CN106018388A - Kit for testing PG (pepsinogen) I or II and preparation method of kit - Google Patents

Abstract

The invention provides a kit for testing PG (pepsinogen) I or II. The kit comprises magnetic particles for testing of PG I/II, tracing conjugates for testing of PG I/II and a PG I/II calibrator, wherein the magnetic particles for testing of PG I/II are magnetic microspheres labeled with PG I/II monoclonal antibodies and a magnetic particle preserving fluid; the tracing conjugates for testing of PG I/II are tracing markers labeled with PG I/II antibodies and tracing conjugate diluent, and the tracing markers are acridinium ester and derivatives of acridinium ester; the PG I/II calibrator comprises a PG I/II solution and is used for calibrating a principal storage curve in a tester. The kit is combined with a high-sensitivity chemiluminiscence testing technique and a magnetic particle separation technique on the basis of enzyme immnunoassay, paramagnetic particles are taken as solid-phase carriers, the reaction area is increased due to the small particle size and the large specific area, and the sensitivity is greatly improved; a full-automatic instrument and matched reagents are used, artificial factors are reduced to the largest extent, the method stability and the result repeatability are improved, and the difference in a batch and the difference between batches are smaller.

Description

A kind of pepsinogen Cgene or II measures test kit and preparation method thereof

Technical field

The invention belongs to immune diagnostic technique field, be specifically related to a kind of micro-for measuring the magnetic of pepsinogen (PG I/II) Grain chemiluminescence immunoassay kit, particularly relates to preparation and the assay method of this test kit.

Background technology

Pepsinogen (pepsinogen, PG) is the pepsic predecessor secreted by gastric mucosa, belongs to Aspartic Acid Protease family, molecular weight is 42kDa, different by its immunogenicity, can be divided into two subgroups, i.e. pepsinogen Cgene and Pepsinogen II.The PG of the most about 1% enters blood circulation, and the amount of entrance is sufficiently stable, when gastric mucosa occurs disease During reason change, serum PG content changes the most therewith, therefore serum PG reflection gastric mucosa body of gland and the quantity of cell, also between Connect the secretory function of reflection gastric mucosa different parts, so PG is referred to as " the serology biopsy " of gastric mucosa.PG I is main By chief cell and the secretion of mucus neck cell of gastric mucosa, increasing or reducing of its secretion all can be as the one of gastric mucosa pathology Index, thus PG I to detect in the diagnosis and early screening of current gastropathy be highly important work, PG II is by gastric mucosa Chief cell and the secretion of mucus neck cell, it is possible to being produced by pyloric gland and duodenal gland, increasing of its secretion can be as at the bottom of stomach One index of mucosa infection.

The traditional detection method of PG I/II includes colloidal gold method, radioimmunology, euzymelinked immunosorbent assay (ELISA) and general chemistry luminescence method Deng, but colloidal gold method sensitivity is on the low side and can only realize the detection of single person-portion every time；The advantage of radioimmunology be sensitive, Special, simple and easy to do, few etc. by sample amount, but the half-life section of reagent is costly, needs special with scintillation counter etc. Equipment, radionuclide also exists certain potentially hazardous property to human body, tests refuse difficult treatment, radionuclide Labelling changes the physiologically active of some biological substance sometimes；Euzymelinked immunosorbent assay (ELISA) and chemoluminescence method detection time are long, mainly Relying on the series troublesome operation such as pure manual sample-adding, efficiency is low, causes the easy error of experimental result big, and enzymatic reaction is the most thorough The end, and easily affected by external interference factor, such as temperature, time and material concentration etc., therefore during detection, specificity is low, clever Sensitivity is poor, detection range is narrow.Therefore, this area is needed badly and a kind of is operated more while ensureing highly sensitive, good stability Add simplicity, rapid, pollution-free, it is possible to realize, by analytical tool, the mensuration test kit that detection process is full-automatic.

Number of patent application: 201110257438.4 disclose the quantitative determination reagent kit of a kind of pepsinogen I (PGI), its Being characterised by, this test kit comprises PGI Magneto separate reagent, enzyme reaction thing, increased response agent, diluent, PGI calibration object, PGI quality-control product, cleanout fluid concentrated solution and substrate solution.This invention also discloses the preparation method of this test kit, its generalization Learn luminescence technology to combine with immunity magnetic particle, it is provided that a kind of close to homogeneous reaction system, compared with prior art, This test kit has higher detection sensitivity and a specificity, simultaneously go out hinge structure on the result time shorten to Few 10 minutes, and reached preferably performance parameter, and greatly reduce product cost.

Number of patent application: 201110257440.1 disclose the quantitative determination reagent kit of a kind of pepsinogen I I (PGII), It is characterized in that, this test kit comprises PGII Magneto separate reagent, enzyme reaction thing, increased response agent, diluent, PGII Calibration object, PGII quality-control product, cleanout fluid concentrated solution and substrate solution, which also discloses the preparation method of this test kit. Chemiluminescence is combined by this test kit with immunity magnetic particle, it is provided that a kind of close to homogeneous reaction system, with existing Having technology to compare, this test kit has higher detection sensitivity and specificity, and has reached preferably performance parameter, and And greatly reduce product cost.

Number of patent application: 201410441604.X discloses a kind of pepsinogen I I (PGII) quantitative determination reagent kit, should Test kit includes calibration object, quality-control product, anti-reagent, magnetic particle reagent and luminous substrate.This patent also discloses this reagent The preparation method of box and the method using this test kit detection pepsinogen I I (PGII), with marked by fluorescein isothiocyanate Pepsinogen I I (PGII) coated antibody and pepsinogen I I (PGII) traget antibody of alkali phosphatase enzyme mark prepare Anti-reagent, prepares magnetic particle reagent with anti-Fluorescein isothiocyanate antibody coupling carboxyl magnetic bead, makes immunoreation be easier to mixed Even separation, and substantially increase response speed, with new chemical luminous substrate APLS as substrate, improve test kit Sensitivity and specificity performance.This detection kit dependable performance, highly sensitive, range of linearity width, can coordinate half certainly Dynamic, full-automatic instrument uses.

Summary of the invention

It is an object of the invention to provide one and there is high sensitivity and specificity, be suitable for the corpusculed of clinical assistant diagnosis Learn luminescence method pepsinogen (PG I/II) and measure test kit and preparation method thereof.The present invention ties on the basis of EIA enzyme immunoassay Close highly sensitive chemical luminescent detecting technology and magnetic particle isolation technics, relatively additive method have many unique advantages, First it is by paramagnetic particles as solid phase carrier, and owing to particle volume is little, surface area is big, expands response area, greatly Improve greatly sensitivity, secondly because use full-automatic instrument and matched reagent, make anthropic factor reduce to minimum, improve The stability of method and the repeatability of result, the most also make batch interior difference the least with differences between batches.

The technical scheme is that

A kind of pepsinogen Cgene/II measures test kit, including surveying PG I/II magnetic particle, surveying PG I/II spike conjugate With PG I/II calibration object, described survey PG I/II magnetic particle is the magnetic being marked with pepsinogen Cgene/II monoclonal antibody Microsphere and magnetic particle preserve liquid；Surveying PG I/II spike conjugate is the trace labelling being marked with pepsinogen Cgene/II antibody Thing and spike conjugate diluent；PG I/II calibration object includes pepsinogen Cgene/II solution, stores in analyzer The correction of principal curve.

The present invention is to utilize pepsinogen in magnetic microparticle chemiluminescence immune assay technology quantitative determination human serum or blood plasma The content of (PG I/II).Covalent labeling pepsinogen (PG I/II) antibody on magnetic particle, adds sample to be tested, First step reaction forms magnetic particle traget antibody-antigen conjugates, with the pepsin that second step reacts the tracer-labelling added Proenzyme (PG I/II) antibody, forms magnetic particle traget antibody-antigen-tracer-labelling antibody complex, fully after washing, Adding exciting liquid, catalytic luminescence, relative luminous intensity (RLU) is proportionate with PG I/II content in human serum/slurry, The content of PG I/II in sample can be calculated according to standard curve.

Described trace labelling thing is acridinium ester and derivant thereof.

Acridinium ester relatively other chemiluminescent labels, have signal to noise ratio high, and interference factor is few, and luminous efficiency is high, intensity is big, It is prone to protein-crosslinking, sensitivity advantages of higher.

Described magnetic microsphere is the complex of Fe2O3 or Fe3O4 magnetic nano-particle and high-molecular organic material.

Described magnetic microsphere by surface modification with one or more activity functional groups, described activity functional groups bag Include but be not limited to-OH ,-COOH ,-NH2 ,-CHO ,-SO3H.

Described pepsinogen Cgene/II antibody is one or more monoclonal antibodies and/or polyclonal antibody.

Described PG I/II calibration object also includes PG I/II antigen and protected protein solution, described protected protein comprise but not It is limited to new-born calf serum, bovine serum albumin, casein.

Protected protein can provide well stablizes reaction environment, makes antigen/antibody keep native conformation, can select above-mentioned Stabilizer one of which.

In described PG I/II calibration object, protected protein mass concentration is 1-30%.

In calibration object, protein concentration is low, easy in inactivation, and the protected protein of 1-30% mass concentration can reduce the polarity of solution, The hydrophobic environment that can preserve steadily in the long term is provided for albumen.

Described magnetic particle preserves liquid and spike conjugate diluent includes that stabilizer, surfactant, preservative and pH are slow Electuary.

Described pH buffer agent includes that TBS buffer system, HAC-NAC buffer system, PBS system, MES buffer One or more in system, HEPES buffer system, the buffer capacity of described pH buffer agent is regulation pH value 6.0-8.5, The concentration range of described pH buffer agent is 0.01-0.2mol/L.

Described surfactant is that TWEEN Series (includes polysorbas20 (TWEEN-20), tween 21 (TWEEN-21), tween 40 (TWEEN-40), polysorbate60 (TWEEN-60), Tween61 (TWEEN-61), Tween 80 (TWEEN-80), tween 81 (TWEEN-81), polysorbate85 (TWEEN-85)), at least one in Triton X-100, CTAC；

The most described surfactant be mass concentration scope be the polysorbas20 of 0.1-1%.

The most easily there is non-specific adsorption in protein conjugates, the polysorbas20 of 0.1-1% can weaken non-specific adsorption energy Power, affects specific adsorption effect after polysorbas20 concentration is higher than 1%, reduces reaction sensitivity.

At least one in thimerosal, sodium azide, antibiotics, proclin of described preservative；Described thimerosal Concentration is not more than 1 ‰；The concentration of described sodium azide is not more than 1 ‰；Described antibiotics includes gentamycin etc.；Described The concentration of proclin is not more than 2 ‰.

Surfactant can reduce surface tension when reagent joins reaction system, improves dispersibility and stability.

A kind of described pepsinogen PG I/II measures the preparation method of test kit, specifically comprises the following steps that

1) survey the preparation of PG I/II magnetic particle, preserve preparation and magnetic particle labelling PG I/II antibody of liquid including magnetic particle Preparation；

2) preparation of PG I/II spike conjugate is surveyed, including preparation and the acridinium ester label PG I/II of tracer diluent The preparation of antibody；

3) preparation of PG I/II calibration object, including preparation and the preparation of calibration object of calibration object diluent.

Described step 1) in magnetic particle preserve liquid preparation comprise the steps:

1.1 weigh disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g in the container of 1L, add Appropriate purified water stirring makes it be completely dissolved；

1.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved State in the container of 1L, add purified water and to 900mL and stir；

1.3 regulation PH measurement amount solution PHs so that it is control between 7.2-7.4；

1.4 weigh casein 10g adds in above-mentioned 1L container, stirs so that it is fully dissolve；

1.5 are settled to 1L, filter with 0.2 μm filter, post label and store under 2-8 DEG C of gnotobasis after filtration.

Described step 1) in the preparation of magnetic particle labelling PG I/II antibody comprise the steps:

The ratio of the magnetic particle of band carboxyl with carbodiimide (EDC) difference in mass ratio 1: 1-3 is mixed by 1.6；

1.7 are separately added into ratio addition PG I/II monoclonal antibody of 5-40 μ g antibody, magnetic particle and antibody in every milligram of magnetic particle Mass ratio is 100:1；

1.8 in the case of mixing 20-28 DEG C of respectively labelling 0.5-2 hour；

Unnecessary site closed by the glycine using final concentration 25mM after 1.9 labellings, and 22-26 DEG C is reacted 0.5 hour, extremely Washing three times less, preserve liquid with magnetic particle and preserve；

1.10 use square formation titrimetry debugging optium concentration, preserve liquid with magnetic particle and dilute magnetic particle labelling according to optium concentration PG I/II obtains surveying PG I/II magnetic particle, in 2-8 DEG C of storage；

Described step 2) in the preparation of tracer diluent comprise the steps:

2.1 weigh disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g in the container of 1L, add Appropriate purified water stirring, pipettes 1mL Tween-20 stirring with pipettor and makes it be completely dissolved；

2.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved In the container of above-mentioned 1L, add purified water and to 900mL and stir；

2.3 regulation PH measurement amount solution PHs so that it is PH controls between 7.2-7.4；

2.4 weigh casein 10g adds in above-mentioned 1L container, stirs so that it is fully dissolve；

2.5 are settled to 1L, filter with 0.2 μm filter, post label and store under 2-8 DEG C of gnotobasis after filtration；

Described step 2) in the preparation of acridinium ester label PG I/II antibody comprise the steps:

2.6 take 10mol PG I/II monoclonal antibody respectively, are separately added into acridinium ester 25-200mol mark according to every 10mol antibody Note；

2.7 addition glutaraldehydes are 0.75-1.5% to glutaraldehyde mass concentration, react 0.5-2 hour under the conditions of 20-28 DEG C；

Glutaraldehyde is double-functional group reagent, connects monoclonal antibody and acridinium ester by amino, and acridinium ester typically uses the method labelling.

2.8, with the 0.01M PBS of pH 7.2-7.4, at least change liquid three times, add equal-volume glycerol-20 after dialysis DEG C deposit；

2.9 use square formation titrimetry to determine the suitableeest working concentration of acridinium ester label PG I/II antibody, use spike conjugate Diluent is diluted, and mixing i.e. obtains surveying PG I/II spike conjugate, puts 2-8 DEG C and deposits.

Described step 3) preparation of alignment product diluent comprises the steps:

3.1 weigh disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g in the container of 1L, add Appropriate purified water stirring, pipettes 1mL Tween-20 stirring with pipettor and makes it be completely dissolved；

3.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved State in the container of 1L, add purified water and to 900mL and stir；

3.3 regulation PH measurement amount solution PHs so that it is PH controls between 7.2-7.4；

3.4 weigh bovine serum albumin 10g adds in above-mentioned 1L container, stirs so that it is fully dissolve；

3.5 are settled to 1L, filter with 0.2 μm filter, post label and store under 2-8 DEG C of gnotobasis after filtration.

Described step 3) preparation of alignment product comprises the steps:

3.6 is 0.5-500ng/mL by the working concentration scope of calibration object diluted PG I sterling；

The working concentration scope of 3.7 dilution PG II sterlings is 0.1-100ng/mL, is made into PG I/II calibration object 1 and PG I/II calibration object 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, the concentration range of PG I calibration object 2 is: 291.300-365.103ng/mL；PG II calibrates The concentration range of product 1 is: 8.788-11.018ng/mL, PG II the concentration range of calibration object 2 be: 53.289-67.494 ng/mL。

Preferably, adding glutaraldehyde to glutaraldehyde mass concentration in described step 2.7 is 1.25%, 22-26 DEG C of condition Lower reaction 1 hour.

The advantage of test kit of the present invention is to have employed microparticle chemiluminescence immunoassay technology, eliminates artificial and environment Impact, improves reagent accurate degree；Doing solid phase carrier by micropartical, expand reaction table area, the range of linearity is wider； Full-automation, its operation is more simple and easy to do, and the test kit of the present invention can with chemical illumination immunity analysis instrument especially simultaneously Be sequence of chemical luminescence immunoassay instrument (AutolumiS 500, AutolumiS 1000, AutolumiS 2000, AutolumiS 3000 Full-automatic chemiluminescence analyzer) support the use, during sample measures, achieve full-automation, The detection making concentration simply, easily and fast, carry out in bulk, can ensure that the systematic error of detection is less simultaneously.

Detailed description of the invention

Embodiment 1

One, magnetic particle preserves the preparation steps (as a example by preparing 1L, formula is shown in Table 1) of liquid

1. weighing disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g, in the container of 1L, adds Entering appropriate purified water stirring makes it be completely dissolved；

2. pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved State in the container of 1L, add purified water and to 900mL and stir；

3. regulation PH measurement amount solution PH, adds HCL or NaOH and regulates PH so that it is control between 7.2-7.4.

4. weigh casein 10g to add in above-mentioned 1L container, stir so that it is fully dissolve；

5. it is settled to 1L, filters with 0.2 μm filter, post label after filtration and store under 2-8 DEG C of gnotobasis.

Table 1 pepsinogen (PG I/II) magnetic particle preserves formula of liquid

Two, the preparation of magnetic particle labelling PG I/II antibody

The most first the magnetic particle of band carboxyl is mixed with carbodiimide (EDC) difference in mass ratio 1: 1,1: 2,1: 3, Optimum quality ratio is 1:2；

The ratio addition PG I/II of 5 μ g, 10 μ g, 20 μ g, 40 μ g antibody it is separately added into the most again in every milligram of magnetic particle Monoclonal antibody, magnetic particle and antibody optimum quality ratio are 100:1；

3. in the case of mixing 20 DEG C, 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C respectively labelling 0.5 hour, 1 hour, 2 Hour, optimum mark temperature is 22-26 DEG C, and the optimum mark time is 1 hour；

4. unnecessary site closed by the glycine using final concentration 25mM after labelling, and 22～26 DEG C are reacted 0.5 hour, At least washing three times, preserves liquid with magnetic particle and preserves；

5. use square formation titrimetry debugging optium concentration, preserve liquid with magnetic particle and dilute magnetic particle mark according to optium concentration Note PG I/II obtains surveying PG I/II magnetic particle, in 2～8 DEG C of storages；

6. survey PG I/II magnetic particle placement 37 DEG C to accelerate the failure 6 days, have good stability.

Embodiment 2: survey the preparation of PG I/II spike conjugate

One, the preparation steps (as a example by preparing 1L, formula is shown in Table 2) of tracer diluent

1. weighing disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g, in the container of 1L, adds Enter appropriate purified water stirring, pipette 1mL Tween-20 stirring with pipettor and make it be completely dissolved；

2. pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved State in the container of 1L, add purified water and to 900mL and stir；

3. regulation PH measurement amount solution PH so that it is PH controls between 7.2-7.4；

4. weigh casein 10g to add in above-mentioned 1L container, stir so that it is fully dissolve；

5. it is settled to 1L, filters with 0.2 μm filter, post label after filtration and store under 2-8 DEG C of gnotobasis.

Table 2 pepsinogen (PG I/II) tracer diluent formula

Two, the preparation of acridinium ester label PG I/II antibody

Take 10mol PG I/II monoclonal antibody the most respectively, according to every 10mol antibody be separately added into 25mol, 50mol, 100 The ratio of mol, 200mol adds acridinium ester label, and antibody is 1:10 with the optimum mark ratio of acridinium ester；

2. glutaraldehyde concentration is respectively 0.75%, 1.0%, 1.25%, 1.5%, respectively 20 DEG C, 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C react 0.5 hour, 1 hour, 1.5 hours, 2 hours, glutaraldehyde optimal Concentration is 1.25%, and optimal reaction temperature is 22-26 DEG C, and optimum reacting time is 1 hour；

3. with the 0.01M PBS of PH 7.2-7.4, at least change liquid three times, after dialysis, add equal-volume glycerol-20 DEG C Deposit；

4. use square formation titrimetry to determine the suitableeest working concentration of acridinium ester label PG I/II antibody, combine with spike Thing diluent is diluted, and mixing i.e. obtains surveying PG I/II spike conjugate, puts 2-8 DEG C and deposits；

5. surveying PG I/II spike conjugate 37 DEG C to accelerate the failure 6 days, its activity keeps more than 90%, illustrates stable Property is good.

The preparation of embodiment 3:PG I/II calibration object

One, the preparation steps (as a example by preparing 1L, formula is shown in Table 3) of calibration object diluent

1. weighing disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g, in the container of 1L, adds Enter appropriate purified water stirring, pipette 1mL Tween-20 stirring with pipettor and make it be completely dissolved；

2. pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into after being completely dissolved State in the container of 1L, add purified water and to 900mL and stir；

3. regulation PH measurement amount solution PH so that it is PH controls between 7.2-7.4；

4. weigh bovine serum albumin 10g to add in above-mentioned 1L container, stir so that it is fully dissolve；

5. it is settled to 1L, filters with 0.2 μm filter, post label after filtration and store under 2-8 DEG C of gnotobasis.

Table 3 pepsinogen (PG I/II) calibration object diluent formula

Two, the preparation of PG I/II calibration object

It is 0.5-500ng/mL by the working concentration scope of calibration object diluted PG I sterling, dilution PG II sterling Working concentration scope is 0.1-100ng/mL, is made into PG I/II calibration object 1 (low value calibration object) and PG I/II calibration object 2 (high level calibration objects), calibration object measured value should within the scope of.PG I calibration object 1:47.832-60.833ng/mL, PG I calibration object 2:291.300-365.103ng/mL；PG II calibration object 1:8.788-11.018ng/mL, PG II calibration object 2:53.289-67.494ng/mL.

The above-mentioned various raw-material selections of the present invention require as follows:

The selection of magnetic particle

By the outward appearance to magnetic particle, the ratio of labelled protein, magnetic responsiveness, magnetic particle adsorbs the aspects such as concordance to be carried out Analyze, through repeatedly analyzing and researching, magnetic particle is mixed, observe under light, easily disperse, without assembling, foreign； Albumen uses different methods be marked, and mark rate should be greater than 90%；Magnetic particle is put the magnetic of 370-380 tesla On ferrum, observing the aggregation velocity of magnetic particle, finely dispersed magnetic particle was assembled in 10 seconds completely；Magnetic particle absorption one Cause property CV≤10%.Result of study shows the magnetic particle of a diameter of 0.90-1.10 μm, and containing carboxylic group, mark rate is the highest, Can be used for the preparation of diagnostic kit of the present invention.

The selection of acridinium ester

Acridinium ester DMSO (dimethyl sulfoxide) is dissolved, is diluted the amount to 1.0ng/mL by purified water, adds Enter 10 μ L acridinium ester liquid, each addition 200 μ L exciting liquids, measure its luminous value, luminous value answers >=1000000, passes through Research, uses the acridinium ester that Mei Kaite biology provides as luminous raw material.

The selection of pepsinogen (PG I/II) antibody of magnetic particle labelling

First verifying with regard to the outward appearance of antibody, concentration, purity, titer, result antibody is the supernatant liquid of micro-strip opalescence, It is visible by naked eyes foreign body, without shaking the precipitation not dissipated, detects its protein content with ultraviolet absorption method and should be not less than 2.0mg/mL, Titer should be not less than sign titer and be not less than 1:10000, and SDS-PAGE detection purity answers master tape clear, without the most miscellaneous Band.

The acridinium ester label selection of pepsinogen (PG I/II) antibody

The most still verifying with regard to the outward appearance of antibody, concentration, purity, titer, result antibody is the clear liquor of micro-strip opalescence Body, is visible by naked eyes foreign body, and without shaking the precipitation not dissipated, detecting its protein content with ultraviolet absorption method should be not less than 2.0mg/mL, titer should be not less than sign titer and be not less than 1:10000, and SDS-PAGE detection purity answers master tape clear, Without obvious miscellaneous band.

Other matched reagents related in implementation process of the present invention:

Cleanout fluid, excite A liquid, excite the preparation of B liquid

Being mainly composed of of cleanout fluid: disodium hydrogen phosphate 28.6g/L, sodium dihydrogen phosphate 3.9g/L, sodium chloride 160g/L, Tween20 10mL/L, uses according to 10 times of dilutions by purified water.Exciting A liquid is molten containing 0.35mol/L NaOH Liquid (wherein contains the potassium ferricyanide of 1mM), excites B liquid for containing 1.32%w/v H₂O₂Buffer.

The evaluation of methodology method of this test kit

This product test kit and prior art products contrast experiment's scheme

1. minimum detectability

Detect as sample with zero-dose enterprise linear reference product, replication 10 times, draw 10 measurement results Relative light unit, calculate its meansigma methods (M) and standard deviation (SD), draw M+2SD, according to zero-dose enterprise Concentration-RLU value result between linear reference product and adjacent calibration object carries out 2 regression fits and draws linear function, will The RLU value of M+2SD is brought in above-mentioned equation, obtains the concentration value of correspondence, is minimum detectability.

2. accuracy

Finite concentration pepsinogen Cgene/pepsinogen II (PG I/PG II) liquid (A) is added in serum B, institute The volume ratio added between PG I/PG II and serum B is 1:9, and each duplicate detection three times is averaged, according to formula (1) result of calculation.

$R=\frac{C\×(V_0+Vs)-C_0\×V_0}{Vs\×Cs}\×100\%\mathrm{...}\left(1\right)$

In formula: the R response rate；

Vs adds A liquid and amasss；

V₀The volume of blood serum sample B；

C blood serum sample adds the detectable concentration after A liquid；

C₀The detectable concentration of blood serum sample B；

The concentration of Cs A liquid.

The most linear

High level sample close to the range of linearity upper limit is diluted, by these 8 sample weights with Sample dilution according to a certain percentage Recheck and survey 2 times, calculate mean concentration, result meansigma methods and dilution ratio method of least square are carried out fitting a straight line, And calculate linearly dependent coefficient r.

4. repeatability

By each duplicate detection of the sample of high-concentration and low-concentration 10 times, calculate meansigma methods (M) and the standard deviation of 10 measurement results (SD), the coefficient of variation (CV) is drawn according to formula (2).

CV=SD/M × 100% ... ... ... ... ... ... ... ... ... ... (2)

In formula: the CV coefficient of variation；

The meansigma methods 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

Test kit in the market according to product different exist full-automatic sample-adding and semi-automatic manual sample-adding pattern point, Automatically it is loaded pattern owing to this product test kit uses, relative to the test kit of other manual sample-addings, eliminates and artificially add The error that sample exists, is preferably minimized artificial disturbance factor to a certain extent.Examining report data show, this product tries Agent box all meets quality testing standard in minimum detectability, accuracy, the index such as linear and repeated.And pepsinogen The minimum detectability of I, accuracy, repeatability and the minimum detectability of pepsinogen II, accuracy, the performance such as linear Index is superior to reference product test kit, and exists in two indexs of gap, its gap still in the range of quality testing standard, Have no effect on the properties of product of test kit.Comprehensive the most provable product test kit of indices has highly sensitive, repetition The advantage such as property is by force, the most excellent, artificial disturbance factor is little.

Above embodiments of the invention are described in detail, but described content has been only presently preferred embodiments of the present invention, no The practical range for limiting the present invention can be considered.All impartial changes made according to the present patent application scope and improvement etc., Within all should still belonging to the patent covering scope of the present invention.

Claims (10)

1. pepsinogen Cgene/II measures a test kit, including surveying PG I/II magnetic particle, surveying PG I/II spike combination Thing and PG I/II calibration object, it is characterised in that: described survey PG I/II magnetic particle is single for being marked with pepsinogen Cgene/II The magnetic microsphere of clonal antibody and magnetic particle preserve liquid；Survey PG I/II spike conjugate for being marked with pepsinogen Cgene/II The trace labelling thing of antibody and spike conjugate diluent, described trace labelling thing is acridinium ester and derivant thereof；PGⅠ/Ⅱ Calibration object includes pepsinogen Cgene/II solution, stores the correction of principal curve in analyzer.

A kind of pepsinogen Cgene/II the most according to claim 1 measures test kit, it is characterised in that: described magnetic Property microsphere is Fe₂O₃Or Fe₃O₄Magnetic nano-particle and the complex of high-molecular organic material.

A kind of pepsinogen PG I/II the most according to claim 2 measures test kit, it is characterised in that: described Magnetic microsphere is by surface modification with one or more activity functional groups, and described activity functional groups includes but do not limits In-OH ,-COOH ,-NH₂,-CHO ,-SO₃H。

A kind of pepsinogen PG I/II the most according to claim 1 measures test kit, it is characterised in that: described Pepsinogen Cgene/II antibody is one or more monoclonal antibodies and/or polyclonal antibody.

A kind of pepsinogen PG I/II the most according to claim 1 measures test kit, it is characterised in that: described PG I/II calibration object also includes PG I/II antigen and protected protein solution, and described protected protein is including but not limited to new born bovine Serum, bovine serum albumin, casein.

A kind of pepsinogen PG I/II the most according to claim 5 measures test kit, it is characterised in that: described In PG I/II calibration object, protected protein mass concentration is 1-30%.

A kind of pepsinogen PG I/II the most according to claim 1 measures test kit, it is characterised in that: described Magnetic particle preserves liquid and spike conjugate diluent includes stabilizer, surfactant, preservative and pH buffer agent.

A kind of pepsinogen PG I/II the most according to claim 7 measures test kit, it is characterised in that: described PH buffer agent includes TBS buffer system, HAC-NAC buffer system, PBS system, MES buffer system, HEPES One or more in buffer system, the buffer capacity of described pH buffer agent is slow for regulation pH value 6.0-8.5, described pH The concentration range of electuary is 0.01-0.2mol/L.

9. pepsinogen PG I/II described in claim 1-8 any one measures a preparation method for test kit, tool Body step is as follows:

1) survey the preparation of PG I/II magnetic particle, preserve preparation and magnetic particle labelling PG I/II antibody of liquid including magnetic particle Preparation；

2) surveying the preparation of PG I/II spike conjugate, preparation and acridinium ester label PG I/II including tracer diluent resist The preparation of body；

3) preparation of PG I/II calibration object, including preparation and the preparation of calibration object of calibration object diluent.

A kind of pepsinogen PG I/II the most according to claim 9 measures the preparation method of test kit, its feature Be: described step 1) in magnetic particle preserve liquid preparation comprise the steps:

1.1 weigh disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g in the container of 1L, add Appropriate purified water stirring makes it be completely dissolved；

1.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into above-mentioned after being completely dissolved In the container of 1L, add purified water and to 900mL and stir；

1.3 regulation PH measurement amount solution PHs so that it is control between 7.2-7.4；

1.4 weigh casein 10g to add in above-mentioned 1L container, stir so that it is fully dissolve；

1.5 are settled to 1L, filter with 0.2 μm filter, post label and store under 2-8 DEG C of gnotobasis after filtration.

Described step 1) in the preparation of magnetic particle labelling PG I/II antibody comprise the steps:

The ratio of the magnetic particle of band carboxyl with carbodiimide (EDC) difference in mass ratio 1: 1-3 is mixed by 1.6；

1.7 are separately added into ratio addition PG I/II monoclonal antibody of 5-40 μ g antibody, magnetic particle and antibody in every milligram of magnetic particle Mass ratio is 100:1；

1.8 in the case of mixing 20-28 DEG C of respectively labelling 0.5-2 hour；

Unnecessary site closed by the glycine using final concentration 25mM after 1.9 labellings, and 22-26 DEG C is reacted 0.5 hour, at least Wash three times, preserve liquid with magnetic particle and preserve；

1.10 use square formation titrimetry debugging optium concentration, preserve liquid with magnetic particle and dilute magnetic particle labelling according to optium concentration PG I/II obtains surveying PG I/II magnetic particle, in 2-8 DEG C of storage；

Described step 2) in the preparation of tracer diluent comprise the steps:

2.1 weigh disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g in the container of 1L, add Appropriate purified water stirring, pipettes 1mL Tween-20 stirring with pipettor and makes it be completely dissolved；

2.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into above-mentioned after being completely dissolved In the container of 1L, add purified water and to 900mL and stir；

2.3 regulation PH measurement amount solution PHs so that it is PH controls between 7.2-7.4；

2.4 weigh casein 10g to add in above-mentioned 1L container, stir so that it is fully dissolve；

2.5 are settled to 1L, filter with 0.2 μm filter, post label and store under 2-8 DEG C of gnotobasis after filtration；

Described step 2) in the preparation of acridinium ester label PG I/II antibody comprise the steps:

2.6 take 10mol PG I/II monoclonal antibody respectively, are separately added into acridinium ester 25-200mol mark according to every 10mol antibody Note；

2.7 glutaraldehyde concentration is respectively 0.75-1.5%, react 0.5-2 hour at 20-28 DEG C respectively；

2.8, with the 0.01M PBS of pH 7.2-7.4, at least change liquid three times, add equal-volume glycerol-20 after dialysis DEG C deposit；

2.9 use square formation titrimetry to determine the suitableeest working concentration of acridinium ester label PG I/II antibody, dilute with spike conjugate Releasing liquid to be diluted, mixing i.e. obtains surveying PG I/II spike conjugate, puts 2-8 DEG C and deposits.

Described step 3) preparation of alignment product diluent comprises the steps:

3.1 weigh disodium hydrogen phosphate 2.68g, sodium dihydrogen phosphate 0.39g, sodium chloride 8.5g in the container of 1L, add Appropriate purified water stirring, pipettes 1mL Tween-20 stirring with pipettor and makes it be completely dissolved；

3.2 pipette 1mL proclin 300 in the beaker of 10mL purified water with pipettor, pour into above-mentioned after being completely dissolved In the container of 1L, add purified water and to 900mL and stir；

3.3 regulation PH measurement amount solution PHs so that it is PH controls between 7.2-7.4；

3.4 weigh bovine serum albumin 10g to add in above-mentioned 1L container, stir so that it is fully dissolve；

3.5 are settled to 1L, filter with 0.2 μm filter, post label and store under 2-8 DEG C of gnotobasis after filtration.

Described step 3) preparation of alignment product comprises the steps:

3.6 is 0.5-500ng/mL by the working concentration scope of calibration object diluted PG I sterling；

The working concentration scope of 3.7 dilution PG II sterlings is 0.1-100ng/mL, is made into PG I/II calibration object 1 and PG I/II calibration object 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, the concentration range of PG I calibration object 2 is: 291.300-365.103ng/mL；PG II calibrates The concentration range of product 1 is: 8.788-11.018ng/mL, PG II the concentration range of calibration object 2 be: 53.289-67.494 ng/mL。