg/L, Monitoring lower-cut 0.5mg/L.
2, repeated assessment
The c reactive protein solution for being 10mg/L and 0mg/L by concentration, each concentration repeat detection 10 times, calculate variation lines
Number CV.The result shows that CV is less than 10%.
The serum amyloid A protein solution for being 0mg/L and 0mg/L by concentration, each concentration repeat detection 10 times, calculate and become
Different coefficient CV.The result shows that CV is less than 10%.
3, the assessment of accuracy
It is 1mg/L c reactive protein solution as basic sample using concentration, the c reactive protein of same volume various concentration is added
Human serum is configured to the c reactive protein solution that concentration is 20mg/L and 10mg/L；The feminine gender of another sample addition same volume
Human serum repeats detection 3 times to recycling sample and basic sample, calculates the rate of recovery.The result shows that the rate of recovery is in 90%-110%
In range.
It is 1mg/L serum amyloid A protein solution as basic sample using concentration, the serum of same volume various concentration is added
Amyloid A human serum is configured to the serum amyloid A protein solution that concentration is 20mg/L and 10mg/L；Another sample
The negative human serum of same volume is added, detection 3 times is repeated to recycling sample and basic sample, calculates the rate of recovery.The result shows that
The rate of recovery is within the scope of 90%-110%.
4 test sample of embodiment
The CRP reagent and mating detection system, depth of this kit and Mairui Biological Medical Electronic Co., Ltd., Shenzhen
Zhen Guosai Bioisystech Co., Ltd SAA reagent and mating detection system carry out test sample, and table 1- table 2 is kit of the present invention
Measurement result and Siemens's instrument and matched reagent box measurement result correlation coefficient r are respectively 0.9945 and 0.9990, correlation
Well, it can be used for clinical diagnosis use.
1 CRP clinical sample comparison result of table
2 SAA clinical sample comparison result of table
The experiment of 5 stabilization of kit of embodiment
This kit is measured after being stored in 2 DEG C -30 DEG C, 18 months with a sample, and the accuracy of kit, which is all satisfied, to be wanted
Ask (table 3).Kit is stored in 37 DEG C and 45 DEG C of insulating boxs save, observes measurement in 7 days with a sample, kit it is accurate
Degree meets the requirements (table 4).
3 kit of table, 2 DEG C of -30 DEG C of storage stability results
4 kit of table investigates result in 37 DEG C and 45 DEG C of accelerated stabilities
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1.CRP and SAA combined detection test paper, which is characterized in that including sample pad, bonding pad, analyzing film, absorption pad and bottom
Plate, the analyzing film are equipped with 2 detection lines and 1 nature controlling line, and the sample pad, bonding pad, analyzing film and absorption pad are successively
It is pasted on bottom plate and partially overlaps respectively to overlap joint；
Wherein, 2 detection lines are close to bonding pad one end, and nature controlling line is close to water absorption pad one end；
2 detection lines are respectively CRP detection line, SAA detection line；The CRP detection line is coated with CRP detection antibody, the SAA
Detection line is coated with SAA detection antibody；
The bonding pad is coated with CRP antibody mark fluorescent microballoon and SAA antibody mark fluorescent microballoon；
The coated antibody of detection line is respectively provided with different antigen binding sites from the coated antibody of the bonding pad.
2. test strips according to claim 1, which is characterized in that the sample pad is glass fibre element film or polyester fiber
Plain film；And/or
The bonding pad is glass fibre element film or polyester cellulose film；And/or
The analyzing film is nitrocellulose filter；And/or
The material of the bottom plate is PVC.
3. test strips according to claim 1, which is characterized in that fluorescent microsphere used is the polyphenyl second containing fluorescent material
Alkene microballoon, the fluorescent material be selected from fluorescein isothiocynate, rhodamine, tetramethylisothiocyanate rhodamine, TO, Cy3, Cy5,
At least one of phycoerythrin, quantum dot；
The partial size of the fluorescent microsphere is 20nm~600nm, and launch wavelength is 500nm~700nm, the use concentration of fluorescent microsphere
For 0.00001mg/ml~0.1mg/ml；The surface modification of the fluorescent microsphere has functional group, and the functional group is selected from carboxyl, ammonia
At least one of base or aldehyde radical.
4. test strips according to claim 1, which is characterized in that the CRP detection antibody is the production of Abgree company
Antibody 801001, the SAA detection antibody are the antibody 2203 of Medix Biochemica company production；And/or
CRP antibody used in the CRP antibody mark fluorescent microballoon is the antibody 801002 of Abgree company production, described
SAA antibody used in SAA antibody mark fluorescent microballoon is the antibody 2201 of Medix Biochemica company production；And/or
The nature controlling line is coated with secondary antibody, and the secondary antibody is selected from least one of sheep anti mouse, goat-anti rabbit, rabbit-anti goat-anti body.
5. test strips according to claim 1, which is characterized in that the CRP antibody mark fluorescent microballoon be by EDC and
NHS connect CRP antibody with the functional group on fluorescent microsphere；And/or
The SAA antibody mark fluorescent microballoon is to connect SAA antibody with the functional group on fluorescent microsphere by EDC and NHS.
6. test strips according to claim 1-5, which is characterized in that the length of the test strips is 5~10cm,
Width is 3~5mm.
7.CRP and SAA combined detection kit, which is characterized in that the kit includes described in any one of claims 1-6
Test strips, optionally comprising for loading getting stuck for the test strips.
The preparation method of 8.CRP and SAA combined detection test paper, which comprises the steps of:
(1) microballoon activates: it is added into 1~5ml of 1mg/ml fluorescent microsphere and uses 50mM, 0.1~10mg of pH6.1 MES dissolution
EDC and 10mg NHS activates 10~60min, and 7000~15000rpm is centrifuged 30min, abandons supernatant, precipitating pH7.0~8.0
50mM PBS is resuspended, and obtains the fluorescent microsphere solution of 1mg/ml activation；
Wherein, the fluorescent microsphere is using fluorescent microsphere described in claim 3；
(2) coupling of CRP antibody: 0.2mg CRP labelled antibody is added in the fluorescent microsphere solution 2ml of 1mg/ml activation, room
Temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating closes 60min with the 50mM PBS solution of the BSA containing 5mg,
It stirs and evenly mixs, 10000rpm is centrifuged 30min, and the precipitating of collection is stored in 20mM Tris buffer of the pH7.8 containing 0.5%BSA
In, obtaining concentration is 0.1mg/ml CRP antibody mark fluorescent microspheres solution；
Wherein, the CRP labelled antibody is the antibody 801002 of Abgree company production；
(3) coupling of SAA antibody: 0.4mg SAA labelled antibody is added in the fluorescent microsphere solution 2ml of 1mg/ml activation, room
Temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating is closed 60min with the 50mM PBS solution containing 1%BSA, stirred
Mixing is mixed, 10000rpm is centrifuged 30min, and the precipitating of collection is stored in 20mM Tris buffer of the pH7.8 containing 0.5%BSA,
Obtaining concentration is 0.1mg/ml SAA antibody mark fluorescent microspheres solution；
Wherein, the SAA labelled antibody is the antibody 2201 of Medix Biochemica company production；
(4) by fluorescent microsphere solution obtained by step (2) and step (3), 1:0.1~1:10 is mixed by volume；
(5) preparation of bonding pad: using glass fibre element film or polyester cellulose film as bonding pad, by the glimmering of step (4) preparation
Light microballoon mixed liquor is sprayed on film by 0.25~1.5 μ L/cm, drying for standby；
(6) preparation of analyzing film: CRP is detected into antibody, SAA detection antibody and secondary antibody and is diluted to 0.8-1mg/ml, with 0.8-1.2
The discharge rate of μ L/cm is sprayed on nitrocellulose filter, forms CRP detection line, SAA detection line and nature controlling line, as analyzing film, is done
It is dry spare；
Wherein, the CRP detection antibody is the antibody 801001 of Abgree company production, and the SAA detection antibody is Medix
The antibody 2203 of Biochemica company production, the secondary antibody are sheep anti-mouse igg；
(7) assembling of test strips: sample pad, bonding pad, analyzing film and water absorption pad being pasted into successively overlapping on PVC bottom plate and
It partially overlaps respectively, cutting into length is 5~10cm, and width is the paper slip of 3~5mm to get CRP and SAA joint-detection test paper
Item；
Wherein, the sample pad is glass fibre element film or polyester cellulose film.
9. according to the method described in claim 8, it is characterized by comprising the following steps:
(1) microballoon activates: being to be added to use in carboxyl fluorescent microsphere 1ml that 100nm launch wavelength is 535nm to 1mg/ml partial size
1mg EDC and the 10mg NHS of 50mM, pH6.1 MES dissolution, activates 30min, and 10000rpm is centrifuged 30min and abandons supernatant, precipitating
It is resuspended with the 50mM PBS of pH7.4, obtains the fluorescent microsphere solution of 1mg/ml activation；
(2) the activation fluorescent microsphere solution that 2ml concentration is 1mg/ml the coupling of CRP antibody: is added in 0.2mgCRP labelled antibody
In, room temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating is closed with the 50mM PBS solution of the BSA containing 5mg
60min is stirred and evenly mixed, and 10000rpm is centrifuged 30min, and it is slow that the precipitating of collection is stored in 20mM Tris of the pH7.8 containing 0.5%BSA
In fliud flushing, obtaining concentration is 0.1mg/ml CRP antibody mark fluorescent microspheres solution；
(3) the activation fluorescent microsphere solution that 2ml concentration is 1mg/ml the coupling of SAA antibody: is added in 0.4mgSAA labelled antibody
In, room temperature mixes 2~4h, and 10000rpm is centrifuged 30min, abandons supernatant, and precipitating is closed with the 50mM PBS solution containing 1%BSA
60min is stirred and evenly mixed, and 10000rpm is centrifuged 30min, and it is slow that the precipitating of collection is stored in 20mM Tris of the pH7.8 containing 0.5%BSA
In fliud flushing, obtaining concentration is 0.1mg/ml SAA antibody mark fluorescent microspheres solution；
(4) by fluorescent microsphere solution obtained by step (2) and step (3), 1:1 is mixed by volume；
(5) preparation of bonding pad: using glass fibre membrane as bonding pad, fluorescent microsphere mixed liquor prepared by step (4) is used a little
Film instrument is sprayed on pretreated glass fibre element film with 0.8 μ L/cm, drying for standby；
(6) preparation of analyzing film: detecting antibody, SAA detection antibody and secondary antibody sheep anti-mouse igg for CRP and be diluted to 0.8mg/ml, with
The discharge rate of 1 μ L/cm is sprayed on nitrocellulose filter, is respectively formed CRP detection line, SAA detection line and nature controlling line, is as analyzed
Film, drying for standby；
(7) assembling of test strips: sample pad, bonding pad, analyzing film and water absorption pad being pasted into successively overlapping on PVC bottom plate and
It partially overlaps respectively, cutting into length is 8cm, and width is the paper slip of 4mm to get CRP and SAA combined detection test paper.
The preparation method of 10.CRP and SAA combined detection kit, which is characterized in that by any one of the claim 1-6 examination
Paper slip, or be loaded into according to test strips prepared by claim 8 or 9 the methods get stuck in get.
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| CN110927388A (en) * | 2019-11-25 | 2020-03-27 | 益善生物技术股份有限公司 | Preparation method of CRP and SAA combined detection test strip, and prepared test strip, reagent card and kit |
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