CN108445218A - The kit and preparation method thereof of joint-detection CRP, PCT and SAA - Google Patents
The kit and preparation method thereof of joint-detection CRP, PCT and SAA Download PDFInfo
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- CN108445218A CN108445218A CN201810212180.8A CN201810212180A CN108445218A CN 108445218 A CN108445218 A CN 108445218A CN 201810212180 A CN201810212180 A CN 201810212180A CN 108445218 A CN108445218 A CN 108445218A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of kit and preparation method thereof of joint-detection CRP, PCT and SAA, which includes：C reactive protein monoclonal antibody solution, Procalcitonin monoclonal antibody solution and the serum amyloid A protein monoclonal antibody solution marked respectively with magnetic particle；It is coated with the carrier of c reactive protein monoclonal antibody, the carrier for being coated with Procalcitonin monoclonal antibody, the carrier for being coated with serum amyloid A protein monoclonal antibody and the carrier for being coated with sheep anti-mouse antibody；Nitrocellulose filter test strips.Preparation method includes：C reactive protein monoclonal antibody, Procalcitonin monoclonal antibody and the serum amyloid A protein monoclonal antibody marked respectively with magnetic particle；C reactive protein monoclonal antibody, Procalcitonin monoclonal antibody, serum amyloid A protein monoclonal antibody and sheep anti-mouse antibody is used to be coated with carrier respectively；It dispenses and is assembled into finished product.This kit can quick simultaneously, quantitative accurately three kinds of markers of detection.
Description
Technical field
The present invention relates to biomedical sector, more particularly to a kind of the kit and its system of joint-detection CRP, PCT and SAA
Preparation Method.
Background technology
C reactive protein（C-neactveprotein, abbreviation CRP）, earlier than nineteen thirty find, be it is a kind of can be with pneumonia ball
Bacterium C polysaccharide precursor reactants form the Acute reaction protein of compound.And it is infected in body or blood plasma when tissue damage
In some protein for steeply rising（Acute protein）, activating complement and reinforce phagocyte phagocytosis and rise opsonic action, clearly
Pathogenic microorganism except invasion body and damage, necrosis, the histocyte of apoptosis.In recent years, it due to the update of detection technique, surveys
Quick, the easy and reliable method for determining CRP is established rapidly.CRP is set to be greatly increased in clinical application field, medically
Value just tested and just and recognized extensively.CRP starts a few hours in inflammation and just increases, 48 hours can peaking, with disease
Normal level is down in the recovery for becoming recession, tissue, structure and function.This reaction is not by radiotherapy, the shadow of chemotherapy, corticosteroid therapy
It rings.Therefore, the detection of CRP is quite extensive in clinical application, includes the diagnosis and differential diagnosis of acute infectious diseases, after operation
The monitoring of infection；The observation of antibiotic curative effect；Course of disease detection and Index for diagnosis etc..
Procalcitonin（Procalcitonin, PCT）It is a kind of protein, in normal and healthy individual, serum is dense
Spend extremely low, only 10-50pg/ml, conventional method can't detect.In systemic bacterial, fungi and parasitic infection, system inflammation
The blood-serum P CT levels for reacting the patients such as syndrome, septicemia, acute and chronic pneumonia, acute pancreatitis, active hepatitis, wound are different
Often increase, even up to ten thousand times of several times of concentration University of Science and Technology normal level；It is infected in virus, autoimmune disease, organ transplant
The serum-concentration for excluding the patients such as reaction does not increase or slightly increases.This just determines the high degree of specificity of PCT, therefore can be used for
The antidiastole of such disease, in the side such as systemic bacterial infections and the antidiastole of pyemia auxiliary, Index for diagnosis, observation of curative effect
There is very high clinical value in face, can be widely used for the wards ICU, hematology, surgery, internal medicine, organ transplant section, Experiment on therapy room
Deng.
Serum amyloid A protein（Serum amyloid A, SAA) it is a kind of acute time limit reactive protein, belong to and carries fat egg
Heterogeneous proteinoid in white family, relative molecular weight about 12 000.In the acute time limit reacts, pierced through IL-1, IL-6 and TNF
Swash, SAA is synthesized in liver by the macrophage and fibroblast that are activated, can be increased to the 100-1000 of initial concentration
Times, but half-life period is extremely short, and only 50 minutes or so.Serum amyloid A protein is related with high-density lipoprotein (HDL), its energy
The metabolism of high-density lipoprotein is adjusted during inflammation.One especially important characteristic of serum amyloid A protein is its degradation
Product can be deposited in a manner of amyloid A (AA) fibrinogen in different organs, this is in chronic inflammatory diseases
A kind of serious complication.
The method that the prior art detects serological index includes mainly radioactive isotope immunoassay, biochemical immunity than turbid
Method analysis, colloidal gold immunochromatographimethod, Enzyme-linked Immunosorbent Assay method and chemiluminescence immune assay.Radiommunoassay palpus
With125The radioactive elements such as I mark, and detection device is complicated, the half-life short of radionuclide, are unable to long-term preservation, measure knot
Fruit is unstable, while also bringing radiation damage to experiment operator.The method of immunoturbidimetry, this method detection sensitivity
Low, solution is protected from environmental.Colloidal gold immunity chromatography detects, quickly, convenient, single part operation, but a disadvantage is that only
It can realize sxemiquantitative, can only indicate a probable ranges, cannot achieve accurate quantification.ELISA detection sensitivity is inadequate
Height, detection range is narrow, and influence factor is more, easily causes false negative and false positive.Chemiluminescence immune assay detection sensitivity compared with
Height, but large automatic instrument is needed, it is high to laboratory and related experiment personnel requirement, it charges the shortcomings of higher.
From the foregoing, it will be observed that the marker of inflammation such as joint-detection CRP, PCT and SAA, quick for the early stage of infection, pyemia etc.
Precise Diagnosis, course of disease monitoring, curative effect tracking etc. all have important support effect.It can be quick simultaneously, fixed however, still lacking at present
Amount accurately detects the joint inspection reagent of above-mentioned three kinds of indexs.
Invention content
The present invention provides a kind of kit and preparation method thereof of joint-detection CRP, PCT and SAA, overcomes the prior art
It cannot quick simultaneously, quantitative the shortcomings that accurately detecting the joint inspection reagent of above-mentioned three kinds of indexs.
On the one hand, the present invention provides a kind of kit of joint-detection CRP, PCT and SAA, kit packets of the invention
It includes：
（1）C reactive protein monoclonal antibody solution, Procalcitonin monoclonal antibody solution and the serum marked respectively with magnetic particle
Amyloid A monoclonal antibody solution；
（2）It is coated with the carrier of c reactive protein monoclonal antibody, the carrier for being coated with Procalcitonin monoclonal antibody, is coated with
The carrier of serum amyloid A protein monoclonal antibody and the carrier for being coated with sheep anti-mouse antibody；
（3）Nitrocellulose filter（NC films）Test strips.
Wherein, the composition of NC films test strips includes：It is coated with the inspection of the detection line, Procalcitonin antibody of c reactive protein antibody
Survey line, the detection line of serum amyloid A protein antibody and nature controlling line.
Wherein, it is coated with sheep anti-mouse antibody on nature controlling line.
Wherein, nitrocellulose filter test strips include bottom plate and nitrocellulose filter.
Wherein, nitrocellulose filter test strips include the first detection zone, the second detection zone, third detection zone and the 4th detection
Area；
First detection zone has the detection line of coating c reactive protein antibody, on the detection line region of the c reactive protein antibody
Coated antibody can specifically bind c reactive protein antigen；
Second detection zone has the detection line of coating Procalcitonin antibody, the detection line region of the Procalcitonin antibodies Antibodies
On coated antibody can specifically bind Procalcitonin antibody antigen；
The third detection zone has the detection line of coating serum amyloid A protein antibody, the serum amyloid A protein antibody
Coated antibody on detection line region can specifically bind serum amyloid A protein antigen；
4th detection zone has the nature controlling line of coating sheep anti-mouse antibody, and the coated antibody on the nature controlling line region can
Specifically bind c reactive protein antibody, Procalcitonin antibody and serum amyloid A protein antibody.
On the other hand, the present invention provides a kind of preparation method of the reagent of joint-detection CRP, PCT and SAA, this method
Including：
（1）Mark c reactive protein monoclonal antibody, Procalcitonin monoclonal antibody and serum amyloid protein respectively with magnetic particle
A monoclonal antibodies；
（2）C reactive protein monoclonal antibody, Procalcitonin monoclonal antibody, serum amyloid A protein monoclonal antibody are used respectively
It is coated with carrier with sheep anti-mouse antibody；
（3）Dispense above-mentioned c reactive protein monoclonal antibody, Procalcitonin monoclonal antibody and the serum marked respectively with magnetic particle
Amyloid A monoclonal antibody；
（4）It is assembled into finished product.
In above-mentioned kit according to the present invention and preparation method thereof, the solid phase carrier is nitrocellulose filter（NC
Film）, microwell plate, plastic tube or plastic bead；
The carrier is preferably NC films in one of the embodiments, have it is at low cost, be easy to close non-specific binding, have
The advantages of lower background noise.
Preferably, the magnetic particle, grain size have large specific surface area, absorption potent antibodies are more, need not add at 1-3 μm
The solution such as substrate can directly read magnetic signal, advantage easy to operate.
Preferably, on the carrier NC films, detection line is located at from the nearlyr side in sample-adding end, nature controlling line be located at from sample-adding end compared with
Remote side.
In the method according to the invention, wherein the step（1）Mark c reactive protein monoclonal anti-respectively with magnetic particle
Body, Procalcitonin monoclonal antibody and serum amyloid A protein monoclonal antibody, including following procedure：
（1）Prepare the magnetic particle containing c reactive protein antibody；
（2）Prepare the magnetic particle containing Procalcitonin antibody；
（3）Prepare the magnetic particle containing serum amyloid A protein antibody；
（4）By three kinds of magnetic particles according to 1:1:1 volume ratio mixing.
In the method according to the invention, wherein the step（2）In respectively use c reactive protein monoclonal antibody, drop
The former monoclonal antibody of calcium element, serum amyloid A protein monoclonal antibody and sheep anti-mouse antibody are coated with carrier, including following procedure：
（1）With 0.05M, the phosphate buffer of pH7.4（PB）C reactive protein/Procalcitonin/serum is formed sediment for coating buffer solution
Powder sample protein A antibody is diluted to 0.5mg/ml, and the secondary antibody of sheep anti mouse is diluted to 1mg/ml.
（2）Aforesaid liquid using quantitatively spray film device respectively with the amount of 1 μ l/cm with the uniform spray printing in the interval of 0.5cm in width
Degree is on the NC films of 3.5cm.
（3）Room temperature dry 30 minutes after in pH7.4, contain 1.0% bovine serum albumin(BSA) of mass ratio（BSA）0.05M phosphorus
Phthalate buffer（PBS）Confining liquid in impregnate after ten minutes in 25-35 DEG C dry 8 hours, be added drier seal up for safekeeping it is spare.
（4）NC films after drying are cut into the finished product test strips of 0.4cm wide using cutting machine, are packed into black plastic cylinder, 20
Item/cylinder.
" kit and preparation method thereof of joint-detection CRP, PCT and SAA " of the invention can simultaneously accurately quantify inspection
Measure the content of patient's c reactive protein/Procalcitonin/serum amyloid A protein, can according to c reactive protein/Procalcitonin/
Serum amyloid A protein content, the state of an illness of acute infection is monitored, the judgement of therapeutic evaluation and prognosis it is significant.
It has many advantages, such as that high specific, high sensitivity, simplicity are quick.
Further, the present invention is combined without multiple film item, and a film item can detect three markers simultaneously.
Further, reagent proportioning screen and optimized in the present invention, reagent proportioning will influence the steady of signal
It is qualitative, signal strength；The final sensitivity and specificity for influencing kit.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Obtain other attached drawings according to these attached drawings.
Fig. 1 is NC film test strips, bottom plate（1）Upper stickup NC films（2）The test strips assembled, NC films（2）On be coated with C
Reactive protein monoclonal antibody detection line（3）, Procalcitonin monoclonal antibody detection line（4）, serum amyloid A protein monoclonal
Antibody detection line（5）And nature controlling line（6）.
Fig. 2 is the flow chart of reagent box preparation method.
Specific implementation mode
Illustrate that embodiments of the present invention, those skilled in the art can be by this specification below by way of specific specific example
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under the spirit of the present invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless literary
In in addition explicitly point out, singulative "one", " one " and " this " include plural form.When embodiment provides numberical range
When, it should be appreciated that except non-present invention is otherwise noted, between two endpoints and two endpoints of each numberical range any one
Numerical value can be selected.Unless otherwise defined, all technical and scientific terms used in the present invention and the art technology
The normally understood meaning of personnel is identical.Except used in embodiment specific method, equipment, in addition to material, according to the art
Record of the technical staff to the grasp of the prior art and the present invention, can also use and the side described in the embodiment of the present invention
Any method, equipment and the material of the similar or equivalent prior art of method, equipment, material realizes the present invention.
The preparation of 1 coated film of embodiment：
It is coated with the preparation of buffer solution：The phosphate buffer of 0.05M pH 7.4（PB）To be coated with buffer solution, 0.22 μm of miillpore filter
4 DEG C of filtration sterilization postposition saves backup.
The preparation of confining liquid：Containing 1.0% bovine serum albumin(BSA) of mass ratio（BSA）0.05M, the phosphate-buffered of pH7.4
Liquid（PBS）, 0.22 μm of filtering with microporous membrane degerming is placed on 4 DEG C and saves backup.
The preparation of coated film：With 0.05M phosphate buffers（PB）, the coating buffer solution of pH7.4 is by c reactive protein/drop calcium
Plain original/serum amyloid A protein antibody is diluted to 0.5mg/ml, and the secondary antibody of sheep anti mouse is diluted to 1mg/ml, uses quantitative spray film
Device with the amount of 1 μ l/cm by with the uniform spray printing in the interval of 0.5cm on 3.5cm width NC films, room temperature dry 30 minutes after in envelope
Close liquid（0.05M PBS, pH7.4 containing 1.0%BSA）Middle immersion is dried 8 hours in 25-35 DEG C after ten minutes, and drier is added
It seals up for safekeeping spare.
The preparation of 2 magnetic particle labelled antibody of embodiment：
The preparation of citrate buffer solution：Weigh sodium citrate 14.7g and citric acid 10.5g is with purifying water dissolution secure ph
The Tween-20 of volume ratio 0.05% is added, with 0.22 μm of miillpore filter mistake in the citrate buffer solution 1L of 4.6, a concentration of 0.05M
It filters out and saves backup for 4 DEG C after bacterium.
Boric acid preserves the preparation of buffer solution：It is 9.0 to weigh boric acid 0.62g and borax 3.81g purifying water dissolution preparations pH,
The povidone of mass ratio 1% is added in the borate buffer 1L of final concentration of 0.05M（PVP）, the casein of mass ratio 0.5%
(Casein), the Tween-20 of volume ratio 0.5%, the sucrose of mass ratio 5%, 4 DEG C of preservations after 0.22 μm of filtering with microporous membrane degerming
It is spare.
The preparation of magnetic particle：
1. the preparation of the magnetic particle containing CRP antibody：
（1）Using the 0.05M containing volume ratio 0.1%Tween-20, pH4.6 citrate buffer solutions wash magnetic particle, and carbon is added
Changing diimine (EDC) and n-hydroxysuccinimide (NHS) makes the two final concentration be 0.05M, reacts at room temperature 1 hour；
（2）Fully after washing magnetic particle, the ratio that CRP antibody and magnetic particle is added is 1：5（Molar ratio）, react at room temperature 2 hours；
（3）The 0.05M containing mass ratio 1.0%BSA is added, the PBS room temperatures of pH7.4 are closed 30 minutes, using containing volume ratio
The citrate buffer solution of the 0.05M of 0.1%Tween-20, pH4.6 wash magnetic particle；
（4）Using containing mass ratio 1%PVP, mass ratio 1%Casein, volume ratio 0.5%Tween-20,5% sucrose of mass ratio
9.0 borate buffers of 0.05M pH redissolve magnetic particle, and 4 DEG C save backup.
2. the preparation of the magnetic particle containing PCT antibody：
（1）Using the 0.05M containing volume ratio 0.1%Tween-20, pH4.6 citrate buffer solutions wash magnetic particle, and EDC is added
Make the two final concentration be 0.05M with NHS, reacts at room temperature 1 hour；
（2）Fully after washing magnetic particle, the ratio that PCT antibody and magnetic particle is added is 1：5（Molar ratio）, react at room temperature 2 hours；
（3）The 0.05M containing mass ratio 1.0%BSA is added, the PBS room temperatures of pH7.4 are closed 30 minutes, using containing volume ratio
The citrate buffer solution of the 0.05M of 0.1%Tween-20, pH4.6 wash magnetic particle；
（4）Using containing mass ratio 1%PVP, mass ratio 1%Casein, volume ratio 0.5%Tween-20,5% sucrose of mass ratio
9.0 boric acid of 0.05M pH preserves buffer solution and redissolves magnetic particle, and 4 DEG C save backup.
3. the preparation of the magnetic particle containing SAA antibody：
（1）Using the 0.05M containing volume ratio 0.1%Tween-20, pH4.6 citrate buffer solutions wash magnetic particle, and EDC is added
Make the two final concentration be 0.05M with NHS, reacts at room temperature 1 hour；
（2）Fully after washing magnetic particle, the ratio that SAA antibody and magnetic particle is added is 1：5（Molar ratio）, react at room temperature 2 hours；
（3）The 0.05M containing mass ratio 1.0%BSA is added, the PBS room temperatures of pH7.4 are closed 30 minutes, using containing volume ratio
The citrate buffer solution of the 0.05M of 0.1%Tween-20, pH4.6 wash magnetic particle；
（4）Using containing mass ratio 1%PVP, mass ratio 1%Casein, volume ratio 0.5%Tween-20,5% sucrose of mass ratio
9.0 boric acid of 0.05M pH preserves buffer solution and redissolves magnetic particle, and 4 DEG C save backup.
4. by three kinds of magnetic particles according to 1:1:1 volume ratio mixes spare.
3 test strips of embodiment and finished product assembling
Following all operating environments require：Humidity is less than 20%, 18-26 DEG C of temperature.
The assembling of test paper plate：Instrument is assembled as requested by 3.5cm coated films using BioDot LM5000 types, is assembled in
On 9.8cm width transparent plastic bottom plates, upper layer transparent plastic cover board is covered, test paper plate is assembled into.
Test strips are cut：Assembled test paper plate is cut into the examination of 0.5cm wide using BioDot CM4000 types cutting machines
Then paper slip dresses up 20/tin, room temperature preservation.
D, the packing of magnetic particle labelled antibody
The magnetic particle containing three kinds of antibody prepared is packed as 25mL/ bottles, 2-8 DEG C of preservation.
E, finished product packing
By a wound packages at the vial of the cylinder and a bottled 25mL mixing magnetic particle labelled antibody of 20 test strips, packaging is placed in one
In a packing box, paper box is made in a box portion specification, and for the paper box in 2-8 DEG C of preservation, the shelf-life is 18 months.
4 kit forms of embodiment
1. the molar ratio of the magnetic particle labelled antibody in the present invention will influence the stability of signal, signal strength；It is final to influence
The sensitivity and specificity of kit.
Part of test results is as follows, by the molar ratio of CRP antibody and magnetic particle according to 1:2,1:5,1:10,1:20 carry out instead
It answers, good berth liquid is respectively configured in label after reaction, carries out the detection of reference material Q0 and Q5, and testing result is as follows.
According to the above experimental result it is found that in several molar ratio reactions, 1:5 molar ratio its signal-to-noise ratio Q5 RMU/Q0
RMU values are maximum, and sensitivity is best, therefore select 1:5 optimum mole ratio as CRP antibody and magnetic particle.
Part of test results is as follows, by the molar ratio of PCT antibody and magnetic particle according to 1:2,1:5,1:10,1:20 carry out instead
It answers, good berth liquid is respectively configured in label after reaction, carries out the detection of reference material Q0 and Q5, and testing result is as follows.
According to the above experimental result it is found that in several molar ratio reactions, 1:5 molar ratio its signal-to-noise ratio Q5 RMU/Q0
RMU values are maximum, and sensitivity is best, therefore select 1:5 optimum mole ratio as PCT antibody and magnetic particle.
Part of test results is as follows, by the molar ratio of SAA antibody and magnetic particle according to 1:2,1:5,1:10,1:20 carry out instead
It answers, good berth liquid is respectively configured in label after reaction, carries out the detection of reference material Q0 and Q5, and testing result is as follows.
According to the above experimental result it is found that in several molar ratio reactions, 1:5 molar ratio its signal-to-noise ratio Q5 RMU/Q0
RMU values are maximum, and sensitivity is best, therefore select 1:5 optimum mole ratio as SAA antibody and magnetic particle.
2. the confining liquid buffer system of coated film in the present invention, will influence the background of the NC films of signal, to influence reagent
The sensitivity and specificity of box.
Part of test results is as follows, and coated film is used 4.6 citrate buffers of 0.05M pH, 0.05M pH respectively
7.4 phosphate buffers, 9.6 carbonate buffer solutions of 0.05M pH are closed, and after preparing microwell plate, carry out reference material Q0
With the detection of Q5, testing result is as follows：
1. CRP testing results
2. PCT testing results
3. SAA testing results
According to the above experimental result it is found that three projects are using 0.05M pH its signal-to-noise ratio of 7.4 phosphate buffer Q5
RMU/Q0 RMU ratios are preferable compared with other Block buffers, therefore select 7.4 phosphate buffer liquid of 0.05M pH as coating
The confining liquid buffer system of film.
The methodology identification of the kit of 5 present invention of embodiment
It requires to examine and determine the kit prepared in embodiment 2 according to the technical standard of the product：
（1）Kit sensitivity experiment
20 hole replications are carried out with S0 calibration objects, average value brings the concentration value obtained by curvilinear equation into plus twice of standard deviation
For the sensitivity of kit, the sensitivity of CRP, PCT, SAA of three projects be respectively 0.81mg/L, 0.05 ng/mL,
0.75 ng/mL；
Wherein, the detection sensitivity data of the detection kit of single marker are as follows：
The detection sensitivity of the independent reagents of CRP, PCT, SAA tri- is respectively 0.85mg/L, 0.06 ng/mL, 0.76 ng/
mL；
Experiment proves that kit of the invention not only can be used a film item while detect three kinds of markers, and kit
Sensitivity is essentially identical to single detection kit.
（2）Kit specificity experiments
Make cross reaction experiment, cross reacting rate with its analog<0.01%；
Wherein, the detection kit of single marker with its analog makees cross reaction experiment, cross reacting rate<0.01%；
Experiment proves that kit of the invention not only can be used a film item while detect three kinds of markers, and kit
Specificity is essentially identical to single detection kit.
（3）Kit accuracy is tested
Variation within batch
It takes two horizontal quality-control products to carry out 10 hole parallel laboratory tests respectively, calculates the average value of measured value（）And standard deviation（s）.It presses
Formula CV=s/× 100% calculates the coefficient of variation, and variation within batch coefficient CV is respectively less than 10%；
The variation within batch coefficient CV of the detection kit of single marker is less than 10%；
Batch variation
It selects the blood serum sample of 5 parts of various concentrations to carry out 3 replications to every part of serum, calculates its interassay coefficient of variation
（CV%）, batch variation CV is respectively less than 5%；
The batch variation CV of the detection kit of single marker is respectively less than 5%；
Experiment proves that kit of the invention not only can be used a film item while detect three kinds of markers, and kit
Accuracy is essentially identical to single detection kit.
（4）Kit accuracy is tested
By the calibration object raw material of high concentration, four various concentration values are diluted to normal human serum, each concentration does the parallel reality in 5 holes
It tests, calculates separately the rate of recovery within the scope of 90-109%；
The rate of recovery of the detection kit of single marker is within the scope of 92-108%；
Experiment proves that kit of the invention not only can be used a film item while detect three kinds of markers, and kit
Accuracy is essentially identical to single detection kit.
（5）Stabilization of kit is tested
Kit storage temperature is 2-80 DEG C, and the indices by 15 months assay kits are satisfied by requirement, it is contemplated that
Influence during transport and use to kit, we carry out 37 DEG C, 7 days Acceleration studies, the experimental results showed that kit
Indices comply fully with requirement.
Experiment proves that kit of the invention not only can be used a film item while detect three kinds of markers, and reagent
Box has good stability, and is essentially identical to single detection kit.
Illustrate the sensitivity of " kit of joint-detection CRP, PCT and SAA and ", specificity, accuracy, accuracy and steady
Qualitative is complete qualified.
" kit and preparation method thereof of joint-detection CRP, PCT and SAA " of the invention can simultaneously accurately quantify inspection
Measure the content of patient's c reactive protein/Procalcitonin/serum amyloid A protein, can according to c reactive protein/Procalcitonin/
Serum amyloid A protein content, the state of an illness of acute infection is monitored, the judgement of therapeutic evaluation and prognosis it is significant.
It has many advantages, such as that high specific, high sensitivity, simplicity are quick.
Further, reagent proportioning screen and optimized in the present invention, reagent proportioning will influence the stabilization of signal
Property, signal strength；The final sensitivity and specificity for influencing kit.
Above-described specific implementation mode has carried out further the purpose of the present invention, technical solution and advantageous effect
It is described in detail, it should be understood that the foregoing is merely the specific implementation mode of the present invention, is not intended to limit the present invention
Protection domain, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of kit of joint-detection CRP, PCT and SAA, which is characterized in that the kit includes：
（1）C reactive protein monoclonal antibody solution, Procalcitonin monoclonal antibody solution and the serum marked respectively with magnetic particle
Amyloid A monoclonal antibody solution；
（2）It is coated with the carrier of c reactive protein monoclonal antibody, the carrier for being coated with Procalcitonin monoclonal antibody, is coated with
The carrier of serum amyloid A protein monoclonal antibody and the carrier for being coated with sheep anti-mouse antibody；
（3）Nitrocellulose filter test strips.
2. kit according to claim 1, which is characterized in that the nitrocellulose filter test strips include：It is anti-to be coated with C
Answer protein antibodies detection line, be coated with Procalcitonin antibody detection line, be coated with serum amyloid A protein antibody detection line and
Nature controlling line.
3. kit according to claim 1, which is characterized in that on the nitrocellulose filter, detection line is located at from adding
The nearlyr side in sample end, nature controlling line are located at from sample-adding end side farther out；It is coated with sheep anti-mouse antibody on the nature controlling line.
4. kit according to claim 2, which is characterized in that the nitrocellulose filter test strips include the first detection
Area, the second detection zone, third detection zone and the 4th detection zone；
First detection zone has the detection line of coating c reactive protein antibody, on the detection line region of the c reactive protein antibody
Coated antibody can specifically bind c reactive protein antigen；
Second detection zone has the detection line of coating Procalcitonin antibody, the detection line region of the Procalcitonin antibodies Antibodies
On coated antibody can specifically bind Procalcitonin antibody antigen；
The third detection zone has the detection line of coating serum amyloid A protein antibody, the serum amyloid A protein antibody
Coated antibody on detection line region can specifically bind serum amyloid A protein antigen；
4th detection zone has the nature controlling line of coating sheep anti-mouse antibody, and the coated antibody on the nature controlling line region can
Specifically bind c reactive protein antibody, Procalcitonin antibodies Antibodies and serum amyloid A protein antibody.
5. kit according to claim 1, which is characterized in that the magnetic particle, grain size is at 1-3 μm.
6. a kind of reagent box preparation method of joint-detection CRP, PCT and SAA, which is characterized in that including：
（1）Mark c reactive protein monoclonal antibody, Procalcitonin monoclonal antibody and serum amyloid protein respectively with magnetic particle
A monoclonal antibodies；
（2）C reactive protein monoclonal antibody, Procalcitonin monoclonal antibody, serum amyloid A protein monoclonal antibody are used respectively
It is coated with carrier with sheep anti-mouse antibody；
（3）Dispense above-mentioned c reactive protein monoclonal antibody, Procalcitonin monoclonal antibody and the serum marked respectively with magnetic particle
Amyloid A monoclonal antibody；
（4）It is assembled into finished product.
7. according to the method described in claim 6, it is characterized in that, it is described with magnetic particle label c reactive protein monoclonal antibody,
Procalcitonin monoclonal antibody and serum amyloid A protein monoclonal antibody, including following procedure：
（1）Prepare the magnetic particle containing c reactive protein monoclonal antibody；
（2）Prepare the magnetic particle containing Procalcitonin monoclonal antibody；
（3）Prepare the magnetic particle containing serum amyloid A protein monoclonal antibody；
（4）By three kinds of magnetic particles according to 1:1:1 volume ratio mixing.
8. the method according to the description of claim 7 is characterized in that the magnetic particle of the preparation containing c reactive protein antibody, packet
Include following steps：
（1）Using the 0.05M containing volume ratio 0.1%Tween-20, the citrate buffer solution of pH4.6 washs magnetic particle, is added
Carbodiimides and n-hydroxysuccinimide make the two final concentration be 0.05M, react at room temperature 1 hour；
（2）Fully after washing magnetic particle, the molar ratio that c reactive protein antibody and magnetic particle is added is 1：5, it reacts at room temperature 2 hours；
（3）The 0.05M containing 1.0% bovine serum albumin(BSA) of mass ratio is added, the phosphate buffer room temperature of pH7.4 closes 30 points
Clock, using the 0.05M containing volume ratio 0.1%Tween-20, the citrate buffer solution of pH4.6 washs magnetic particle；
（4）Using containing 1% povidone of mass ratio, 1% casein of mass ratio, volume ratio 0.5%Tween-20,5% sucrose of mass ratio
9.0 borate buffers of 0.05M pH redissolve magnetic particle, 4 DEG C save backup；
The magnetic particle of the preparation containing Procalcitonin antibody, includes the following steps：
（1）Using the 0.05M containing volume ratio 0.1%Tween-20, pH4.6 citrate buffer solutions wash magnetic particle, and carbon is added
Changing diimine and n-hydroxysuccinimide makes the two final concentration be 0.05M, reacts at room temperature 1 hour；
（2）Fully after washing magnetic particle, the molar ratio that Procalcitonin antibody and magnetic particle is added is 1：5, it reacts at room temperature 2 hours；
（3）The 0.05M containing 1.0% bovine serum albumin(BSA) of mass ratio is added, the phosphate buffer room temperature of pH7.4 closes 30 points
Clock, using the 0.05M containing volume ratio 0.1%Tween-20, the citrate buffer solution of pH4.6 washs magnetic particle；
（4）Using containing 1% povidone of mass ratio, 1% casein of mass ratio, volume ratio 0.5%Tween-20,5% sucrose of mass ratio
9.0 boric acid of 0.05M pH preserve buffer solution, redissolve magnetic particle, 4 DEG C save backup；
The magnetic particle of the preparation containing serum amyloid A protein antibody, includes the following steps：
（1）Using the 0.05M containing mass ratio 0.1%Tween-20, pH4.6 citrate buffer solutions wash magnetic particle, and carbon is added
Changing diimine and n-hydroxysuccinimide makes the two final concentration be 0.05M, reacts at room temperature 1 hour；
（2）Fully after washing magnetic particle, the ratio that serum amyloid A protein antibody and magnetic particle is added is 1：5（Molar ratio）, room
Temperature reaction 2 hours；
（3）Enter the 0.05M containing 1.0% bovine serum albumin(BSA) of mass ratio, the phosphate buffer room temperature of pH7.4 closes 30 points
Clock, using the 0.05M containing volume ratio 0.1%Tween-20, the citrate buffer solution of pH4.6 washs magnetic particle；
（4）Using containing 1% povidone of mass ratio, 1% casein of volume ratio, volume ratio 0.5%Tween-20,5% sucrose of volume ratio
9.0 boric acid of 0.05M pH preserve buffer solution, redissolve magnetic particle, 4 DEG C save backup.
9. according to the method described in claim 6, it is characterized in that, c reactive protein monoclonal antibody, the Procalcitonin list
Clonal antibody, serum amyloid A protein monoclonal antibody are coated with carrier, include the following steps：
（1）With 0.05M, the phosphate buffer of pH7.4 is to be coated with buffer solution by c reactive protein, Procalcitonin, serum amyloid sample egg
White A antibody is diluted to 0.5mg/ml respectively, and the secondary antibody of sheep anti mouse is diluted to 1mg/ml；
（2）Using quantitative spray film device respectively with the amount of 1 μ l/cm with the uniform spray printing in the interval of 0.5cm in width be 3.5cm's
On NC films；
（3）Room temperature dry 30 minutes after in the 0.05M phosphate buffers containing 1.0% bovine serum albumin of mass ratio, pH7.4's
It impregnates in confining liquid and is dried 8 hours in 25-35 DEG C after ten minutes, addition drier is sealed up for safekeeping spare；
（4）NC films after drying are cut into the finished product test strips of 0.4cm wide using common cutting machine, are packed into black plastic cylinder,
20/tin.
10. according to the method described in claim 8, it is characterized in that, the preparation method of the citrate buffer solution includes：It weighs
Sodium citrate 14.7g and citric acid 10.5g purifying water dissolution secure phs are 4.6, the citrate buffer solution of a concentration of 0.05M
1L is added the Tween-20 of volume ratio 0.05%, is saved backup with 4 DEG C after 0.22 μm of filtering with microporous membrane degerming；
The preparation method that the boric acid preserves buffer solution includes：Boric acid 0.62g and borax 3.81g is weighed to be prepared with purifying water dissolution
PH is 9.0, the borate buffer 1L of final concentration of 0.05M, the povidone of addition mass ratio 1%, the casein of mass ratio 0.5%,
The Tween-20 of volume ratio 0.5%, the sucrose of mass ratio 5% save backup for 4 DEG C after 0.22 μm of filtering with microporous membrane degerming.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109055319A (en) * | 2018-09-18 | 2018-12-21 | 四川迈克生物新材料技术有限公司 | Anti- C reactive protein monoclonal antibody, its hybridoma cell strain and application |
| CN109633163A (en) * | 2018-11-28 | 2019-04-16 | 浙江聚康生物工程有限公司 | The two-in-one detection kit of Procalcitonin/c reactive protein |
| CN110646620A (en) * | 2019-09-29 | 2020-01-03 | 湖北新纵科病毒疾病工程技术有限公司 | Method for simultaneously detecting four inflammation markers and kit thereof |
| US20210263029A1 (en) * | 2018-06-20 | 2021-08-26 | Xiamen Innodx Biotech Co. Ltd | Method for full-range detection of c-reactive protein and corresponding kit |
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