CN106814190A - PCT and CRP joint inspection test strips and preparation method thereof - Google Patents
PCT and CRP joint inspection test strips and preparation method thereof Download PDFInfo
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- CN106814190A CN106814190A CN201710041314.XA CN201710041314A CN106814190A CN 106814190 A CN106814190 A CN 106814190A CN 201710041314 A CN201710041314 A CN 201710041314A CN 106814190 A CN106814190 A CN 106814190A
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- G—PHYSICS
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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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
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Abstract
The invention discloses a kind of PCT and CRP joint inspection test strips and preparation method thereof, the PCT includes a supporting pad with CRP joint inspections test strips, located at the coated film of the supporting pad, part covers the gold pad of one end of the coated film, part covers second sample pad of the gold pad away from one end of coated film, part covers first sample pad of second sample pad away from one end of gold pad, the coated film includes porous matrix and is sequentially arranged in the CRP detection bands of the porous matrix, PCT detection bands and quality control band, the CRP detection bands are near gold pad, the CRP detection bands are coated with CRP antibody, the PCT detection bands are coated with PCT antibody, the quality control band is coated with rabbit anti-mouse igg antibody, second sample pad is the glass fibre element film for being closed with CRP antibody.The PCT can simultaneously detect PCT and CRP with CRP joint inspections test strips, and sensitivity is high.
Description
Technical field
The present invention relates to technical field of clinical medical detection, particularly a kind of PCT and CRP joint inspections test strips and its preparation side
Method.
Background technology
Procalcitonin (Procalcitonin, PCT) is a kind of SGP, is made up of 116 amino acid, is blood
The preceding leach protein of clear calcitonin (Calcitonin, CT).As a kind of immune modulator, PCT (Procalcitonin, PCT)
It is a kind of metastable albumen in blood sample, PCT (Procalcitonin, PCT) releases is can induce during bacterium infection, makes blood
Slurry Procalcitonin (Procalcitonin, PCT) level is raised.Recent study shows, Procalcitonin (Procalcitonin,
PCT) detect compared with traditional index such as white blood cell count(WBC), erythrocyte sedimentation rate, c reactive protein, Bacteria Culture, with spirit higher
Sensitivity and specificity.It is used to differentiate septicopyemia and systemic inflammatory responses syndrome, current Procalcitonin in clinic
(Procalcitonin, PCT) is one of most useful lab index.How Using adapted Antibios have become global doctor
Treat the common issue on boundary.Procalcitonin (Procalcitonin, PCT) detection can help doctor to provide antibiotic to infected patient
Therapeutic scheme, and whether effectively correct reaction antibiotic therapy, reduces the possibility that patient produces drug resistance to antibiotic in time.
C reactive protein (C-reactionprotein, CRP) is synthesized by liver cell, when body is infected or tissue damaged
Macrophage and other leucocytes etc. are activated when hindering, and produce interleukin-6 (IL-6), interleukin 1 (IL-1), swell
The cell factors such as tumor necrosis factor TNF-a and other mediators, these cell factors and mediators reach liver, cell cultured supernatant
Synthesize C reactive protein (C-reactionprotein, CRP) with epithelial cell.In structure, C reactive protein (C-
Reactionprotein, CRP) contain 5 polypeptide chain subunits, dish type polymer is noncovalently combined into, molecular weight is 11.5
Ten thousand -14 ten thousand, C reactive protein (C-reactionprotein, CRP) is a kind of typical acute phase protein.C reactive protein
(C-reactionprotein, CRP) is determined and be can be used to evaluate infection, tissue damage and diseases associated with inflammation.In healthy population blood
Middle C reactive protein (C-reactionprotein, CRP) level is less than 5 mg/litres, and under various conditions, acute inflammation 4-
In 8 hours, C reactive protein (C-reactionprotein, CRP) value reaches about 20 to 500 mg/litres.C reactive protein (C-
Reactionprotein, CRP) as acute inflammation evaluation index than erythrocyte sedimentation rate (ESR) (Erythrocyte
Sedimentation Rate, ESR) and white blood cell count(WBC) it is more sensitive, more reliable.
At present, above-mentioned two detection project is main is widely used within the hospital in the form of individual event detection, and market application is
It is quite ripe.But PCT (Procalcitonin, PCT) and C reactive protein (C-reactionprotein, CRP) joint inspection product
It is also phoenix feathers and unicorn horns in market is applied.It is main reason is that Procalcitonin (Procalcitonin, PCT) reacts egg with C-
(C-reactionprotein, CRP) is different in body burden's rank in vain, Procalcitonin (Procalcitonin, PCT) it is sensitive
Degree is 0.1ng/ml, and the sensitivity of C reactive protein (C-reactionprotein, CRP) is 0.5mg/L, and both differ near
5000 times.After needing to 100-300 times of Sample Dilution in C reactive protein (C-reactionprotein, CRP) individual event detection again
Detected, do not dilute or extension rate is improper can all a hook effect (HOOK effects), detected inaccurate, so in calcitonin
Sample should be first solved in former (Procalcitonin, PCT) and C reactive protein (C-reactionprotein, CRP) joint inspection product
The problem of this sample-adding.
The content of the invention
It is a primary object of the present invention to provide a kind of PCT and CRP joint inspection test strips, it is intended to improve the accuracy of detection,
Realize detecting PCT and CRP simultaneously in a test paper.
To achieve the above object, the PCT that the present invention is provided is with CRP joint inspections test strips including a supporting pad, located at the branch
The coated film of stake pad, partly cover the coated film one end gold pad, partly cover the one end of the gold pad away from coated film
The second sample pad, partly cover first sample pad of second sample pad away from one end of gold pad, the coated film includes
Porous matrix and the CRP detection bands, PCT detection bands and the quality control band that are sequentially arranged in the porous matrix, the CRP detection bands are close to
Gold pad, the CRP detection bands are coated with CRP antibody, and the PCT detection bands are coated with PCT antibody, and the quality control band is coated with rabbit
Dynamics, second sample pad is the glass fibre element film for being closed with CRP antibody.
Preferably, the spacing distance between the CRP detection bands and PCT detection bands is 3~10mm, the PCT detection bands
Spacing distance and quality control band between is 3~10mm.
Preferably, the porous matrix is nitrocellulose filter, and the aperture of the porous matrix is 8~20 μm, and film is a width of
20~25mm.
Preferably, the gold pad is coated with the poly- of CRP antibody colloidal golds label and PCT antibody colloidal gold labels
Ester pad, the width of the gold pad is 8~15mm.
Preferably, first sample pad is the hemofiltration film for being closed with blocker, and the width of first sample pad is 10
~20mm.
Preferably, the PCT also includes that part covers the coated film away from the one of the gold pad with CRP joint inspections test strips
The adsorptive pads at end.
The present invention also provides the preparation method of a kind of PCT and CRP joint inspection test strips, the PCT and CRP joint inspection test strips
Preparation process is as follows：
Prepare coated film：One porous matrix is provided, bag is respectively adopted to CRP antibody, PCT antibody and rabbit anti-mouse igg antibody
It is buffered liquid to be diluted, the CRP antibody after dilution, PCT antibody and rabbit anti-mouse igg antibody is sprayed at porous matrix respectively
One surface, CRP antibody penetrates into porous matrix and forms CRP detection bands, and PCT antibody penetrates into porous matrix and forms PCT detection bands, rabbit-anti
Mouse IgG antibody is penetrated into porous matrix and forms quality control band, and the CRP detection bands and quality control band are respectively positioned at the both sides of PCT detection bands；
Treatment is dried to the porous matrix after spray treatment, coated film is obtained final product；
Prepare the second sample pad：Sealing pores are carried out using glass fibre element membrane closure liquid to glass fibre element film, it is described
Glass fibre element membrane closure liquid includes the BSA that mass percent is 3%~8%, and percent by volume is 0.1%~0.5%
Tween-20, mass percent is 2%~5% trehalose, the CRP antibody of 0.01~0.03mg/ml, 0.01~0.02Mol,
PH is 6.5~8.0 PBS solution, to being dried treatment through the glass fibre element film after sealing pores, obtains final product the second sample
Pad；
Prepare gold pad；
Prepare the first sample pad；
The quantitative joint inspection test strips of assembling PCT and CRP：Coated film is bonded on supporting pad, gold pad is then adhered to coating
One end of film, the one end of gold pad away from coated film is adhered to by the second sample pad, and the first sample pad is adhered into the second sample pad
Away from one end of gold pad.
Preferably, the coating buffer solution includes the methyl alcohol that percent by volume is 1~5%, mass percent is 2%~
5% trehalose, 0.01~0.02Mol, pH are 6.5~8.0 PBS solution；The concentration of the CRP antibody is 0.5~2mg/
Ml, the concentration of the PCT antibody is 0.5~2mg/ml, and the concentration of the rabbit anti-mouse igg antibody is 0.5~1mg/ml, described
The coating buffer solution of CRP detection bands spraying and the consumption of CRP antibody are 0.7~1.2 μ l/cm, the bag of the PCT detection bands spraying
It is 0.7~1.2 μ l/cm, the coating buffer solution and rabbit anti-mouse igg of the quality control band spraying to be buffered the consumption of liquid and PCT antibody
The consumption of antibody is 0.7~1.2 μ l/cm.
Preferably, the preparation process of the gold pad includes：
(1) collaurum coating buffer, is prepared：The collaurum coating buffer includes CRP antibody colloidal golds label, PCT antibody
Colloid gold label thing and liquid is redissolved, the volume ratio of the CRP antibody colloidal golds label and PCT antibody colloidal gold labels is：
15~20:15~30；
The preparation of a, CRP antibody colloidal gold label：The antibody labeling amount of the CRP antibody colloidal golds label is 4~7
μ g/ml, 6.5~8.5 are adjusted to the solution of potassium carbonate of 0.1~0.3Mol by the pH value of collaurum, add CRP antibody stirring 10
~30 minutes, the BSA for adding mass percent to be 0.1%~0.5% carried out Seal treatment, is then centrifuged for purification process and obtains CRP
Antibody colloidal gold label；
The preparation of b, PCT antibody colloidal gold label：The antibody labeling amount of the PCT antibody colloidal golds label is 4~7
μ g/ml, 6.5~8.5 are adjusted to the solution of potassium carbonate of 0.1~0.3Mol by the pH value of collaurum, add PCT antibody stirring 10
~30 minutes, the BSA for adding mass percent to be 0.1%~0.5% carried out Seal treatment, and centrifugal purification processes to obtain PCT antibody
Colloid gold label thing；
Wherein, the preparation of CRP antibody colloidal golds label and collaurum in the preparation process of PCT antibody colloidal gold labels
Preparation method be：The gold chloride that 100ml, mass percent are 0.01% is heated to boiling, 0.75~1.5ml, matter is added
Measure the sodium solution of citric acid three that percentage is 1% to be reacted, obtain the collaurum that particle size is 30~60nm；
C, mixing CRP antibody colloidal golds label, PCT antibody colloidal golds label and redissolution liquid, the redissolution liquid include：
Mass percent is 3%~8% BSA, and mass percent is 3%~8% trehalose, and mass percent is 0.1%~1%
Casein, percent by volume is 0.1%~0.5% Tween-20, the boric acid of 0.002~0.01Mol, and mass percent is
0.1%~0.5% PEG, pH is 8.0~9.0；
(2) polyester mat confining liquid, is provided, polyester mat confining liquid includes the casein that mass percent is 0.1%~1%,
Mass percent is 1%~5% trehalose, and percent by volume is 0.1%~1% tween20,0.002~0.01Mol's
Boric acid, mass percent be 0.1%~0.5% PEG, pH be 8.0~9.0；Polyester mat is provided, polyester mat is immersed into polyester mat
Confining liquid carries out Seal treatment to polyester mat, to being dried treatment by the polyester mat after Seal treatment, collaurum is coated with
Liquid is sprayed at polyester mat, and collaurum coating buffer penetrates into polyester mat, then is dried treatment, obtains final product gold pad.
Preferably, the preparation process of first sample pad includes：Hemofiltration film is provided；Hemofiltration membrane closure liquid, hemofiltration are provided
Membrane closure liquid includes the BSA that mass percent is 0.5~2%, and percent by volume is 0.1~1% Tween-20,0.005~
0.02Mol, pH are 6.5~8.0 PBS solution；Blocking reagent is provided, blocking reagent is dissolved in hemofiltration membrane closure liquid；It is right
Being dissolved with the hemofiltration membrane closure liquid of blocking reagent carries out immersion treatment to hemofiltration film, and blocking reagent penetrates into hemofiltration film, to soaking
Hemofiltration film after treatment is dried treatment.
PCT of the invention and CRP joint inspections test strips add CRP antibody to be processed by when the second sample pad is prepared,
The second sample pad for obtaining can neutralize CRP immunizing antigens to be detected, and method is simple, finally reaches the CRP ranges of linearity
0.1-100mg/L, while having also complied with PCT sensitivity 0.1ng/ml requirements.
The present invention has following remarkable advantage：PCT of the invention and CRP joint inspection test strips are provided with supporting pad supporting pad and are provided with
Coated film, coated film one end is provided with gold pad, and gold pad one end is provided with the second sample pad, and the second sample pad is provided with the first sample pad, bag
Envelope is sequentially provided with CRP detection bands, PCT detection bands and quality control band, and CRP detection bands are preparing the second sample near the second sample pad
Add CRP antibody to be processed during product pad, it is possible to achieve detection people whole blood, blood plasma and in serum PCT and CRP content, PCT's
Detection range is 0.1~50ng/ml, and the detection range of CRP is 0.5~100mg/L；The sensitivity of PCT reaches 0.1ng/ml,
CRP sensitivity reaches 0.5mg/L.
Brief description of the drawings
Fig. 1 is the side structure schematic diagram of one embodiment of the invention PCT and CRP joint inspection test strips；
Fig. 2 is the linear dependence diagram of one embodiment of the invention PCT and PCT in CRP joint inspection test strips；
Fig. 3 is the linear dependence diagram of one embodiment of the invention PCT and CRP in CRP joint inspection test strips.
Drawing reference numeral explanation：
The realization of the object of the invention, functional characteristics and advantage will be described further referring to the drawings in conjunction with the embodiments.
Specific embodiment
It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not intended to limit the present invention.
Fig. 1 is refer to, in one embodiment of the invention, Procalcitonin (Procalcitonin, PCT) and C reactive protein
(C-reactionprotein, CRP) joint inspection test strips 100 include a supporting pad 10, the coated film located at the supporting pad 10
20th, partly cover the gold pad 30 of one end of the coated film 20, partly cover the gold pad 30 away from one end of coated film 20
Second sample pad 40, partly cover first sample pad 50 of second sample pad 40 away from one end of gold pad 30, the coating
Film 20 includes porous matrix and is sequentially arranged in CRP detection bands 21, PCT detection bands 22 and the quality control band 23 of the porous matrix, institute
CRP detection bands 21 are stated near gold pad 30, the CRP detection bands 21 are coated with CRP antibody, and the PCT detection bands 22 are coated with PCT
Antibody, the quality control band 23 is coated with rabbit anti-mouse igg antibody, and second sample pad 40 is the glass fibers for being closed with CRP antibody
The plain film of dimension.
Specifically, PCT antibody be can with antigen PCT to be detected specifically bind antibody, CRP antibody can be with it is to be detected
The antibody of antigens c RP specific bindings, antigen PCT to be detected is the PCT of people, and antigens c RP to be detected is the CRP of people.PCT antibody
It is PCT monoclonal antibodies, or is PCT polyclonal antibodies, CRP antibody is CRP monoclonal antibody, or is CRP polyclonal antibodies, rabbit
Dynamics are rabbit anti-mouse igg monoclonal antibody, or rabbit anti-mouse igg polyclonal antibody.Preferred PCT in the embodiment of the present invention
Antibody is PCT monoclonal antibodies, and CRP antibody is CRP monoclonal antibody, and rabbit anti-mouse igg antibody is rabbit anti-mouse igg Anti-TNF-α
Body.One surface of supporting pad 10 is provided with viscose glue, and the surface for being provided with viscose glue is provided with coated film 20, gold pad 30, the and of the second sample pad 40
First sample pad 50 is adhered to supporting pad 10.The width of the second sample pad 40 is 5~15mm, and the second neutralization of sample pad 40 is treated
The effect of detection antigens c RP.Coated film 20 is the supporting body of CRP detection bands 21, PCT detection bands 22 and quality control band 23, is also anti-
Answer area.Porous matrix is nitrocellulose filter, and the aperture of the porous matrix is 8~20 μm, a width of 20~25mm of film.
Procalcitonin (Procalcitonin, PCT) of the invention and C reactive protein (C-reactionprotein,
CRP) supporting pad 10 of joint inspection test strips 100 is provided with coated film 20, and one end of coated film 20 is bonded with gold pad 30, the second sample successively
The sample pad 50 of product pad 40 and first, the second sample pad 40 is closed with the glass fibre element film of CRP antibody, the Procalcitonin
(Procalcitonin, PCT) can be detected simultaneously with C reactive protein (C-reactionprotein, CRP) joint inspection test strips 100
Procalcitonin (Procalcitonin, PCT) and C reactive protein (C-reactionprotein, CRP), detection sensitivity is high.
Second sample pad 40 is coated in the first seal of glass fibre element film, and hermetically be coated with CRP antibody by first seal
In glass fibre element film.
Further, the spacing distance between CRP detection bands 21 and PCT detection bands 22 is 3~10mm, the PCT detections
It is 3~10mm with the spacing distance between 22 and quality control band 23.PCT detection bands 22 can detect PCT projects in antigen to be detected
Effect；CRP detection bands 21 can detect the effect of CRP projects in antigen to be detected；Whether quality control band 23 can judge quality control reagent card
Effectively, there is red, as a result for interpretation effectively, redfree knot fruit is invalid.
Further, gold pad 30 is coated with the poly- of CRP antibody colloidal golds label and PCT antibody colloidal gold labels
Ester pad, the width of the gold pad 30 is 8~15mm.Wherein gold pad 30 carries CRP antibody colloidal golds label and PCT antibody glue
The effect of body gold label.Gold pad 30 is coated in the second seal of polyester mat, and second seal is by CRP antibody colloidal golds
Label and PCT antibody colloidal gold labels are hermetically coated in polyester mat.
Further, the first sample pad 50 is the hemofiltration film for being closed with blocker, and the width of first sample pad 50 is
10~20mm.First sample pad 50 plays nonspecific proteins and red blood cell in treatment antigen to be detected.First sample pad
The blocker is hermetically coated in hemofiltration film by 50 the 3rd seals for being coated in hemofiltration film, the 3rd seal.
Further, Procalcitonin (Procalcitonin, PCT) and C reactive protein (C-reactionprotein,
CRP) joint inspection test strips also include that part covers adsorptive pads 60 of the coated film 20 away from one end of the gold pad 30.Adsorptive pads
60 absorb reacted waste liquid.
The invention also discloses a kind of Procalcitonin (Procalcitonin, PCT) and C reactive protein (C-
Reactionprotein, CRP) joint inspection test strips preparation method, Procalcitonin in one embodiment of the invention
The preparation process of (Procalcitonin, PCT) and C reactive protein (C-reactionprotein, CRP) joint inspection test strips is such as
Under：
Prepare coated film 20：One porous matrix is provided, CRP antibody, PCT antibody and rabbit anti-mouse igg antibody are respectively adopted
Coating buffer solution is diluted, and the CRP antibody after dilution, PCT antibody and rabbit anti-mouse igg antibody are sprayed at into porous matrix respectively
A surface, CRP antibody penetrates into porous matrix and forms CRP detection bands 21, and PCT antibody penetrates into porous matrix and forms PCT detection bands
22, rabbit anti-mouse igg antibody penetrates into porous matrix and forms quality control band 23, and the CRP detection bands 21 and quality control band 23 are located at PCT respectively
The both sides of detection band 22；Treatment is dried to the porous matrix after spray treatment, coated film 20 is obtained final product；
Prepare the second sample pad 40：Sealing pores, institute are carried out using glass fibre element membrane closure liquid to glass fibre element film
Stating glass fibre element membrane closure liquid includes the BSA that mass percent is 3%~8%, and percent by volume is 0.1%~0.5%
Tween-20, mass percent is 2%~5% trehalose, the CRP antibody of 0.01~0.03mg/ml, 0.01~0.02Mol,
PH is 6.5~8.0 PBS solution, to being dried treatment through the glass fibre element film after sealing pores, obtains final product the second sample pad
40；
Prepare gold pad 30；
Prepare the first sample pad 50；
Assembling Procalcitonin (Procalcitonin, PCT) is fixed with C reactive protein (C-reactionprotein, CRP)
Amount joint inspection test strips：Coated film 20 is bonded on supporting pad 10, then gold pad 30 is adhered to one end of coated film 20, by second
Sample pad 40 is adhered to the one end of gold pad 30 away from coated film 20, and the first sample pad 50 is adhered into the second sample pad 40 away from gold
One end of pad 30.
Specifically, in step, coating buffer solution includes the methyl alcohol that percent by volume is 1~5%, and mass percent is 2%
~5% trehalose, 0.01~0.02Mol, pH are 6.5~8.0 PBS solution；The concentration of the CRP antibody be 0.5~
2mg/ml, the concentration of the PCT antibody is 0.5~2mg/ml, and the concentration of the rabbit anti-mouse igg antibody is 0.5~1mg/ml, institute
It is 0.7~1.2 μ l/cm to state the coating buffer solution of the spraying of CRP detection bands 21 and the consumption of CRP antibody, and the PCT detection bands 22 are sprayed
The coating buffer solution of painting and the consumption of PCT antibody are 0.7~1.2 μ l/cm, the coating buffer solution of the spraying of the quality control band 23 and rabbit
The consumption of dynamics is 0.7~1.2 μ l/cm.Prepare coated film 20, the second sample pad 40, gold pad 30, the step of gold pad 30
Suddenly can simultaneously carry out successively carrying out, be limited without order.
Procalcitonin (Procalcitonin, PCT) of the invention and C reactive protein (C-reactionprotein,
CRP the concentration of addition CRP antibody, finally makes CRP when) preparation method of joint inspection test strips is by adjusting the second sample pad 40 for the treatment of
(C-reactionprotein, CRP) range of linearity reaches 0.1~100mg/L, while having also complied with PCT
(Procalcitonin, PCT) sensitivity 0.1ng/ml requirements.And detection sensitivity is high.
Further, the preparation process of gold pad 30 includes：
(1) collaurum coating buffer, is prepared：The collaurum coating buffer includes CRP antibody colloidal golds label, PCT antibody
Colloid gold label thing and redissolution liquid；The volume ratio of the CRP antibody colloidal golds label and PCT antibody colloidal gold labels is：
15~20:15~30；
The preparation of a, CRP antibody colloidal gold label：The antibody labeling amount of the CRP antibody colloidal golds label is 4~7
μ g/ml, 6.5~8.5 are adjusted to the solution of potassium carbonate of 0.1~0.3Mol by the pH value of collaurum, add CRP antibody stirring 10
~30 minutes, the BSA for adding mass percent to be 0.1%~0.5% carried out Seal treatment, is then centrifuged for purification process and obtains CRP
Antibody colloidal gold label；
The preparation of b, PCT antibody colloidal gold label：The antibody labeling amount of the PCT antibody colloidal golds label is 4~7
μ g/ml, 6.5~8.5 are adjusted to the solution of potassium carbonate of 0.1~0.3Mol by the pH value of collaurum, add PCT antibody stirring 10
~30 minutes, the BSA for adding mass percent to be 0.1%~0.5% carried out Seal treatment, and centrifugal purification processes to obtain PCT antibody
Colloid gold label thing；
Wherein, the preparation of CRP antibody colloidal golds label and collaurum in the preparation process of PCT antibody colloidal gold labels
Preparation method be：The gold chloride that 100ml, mass percent are 0.01% is heated to boiling, 0.75~1.5ml, matter is added
Measure the sodium solution of citric acid three that percentage is 1% to be reacted, obtain the collaurum that particle size is 30~60nm；
C, mixing CRP antibody colloidal golds label, PCT antibody colloidal golds label and redissolution liquid, the redissolution liquid include：
Mass percent is 3%~8% BSA, and mass percent is 3%~8% trehalose, and mass percent is 0.1%~1%
Casein, percent by volume is 0.1%~0.5% Tween-20, the boric acid of 0.002~0.01Mol, and mass percent is
0.1%~0.5% PEG, pH is 8.0~9.0；
(2) polyester mat confining liquid, is provided, polyester mat confining liquid includes the casein that mass percent is 0.1%~1%,
Mass percent is 1%~5% trehalose, and percent by volume is 0.1%~1% tween20,0.002~0.01Mol's
Boric acid, mass percent be 0.1%~0.5% PEG, pH be 8.0~9.0；Polyester mat is provided；Polyester mat is immersed into polyester mat
Confining liquid carries out Seal treatment to polyester mat, to being dried treatment by the polyester mat after Seal treatment, collaurum is coated with
Liquid is sprayed at polyester mat, and collaurum coating buffer penetrates into polyester mat, then is dried treatment, obtains final product gold pad 30.
Further, the preparation process of the first sample pad 50 includes：Hemofiltration film is provided；Hemofiltration membrane closure liquid, hemofiltration are provided
Membrane closure liquid includes the BSA that mass percent is 0.5~2%, and percent by volume is 0.1~1% Tween-20,0.005~
0.02Mol, pH are 6.5~8.0 PBS solution；The HBR and 0.1mg/ml~0.5mg/ml of 0.1mg/ml~1mg/ml are provided
Anti- RBC, blocking reagent is dissolved in hemofiltration membrane closure liquid；Hemofiltration membrane closure liquid to being dissolved with blocking reagent enters to hemofiltration film
Row immersion treatment, blocking reagent penetrates into hemofiltration film, and treatment is dried to the hemofiltration film after soaking treatment.Hindered in the present embodiment
Disconnected reagent is anti-RBC (people's anti erythrocyte antibody) and HBR solution, and being processed the first sample pad 50 and acted on by hemofiltration membrane closure liquid is
Non-specific sites are removed, antijamming capability is lifted, the sensitivity of detection is improved.
The present invention using Procalcitonin (Procalcitonin, PCT) with C reactive protein (C-reactionprotein,
CRP) during joint inspection test strips 100, antigen to be detected is dropped in into the first sample pad 50, the nonspecific proteins in antigen to be detected and
Red blood cell is processed by the first sample pad 50, with the effect of CRP immunizing antigens in antigen to be detected, gold pad in the second sample pad 40
30 carry colloidal gold reaction things, are reacted on coated film 20, i.e., the PCT antigens in antigen to be detected and PCT detection bands 22
In PCT antibody specificities combine, CRP antigens are combined with the CRP antibody specificities in CRP detection bands 21, and quality control band 21 judges
Whether effectively result, there is red, and as a result for interpretation effectively, redfree knot fruit is invalid, and adsorptive pads inhale reacted waste liquid
Receive.
The preparation method of one embodiment of the invention is specific as follows, comprises the steps of：
Prepare coated film 20：Aperture from nitrocellulose filter is 15 μm, film 25mm wide.CRP detection bands 21, PCT are examined
Coated antibody is prepared using coating buffer solution on measuring tape 22 and quality control band 23, and coating buffer solution includes percent by volume
It is 5% methyl alcohol, mass percent is 5% trehalose, and 0.01Mol, pH are 6.5~8.0 PBS；The concentration of CRP antibody is
The concentration of 1mg/ml, PCT antibody is 1mg/ml, and the concentration of rabbit anti-mouse igg antibody is 0.8mg/ml, the spraying of CRP detection bands 21
The consumption of coating buffer solution and CRP antibody is the coating buffer solution of the spraying of 0.8 μ l/cm, PCT detection band 22 and the use of PCT antibody
It is 0.8 μ l/cm to measure, and the coating buffer solution of the spraying of quality control band 23 and the consumption of rabbit anti-mouse igg antibody are 0.8 μ l/cm.CRP is detected
It is 3mm with spaced between 21, PCT detection bands 22 and quality control band 23, is made coated film 20.Coated film 20 is put into 45 DEG C
Drying box is toasted 24~48 hours.
Prepare gold pad 30：Gold pad 30 includes collaurum coating buffer and polyester mat.Collaurum coating buffer is by 20/125/ml's
CRP monoclonal antibody colloid gold label thing, the PCT monoclonal antibody colloid gold label things of 30/134/ml and surplus redissolve liquid group
Into.Colloid gold label thing is diluted using liquid is redissolved, and the redissolution liquid includes：5%BSA (bovine serum albumin(BSA)), 5% marine alga
Sugar, 0.5% casein, the boric acid of 0.002~0.01Mol, 0.1% PEG, pH are 9.0.Polyester mat application confining liquid is located in advance
Reason, fully dries, and cuts into 10mm polyester mats wide.Film instrument is drawn with HM-3030 three-dimensional metal sprayings to be coated on collaurum coating buffer
In polyester mat, consumption is 8 μ l/cm.
Wherein, the preparation of colloid gold label thing includes：CRP monoclonal antibody colloid gold label thing and PCT monoclonal antibodies
Colloid gold label thing is formed with colloid gold label albumen.
Collaurum is, for the gold chloride of 100ml 0.01% is heated to boiling, to be rapidly added quality percentage by percent by volume
Than being 1ml, 1% citric acid trisodium is reacted, and obtains the collaurum that particle size is 50nm.
The antibody labeling amount of CRP monoclonal antibody colloid gold label thing is 5 μ g/ml, with the solution of potassium carbonate of 0.1M by glue
The pH value of body gold is adjusted to 8.0, and antibody is stirred 10 minutes needed for adding, and adds a certain amount of 0.2% BSA (bovine serum albumin(BSA))
Seal treatment is carried out, centrifugal purification processes to obtain CRP monoclonal antibody colloid gold label thing, uses UV spectrophotometer measuring wavelength
531nm OD values (OD) are 125.
The antibody labeling amount of PCT monoclonal antibody colloid gold label things is 5 μ g/ml, with the solution of potassium carbonate of 0.1M by glue
The pH value of body gold is adjusted to 8.0, and antibody is stirred 10 minutes needed for adding, and adds a certain amount of 0.2% BSA (bovine serum albumin(BSA))
Seal treatment is carried out, centrifugal purification processes to obtain PCT monoclonal antibody colloid gold label things, uses UV spectrophotometer measuring wavelength
Highest optical density value (OD) 134 between 532nm.
Prepare the second sample pad 40：Second sample pad confining liquid is BSA (bovine serum albumins that mass percent is 5%
In vain), mass percent is 0.5% Tween-20,3% trehalose, 0.01mg/mlCRP monoclonal antibodies, balance of 0.01M
The PBS (Phosphate buffer saline, PBS) of pH7.4.Second sample pad 40 is sealed with the second sample pad
Liquid Seal treatment is closed, is dried 12 hours, cut into 10mm the second sample pads 40 wide.
Prepare the first sample pad 50:First sample pad confining liquid is BSA (bovine serum albumins that mass percent is 1%
In vain), percent by volume is 0.3% Tween-20, and percent by volume is the anti-RBC (people's anti erythrocyte antibody) of 0.5mg/ml,
The PBS (Phosphate buffer saline, PBS) of 0.3mg/mlHBR, balance of 0.01M pH7.4.The
One sample pad 50 is dried 12 hours with the first sample pad confining liquid Seal treatment, cuts into 16mm the first sample pads 50 wide.
Assembling Procalcitonin (Procalcitonin, PCT) is fixed with C reactive protein (C-reactionprotein, CRP)
Amount joint inspection test strips：Overlapped on supporting pad 10 and paste coated film 20, in one end of the close CRP detection bands 21 of coated film 20 successively
Overlap joint pastes gold pad 30, the second sample pad 40 and the first sample pad 50, viscous near one end overlap joint of quality control band 23 in coated film 20
Patch adsorptive pads 60, obtain final product the big plate of test paper, and the test strips of proper width are cut into as requested.
First sample pad 50 and the second sample pad 40 of the preparation method of another embodiment of the present invention, comprise the steps of：
Second sample pad confining liquid is the BSA (bovine serum albumin(BSA)) that mass percent is 5%, and percent by volume is
0.5% Tween-20, mass percent is 3% trehalose, and percent by volume is 0.03mg/mlCRP monoclonal antibodies, remaining
Measure the PBS (Phosphate buffer saline, PBS) for 0.01Mol, pH7.4.Second sample pad 40 is used
Second sample pad confining liquid Seal treatment, dries 12 hours, cuts into 10mm the second sample pads 40 wide.
First sample pad confining liquid is the BSA that mass percent is 1%, and percent by volume is 0.3% Tween-20, body
Product percentage is anti-RBC (people's anti erythrocyte antibody), the 0.3mg/mlHBR of 0.1mg/ml, balance of 0.01Mol, pH7.4's
PBS (Phosphate buffer saline, PBS).First sample pad 50 closes fluid-tight with the first sample pad
Treatment is closed, is dried 12 hours, cut into 16mm the first sample pads 50 wide.
Embodiments of the invention are evaluated, C reactive protein (C-reactionprotein, CRP) selects Shenzhen
Guo Sai Bioisystech Co., Ltd production Nephstar specific proteins analyzer and《Acute-phase response specific protein determines ratio
Turbid kit》It is reference standard；Procalcitonin (Procalcitonin, PCT) selects Roche cobas e601 and Procalcitonin
(PCT, Procalcitonin) Electrochemiluminescince immue quantitative detection reagent box is reference.
Table one is for of the invention with above-mentioned two instrument and the accuracy Comparability test result of reagent
Table one
As seen from the table, according to《Colloidal gold immunochromatographimethod determines reagent (box) drug evaluation specification》, the degree of accuracy is relatively inclined
Difference answers ± 15% standard, using the test strips of embodiment one high to C reactive protein (C-reactionprotein, CRP)
When value sample is detected, relative deviation is larger.It is relatively inclined to Procalcitonin (Procalcitonin, PCT) pattern detection result
Difference is good.Carried out to C reactive protein (C-reactionprotein, CRP) high level sample using the test strips of embodiment two
During detection, relative deviation has clear improvement.Illustrate that test strips of the invention can realize that accurate quantitative analysis are detected.
Table two is the accuracy result of the test of test paper of the present invention
Table two
As seen from the table, according to《Colloidal gold immunochromatographimethod determines reagent (box) drug evaluation specification》, accuracy variation lines
Number (CV%) should be not more than 20% standard, and test strips accuracy of the present invention is good.
Fig. 2 and Fig. 3 is refer to, according to following data：
Table three is the linear correlation data of PCT and CRP
Table three
Fig. 2 and Fig. 3 show, according to《Colloidal gold immunochromatographimethod determines reagent (box) drug evaluation specification》, contrast agent phase
Relation number (r) answers >=0.95 standard, the equal ＞ 0.99 of two correlation coefficient rs of project 2 of test strips of the invention, therefore the present invention
Test strips are in C reactive protein (C-reactionprotein, CRP) 0.5~100mg/L and Procalcitonin
Linear dependence is good during (Procalcitonin, PCT) 0.1~50ng/ml.
The preferred embodiments of the present invention are these are only, the scope of the claims of the invention is not thereby limited, it is every to utilize this hair
Equivalent structure or equivalent flow conversion that bright specification and accompanying drawing content are made, or directly or indirectly it is used in other related skills
Art field, is included within the scope of the present invention.
Claims (10)
1. a kind of PCT and CRP joint inspection test strips, it is characterised in that including a supporting pad, the coated film located at the supporting pad,
Part cover the coated film one end gold pad, partly cover the gold pad away from one end of coated film the second sample pad,
Part covers first sample pad of second sample pad away from one end of gold pad, and the coated film is including porous matrix and successively
CRP detection bands, PCT detection bands and quality control band located at the porous matrix, the CRP detection bands are near gold pad, the CRP inspections
Measuring tape is coated with CRP antibody, and the PCT detection bands are coated with PCT antibody, and the quality control band is coated with rabbit anti-mouse igg antibody, institute
It is the glass fibre element film for being closed with CRP antibody to state the second sample pad.
2. PCT as claimed in claim 1 and CRP joint inspection test strips, it is characterised in that the CRP detection bands and PCT detection bands
Between spacing distance be 3~10mm, spacing distance between the PCT detection bands and quality control band is 3~10mm.
3. PCT as claimed in claim 1 and CRP joint inspection test strips, it is characterised in that the porous matrix is nitrocellulose
Film, the aperture of the porous matrix is 8~20 μm, a width of 20~25mm of film.
4. the PCT as described in any one of claims 1 to 3 and CRP joint inspection test strips, it is characterised in that the gold pad includes bag
There is the polyester mat of CRP antibody colloidal golds label and PCT antibody colloidal gold labels, the width of the gold pad is 8~15mm.
5. the PCT as described in any one of claims 1 to 3 and CRP joint inspection test strips, it is characterised in that first sample pad
To be closed with the hemofiltration film of blocker, the width of first sample pad is 10~20mm.
6. the PCT as described in any one of claims 1 to 3 and CRP joint inspection test strips, it is characterised in that the PCT and CRP joins
Inspecting paper slip also includes that part covers adsorptive pads of the coated film away from one end of the gold pad.
7. a kind of preparation method of PCT and CRP joint inspection test strips, it is characterised in that the system of the PCT and CRP joint inspection test strips
Standby step is as follows：
Prepare coated film：One porous matrix is provided, coating is respectively adopted to CRP antibody, PCT antibody and rabbit anti-mouse igg antibody slow
Fliud flushing is diluted, and the CRP antibody after dilution, PCT antibody and rabbit anti-mouse igg antibody are sprayed at a table of porous matrix respectively
Face, CRP antibody penetrates into porous matrix and forms CRP detection bands, and PCT antibody penetrates into porous matrix and forms PCT detection bands, rabbit-anti mouse
IgG antibody is penetrated into porous matrix and forms quality control band, and the CRP detection bands and quality control band are respectively positioned at the both sides of PCT detection bands；It is right
Porous matrix after spray treatment is dried treatment, obtains final product coated film；
Prepare the second sample pad：Sealing pores, the glass are carried out using glass fibre element membrane closure liquid to glass fibre element film
Cellulose membrane confining liquid includes the BSA that mass percent is 3%~8%, and percent by volume is 0.1%~0.5% Tween-
20, mass percent is 2%~5% trehalose, the CRP antibody of 0.01~0.03mg/ml, and 0.01~0.02Mol, pH are
6.5~8.0 PBS solution, to being dried treatment through the glass fibre element film after sealing pores, obtains final product the second sample pad；
Prepare gold pad；
Prepare the first sample pad；
The quantitative joint inspection test strips of assembling PCT and CRP：Coated film is bonded on supporting pad, gold pad is then adhered to coated film
One end, the one end of gold pad away from coated film is adhered to by the second sample pad, by the first sample pad be adhered to the second sample pad away from
One end of gold pad.
8. the preparation method of PCT as claimed in claim 7 and CRP joint inspection test strips, it is characterised in that the coating buffer solution
Including the methyl alcohol that percent by volume is 1~5%, mass percent is 2%~5% trehalose, and 0.01~0.02Mol, pH are
6.5~8.0 PBS solution；The concentration of the CRP antibody is 0.5~2mg/ml, and the concentration of the PCT antibody is 0.5~2mg/
Ml, the concentration of the rabbit anti-mouse igg antibody is 0.5~1mg/ml, and the coating buffer solution and CRP of the CRP detection bands spraying are anti-
The consumption of body is 0.7~1.2 μ l/cm, the coating buffer solution of the PCT detection bands spraying and the consumption of PCT antibody for 0.7~
1.2 μ l/cm, the coating buffer solution of the quality control band spraying and the consumption of rabbit anti-mouse igg antibody are 0.7~1.2 μ l/cm.
9. the preparation method of PCT as claimed in claim 7 and CRP joint inspection test strips, it is characterised in that the preparation of the gold pad
Step includes：
(1) collaurum coating buffer, is prepared：The collaurum coating buffer includes CRP antibody colloidal golds label, PCT Antibody Golds
Golden label and liquid is redissolved, the volume ratio of the CRP antibody colloidal golds label and PCT antibody colloidal gold labels is：15~
20:15~30；
The preparation of a, CRP antibody colloidal gold label：The antibody labeling amount of the CRP antibody colloidal golds label is 4~7 μ g/
Ml, 6.5~8.5 are adjusted to the solution of potassium carbonate of 0.1~0.3Mol by the pH value of collaurum, add CRP antibody stirring 10~
30 minutes, it was that 0.1%~0.5% BSA carries out Seal treatment to add mass percent, was then centrifuged for purification process and obtains CRP resisting
Body colloid gold label thing；
The preparation of b, PCT antibody colloidal gold label：The antibody labeling amount of the PCT antibody colloidal golds label is 4~7 μ g/
Ml, 6.5~8.5 are adjusted to the solution of potassium carbonate of 0.1~0.3Mol by the pH value of collaurum, add PCT antibody stirring 10~
30 minutes, the BSA for adding mass percent to be 0.1%~0.5% carried out Seal treatment, and centrifugal purification processes to obtain PCT antibody glue
Body gold label；
Wherein, CRP antibody colloidal golds label preparation and PCT antibody colloidal gold labels preparation process in collaurum system
Preparation Method is：The gold chloride that 100ml, mass percent are 0.01% is heated to boiling, 0.75~1.5ml, quality hundred is added
Divide and reacted than the sodium solution of citric acid three for 1%, obtain the collaurum that particle size is 30~60nm；
C, mixing CRP antibody colloidal golds label, PCT antibody colloidal golds label and redissolution liquid, the redissolution liquid include：Quality
Percentage is 3~8% BSA, and mass percent is 3%~8% trehalose, and mass percent is 0.1%~1% junket egg
In vain, percent by volume is 0.1%~0.5% Tween-20, and the boric acid of 0.002~0.01Mol, mass percent is 0.1%
~0.5% PEG, pH is 8.0~9.0；
(2) polyester mat confining liquid, is provided, polyester mat confining liquid includes the casein that mass percent is 0.1%~1%, quality
Percentage is 1%~5% trehalose, and percent by volume is 0.1%~1% tween20, the boron of 0.002~0.01Mol
Acid, mass percent be 0.1%~0.5% PEG, pH be 8.0~9.0；Polyester mat is provided；By polyester mat immersion polyester mat envelope
Close liquid carries out Seal treatment to polyester mat, to being dried treatment by the polyester mat after Seal treatment, by collaurum coating buffer
Polyester mat is sprayed at, collaurum coating buffer penetrates into polyester mat, then is dried treatment, obtains final product gold pad.
10. the preparation method of PCT as claimed in claim 7 and CRP joint inspection test strips, it is characterised in that first sample
The preparation process of pad includes：Hemofiltration film is provided；Hemofiltration membrane closure liquid is provided, hemofiltration membrane closure liquid includes that mass percent is 0.5
~2% BSA, percent by volume is 0.1~1% Tween-20,0.005~0.02Mol, pH be 6.5~8.0 PBS it is molten
Liquid；Blocking reagent is provided, blocking reagent is dissolved in hemofiltration membrane closure liquid；Hemofiltration membrane closure liquid to being dissolved with blocking reagent
Immersion treatment is carried out to hemofiltration film, blocking reagent penetrates into hemofiltration film, treatment is dried to the hemofiltration film after soaking treatment.
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| CN115308406A (en) * | 2022-07-19 | 2022-11-08 | 杭州协合医疗用品有限公司 | Double-sample pad colloidal gold immunochromatographic test strip |
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Application publication date: 20170609 |