CN108802378A - A kind of test strips that the nature controlling line of test strips is coated with solution and is prepared with this solution - Google Patents
A kind of test strips that the nature controlling line of test strips is coated with solution and is prepared with this solution Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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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Abstract
The present invention provides a kind of coating solution of nature controlling line of test strips, the preparation method of this coating solution and using the Test paper of this nature controlling line coating solution preparation, wherein the present invention is illustrated using HCG Test papers, the coating solution includes following component：Antigen and antibody corresponding with the antigen.The present invention provides the new methods that one kind overcoming " HOOK " effect, and the ELISA test strip concentration range that nature controlling line coating solution using the present invention is prepared is wider, and its detection method is simpler, and required detection time is short, and testing cost is small and stability is also more preferable.
Description
Technical field
The present invention designs technical field of immunoassay more particularly to immune chromatography test paper.
Background technology
Nitrocellulose filter is also known as NC films, and the supporting body of C/T lines is used as in colloidal gold or fluorescent test paper, is also simultaneously
The point of immune response.Antigen or antibody are fixed on the technique on NC films, we term it coatings.Coated effect is direct
The performance, including sensitivity, stability specificity etc. of test strips are influenced, while also directly affecting production cost.Existing nature controlling line
Coating technique mainly be coated with sheep it is mostly anti-based on, such method forms more constant nature controlling line, but when antigen in sample
When concentration is higher, excessive antigen is combined with gold labeling antibody, forms excessive antigen-gold labeling antibody compound, and this is excessive
Compound will not be combined with the antibody in detection line.At this point, the signal value as read detection line as usual, can cause testing result inclined
Low, this is because hook-shaped phenomenon occurs in calibration curve after culminating, when antigen is sufficiently high, detection line can even be examined and not measured
Signal value, occur false negative as a result, this phenomenon is known as " HOOK effects ".Generally pass through the dense of diluted sample in the prior art
Degree or overcome " HOOK effects " using the monoclonal antibody of high-affinity, but when hospital in emergency circumstances, need
In the case of rapid accurately measurement patient's states, if necessary to overcome " HOOK effects " by the concentration of diluted sample,
It is so less to allow such way in the case of this urgent, if wherein using the monoclonal of high-affinity
Antibody also increases the burden of patient then virtually increasing the cost of detection, so being badly in need of developing another energy
Enough overcome the method for " HOOK effects ".
Have at present and overcome the influence of HOOK effects using antigen as nature controlling line, widens the detection range of product.It is such as right
In this project of HCG, patent (104991078A) discloses a kind of HCG colloid gold immunes lateral chromatography test strips and its detection side
Method, this method are coated with the antigen of HCG on nature controlling line, can widen detection range to 200000mIU/mL.But the coating of antigen
Compare for antibody less ripe, and different item purpose antigen, property also can be multifarious, coated condition disunity, and
And it may cover the binding site of this antigen and gold labeling antibody when antigen and NC films are coated with or lead to this antigen
Antigenic determinant changes, and causes gold labeling antibody low with the joint efficiency of antigen or can not even combine, furthermore subitem
Purpose antigen coating difficulty is larger, the problems such as stability is poor or joint efficiency is low easily occurs.So be badly in need of exploitation one kind can gram
Take " HOOK " effect, easy to operate, the test strips that required detection time is short and stability is good.
Invention content
In order to overcome above-mentioned technical problem, the present invention provides a kind of easy to operate, required detection time is short and stablizes
Property the test strips that prepare of good nature controlling line coating solution and application this nature controlling line coating solution.
The present invention provides a kind of nature controlling lines of test strips to be coated with solution, and the nature controlling line is coated with solution mainly by with the following group
It is grouped as：Antigen and antibody corresponding with antigen；
Further, the antigen is a concentration of：0.2-3mg/mL, the corresponding antibody of the antigen it is a concentration of：0.2-
3mg/mL；
Further, a concentration of 0.5mg/mL of the antigen, a concentration of 1mg/mL of the antibody of the corresponding antigen；
Further, nature controlling line coating solution further includes coating buffer mixture early period, and the coating early period is slow
It includes following component to rush mixed liquor mainly：0.01~20mmol/L buffer solutions, 0.1~5g/100mL protective agents and 0.01~
1g/100mL preservatives；
Further, coating buffer solution early period includes mainly following component：10mmol/L buffer solutions, 1g/100mL are protected
Protect agent and 0.05g/100mL preservatives；
Further, the buffer solution is PBS buffer solution, Tris-HCI buffer solutions, glycine buffer, boric acid salt buffer
It is one or more in liquid and citrate buffer solution；
Further, the preservative is one kind or more in Sodium azide, thimerosal, Proclin300 and Proclin200
Kind；
Further, the protective agent is one or more in bovine serum albumin(BSA) BSA, ovalbumin OVA and gelatin；
Further, the test strips are detection HCG test strips or detection CRP test strips；
Further, the HCG is β-HCG or free β-HCG.
The present invention also provides a kind of methods that the nature controlling line preparing the test strips is coated with solution, it includes following step
Suddenly：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：Buffer solution,
Protective agent and preservative；
B, premixed liquid is prepared：The antibody is added in buffer mixture early period；
C, nature controlling line coating solution is prepared：The antigen is mixed with premixed liquid, is stood, it is molten to eventually form nature controlling line coating
Liquid.
The present invention also provides a kind of test strip, including PVC backer boards and it is connected successively and is arranged in PVC backings
Sample pad, bonding pad, chromatographic film on plate and absorbent filter are provided with detection line and nature controlling line, the matter in the chromatographic film
Control line is coated with coating solution above-mentioned；
The present invention provides detection HCG test strips, including PVC backer boards and are connected successively and are arranged in PVC backer boards
On sample pad, bonding pad, chromatographic film and absorbent filter, be provided with detection line and nature controlling line, the Quality Control in the chromatographic film
Line is coated with coating solution above-mentioned；
The present invention provides detection CRP test strips, including PVC backer boards and are connected successively and are arranged in PVC backer boards
On sample pad, bonding pad, chromatographic film and absorbent filter, be provided with detection line and nature controlling line, the Quality Control in the chromatographic film
Line is coated with coating solution above-mentioned；
Further, the bearing capacity of coating solution is 0.5-1 μ l/ on the detection line in the test strips and nature controlling line
cm；
Further, the bearing capacity of coating solution is 0.7 μ l/cm on the detection line in the test strips and nature controlling line；
Further, the corresponding antibody of the antigen that the coating solution of detection line is a concentration of 0.5-2.5mg/mL is molten
Liquid；
Further, the corresponding antibody-solutions of the antigen that the coating solution of detection line is a concentration of 2mg/mL.
The present invention also provides a kind of methods preparing test strips, it includes the following steps：
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：0.01~
20mmol/L buffer solutions, 0.1~5g/100mL protective agents and 0.01~1g/100mL preservatives；
B, premixed liquid is prepared：The corresponding antibody of the antigen is added in buffer mixture early period, the antigen is made to correspond to
Antibody a concentration of 0.2~3mg/mL；
C, nature controlling line coating solution is prepared：The antigen is mixed with premixed liquid, make the antigen a concentration of 0.2~
3mg/mL；
5-30min is stood, nature controlling line coating solution is eventually formed；
Detection line is coated with the preparation of solution:Concentration in the corresponding antibody spraying of the antigen of 0.5-2.5mg/ml and is examined
On survey line.
Further, coating buffer mixture early period includes mainly following component：10mmol/L buffer solutions, 1g/100mL
Protection
Agent and 0.05g/100mL preservatives；
Further, the method for preparing test strips further includes following steps：
The preparation of the gold-labelled pad：Unlabelled colloidal gold solution is added in the corresponding antibody of the antigen to be marked
In, after standing 5-30min after mixing, the BSA solution that 0.1%-1% is added is closed, and removes supernatant, addition and colloidal gold etc.
The buffer solution of volume toasts after mixing, after drying 4-30 DEG C of kept dry to get；
The preparation of the sample pad：Isometric buffer solution is added in the BSA solution of 0.1%-1%, is toasted after mixing, is dried
After dry 4-30 DEG C of kept dry to get.
Further, the method for preparing the test paper of detection HCG further includes following steps：
The preparation of the gold-labelled pad：HCG monoclonal antibodies to be marked are added in unlabelled colloidal gold solution, mixing
After standing 10min afterwards, the BSA solution for being added 0.5% is closed, and removes supernatant, addition and the isometric buffer solution of colloidal gold,
Toasted after mixing, 4-30 DEG C after drying, kept dry to get；
The preparation of the sample pad：Isometric buffer solution is added in 0.5% BSA solution, is toasted after mixing, after drying
4-30 DEG C of kept dry to get.
In the present invention, the buffer solution is PBS buffer solution, Tris-HCI buffer solutions, glycine buffer, boric acid salt buffer
It is one or more in liquid and citrate buffer solution；
In the present invention, the preservative is one kind or more in Sodium azide, thimerosal, Proclin300 and Proclin200
Kind；
In the present invention, the protective agent is one or more in bovine serum albumin(BSA) BSA, ovalbumin OVA and gelatin；
Compared with the existing technology, the present invention has the advantages that：It is prepared by nature controlling line coating solution using the present invention
Obtained ELISA test strip concentration range is wider, can overcome " HOOK effects ", and its detection method is simple, when required detection
Between the time is short, testing cost is small, precision is more preferable and stability is good.
Nature controlling line coating solution in the present invention is not only applicable to HCG, CRP test strip, other all uses
Test strips prepared by this nature controlling line coating solution are all within the scope of the present invention.
Test strips in the present invention are suitable for detecting various biological samples, such as：Cell, bacterium solution, whole blood, animal tissue's homogenate
Liquid, plant tissue homogenate liquid, serum, blood plasma, tissue extract, swab washing lotion, urine, virus-culturing fluid.
Obviously, the above according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific implementation mode of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Specific implementation mode
The present invention and its technique effect are illustrated using following examples
The screening of 1 nature controlling line of embodiment and detection line coating solution concentration
1, the preparation of gold-labelled pad
HCG monoclonal antibodies to be marked are added in unlabelled colloidal gold solution, after standing 10min after mixing, are added
The BSA solution for entering 0.5% is closed, centrifugation removal supernatant, addition and the isometric citrate buffer solution of colloidal gold, after mixing
Be laid on glass fibre, 37 DEG C are toasted, after drying 30 DEG C of kept dries to get；
2, the preparation of sample pad：
Isometric citrate buffer solution is added in 0.5% BSA solution, is laid on glass fibre after mixing, 37 DEG C into
Row baking, 4 DEG C drying after kept dry to get.
3, the preparation of nature controlling line and detection line coating solution
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：10mmol/
LPBS buffer solutions, 1g/100mL bovine serum albumin(BSA) BSA and 0.05g/100mL Sodium azides；
B, nature controlling line and detection line concentration are pressed noodles part and are prepared
B1, screening nature controlling line solution optimal proportion：HCG antigens and the concentration ratio of HCG monoclonal antibodies on nature controlling line are set
It is set to：1:20；1:15；1:10；1:5；1:2；1:1；2:1；5:1；10:1；15:1；20:1, the HCG monoclonals in detection line are anti-
A concentration of 2.0mg/mL of body；
B2, screening nature controlling line optium concentration：HCG antibody concentrations：HCG antigen concentrations ratio=2：1, detection line is a concentration of
2.0mg/mL is coated with；
B3, selective mechanisms line optium concentration：In nature controlling line HCG antibody concentrations be 1.0mg/mL, HCG antigens it is a concentration of
Five concentration are arranged in 0.5mg/mL, the concentration in detection line：0.5,1,2,2.5 is coated with 3mg/mL.
4, the coating of nature controlling line and detection line：It is 3.5mm, set-point film by the debugging of the distance between detection line and nature controlling line
Parameter starts and draws film instrument, and the coating solution of nature controlling line and detection line is coated on nature controlling line and detection line, nature controlling line and detection
The dosage of coating solution on line is 0.7 μ l/cm.
5, the test strips of detection HCG are prepared：Upper sample pad, sample are pasted in one end on the PVC backer boards of test strip
One end of pad closely crimps the colloidal gold pad that the gold mark marked containing HCG monoclonal antibodies makes, and colloidal gold pad one end is tight
Close to crimp NC films, the NC film other ends connect sample suction pad.
6, detection method
Each concentration calibration product are added 70ul, the interpretation on immune quantitative analyzer after 15min, record C T line signal values
7, experimental data and result：
Table 1a nature controlling lines are coated with the concentration optimal proportion the selection result of solution
Table 1b nature controlling lines are coated with the concentration optimal proportion the selection result of solution
Experimental result：HCG antigens and the concentration ratio of HCG monoclonal antibodies on nature controlling line in this experiment are set as：1:
20；1:15；1:10；1:5；1:2；1:1；2:1；5:1；10:1；15:1；20:1, and HCG antigens and HCG monoclonal antibodies
Concentration range is all：0.2-4mg/mL is obtained from the absorbance value on C lines, when the HCG antigens and HCG monoclonals on nature controlling line
The concentration ratio of antibody is 1:When 20, the span scope of the value on C lines is small compared to the span scope of other ratios, and works as matter
The concentration ratio for controlling HCG antigens and HCG monoclonal antibodies on line is 20:When 1, the detection range of C lines is compared to other ratios
Detection range it is small, so the concentration ratio of the HCG antigens and HCG monoclonal antibodies on the nature controlling line is 1:15；1:10；1:5；1:
2；1:1；2:1；5:1；10:1；15:When 1, the method for the present invention is applicable in, and the concentration of HCG antigens and HCG monoclonal antibodies all exists
Within the scope of 0.2-3mg/mL, it follows that HCG antigens and the effective concentration of HCG monoclonal antibodies are：0.2-3mg/mL works as matter
The concentration ratio for controlling HCG antigens and HCG monoclonal antibodies on line is 1:When 2, C lines and the span of the absorbance value on T lines are maximum,
Effect is best.
2 nature controlling line optium concentration the selection result of table
Experimental result：It is obtained from the absorbance value of the C lines in table 2, it is a concentration of when HCG antigens and HCG monoclonal antibodies：
When 0.5mg/mL is with 1.0mg/mL, 1.0mg/mL and 2.0mg/mL, the span of the absorbance value on C lines is compared to other concentration
Span scope it is larger, it is contemplated that cost problem, the concentration optimum value of HCG antigens of the invention and HCG monoclonal antibodies
It is chosen to be：0.5mg/mL and 1.0mg/mL.
3 detection line optium concentration the selection result of table
Experimental result：Obtained from the absorbance value on the T lines in table 3, when on T lines coating solution a concentration of 2.0 with
When 2.5mg/mL, the span scope of the span scope of the absorbance value on T lines all than the absorbance value of other concentration is big, still
In view of cost problem, the optium concentration of the coating solution on T lines in the present invention is chosen to be 2.0mg/mL.
A kind of method for the test strips preparing detection HCG of embodiment 2
1, the preparation of gold-labelled pad
HCG monoclonal antibodies to be marked are added in unlabelled colloidal gold solution, after standing 5min after mixing, are added
0.1% BSA solution is closed, centrifugation removal supernatant, addition and the isometric citrate buffer solution of colloidal gold, is spread after mixing
In on glass fibre, 37 DEG C are toasted, after drying 4 DEG C of kept dries to get；
2, the preparation of sample pad：
Isometric citrate buffer solution is added in 0.1% BSA solution, is laid on glass fibre after mixing, 37 DEG C into
Row baking, 4 DEG C drying after kept dry to get.
3, the preparation of nature controlling line and detection line coating solution
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：
0.01mmol/LPBS buffer solutions, 0.1g/100mL bovine serum albumin(BSA) BSA and 0.01g/100mL Sodium azides；
B, premixed liquid is prepared：HCG monoclonal antibodies are added in buffer mixture early period, a concentration of of the antibody is made
0.2mg/mL；
C, nature controlling line coating solution is prepared：HCG is mixed with premixed liquid, makes a concentration of 0.2mg/mL of HCG；It stands
5min eventually forms nature controlling line coating solution.
Detection line is coated with the preparation of solution:The HCG monoclonal antibody solutions that the coating solution is a concentration of 0.5mg/ml.
4, the coating of nature controlling line and detection line：It is 3.5mm, set-point film by the debugging of the distance between detection line and nature controlling line
Parameter starts and draws film instrument, and the coating solution of nature controlling line and detection line is coated on nature controlling line and detection line, nature controlling line and detection
The dosage of coating solution on line is 0.5 μ l/cm.
5, the test strips of detection HCG are prepared：Upper sample pad, sample pad are pasted in one end on the PVC backer boards of Test paper
One end closely crimp the colloidal gold pad that the gold mark marked containing HCG monoclonal antibodies makes, colloidal gold pad one end is close
NC films are crimped, the NC film other ends connect sample suction pad.
A kind of method for the test strips preparing detection HCG of embodiment 3
1, the preparation of gold-labelled pad
HCG monoclonal antibodies to be marked are added in unlabelled colloidal gold solution, after standing 30min after mixing, are added
The BSA solution for entering 1% is closed, centrifugation removal supernatant, addition and the isometric citrate buffer solution of colloidal gold, is spread after mixing
In on glass fibre, 37 DEG C are toasted, after drying 30 DEG C of kept dries to get；
2, the preparation of sample pad：
Isometric citrate buffer solution is added in 1% BSA solution, is laid on glass fibre after mixing, 37 DEG C of progress
Baking, 30 DEG C drying after kept dry to get.
3, the preparation of nature controlling line and detection line coating solution
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：20mmol/
LTris-HCI buffer solutions, 5g/100mL ovalbumin OVA and 1g/100mL thimerosals；
B, premixed liquid is prepared：HCG monoclonal antibodies are added in buffer mixture early period, a concentration of of the antibody is made
3mg/mL；
C, nature controlling line coating solution is prepared：HCG is mixed with premixed liquid, makes a concentration of 3mg/mL of HCG；30min is stood,
Eventually form nature controlling line coating solution.
Detection line is coated with the preparation of solution:The HCG monoclonal antibody solutions of a concentration of 2mg/ml are sprayed in detection line.
4, the coating of nature controlling line and detection line：It is 3.5mm, set-point film by the debugging of the distance between detection line and nature controlling line
Parameter starts and draws film instrument, and the coating solution of nature controlling line and detection line is coated on nature controlling line and detection line, nature controlling line and detection
The dosage of coating solution on line is 1 μ l/cm.
5, the test strips of detection HCG are prepared：Upper sample pad, sample pad are pasted in one end on the PVC backer boards of Test paper
One end closely crimp the colloidal gold pad that the gold mark marked containing HCG monoclonal antibodies makes, colloidal gold pad one end is close
NC films are crimped, the NC film other ends connect sample suction pad.
A kind of method for the test paper preparing detection HCG of embodiment 4
1, the preparation of gold-labelled pad
HCG monoclonal antibodies to be marked are added in unlabelled colloidal gold solution, after standing 10min after mixing, are added
The BSA solution for entering 0.5% is closed, centrifugation removal supernatant, addition and the isometric citrate buffer solution of colloidal gold, after mixing
Be laid on glass fibre, 37 DEG C are toasted, after drying 30 DEG C of kept dries to get；
2, the preparation of sample pad：
Isometric citrate buffer solution is added in 0.5% BSA solution, is laid on glass fibre after mixing, 37 DEG C into
Row baking, after drying 25 DEG C of kept dries to get.
3, the preparation of nature controlling line and detection line coating solution
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：10mmol/
L glycine buffers, 21g/100mL gelatin and 0.05g/100mL Proclin300；
B, premixed liquid is prepared：HCG monoclonal antibodies are added in buffer mixture early period, a concentration of of the antibody is made
1mg/mL；
C, nature controlling line coating solution is prepared：HCG is mixed with premixed liquid, makes a concentration of 0.5mg/mL of HCG；It stands
10min eventually forms nature controlling line coating solution.
Detection line is coated with the preparation of solution:The HCG monoclonal antibody solutions of a concentration of 2mg/ml are sprayed in detection line.
4, the coating of nature controlling line and detection line：It is 3.5mm, set-point film by the debugging of the distance between detection line and nature controlling line
Parameter starts and draws film instrument, and the coating solution of nature controlling line and detection line is coated on nature controlling line and detection line, nature controlling line and detection
The dosage of coating solution on line is 0.7 μ l/cm.
5, the test strips of detection HCG are prepared：Upper sample pad, sample pad are pasted in one end on the PVC backer boards of Test paper
One end closely crimp the colloidal gold pad that the gold mark marked containing HCG monoclonal antibodies makes, colloidal gold pad one end is close
NC films are crimped, the NC film other ends connect sample suction pad.
5 comparative example of embodiment
(1) range of linearity
1) experiment material：Calibration object, experimental group test strips, control group test strips, experimental setup such as table 4
2) detection device：Immune quantitative analyzer
Table 4HCG test strips range of linearity experimental setup tables
3) detection method：
Each concentration calibration product are added 70ul, the interpretation on immune quantitative analyzer after 15min, record C T line signal values.
4) experimental result
Table 5HCG range of linearity experimental result tables
5) conclusion：It can show that the coating mode of the nature controlling line in the present invention can improve C lines letter from the experimental data of table 6
Number, and this coating mode does not interfere with the signal of T lines, and the detection range of reagent can be widened while overcoming HOOK effects.
(2) precision
1) experiment material：Quality-control product (target value 12mIU/mL), experimental group test strips, control group test strips, experimental setup is such as
Table 6
2) detection device：Immune quantitative analyzer
Table is arranged in table 6HCG Precision Experiments
3) detection method：
Each concentration calibration product are added 70ul, the interpretation on immune quantitative analyzer after 15min, record C T line signal values
4) experimental result
Table 7HCG Precision Experiment result tables
5) conclusion：It can show that the coating mode of the nature controlling line in the present invention improves C line signals from the experimental data of table 7
Its precision is not interfered with simultaneously, or even precision smaller can be made.
(3) stability
1) experiment material：Calibration object, experimental group test strips, control group test strips, experimental setup see the table below 8
2) detection device：Immune quantitative analyzer
Table 8HCG stability experimental setup tables
3) detection method：
Experimental group and control group are put into 37 DEG C of baking ovens, taken out within 7 days compared with being carried out at the same time with the reagent of room temperature.
Each concentration calibration product are added 70ul, the interpretation on immune quantitative analyzer after 15min, record C T line signal values, this
The stability of C lines coating object is only investigated in secondary experiment, and C line signals are only embodied in following data.
4) experimental result：
Table 9HCG stability experiment result tables
5) conclusion：It can show that the coating mode of the nature controlling line in the present invention exists from the accelerated stability experimental data of table 9
During accelerated stability, the signal value of nature controlling line is illustrated without significant change after being coated with to nature controlling line using this coating buffer,
Stability is more preferable.
A kind of method for the test strips preparing detection CRP of embodiment 6
1, the preparation of gold-labelled pad
CRP monoclonal antibody to be marked is added in unlabelled colloidal gold solution, after standing 10min after mixing, is added
The BSA solution for entering 0.5% is closed, centrifugation removal supernatant, addition and the isometric citrate buffer solution of colloidal gold, after mixing
Be laid on glass fibre, 37 DEG C are toasted, after drying 30 DEG C of kept dries to get；
2, the preparation of sample pad：
Isometric citrate buffer solution is added in 0.5% BSA solution, is laid on glass fibre after mixing, 37 DEG C into
Row baking, after drying kept dry to get.
3, the preparation of nature controlling line and detection line coating solution
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：10mmol/
LPBS buffer solutions, 1g/100mL bovine serum albumin(BSA) BSA and 0.05g/100mL Sodium azides；
B, premixed liquid is prepared：CRP monoclonal antibody is added in buffer mixture early period, a concentration of of the antibody is made
1mg/mL；
C, nature controlling line coating solution is prepared：CRP is mixed with premixed liquid, makes a concentration of 0.5mg/mL of CRP；It stands
10min eventually forms nature controlling line coating solution.
Detection line is coated with the preparation of solution:By the CRP monoclonal antibody solution spraying of a concentration of 2mg/ml in detection line.
4, the coating of nature controlling line and detection line：It is 3.5mm, set-point film by the debugging of the distance between detection line and nature controlling line
Parameter starts and draws film instrument, and the coating solution of nature controlling line and detection line is coated on nature controlling line and detection line, nature controlling line and detection
The dosage of coating solution on line is 0.7 μ l/cm.
5, the test strips of detection CRP are prepared：Upper sample pad, sample pad are pasted in one end on the PVC backer boards of Test paper
One end closely crimp the colloidal gold pad that the gold mark marked containing CRP monoclonal antibody makes, colloidal gold pad one end is close
NC films are crimped, the NC film other ends connect sample suction pad.
A kind of method for the test strips preparing detection CRP of embodiment 7
1, the preparation of gold-labelled pad
CRP monoclonal antibody to be marked is added in unlabelled colloidal gold solution, after standing 5min after mixing, is added
0.1% BSA solution is closed, centrifugation removal supernatant, addition and the isometric citrate buffer solution of colloidal gold, is spread after mixing
In on glass fibre, 37 DEG C are toasted, after drying 4 DEG C of kept dries to get；
2, the preparation of sample pad：
Isometric citrate buffer solution is added in 0.1% BSA solution, is laid on glass fibre after mixing, 37 DEG C into
Row baking, 4 DEG C drying after kept dry to get.
3, the preparation of nature controlling line and detection line coating solution
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：
0.01mmol/LPBS buffer solutions, 0.1g/100mL bovine serum albumin(BSA) BSA and 0.01g/100mL Sodium azides；
B, premixed liquid is prepared：CRP monoclonal antibody is added in buffer mixture early period, a concentration of of the antibody is made
0.2mg/mL；
C, nature controlling line coating solution is prepared：CRP is mixed with premixed liquid, makes a concentration of 0.2mg/mL of CRP；It stands
5min eventually forms nature controlling line coating solution.
Detection line is coated with the preparation of solution:By the CRP monoclonal antibody solution spraying of a concentration of 0.5mg/ml in detection line
On.
4, the coating of nature controlling line and detection line：It is 3.5mm, set-point film by the debugging of the distance between detection line and nature controlling line
Parameter starts and draws film instrument, and the coating solution of nature controlling line and detection line is coated on nature controlling line and detection line, nature controlling line and detection
The dosage of coating solution on line is 0.5 μ l/cm.
5, the test strips of detection CRP are prepared：Upper sample pad, sample pad are pasted in one end on the PVC backer boards of Test paper
One end closely crimp the colloidal gold pad that the gold mark marked containing CRP monoclonal antibody makes, colloidal gold pad one end is close
NC films are crimped, the NC film other ends connect sample suction pad.
A kind of method for the test strips preparing detection CRP of embodiment 8
1, the preparation of gold-labelled pad
CRP monoclonal antibody to be marked is added in unlabelled colloidal gold solution, after standing 30min after mixing, is added
The BSA solution for entering 1% is closed, centrifugation removal supernatant, addition and the isometric citrate buffer solution of colloidal gold, is spread after mixing
In on glass fibre, 37 DEG C are toasted, after drying 30 DEG C of kept dries to get；
2, the preparation of sample pad：
Isometric citrate buffer solution is added in 1% BSA solution, is laid on glass fibre after mixing, 37 DEG C of progress
Baking, 30 DEG C drying after kept dry to get.
3, the preparation of nature controlling line and detection line coating solution
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：20mmol/
LPBS buffer solutions, 5g/100mL bovine serum albumin(BSA) BSA and 1g/100mL Sodium azides；
B, premixed liquid is prepared：CRP monoclonal antibody is added in buffer mixture early period, a concentration of of the antibody is made
3mg/mL；
C, nature controlling line coating solution is prepared：CRP is mixed with premixed liquid, makes a concentration of 3mg/mL of CRP；30min is stood,
Eventually form nature controlling line coating solution.
Detection line is coated with the preparation of solution:By the CRP monoclonal antibody solution spraying of a concentration of 2mg/ml in detection line.
4, the coating of nature controlling line and detection line：It is 3.5mm, set-point film by the debugging of the distance between detection line and nature controlling line
Parameter starts and draws film instrument, and the coating solution of nature controlling line and detection line is coated on nature controlling line and detection line, nature controlling line and detection
The dosage of coating solution on line is 1 μ l/cm.
5, the test strips of detection CRP are prepared：Upper sample pad, sample pad are pasted in one end on the PVC backer boards of Test paper
One end closely crimp the colloidal gold pad that the gold mark marked containing CRP monoclonal antibody makes, colloidal gold pad one end is close
NC films are crimped, the NC film other ends connect sample suction pad.
The test strips Performance Evaluation of embodiment 9CRP
(1) range of linearity
1) experiment material：Calibration object, experimental group test strips, control group test strips, experimental setup such as table 10
2) detection device：Immune quantitative analyzer
Table 10CRP test strips range of linearity experimental setup tables
3) detection method：
Each concentration calibration product are added 70ul, the interpretation on immune quantitative analyzer after 15min, record C T line signal values
4) experimental result
Table 11CRP range of linearity experimental result tables
5) conclusion：It can show that the coating mode of the nature controlling line in the present invention can improve C from the experimental data of upper table 11
Line signal, and this coating mode does not interfere with the signal of T lines, and the detection model of reagent can be widened while overcoming HOOK effects
It encloses.
(2) precision
1) experiment material：Quality-control product (10.0 μ g/mL of target value), experimental group test strips, control group test strips, experimental setup is such as
Table 12；
2) detection device：Immune quantitative analyzer
Table is arranged in table 12CRP Precision Experiments
3) detection method：
Quality-control product (10.0 μ g/mL of target value)) 70ul is added, the interpretation on immune quantitative analyzer after 15min, record C T
Line signal value 4) experimental result
Table 13CRP Precision Experiment result tables
5) conclusion：It can show that the coating mode of the nature controlling line in the present invention improves C lines letter from the experimental data of upper table 13
Number while do not interfere with its precision.
(3) stability
1) experiment material：Calibration object, experimental group test strips, control group test strips, experimental setup see the table below 14
2) detection device：Immune quantitative analyzer
Table 14CRP stability experimental setup tables
3) detection method：
Experimental group and control group are put into 37 DEG C of baking ovens, taken out within 7 days compared with being carried out at the same time with the reagent of room temperature.
Each concentration calibration product are added 70ul, the interpretation on immune quantitative analyzer after 15min, record C T line signal values, this
The stability of C lines coating object is only investigated in secondary experiment, and C line signals are only embodied in following data.
4) experimental result
Table 15CRP stability experiment result tables
4) conclusion：The nature controlling line coating mode in the present invention can be obtained from the accelerated stability experimental data of upper table 15
During accelerated stability, the signal value of nature controlling line is illustrated without significant change after being coated with to nature controlling line using this coating buffer
For its stability without influence.
In conclusion being coated with using the mixed solution of antibody and antigen on nature controlling line, compared to only using antigen
Solution is coated with nature controlling line, the detection range of reagent can be widened while overcoming HOOK effects, and can improve C
It is not interfered with while line signal and detects precision, can be improved it yet and be detected stability.
Claims (10)
1. a kind of nature controlling line of test strips is coated with solution, it is characterised in that：The nature controlling line is coated with solution mainly by following components
Composition：Antigen and the corresponding antibody of antigen.
2. nature controlling line as described in claim 1 is coated with solution, it is characterised in that：The antigen it is a concentration of：0.2-3mg/mL,
The corresponding antibody of the antigen it is a concentration of：0.2-3mg/mL, it is preferred that a concentration of 0.5mg/mL of the antigen, it is described anti-
A concentration of 1mg/mL of former corresponding antibody.
3. the nature controlling line as described in claim 1,2 is coated with solution, it is characterised in that：Before nature controlling line coating solution further includes coating
Phase buffer mixture, coating buffer mixture early period include mainly following component：0.01~20mmol/L buffer solutions, 0.1
~5g/100mL protective agents and 0.01~1g/100mL preservatives, it is preferred that coating buffer solution early period includes mainly such as
Lower component：10mmol/L buffer solutions, 1g/100mL protective agents and 0.05g/100mL preservatives.
4. nature controlling line as claimed in claim 3 is coated with solution, it is characterised in that：The buffer solution is PBS buffer solution, Tris-
HCI buffer solutions, it is one or more in glycine buffer, borate buffer solution and citrate-phosphate salt buffer；It is preferred that
, the preservative is one or more in Sodium azide, thimerosal, Proclin300 and Proclin200；Preferably, described
Protective agent is one or more in bovine serum albumin(BSA) BSA, ovalbumin OVA and gelatin.
5. the nature controlling line as described in claim 1-4 any one is coated with solution, it is characterised in that：The test strips are detection
HCG test strips or detection CRP test strips, it is preferred that the HCG is β-HCG or free β-HCG.
6. a kind of method of the nature controlling line coating solution prepared as described in claim 1-5 any one, it is characterised in that：It is wrapped
Include following steps：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：Buffer solution, protection
Agent and preservative；
B, premixed liquid is prepared：The corresponding antibody of the antigen is added in buffer mixture early period；
C, nature controlling line coating solution is prepared：The antigen is mixed with premixed liquid, is stood, nature controlling line coating solution is eventually formed.
7. a kind of test strip, including PVC backer boards and be connected successively and be arranged on PVC backer boards sample pad, knot
Pad, chromatographic film and absorbent filter are closed, detection line and nature controlling line are provided in the chromatographic film, it is characterised in that：The nature controlling line
It is coated with solution is coated with as shown in claim 1-5 any one.
8. test strips as claimed in claim 7, it is characterised in that：Coating solution in the detection line and nature controlling line is held
Carrying capacity is 0.5-1 μ l/cm, it is preferred that the bearing capacity of the coating solution in the detection line and nature controlling line is 0.7 μ l/
Cm, it is preferred that the corresponding antibody-solutions of the antigen that the coating solution of detection line is a concentration of 0.5-2.5mg/mL, more preferably
, the detection line is coated with the corresponding antibody-solutions of the antigen that solution is a concentration of 2.0mg/mL.
9. a kind of method preparing the test strips as described in claim 7 or 8, it is characterised in that：Include the following steps：
Nature controlling line is coated with the preparation of solution：
A, configuration coating buffer mixture early period：It includes following component that it, which is coated with buffer mixture early period mainly,：0.01~
20mmol/L buffer solutions, 0.1~5g/100mL protective agents and 0.01~1g/100mL preservatives, it is preferred that it is coated with early period
Buffer mixture includes mainly following component：10mmol/L buffer solutions, 1g/100mL protective agents and 0.05g/100mL anti-corrosions
Agent；
B, premixed liquid is prepared：The corresponding antibody of the antigen is added in buffer mixture early period, keeps the antigen corresponding anti-
A concentration of 0.2~3mg/mL of body；
C, nature controlling line coating solution is prepared：The antigen is mixed with premixed liquid, makes a concentration of 0.2~3mg/ of the antigen
mL；It stands, eventually forms nature controlling line coating solution, it is preferred that the time of repose is 10min.
Detection line is coated with the preparation of solution:The solution is that the antigen corresponding antibody of the concentration in 0.5-2.5mg/ml is molten
Liquid.
10. preparation method as claimed in claim 8, it is characterised in that：It further includes following steps：
The preparation of the gold-labelled pad：The corresponding antibody of the antigen to be marked is added in unlabelled colloidal gold solution, is mixed
Stood after even (preferably 10min) after 5-30min, the BSA solution that 0.1%-1% is added is closed, and supernatant is removed, be added with
Colloidal gold isometric buffer solution toasts after mixing, 4-30 DEG C after drying, kept dry to get, it is preferred that will be to be marked
The corresponding antibody of the antigen is added in unlabelled colloidal gold solution, after standing 10min after mixing, be added 0.5% BSA it is molten
Liquid is closed, and is removed supernatant, addition and the isometric buffer solution of colloidal gold, is toasted after mixing, dry to protect 4-30 DEG C after drying
Deposit to get；
The preparation of the sample pad：Isometric buffer solution is added in the BSA solution of 0.1%-1%, is toasted after mixing, after drying
4-30 DEG C of kept dry, it is preferred that isometric buffer solution is added in 0.5% BSA solution, is toasted after mixing, 4- after drying
30 DEG C of kept dries to get.
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