CN106018388B - A kind of pepsinogen Cgene or II assay kit and preparation method thereof - Google Patents
A kind of pepsinogen Cgene or II assay kit and preparation method thereof Download PDFInfo
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- CN106018388B CN106018388B CN201610330619.8A CN201610330619A CN106018388B CN 106018388 B CN106018388 B CN 106018388B CN 201610330619 A CN201610330619 A CN 201610330619A CN 106018388 B CN106018388 B CN 106018388B
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Abstract
The present invention provides a kind of pepsinogen Cgene/II assay kit, including surveying I/II magnetic particle of PG, surveying I/II tracer conjugate of PG and I/II calibration object of PG, surveying I/II magnetic particle of PG is to be marked with pepsinogen Cgene/II monoclonal antibody magnetic microsphere and magnetic particle preservation liquid；Surveying I/II tracer conjugate of PG is to be marked with pepsinogen Cgene/II antibody trace labelling object and tracer conjugate dilution, and the trace labelling object is acridinium ester and its derivative；I/II calibration object of PG includes pepsinogen Cgene/II solution, for storing the correction of principal curve in analyzer.The present invention combines highly sensitive chemical luminescent detecting technology and magnetic particle isolation technics on the basis of EIA enzyme immunoassay, use paramagnetic particles as solid phase carrier, since particle volume is small, surface area is big, expands response area, substantially increases sensitivity, use full-automatic instrument and matched reagent, so that human factor is reduced to minimum, improves the stability of method and the repeatability of result, while but also batch interior difference and differences between batches are all smaller.
Description
Technical field
The invention belongs to immune diagnostic technique fields, and in particular to one kind is for measuring the magnetic of propepsin (PG I/II)
The preparation and measuring method of particulate chemistry luminescent immunoassay kit more particularly to the kit.
Background technique
Propepsin (pepsinogen, PG) is to belong to L-aminobutanedioic acid by the predecessor of the pepsin of stomach lining secretion
Protease family, molecular weight 42kDa can be divided into two subgroups, i.e. pepsinogen Cgene stomach function regulating egg by its immunogenicity difference
White proenzyme II.About 1% PG enters blood circulation under normal circumstances, and the amount of entrance is sufficiently stable, when pathological change occurs for stomach lining
When, serum PG content also changes correspondingly, therefore the quantity of serum PG reflection stomach lining body of gland and cell, also reflects stomach lining indirectly
The secreting function of different parts, so PG is referred to as " the serology biopsy " of stomach lining.PG I mainly by the chief cell of stomach lining and
The secretion of mucus neck cell, secretion increases or reduces the index that can be used as stomach lining pathology, therefore the detection of PG I is current
It is highly important work in the diagnosis of stomach trouble and early screening, PG II is secreted by the chief cell and mucus neck cell of stomach lining,
It can also be generated by pyloric gland and brunner's gland, secretion increases the index that can be used as gastric mucosa lesion.
The traditional detection method of PG I/II includes that colloidal gold method, radioimmunology, enzyme-linked immunization and general chemistry shine
Method etc., but colloidal gold method sensitivity is relatively low and can only realize the detection of single person-portion every time；The advantages of radioimmunology be it is sensitive,
Specifically, simple and easy to do, few etc. with sample amount, but the half-life period section of reagent is costly, needs to be set with scintillation counter etc. is special
Standby, there is certain potentially hazardous property to human body for radionuclide, and test waste processing is difficult, and radioisotope labeling is sometimes
The physiological activity of certain biological substances can be changed；Enzyme-linked immunization and chemoluminescence method detection time are long, rely primarily on pure craft
The serial troublesome operation such as sample-adding, low efficiency causes the easy error of experimental result big, and enzymatic reaction is not thorough enough, and vulnerable to outside
Disturbing factor influences, such as temperature, time and material concentration, therefore specificity is low when detecting, poor sensitivity, detection range are narrow.
Therefore, this field need it is a kind of the operation is more convenient, rapid, pollution-free while guaranteeing that high sensitivity, stability are good, can
The assay kit of detection process full-automation is realized by analysis instrument.
Number of patent application: 201110257438.4 disclose a kind of quantitative determination reagent kit of pepsinogen I (PGI),
It is characterized in that, the kit includes PGI Magneto separate reagent, enzyme reaction object, increased response agent, dilution, PGI calibration object, PGI
Quality-control product cleans concentrated liquor and substrate solution.The invention also discloses the preparation method of the kit, by chemiluminescence
Technology is combined with immune magnetic particle, provide it is a kind of close to homogeneous reaction system, compared with prior art, the kit tool
There are higher detection sensitivity and specificity, while being shortened compared with the prior art on the result time out at least 10 minutes, and
Reach preferable performance parameter, and greatly reduces product cost.
Number of patent application: 201110257440.1 disclose a kind of quantitative determination reagent of pepsinogen I I (PGII)
Box, which is characterized in that the kit includes PGII Magneto separate reagent, enzyme reaction object, increased response agent, dilution, PGII calibration
Product, PGII quality-control product clean concentrated liquor and substrate solution, and which also discloses the preparation methods of the kit.The kit
Chemiluminescence is combined with immune magnetic particle, provide it is a kind of close to homogeneous reaction system, compared with prior art,
The kit has higher detection sensitivity and specificity, and has reached preferable performance parameter, and greatly reduce production
Product cost.
Number of patent application: 201410441604.X discloses a kind of pepsinogen I I (PGII) quantitative determination reagent kit,
The kit includes calibration object, quality-control product, anti-reagent, magnetic particle reagent and luminous substrate.The patent also discloses the kit
Preparation method and using the kit detection pepsinogen I I (PGII) method, with the stomach of marked by fluorescein isothiocyanate
Pepsinogen I I (PGII) labelled antibody of proproteinase II (PGII) coated antibody and alkali phosphatase enzyme mark prepares anti-examination
Magnetic particle reagent is made with anti-fluorescein isothiocynate antibody coupling carboxyl magnetic bead in agent, and immune response is made to be easier to mix and divide
From, and substantially increase reaction speed, using new chemical luminous substrate APLS as substrate, improve kit sensitivity and
Specific performance.The detection kit reliable performance, high sensitivity, the range of linearity are wide, and semi-automatic, full-automatic instrument can be cooperated to make
With.
Summary of the invention
The object of the present invention is to provide one kind to have highly sensitive and specificity, is suitable for the particulate of clinical assistant diagnosis
Chemoluminescence method propepsin (PG I/II) assay kit and preparation method thereof.The present invention ties on the basis of EIA enzyme immunoassay
Highly sensitive chemical luminescent detecting technology and magnetic particle isolation technics are closed, there are many unique advantages compared with other methods, first
First it uses paramagnetic particles as solid phase carrier, and since particle volume is small, surface area is big, expands response area, greatly improves
Sensitivity secondly because making human factor reduce to minimum using full-automatic instrument and matched reagent improves the stabilization of method
Property and result repeatability, while but also batch in difference and differences between batches it is all smaller.
The technical scheme is that
A kind of pepsinogen Cgene/II assay kit, including survey I/II magnetic particle of PG, survey I/II tracer conjugate of PG and
I/II calibration object of PG, survey I/II magnetic particle of PG is to be marked with pepsinogen Cgene/II monoclonal antibody magnetic microsphere and magnetic
Particle saves liquid；Surveying I/II tracer conjugate of PG is to be marked with pepsinogen Cgene/II antibody trace labelling object and tracer combination
Object dilution；I/II calibration object of PG includes pepsinogen Cgene/II solution, for storing the correction of principal curve in analyzer.
The present invention is to utilize pepsin in magnetic microparticle chemiluminescence immune assay technology quantitative determination human serum or blood plasma
The content of former (PG I/II).Sample to be tested is added in covalent labeling propepsin (PG I/II) antibody on magnetic particle, the
Single step reaction forms magnetic particle labelled antibody-antigen conjugates, and the pepsin for the tracer-labelling being added is reacted with second step
Former (PG I/II) antibody forms magnetic particle labelled antibody-antigen-tracer-labelling antibody complex and is added and swashs sufficiently after washing
Lotion, catalytic luminescence, relative luminous intensity (RLU) are positively correlated with I/II content of PG in human serum/slurry, are according to standard curve
The content of PG I/II in sample can be calculated.
The trace labelling object is acridinium ester and its derivative.
Acridinium ester has signal-to-noise ratio high compared with other chemiluminescent labels, and disturbing factor is few, and luminous efficiency is high, intensity is big,
It is easy to protein-crosslinking, the advantages that high sensitivity.
The magnetic microsphere is the complex of Fe2O3 or Fe3O4 magnetic nano-particle and high-molecular organic material.
The magnetic microsphere has one or more activity functional groups, the activity functional groups by the way that surface is modified
Including but not limited to-OH ,-COOH ,-NH2 ,-CHO ,-SO3H.
The pepsinogen Cgene/II antibody is one or more monoclonal antibodies and/or polyclonal antibody.
I/II calibration object of PG further includes I/II antigen of PG and protected protein solution, and the protected protein includes but unlimited
In newborn bovine serum, bovine serum albumin(BSA), casein.
Protected protein can provide good stable reaction environment, so that antigen/antibody is kept native conformation, can choose above-mentioned
Stabilizer is one of.
Protected protein mass concentration is 1-30% in I/II calibration object of PG.
Protein concentration is low in calibration object, easy in inactivation, and the protected protein of 1-30% mass concentration can reduce the polarity of solution,
The hydrophobic environment that can be saved steadily in the long term is provided for albumen.
The magnetic particle saves liquid and tracer conjugate dilution includes stabilizer, surfactant, preservative and pH slow
Electuary.
The pH buffer includes TBS buffer system, HAC-NAC buffer system, PBS buffer solution system, MES buffer system
One or more of system, HEPES buffer system, the buffer capacity of the pH buffer are to adjust pH value 6.0-8.5, the pH
The concentration range of buffer is 0.01-0.2mol/L.
The surfactant is TWEEN Series (including polysorbas20 (TWEEN-20), tween 21 (TWEEN-21), polysorbate40
(TWEEN-40), polysorbate60 (TWEEN-60), Tween61 (TWEEN-61), Tween 80 (TWEEN-80), sorbimacrogol oleate100 (TWEEN-
81), polysorbate85 (TWEEN-85)), Triton X-100, at least one of CTAC；
It is preferred that the surfactant is the polysorbas20 that mass concentration range is 0.1-1%.
Non-specific adsorption easily occurs in the reaction for protein conjugates, and the polysorbas20 of 0.1-1% can weaken non-specific suction
Attached ability influences specific adsorption effect after polysorbas20 concentration is higher than 1%, reduces reaction sensitivity.
The preservative is selected from least one of thimerosal, Sodium azide, antibiotics, proclin；The thimerosal
Concentration is not more than 1 ‰；The concentration of the Sodium azide is not more than 1 ‰；The antibiotics include gentamicin etc.；It is described
The concentration of proclin is not more than 2 ‰.
Surfactant can reduce surface tension when reagent is added to reaction system, improve dispersibility and stability.
A kind of preparation method of I/II assay kit of propepsin PG, the specific steps are as follows:
1) preparation for surveying I/II magnetic particle of PG saves the preparation of liquid including magnetic particle and magnetic particle marks I/II antibody of PG
Preparation；
2) preparation of I/II tracer conjugate of PG is surveyed, preparation and acridinium ester label PG I/II including tracer dilution are anti-
The preparation of body；
3) preparation of I/II calibration object of PG, the preparation of preparation and calibration object including calibration object dilution.
The preparation that magnetic particle saves liquid in the step 1) includes the following steps:
1.1 weigh disodium hydrogen phosphate 2.68g, and sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added suitable in the container of 1L
The purified water stirring of amount makes it completely dissolved；
1.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved above-mentioned
In the container of 1L, purified water is added and to 900mL and stirs；
1.3 adjust PH meter measurement solution PH, make its control between 7.2-7.4；
1.4, which weigh casein 10g, is added in above-mentioned 1L container, stirs evenly, dissolves it sufficiently；
1.5 are settled to 1L, are filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis.
The preparation of magnetic particle label I/II antibody of PG includes the following steps: in the step 1)
1.6 mix the ratio of the magnetic particle with carboxyl and carbodiimide (EDC) difference in mass ratio 1: 1-3；
1.7 are added I/II monoclonal antibody of PG, magnetic particle and antibody in the ratio that every milligram of magnetic particle is separately added into 5-40 μ g antibody
Mass ratio is 100:1；
1.8 in mixing 20-28 DEG C respectively mark 0.5-2 hours；
Extra site is closed using the glycine of final concentration 25mM after 1.9 labels, 22-26 DEG C is reacted 0.5 hour, at least
Washing three times, saves liquid with magnetic particle and is saved；
1.10 debug optium concentration using square matrix titration, save liquid with magnetic particle and dilute magnetic particle mark according to optium concentration
Note PG I/II obtains surveying I/II magnetic particle of PG, stores in 2-8 DEG C；
The preparation of tracer dilution includes the following steps: in the step 2)
2.1 weigh disodium hydrogen phosphate 2.68g, and sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added suitable in the container of 1L
The purified water of amount stirs, and pipettes 1mL Tween-20 stirring with pipettor and makes it completely dissolved；
2.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved above-mentioned
In the container of 1L, purified water is added and to 900mL and stirs；
2.3 adjust PH meter measurement solution PH, make its PH control between 7.2-7.4；
2.4, which weigh casein 10g, is added in above-mentioned 1L container, stirs evenly, dissolves it sufficiently；
2.5 are settled to 1L, are filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis；
The preparation of I/II antibody of acridinium ester label PG includes the following steps: in the step 2)
2.6 take I/II monoclonal antibody of 10mol PG respectively, are separately added into acridinium ester 25-200mol label according to every 10mol antibody；
2.7 addition glutaraldehydes to glutaraldehyde mass concentration is 0.75-1.5%, and it is small that 0.5-2 is reacted under the conditions of 20-28 DEG C
When；
Glutaraldehyde is double-functional group reagent, connects monoclonal antibody by amino and acridinium ester, acridinium ester generally use the method mark
Note.
2.8 are dialysed with the 0.01M PBS of pH 7.2-7.4, at least change liquid three times, -20 DEG C of isometric glycerol is added after dialysis
Storage；
2.9 determine the most suitable working concentration of I/II antibody of acridinium ester label PG using square matrix titration, with tracer conjugate
Dilution is diluted, and is mixed and is obtained surveying I/II tracer conjugate of PG, sets 2-8 DEG C of storage.
The preparation of step 3) the alignment product dilution includes the following steps:
3.1 weigh disodium hydrogen phosphate 2.68g, and sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added suitable in the container of 1L
The purified water of amount stirs, and pipettes 1mL Tween-20 stirring with pipettor and makes it completely dissolved；
3.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved above-mentioned
In the container of 1L, purified water is added and to 900mL and stirs；
3.3 adjust PH meter measurement solution PH, make its PH control between 7.2-7.4；
3.4, which weigh bovine serum albumin(BSA) 10g, is added in above-mentioned 1L container, stirs evenly, dissolves it sufficiently；
3.5 are settled to 1L, are filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis.
The preparation of step 3) the alignment product includes the following steps:
3.6 with the working concentration range of I sterling of calibration object diluted PG be 0.5-500ng/mL；
The working concentration range of 3.7 dilution II sterlings of PG is 0.1-100ng/mL, is made into I/II calibration object 1 of PG and PG I/II
Calibration object 2, calibration object measured value should within the scope of, the concentration range of I calibration object 1 of PG is 47.832-60.833ng/mL, PG I
The concentration range of calibration object 2 are as follows: 291.300-365.103ng/mL；The concentration range of II calibration object 1 of PG are as follows: 8.788-
The concentration range of II calibration object 2 of 11.018ng/mL, PG are as follows: 53.289-67.494ng/mL.
Preferably, it is 1.25% that glutaraldehyde to glutaraldehyde mass concentration is added in the step 2.7, in 22-26 DEG C of condition
Lower reaction 1 hour.
The advantages of kit of the present invention is to use microparticle chemiluminescence immunoassay technology, eliminates artificial and environment
Influence, improve reagent accurate degree；Solid phase carrier is done with particulate, expands reaction surface area, the range of linearity is wider；Entirely certainly
Dynamicization, it is easy that the operation is more convenient, while kit of the invention can be especially serial with chemical illumination immunity analysis instrument
Chemical illumination immunity analysis instrument (AutolumiS 500, AutolumiS 1000, AutolumiS 2000, AutolumiS 3000
Full-automatic chemiluminescence analyzer) it matches, full-automation is realized during sample measures, so that the detection of concentration can
To carry out simply, easily and fast, in bulk, while guaranteeing that the systematic error of detection is smaller.
Specific embodiment
Embodiment 1
One, the preparation steps that magnetic particle saves liquid (for preparing 1L, are formulated and are shown in Table 1)
1. weighing disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added appropriate in the container of 1L
Purified water stirring make it completely dissolved；
2. pipetting 1mL proclin 300 in the beaker of 10mL purified water with pipettor, poured into after being completely dissolved above-mentioned
In the container of 1L, purified water is added and to 900mL and stirs；
3. adjusting PH meter measurement solution PH, adds HCL or NaOH to adjust PH, make its control between 7.2-7.4.
4. weighing casein 10g to be added in above-mentioned 1L container, stirs evenly, dissolve it sufficiently；
5. being settled to 1L, is filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis.
1 propepsin of table (PG I/II) magnetic particle saves formula of liquid
Two, the preparation of magnetic particle label I/II antibody of PG
1. first the magnetic particle with carboxyl in mass ratio 1: 1,1: 2,1: 3 is mixed respectively with carbodiimide (EDC), best matter
Amount is than being 1:2；
2. ratio addition I/II monoclonal antibody of PG of 5 μ g, 10 μ g, 20 μ g, 40 μ g antibody is separately added into every milligram of magnetic particle again,
Magnetic particle and antibody optimum quality ratio are 100:1；
3. in mixing 20 DEG C, 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C of labels 0.5 hour, 1 hour, 2 hours respectively, most
Good label temperature is 22-26 DEG C, and the optimum mark time is 1 hour；
4. close extra site using the glycine of final concentration 25mM after label, 22~26 DEG C are reacted 0.5 hour, at least
Washing three times, saves liquid with magnetic particle and is saved；
5. debugging optium concentration using square matrix titration, liquid is saved according to optium concentration dilution magnetic particle label with magnetic particle
PG I/II obtains surveying I/II magnetic particle of PG, stores in 2~8 DEG C；
Accelerate the failure 6 days 6. surveying I/II magnetic particle of PG and placing 37 DEG C, has good stability.
Embodiment 2: the preparation of I/II tracer conjugate of PG is surveyed
One, (for preparing 1L, 2) formula is shown in Table the preparation steps of tracer dilution
1. weighing disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added appropriate in the container of 1L
Purified water stirring, with pipettor pipette 1mL Tween-20 stirring make it completely dissolved；
2. pipetting 1mL proclin 300 in the beaker of 10mL purified water with pipettor, poured into after being completely dissolved above-mentioned
In the container of 1L, purified water is added and to 900mL and stirs；
3. adjusting PH meter measurement solution PH, make its PH control between 7.2-7.4；
4. weighing casein 10g to be added in above-mentioned 1L container, stirs evenly, dissolve it sufficiently；
5. being settled to 1L, is filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis.
2 propepsin of table (PG I/II) tracer dilutes formula of liquid
Two, the preparation of I/II antibody of acridinium ester label PG
1. take I/II monoclonal antibody of 10mol PG respectively, according to every 10mol antibody be separately added into 25mol, 50mol, 100mol,
The optimum mark ratio of the ratio addition acridinium ester label of 200mol, antibody and acridinium ester is 1:10；
2. glutaraldehyde is respectively 0.75%, 1.0%, 1.25%, 1.5% using concentration, respectively 20 DEG C, 22 DEG C, 24 DEG C,
26 DEG C, 28 DEG C of reactions 0.5 hour, 1 hour, 1.5 hours, 2 hours, the optimal use concentration of glutaraldehyde are 1.25%, most preferably
Reaction temperature is 22-26 DEG C, and optimum reacting time is 1 hour；
3. being dialysed with the 0.01M PBS of PH 7.2-7.4, at least changes liquid three times, -20 DEG C of isometric glycerol is added after dialysis
Storage；
4. the most suitable working concentration of I/II antibody of acridinium ester label PG is determined using square matrix titration, it is dilute with tracer conjugate
It releases liquid to be diluted, mixes and obtain surveying I/II tracer conjugate of PG, set 2-8 DEG C of storage；
5. surveying 37 DEG C of I/II tracer conjugate of PG to accelerate the failure 6 days, activity keeps 90% or more, illustrates that stability is good
It is good.
The preparation of I/II calibration object of embodiment 3:PG
One, (for preparing 1L, 3) formula is shown in Table the preparation steps of calibration object dilution
1. weighing disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added appropriate in the container of 1L
Purified water stirring, with pipettor pipette 1mL Tween-20 stirring make it completely dissolved；
2. pipetting 1mL proclin 300 in the beaker of 10mL purified water with pipettor, poured into after being completely dissolved above-mentioned
In the container of 1L, purified water is added and to 900mL and stirs；
3. adjusting PH meter measurement solution PH, make its PH control between 7.2-7.4；
4. weighing bovine serum albumin(BSA) 10g to be added in above-mentioned 1L container, stirs evenly, dissolve it sufficiently；
5. being settled to 1L, is filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis.
3 propepsin of table (PG I/II) calibration object dilutes formula of liquid
Two, the preparation of I/II calibration object of PG
Working concentration range with I sterling of calibration object diluted PG is 0.5-500ng/mL, dilution II sterling of PG
Working concentration range is 0.1-100ng/mL, is made into I/II calibration object 1 (low value calibration object) of PG and I/II calibration object of PG, 2 (high level
Calibration object), calibration object measured value should within the scope of.I I calibration object 2 of calibration object 1:47.832-60.833ng/mL, PG of PG:
291.300-365.103ng/mL；II II calibration object 2:53.289- of calibration object 1:8.788-11.018ng/mL, PG of PG
67.494ng/mL。
The selection of the above-mentioned various raw material of the present invention requires as follows:
The selection of magnetic particle
Pass through the appearance to magnetic particle, the ratio of labelled protein, magnetic responsiveness, magnetic particle absorption consistency etc. progress
Magnetic particle is mixed, is observed under light, easily dispersed, no aggregation, foreign by repeatedly analyzing and researching by analysis；Albumen is adopted
It is marked with different methods, mark rate should be greater than 90%；On the magnet that magnetic particle is set to 370-380 tesla, magnetic is observed
The aggregation velocity of particle, finely dispersed magnetic particle are assembled completely in 10 seconds；Magnetic particle adsorbs consistency CV≤10%.It grinds
Study carefully the result shows that the magnetic particle that diameter is 0.90-1.10 μm, containing carboxylic group, mark rate highest can be used for diagnosis of the present invention
The preparation of kit.
The selection of acridinium ester
Acridinium ester is dissolved with DMSO (dimethyl sulfoxide), is diluted with purified water to the amount of 1.0ng/mL, is added
Enter 10 μ L acridinium ester liquid, it is each that 200 μ L exciting liquids are added, its luminous value is measured, luminous value answers >=1000000, after study, adopt
Use the acridinium ester of Mei Kaite biology offer as luminous raw material.
The selection of propepsin (PG I/II) antibody of magnetic particle label
The appearance of antibody, concentration, purity, potency are verified first, as a result antibody is the supernatant liquid of micro-strip opalescence,
It is visible by naked eyes foreign matter, without not scattered precipitating is shaken, detecting its protein content with ultraviolet absorption method should be not less than 2.0mg/mL, potency
Not less than mark potency and 1:10000 should be not less than, SDS-PAGE detection purity answers master tape clear, without obvious miscellaneous band.
The selection of acridinium ester label propepsin (PG I/II) antibody
Still the appearance of antibody, concentration, purity, potency are verified first, as a result antibody is the clarified solution of micro-strip opalescence
Body is visible by naked eyes foreign matter, and without not scattered precipitating is shaken, 2.0mg/mL should be not less than by detecting its protein content with ultraviolet absorption method,
Potency should be not less than mark potency and not less than 1:10000, and SDS-PAGE detection purity answers master tape clear, without obvious miscellaneous band.
Other matched reagents involved in implementation process of the present invention:
Cleaning solution, excitation A liquid, the preparation for exciting B liquid
The main component of cleaning solution are as follows: disodium hydrogen phosphate 28.6g/L, sodium dihydrogen phosphate 3.9g/L, sodium chloride 160g/L,
Tween20 10mL/L is used with purified water according to 10 times of dilutions.Excitation A liquid is solution (its containing 0.35mol/L NaOH
In the potassium ferricyanide containing 1mM), excitation B liquid be contain 1.32%w/v H2O2Buffer.
The evaluation of methodology method of this kit
This product kit and prior art products comparative experiments scheme
1. minimum detectability
It uses zero-dose enterprise linear reference product to be detected as sample, replication 10 times, obtains 10 measurement results
Relative light unit, calculate its average value (M) and standard deviation (SD), obtain M+2SD, according to zero-dose enterprise linear reference product and
Concentration-RLU value result between adjacent calibration object carries out two o'clock regression fit and obtains linear function, and the RLU value of M+2SD is brought into
In above-mentioned equation, corresponding concentration value, as minimum detectability are found out.
2. accuracy
A certain concentration pepsinogen Cgene/Pepsinogen II (I/PG of PG II) liquid (A) is added in serum B, PG be added
Volume ratio between I/PG II and serum B is 1:9, each to repeat detection three times, is averaged, according to formula (1) calculated result.
In formula: R --- the rate of recovery；
Vs --- A liquid product is added；
V0--- the volume of blood serum sample B；
The detectable concentration after A liquid is added in C --- blood serum sample；
C0--- the detectable concentration of blood serum sample B；
Cs --- the concentration of A liquid.
3. linear
It will be diluted according to a certain percentage with Sample dilution close to the high level sample of the range of linearity upper limit, by this 8 samples
Detection 2 times is repeated, mean concentration is calculated, result average value and dilution ratio is subjected to straight line fitting with least square method, and
Calculate linearly dependent coefficient r.
4. repeatability
Detection 10 times is respectively repeated with the sample of high-concentration and low-concentration, calculates the average value (M) and standard deviation of 10 measurement results
(SD), the coefficient of variation (CV) is obtained according to formula (2).
CV=SD/M × 100% ... ... ... ... ... ... ... ... ... ... (2)
In formula: the CV-coefficient of variation；
The average value of 10 measurement results of M-；
The standard deviation of 10 measurement results of SD-.
Pepsinogen Cgene (PG I) examining report
Pepsinogen II (PG II) examining report
Kit currently on the market according to product it is different exist full-automatic sample-adding and semi-automatic manual sample-adding mode it
Divide, since this product kit is using full-automatic sample-adding mode, relative to the kit that other are loaded by hand, eliminates artificial add
Human interference factor is preferably minimized by error existing for sample to a certain extent.Examining report data show, this product kit
Meet quality testing standard in the indexs such as minimum detectability, accuracy, linear and repeatability.And pepsinogen Cgene is minimum
Detection limit, accuracy, repeatability and the performance indicators such as the minimum detectability of Pepsinogen II, accuracy, linear are superior to pair
According to product kit, and there are in two indexs of gap, gap has no effect on kit still within the scope of quality testing standard
Properties of product.The comprehensive provable this product kit of indices has high sensitivity, repeatability strong, linear excellent, artificial
The advantages such as disturbing factor is small.
The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention,
It should not be considered as limiting the scope of the invention.Any changes and modifications in accordance with the scope of the present application,
It should still be within the scope of the patent of the present invention.
Claims (8)
1. a kind of pepsinogen I/II assay kit, including survey PG I/II magnetic particle, survey PG I/II tracer conjugate and
PG I/II calibration object, it is characterised in that: the survey PG I/II magnetic particle is to be marked with pepsinogen I/II monoclonal antibody
Magnetic microsphere and magnetic particle save liquid；Surveying PG I/II tracer conjugate is to be marked with the tracer of pepsinogen I/II antibody
Marker and tracer conjugate dilution, the trace labelling object are acridinium ester and its derivative；PG I/II calibration object includes stomach
Proproteinase I/II solution, for storing the correction of principal curve, the pepsinogen I/II assay kit in analyzer
Preparation method, the specific steps are as follows:
1) preparation for surveying PG I/II magnetic particle saves the preparation of liquid and the system of magnetic particle label PG I/II antibody including magnetic particle
It is standby；
2) preparation of PG I/II tracer conjugate, preparation and acridinium ester label PG I/II antibody including tracer dilution are surveyed
Preparation；
3) preparation of PG I/II calibration object, the preparation of preparation and calibration object including calibration object dilution；
The preparation that magnetic particle saves liquid in the step 1) includes the following steps:
1.1 weigh disodium hydrogen phosphate 2.68g, and sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added suitable in the container of 1L
Purified water stirring makes it completely dissolved；
1.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into above-mentioned 1L's after being completely dissolved
In container, purified water is added and to 900mL and stirs；
1.3 adjusting PH meter measurement solution PH, make its control between 7.2-7.4；
1.4, which weigh casein 10g, is added in above-mentioned 1L container, stirs evenly, dissolves it sufficiently；
1.5 are settled to 1L, are filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis；
The preparation of magnetic particle label PG I/II antibody includes the following steps: in the step 1)
1.6 mix the ratio of the magnetic particle with carboxyl and carbodiimide (EDC) difference in mass ratio 1: 1-3；
1.7 are added PG I/II monoclonal antibody, magnetic particle and antibody mass in the ratio that every milligram of magnetic particle is separately added into 5-40 μ g antibody
Than being 100: 1；
1.8 in mixing 20-28 DEG C respectively mark 0.5-2 hours；
Extra site is closed using the glycine of final concentration 25mM after 1.9 labels, 22-26 DEG C is reacted 0.5 hour, is at least washed
Three times, liquid is saved with magnetic particle to be saved；
1.10 debug optium concentration using square matrix titration, save liquid with magnetic particle and mark PG according to optium concentration dilution magnetic particle
I/II obtains surveying PG I/II magnetic particle, stores in 2-8 DEG C；
The preparation of tracer dilution includes the following steps: in the step 2)
2.1 weigh disodium hydrogen phosphate 2.68g, and sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added suitable in the container of 1L
Purified water stirring pipettes 1mL Tween-20 stirring with pipettor and makes it completely dissolved；
2.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into above-mentioned 1L's after being completely dissolved
In container, purified water is added and to 900mL and stirs；
2.3 adjust PH meter measurement solution PH, make its PH control between 7.2-7.4；
2.4, which weigh casein 10g, is added in above-mentioned 1L container, stirs evenly, dissolves it sufficiently；
2.5 are settled to 1L, are filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis；
The preparation of acridinium ester label PG I/II antibody includes the following steps: in the step 2)
2.6 take 10molPG I/II monoclonal antibody respectively, are separately added into acridinium ester 25-200mol label according to every 10mol antibody；
2.7 glutaraldehydes are respectively 0.75-1.5% using concentration, respectively 20-28 DEG C reaction 0.5-2 hours；
2.8 are dialysed with the 0.01M PBS of pH 7.2-7.4, at least change liquid three times, -20 DEG C of isometric glycerol is added after dialysis and deposits
It puts；
2.9 determine the most suitable working concentration of acridinium ester label PG I/II antibody using square matrix titration, are diluted with tracer conjugate
Liquid is diluted, and is mixed and is obtained surveying PG I/II tracer conjugate, sets 2-8 DEG C of storage；
The preparation of step 3) the alignment product dilution includes the following steps:
3.1 weigh disodium hydrogen phosphate 2.68g, and sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g are added suitable in the container of 1L
Purified water stirring pipettes 1mL Tween-20 stirring with pipettor and makes it completely dissolved；
3.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into above-mentioned 1L's after being completely dissolved
In container, purified water is added and to 900mL and stirs；
3.3 adjust PH meter measurement solution PH, make its PH control between 7.2-7.4；
3.4 weighing bovine serum albumin(BSA) 10g to be added in above-mentioned 1L container, stirs evenly, dissolve it sufficiently；
3.5 are settled to 1L, are filtered with 0.2 μm of filter, label is posted after filtering and is stored under 2-8 DEG C of gnotobasis.
The preparation of step 3) the alignment product includes the following steps:
3.6 with the working concentration range of calibration object diluted PG I sterling be 0.5-500ng/mL；
The working concentration range of 3.7 dilution PGII sterlings is 0.1-100ng/mL, is made into PG I/II calibration object 1 and the school PG I/II
Quasi- product 2, calibration object measured value should within the scope of, the concentration range of PG I calibration object 1 is 47.832-60.833ng/mL, PG I
The concentration range of calibration object 2 are as follows: 291.300-365.103ng/mL；The concentration range of PG II calibration object 1 are as follows: 8.788-
The concentration range of 11.018ng/mL, PG II calibration object 2 are as follows: 53.289-67.494ng/mL.
2. a kind of pepsinogen I/II assay kit according to claim 1, it is characterised in that: the magnetic microsphere
For Fe2O3Or Fe3O4The complex of magnetic nano-particle and high-molecular organic material.
3. a kind of pepsinogen I/II assay kit according to claim 2, it is characterised in that: the magnetic microsphere
One or more activity functional groups are had by the way that surface is modified, the activity functional groups include but is not limited to-OH ,-
COOH ,-NH2,-CHO ,-SO3H。
4. a kind of pepsinogen I/II assay kit according to claim 1, it is characterised in that: the pepsin
Former I/II antibody is one or more monoclonal antibodies and/or polyclonal antibody.
5. a kind of pepsinogen I/II assay kit according to claim 1, it is characterised in that: the PG I/II
Calibration object further includes PG I/II antigen and protected protein solution, and the protected protein is including but not limited to newborn bovine serum, ox blood
Pure albumen, casein.
6. a kind of pepsinogen I/II assay kit according to claim 5, it is characterised in that: the school PGI/II
Protected protein mass concentration is 1-30% in quasi- product.
7. a kind of pepsinogen I/II assay kit according to claim 1, it is characterised in that: the magnetic particle is protected
Liquid storage and tracer conjugate dilution include stabilizer, surfactant, preservative and pH buffer.
8. a kind of pepsinogen I/II assay kit according to claim 7, it is characterised in that: the pH buffer
Including one in TBS buffer system, HAC-NAC buffer system, PBS buffer solution system, MES buffer system, HEPES buffer system
Kind is several, and the buffer capacity of the pH buffer is to adjust pH value 6.0-8.5, and the concentration range of the pH buffer is
0.01-0.2mol/L。
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| CN116990513B (en) * | 2023-09-26 | 2023-12-26 | 北京美联泰科生物技术有限公司 | Chemiluminescent detection method of pepsinogen 1 |
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