CN102323417B - Kit for quantitative determination of pesinogen I (PGI) and detection method thereof - Google Patents
Kit for quantitative determination of pesinogen I (PGI) and detection method thereof Download PDFInfo
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for quantitative determination of pesinogen I (PGI) and a detection method thereof. The kit is characterized by comprising a PGI magnetic separation reagent, an enzyme reactant, a reaction reinforcing agent, a diluent, a PGI calibrator, a PGI quality control material, a cleaning liquid concentrate, and a substrate solution. The invention also discloses a preparation method of the kit. By combining a chemiluminescence technology and immunomagnetic particles, the kit of the invention provides a reaction system close to homogeneous. Compared with prior art, the kit provided in the invention has higher detection sensitivity and specificity. Meanwhile, the time for obtaining a result is shortened by at least 10min compared with the present technology, better performance parameters are reached, and also the product cost is substantially reduced.
Description
Technical field
The present invention relates to measure kit and the method for testing thereof of serum, especially relate to kit and the method for testing thereof of measuring pepsinogen I (PGI) content in serum.
Background technology
Propepsin (Pesinogen, PG) be pepsic inactive precursor in gastric juice, people's pepsin can be divided into two kinds of propepsin duck groups that biochemistry is different with immunocompetence feature: press the mobility of propepsin under agargel electrophoresis by fast extremely slow part seven kinds of isozymogens of Pg1-Pg7, wherein Pg1-Pg5 has common immunogenicity, is collectively referred to as PGI (claiming again PGA); Serum PG level has reflected that the morphology and function of different parts gastric mucosa: PGI is the pointer that detects oxyntic gland cell function, and gastric acid secretion increases PGI and raises, and secretion reduces or gastric mucosa body of gland atrophy PGI reduces; The correlativity of PGII and gastric mucosa pathology is large (with respect to antrum), and it raises and rises in value relevant with fundus gland shrink tube, gastric metaplasia or false gland metaplasia, abnormal shape; It is relevant to atrophy of gastric mucosa progress that carrying out property of PGI/II ratio reduces.Therefore, simultaneous determination PGI and PGII ratio can play the effect of fundus gland mucous membrane " serology biopsy ".
Past take and puts the pepsinogen I (PGI) of exempting from as representative and measure kit and be subject to methodological restriction, its sensitivity and antijamming capability wretched insufficiency, have very large drawback, substantially withdraw from the market, applying at present more is enzyme linked immunosorbent detection technology and chemiluminescence.It is continue Enzyme-multiplied immune technique and the emerging technology growing up after putting immune technology the eighties that chemiluminescence rises in eighties of last century, due to its high sensitivity, high specific, while method is easy, quick, mark bond is stable, the features such as "dead" isotope damage and pollution, have obtained develop rapidly in recent years.
Magnetic particle separation enzyme-linked immunoassay technology is a kind ofly to take magnetic particle as solid phase carrier of separating, immune magnetic particle isolation technics is combined with enzyme linked immunosorbent detection technology and a kind of Novel immune detection method of setting up.Traditional E LISA method, the association reaction of antigen, antibody carries out on solid phase (elisa plate reacting hole) surface, and magnetic particle separation enzyme-linked immunoassay, the association reaction of antigen, antibody also carries out under the condition of approximate liquid phase, thereby reaction is fast, thoroughly.Compare with traditional E LISA and have highly sensitively, detect few advantage of used time.
Summary of the invention
The technical issues that need to address of the present invention are to provide a kind of pepsinogen I (PGI) quantitative determination reagent kit and detection method thereof, adopt this kit to carry out PGI detection and there is higher sensitivity and specificity, and the time of shorter acquisition testing result and easier mode of operation.
The invention provides the quantitative determination reagent kit of a kind of pepsinogen I (PGI), its reagent comprising has PGI magnetic separation agent, enzyme reaction thing, increased response agent, dilution, calibration object, quality-control product, cleaning fluid concentrate and substrate solution.
Described magnetic separation agent contains the magnetic microsphere that is marked with anti-PGI monoclonal antibody.
Described enzyme reaction thing is the anti-PGI monoclonal antibody that contains alkali phosphatase enzyme mark.
Described increased response agent is the damping fluid that contains Tris.
Described dilution is to contain BSA solution.
Described calibration object and quality-control product are the BSA protein solutions that contains a certain amount of PGI antigen.
Described cleaning fluid concentrate is the damping fluid that contains TWEEN-20 and Proclin-300.
Described substrate solution is enzyme-catalyzed chemical luminescence substrate solution.
The preparation method of the quantitative determination reagent kit of pepsinogen I of the present invention (PGI), comprises the steps:
The first step: the preparation process of magnetic separation agent
One, magnetic bead buffer solution formulation operations step, preparation 1L:
1, take TRIS 4.58g and NaCl6.81g in 1L container, after taking 0.96g TWEEN-20 and adding suitable quantity of water in 20ml container it is dissolved completely, pour in said vesse;
2, with pipettor, Proclin-300 is measured after 0.2ml dissolves completely in the beaker of 10ml purified water, pour in above-mentioned 1L container, then in above-mentioned 1L container, add 800ml purified water, fully stir;
3, regulate its pH value of PH instrumentation amount, adjust PH, control PH between 7.95-8.05;
4, taking BSA 3g pours in above-mentioned 1L container;
5, be finally settled to 1000ml, with 0.2um filter, filter; After having filtered, post label in 2-8 ℃ of refrigeration house storage.
Two, the preparation process of magnetic separation agent
1,1.0mg disuccinimidyl suberate is dissolved in 50ul DMSO, get in the 0.1mol/L PB damping fluid that the anti-PGI monoclonal antibody of 2mg is dissolved in PH 9.5 to cumulative volume be 1ml;
2, determine the input amount of disuccinimidyl suberate, with liquid-transfering gun, draw disuccinimidyl suberate and join in the antibody-solutions of step 1, put room temperature 90min;
3, step 2 antibody-solutions is joined in Centricon-10 concentration tube, then put in high speed freezing centrifuge under 3000g concentrated 30min to volume be 0.5ml;
4, get 0.5ml magnetic bead, add in 5ml reaction cup, put into test tube rack special, through magnet adsorption, after 2 minutes, draw supernatant;
5, add 1.5ml PH9.5 0.1mol/L PB at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; The antibody-solutions that step 4 is obtained joins in above-mentioned magnetic bead, mixes rear room temperature reaction 4 hours;
6,37 ℃ of the TRIS solution 15 minutes that add 0.3ml 1mol/L;
7, add 1.5ml PH 7.2 0.1mol/L PB to clean the magnetic bead of mark at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times;
8, with 100ml magnetic bead, preserve liquid magnetic bead is proceeded to 125ml vial, be 0.05% PGI magnetic separation agent; It is 0.1%BSA that magnetic bead is preserved formula of liquid, 0.05% Tween-20,0.02%NaN3,20% ethanol, 4 ℃ of preservations.
9, by magnetic bead buffer solution, the ratio according to 1: 1 mixes magnetic separation agent step 9 being obtained, and obtains magnetic separation agent in kit of the present invention.
Second step: enzyme reaction thing preparation process
One, enzyme reaction thing diluent preparing operation steps, preparation 1L:
1, get Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-300 0.2ml and MgCl
20.05g, in flask, then adds 800ml purified water in flask, fully stirs, and reagent is dissolved completely;
2, adjust PH, control PH within the scope of 7.35-7.45;
3, taking BSA 3g pours in above-mentioned beaker;
4, be finally settled to 1000ml, with 0.2um filter, filter and get final product.
Two, the coupling of alkaline phosphatase and anti-PGI monoclonal antibody
1, get 10mgALP and add in 5ml physiological saline, join in concentration tube, centrifugal 20 minutes of 3000RPM, is concentrated into 1 milliliter;
2, to step 1, obtain the 0.1M NaIO that adds 0.2ml in concentrate
4solution, under room temperature, lucifuge stirs 20 minutes, then packs in bag filter, and with the sodium-acetate buffer dialysis of 1mM PH4.4,4 ℃ are spent the night, and collect and retain liquid;
3, to step 3, obtain in reservation liquid and add 0.2M PH9.5 carbonate buffer solution, make PH be elevated to 9.0, then add immediately the anti-PGI monoclonal antibody of 2.5mg, room temperature lucifuge stirs 2 hours gently, then adds the 4mg/ml NaBH of 0.1ml
4solution, mixes, and puts at 4 ℃ 2 hours;
4, above-mentioned liquid is packed in bag filter, to 0.15M PH7.4 PBS dialysis, 4 ℃ are spent the night, and collect and retain liquid;
5, in step 4 to retain liquid, dropwise add equal-volume saturated ammonium sulfate, put 4 ℃ 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment is washed secondary with semi-saturation ammonium sulfate, finally sediment is dissolved in the PBS of 0.15M PH7.4 of 20ml;
6, solution step 5 being obtained packs in bag filter, with the PB buffer saline dialysis of 0.15M PH7.4, within 5 hours, remove ammonium ion, collect and retain after liquid 10,000rpm removes precipitation for centrifugal 30 minutes, collect supernatant, the ratio that is 1: 100 according to volume ratio is added 1M MgCl
2solution, obtain the conjugate of alkaline phosphatase (ALP) and PGI monoclonal antibody, finally by the conjugate of the alkaline phosphatase of collecting (ALP) and PGI monoclonal antibody, with above-mentioned enzyme reaction thing dilution, the volume ratio with 1: 1000 mixes, and obtains enzyme reaction thing.
The 3rd step:
Increased response agent preparation steps, preparation 1L:
1, take TRIS1.56g and NaCl 4.23g in 1L container; With pipettor, Proclin-300 is measured after 0.2ml dissolves completely in the beaker of 10ml purified water, pour in above-mentioned 1L container;
2, with graduated cylinder, measure 800ml purified water in above-mentioned 1L container, fully stir, until dissolve completely, adjust PH, control PH between 7.35-7.45;
3, take Mak33 0.9g in above-mentioned 1L container; Last constant volume 1000ml, after dissolving completely, filters with 0.2um filter.
The 4th step:
Diluent preparing step, preparation 1L:
1, take NaCl9.0g and BSA 60g in the container of 1L;
2, with pipettor, Proclin-300 is measured to 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml, after dissolving completely, filters with 0.2um filter, posts label in 2-8 ℃ of refrigeration house storage, and the term of validity is 12 months.
The 5th step:
The preparation of calibration object and quality-control product:
Calibration object concentration is respectively 5,15,30,80,160ng/ml; Quality-control product concentration is respectively 15,80ng/ml.
The 6th step:
Cleaning concentrate preparation steps, preparation 1L:
1, take TRIS 12.54g and NaCl 325.6g in 1L container;
2, after taking 5g Tween-20 and adding 20ml water in 100ml container it is dissolved completely, pour in above-mentioned 1L container;
3, with pipettor, Proclin-300 is measured after 0.2ml dissolves completely in the beaker that fills 10ml purified water, pour in above-mentioned 1L container;
4, with graduated cylinder, measure 800ml purified water in above-mentioned 1L container, fully stir, until dissolve completely;
5, adjust PH, control its scope between 7.35-7.45;
6, last constant volume 1000ml, filters and get final product with 0.2um filter after dissolving completely.
The 7th step:
Substrate solution preparation steps, preparation 1L:
1, take TRIS 2.35g, NaCl 6.41g, Na
2sO
30.002g and Proclin-300 0.2ml are in 1L beaker;
2, with graduated cylinder, measure 600ml purified water in 1L beaker, fully stir, until dissolve completely, adjust PH, control its scope between 7.95-8.05;
3, add after 250ml Lumi-Phos 480, with 0.2um filter, filter and collect filtrate, by purified water, be settled to 1000ml, after mixing and get final product.
Principle of work of the present invention: this product is a kind of detection method that sandwich method chemiluminescence immunoassay combines with magnetic particle isolation technics.In sample, calibration object and quality-control product, add quantitative enzyme mark PGI monoclonal antibody, in conjunction with PGI monoclonal antibody and the stabilizing reinforcer of magnetic particle.37 ℃ hatch after, the PGI monoclonal antibody of combination on magnetic particle is combined with the different epi-positions of PGI molecule respectively from monoclonal antibody linked with peroxidase, forms " sandwich " structure.Direct precipitation in externally-applied magnetic field, does not need centrifugal separable.Remove supernatant, the compound of washing and precipitating, then adds enzyme-catalyzed chemical luminescence substrate.Substrate is catalyzed cracking under enzyme effect, form unsettled excited state intermediate, when excited state intermediate is got back to ground state, just send photon, form luminescence-producing reaction, the luminous intensity of light-emitting appearance detection reaction can be used, according to typical curve, the PGI content in sample can be calculated.In sensing range, luminous intensity is directly proportional to the PGI concentration in sample.
Main innovation part of the present invention is:
1, kit of the present invention combines chemiluminescence with immune magnetic particle, a kind of reaction system that approaches homogeneous phase is provided, compared with prior art, kit of the present invention has higher detection sensitivity and specificity, simultaneously go out result on the time relatively prior art shortened at least 10 minutes, and reached preferably performance parameter.
2, the invention discloses a kind of new special-purpose stabilizing reinforcer and cleaning fluid concentrate, make course of reaction more reliable and more stable, experimental data is sensitive effectively, when enhancing product performance, and greatly reduces cost of products;
3, the magnetic separation agent in kit, enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning fluid concentrate, dilution and substrate solution are all the optimization formulas under this reaction system, and giving the use effect phase of this kit and detecting performance provides powerful guarantee.
Embodiment
Embodiment 1,
One, magnetic bead buffer solution formulation operations rules: formula, in Table 1, be take and prepared 1L as example:
1, take TRIS 4.58g and NaCl6.81g in 1L container, after taking 0.96g TWEEN-20 and adding suitable quantity of water in 20ml container it is dissolved completely, pour in said vesse;
2, with pipettor, Proclin-300 is measured after 0.2ml dissolves completely in the beaker of a small amount of purified water, pour in above-mentioned 1L container; Then in 1L container, add 800ml purified water, fully stir, reagent is dissolved completely;
3, regulate its pH value of PH instrumentation amount; With HCl or NaOH, adjust PH, measure its PH and meet the requirements between 7.95-8.05;
4, taking BSA (bovine serum albumin(BSA)) 3g pours in said vesse;
5, be finally settled to 1000ml, survey pH value, scope meets the requirements between 7.95-8.05, with 0.2um filter, filters; After having filtered, post label in 2-8 ℃ of refrigeration house storage, the magnetic bead buffer solution term of validity is 12 months;
Magnetic bead buffer solution table 1
Two, the preparation process of magnetic separation agent
1,1.0mg DSS (disuccinimidyl suberate) is dissolved in 50ul DMSO, concentration is 20mg/ml; Get in the 0.1mol/LPB damping fluid that the anti-PGI monoclonal antibody of 2mg (day strong bio-pharmaceuticals (Tianjin) company limited, concentration is 2.9mg/ml) is dissolved in PH 9.5 to cumulative volume be 1ml;
2, calculate the input amount of DSS, according to following formula, calculate: (antibody quality/16000) * 10 * 368/C
dSS), C wherein
dSSrefer to the amount of substance concentration mol/L of DSS;
3, with the DSS of liquid-transfering gun absorption respective volume, join in the antibody-solutions of step 1, put room temperature 90min;
4, step 3 antibody-solutions is joined in Centricon-10 concentration tube, then put in sigma2-16k high speed freezing centrifuge under the centrifugal force of 3000g concentrated about 30min to volume be 0.5ml;
5, get 0.5ml magnetic bead, magnetic bead concentration is 0.5g/ml, and described magnetic bead diameter, between 0.9 μ m, adds in 5ml reaction cup, puts into test tube rack special, through magnet adsorption, after 2 minutes, draws supernatant;
6, add 1.5ml PH9.5 0.1mol/L PB at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; The antibody-solutions that step 4 is obtained joins in magnetic bead, mixes rear room temperature reaction 4 hours, keeps mixing state;
7, add the TRIS solution 37 of 0.3ml 1mol/L to spend 15 minutes, wherein the application of sample amount of TRIS is that 1mg antibody adds 0.15mlTRIS;
8, add 1.5ml PH7.2 0.1mol/L PB to clean the magnetic bead of mark at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times;
9, with 100ml magnetic bead, preserve liquid magnetic bead is proceeded to 125ml vial, be 0.05% PGI magnetic separation agent; It is 0.1%BSA that magnetic bead is preserved formula of liquid, 0.05% Tween-20,0.02%NaN3,20% ethanol, 4 ℃ of preservations.
10, by magnetic bead buffer solution, the ratio according to 1: 1 mixes magnetic separation agent step 9 being obtained;
11, post label in 2-8 ℃ of refrigeration house storage, the magnetic separation agent term of validity is 12 months;
Embodiment 2
One, enzyme reaction thing diluent preparing working specification: formula, in Table 2, be take and prepared 1L as example:
1, get Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-300 0.2ml and MgCl
20.05g is in flask; Then in flask, add 800ml purified water, fully stir, reagent is dissolved completely;
2, with HCl or NaOH, adjust PH, measure and make PH within the scope of 7.35-7.45;
3, taking BSA 3g pours in said vesse;
4, be finally settled to 1000ml, survey pH value, scope meets the requirements between 7.35-7.45, with 0.2um filter, filters; After having filtered, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months;
Enzyme reaction thing dilution table 2
Two, the coupling of alkaline phosphatase (ALP) and anti-PGI monoclonal antibody
1, get 10mgALP and add in 5ml physiological saline, join in Centriprep YM10 concentration tube, centrifugal about 20 minutes of 3000RPM, is concentrated into 1 milliliter.
2, to step 1, obtain the 0.1M NaIO that adds 0.2ml in concentrate
4solution, under room temperature, lucifuge stirs 20 minutes, then packs in bag filter, and with the sodium-acetate buffer dialysis of 1mM PH4.4,4 ℃ are spent the night, and collect and retain liquid;
3, to step 2, obtain in reservation liquid and add 0.2M PH9.5 carbonate buffer solution, make PH be elevated to 9.0～9.5, then add immediately the anti-PGI monoclonal antibody of 2.5mg (day strong bio-pharmaceuticals (Tianjin) company limited, concentration is 2.9mg/ml), room temperature lucifuge stirs 2 hours gently, then adds the 4mg/ml NaBH of 0.1ml
4(sodium borohydride) solution, mixes, then puts at 4 ℃ 2 hours;
4, above-mentioned liquid is packed in bag filter, to 0.15M PH7.4 PBS dialysis, 4 ℃ are spent the night, and collect and retain liquid;
5, in step 4 to retain liquid, dropwise add equal-volume saturated ammonium sulfate, put 4 ℃ 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment is washed secondary with semi-saturation ammonium sulfate, finally sediment is dissolved in the PBS of a small amount of 0.15M PH7.4;
6, solution step 5 being obtained packs in bag filter, by the PB buffer saline of 0.15M PH7.4, dialyse approximately and within 5 hours, to remove ammonium ion, collect and retain after liquid 10,000rpm removes precipitation for centrifugal 30 minutes, collect supernatant, the ratio that is 1: 100 according to volume ratio is added 1M Mgcl
2solution, obtain the conjugate of alkaline phosphatase (ALP) and PGI monoclonal antibody, finally by the conjugate of the alkaline phosphatase of collecting (ALP) and PGI monoclonal antibody, with above-mentioned enzyme reaction thing dilution, the volume ratio with 1: 1000 mixes, and obtains enzyme reaction thing.
Embodiment 3
Increased response agent formulation operations rules: formula is shown in (table 3), take and prepares 1L as example:
1, take TRIS (trishydroxymethylaminomethane, molecular formula: (HOCH2) 3CNH2) 1.56g and NaCl 4.23g are in 1L container; With pipettor, Proclin-300 is measured after 0.2ml dissolves completely in the beaker of a small amount of purified water, pour in above-mentioned 1L container;
2, with graduated cylinder, measure 800ml purified water in 1L container, fully stir, until dissolve completely; With HCL or NaOH, adjust PH, measure its scope between 7.35-7.45;
3, take Mak33 0.9g in above-mentioned 1L container;
4, last constant volume 1000ml, after dissolving completely, surveys pH value, and scope meets the requirements between 7.35-7.45, with 0.2um filter, filters; After having filtered, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months;
The preparation (table 3) of increased response agent
Embodiment 4
Dilution formula is shown in (table 4), take and prepares 1L as example:
1, take NaCl9.0g and BSA 60g in the container of 1L;
2, with pipettor, Proclin-300 is measured to 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml, after dissolving completely, filters with 0.2um filter, posts label in 2-8 ℃ of refrigeration house storage, and the term of validity is 12 months.
The preparation (table 4) of dilution
Embodiment 5
The preparation of calibration object and quality-control product:
1, peak: maximum concentration point is X, impact point concentration is A, B, C, D, E, F, while preparing the solution of V volume, need add the volume of raw material for being respectively: table 5
| Concentration | Add calibration object dilution volume | Add X volume |
| A | V-A*V/X | A*V/X |
| B | V-B*V/X | B*V/X |
| C | V-C*V/X | C*V/X |
| D | V-D*V/X | D*V/X |
| E | V-E*V/X | E*V/X |
| 0F | V-F*V/X | F*V/X |
2, dilution for pepsinogen I (PGI) quantitative determination reagent kit PGI calibration object raw material (be purchased from PPS company, concentration is 5.7mg/ml) (concrete formula is shown in embodiment 4) is mixed with that concentration point is 5,15,30,80,160ng/ml; Quality-control product with the concentration point of diluent preparing be 15,80ng/ml.
3, after dissolving completely, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months.
Embodiment 6:
Cleaning concentrate formulation operations rules: formula is shown in (table 6), take and prepares 1L as example:
1, take TRIS 12.54g and NaCl 325.6g in 1L container;
2, after taking 5g Tween-20 and adding suitable quantity of water in 100ml container it is dissolved completely, pour in said vesse;
3, with pipettor, Proclin-300 is measured after 0.2ml dissolves completely in the beaker that fills 10ml purified water, pour in above-mentioned 1L container;
4, with graduated cylinder, measure 800ml purified water in above-mentioned 1L container, fully stir, until dissolve completely;
5, with HCL or NaOH, adjust PH, measure its scope between 7.35-7.45;
6, last constant volume 1000ml, surveys pH value, and scope meets the requirements between 7.35-7.45, after dissolving completely, with 0.2um filter, filters; After having filtered, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months;
The preparation (table 6) of cleaning concentrate
Embodiment 7
Substrate solution formulation operations rules: formula is shown in (table 7), take and prepares 1L as example:
1, take TRIS 2.35g, NaCl 6.41g, Na
2sO
30.002g and Proclin-300 0.2ml are in 1L beaker;
2, with graduated cylinder, measure 600ml purified water in 1L beaker, fully stir, until dissolve completely; With HCl or NaOH, adjust PH, measure its scope between 7.95-8.05;
3, add after 250ml Lumi-Phos 480, with 0.2um filter, filter and collect filtrate, by purified water, be settled to 1000ml, after mixing, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months.
The preparation (table 7) of chemical luminous substrate
Method of testing of the present invention is as follows
1, add 30 μ l PGI calibration objects, quality-control product, sample to be measured to corresponding test tube bottom.
2, add 30 μ l enzyme reaction things to each test tube.
3, add 30 μ l increased response agent to each test tube.
4, add 30 μ l magnetic separation agents to each test tube.
5, with plastic sheeting, cover test tube, multitube vortex mixer after tube shaken frame 30s, is put 37 ℃ of water-baths 30 minutes gently.
6, test tube frame linking is put to magnetic separator, guarantees that every test tube all contacts with separator surface, precipitates 2 minutes.The separation vessel that reverses is slowly poured out supernatant, and the test tube of reversing is placed on filter paper together with separation vessel, firmly bounces separation vessel bottom to remove all drops that are bonded on tube wall.
7, cleaning fluid concentrate is with after 20 times of purified water dilutions, adds cleaning fluid after 200 μ l dilutions to each test tube, puts on multitube vortex mixer vibration gently and mixes 30s.During application of sample, should avoid application of sample dynamics excessive and cause magnetic bead to spill.Mix and want thoroughly.
8, repeating step is 6,7,6 one times.
9, add 200 μ l substrate solutions and mix 3 seconds to test tube, with ready luminous detector, detect rapidly.
10,, as run into high value HOOK sample, for fear of there is high value HOOK effect, suggestion clinician selects suitable extension rate to dilute sample according to all the other test indexs.
Clinical testing:
1, detect data
Sample picks up from the normal health check-up of 1000 example, blood donor.Serum sample physical examination result is all without liver, brain, kidney, disease of digestive tract, and in half a year, without blood transfusion and major operation history, women is not in the gestational period and lactation.Measured value is carried out to statistical analysis, draw normal serum term of reference: PGI < 70.00ng/ml; PG I/II < 3.The testing result of the kit of producing with external renowned company is compared, and kit of the present invention is more accurate, is accurate to 2 significant digits.
Notebook data is only for reference, and different regions, Different Individual and employing distinct methods detect, and measured PGI level also can be different, advises the range of normal value of each laboratory foundation oneself.The PGI value that can not only draw with this method is made diagnosis, only as intermediate data reference role, should, in conjunction with clinical other Data Analysis Results, comprise patient's concrete condition and treatment situation.PGI concentration value and this kit measurement result in the sample obtaining by additive method do not have direct comparability.The sample that exceeds kit measurement scope, system cannot provide definite numerical value.As wish is measured its definite result, after suggestion dilution, measure again.The testing result of this kit only supplies clinical reference, can not be separately as making a definite diagnosis or the foundation of Excluded cases, for reaching diagnostic purpose, this testing result will be combined with clinical examination, medical history and other inspection.This product can be used for the mensuration of serum sample, for the reliability of other body fluid samples PGI concentration determination, is fully confirmed.
2, kit performance index of the present invention
Sensitivity for analysis is defined as: the mensuration to 20 zero calibration objects, get its mean deviation of 2 times, and its corresponding concentration on typical curve is sensitivity for analysis;
Sensitivity for analysis: 1.00ng/ml; Accuracy: variation within batch CV%≤10.0%; Batch variation CV%≤15.0%; Linear coefficient: r >=0.9900; The range of linearity: 1.00-200ng/ml; Specificity: measure the cross reacting material of high concentration, result is as follows:
Claims (1)
1. a quantitative determination reagent kit of propepsin PGI, is characterized in that, this kit comprises PGI magnetic separation agent, enzyme reaction thing, increased response agent, dilution, PGI calibration object, PGI quality-control product, cleaning fluid concentrate and substrate solution;
Described PGI magnetic separation agent contains the magnetic microsphere that is marked with anti-PGI monoclonal antibody;
Described enzyme reaction thing is the anti-PGI monoclonal antibody that contains alkali phosphatase enzyme mark;
Described increased response agent is the damping fluid that contains Tris;
Described dilution is the solution that contains BSA;
Described calibration object and quality-control product are the BSA solution that contains a certain amount of PGI antigen;
Described cleaning fluid concentrate is the damping fluid that contains Tween-20 and Proclin-300;
Described substrate solution is enzyme-catalyzed chemical luminescence substrate solution;
In described kit, each component makes according to the method for being prepared as follows:
One, the preparation process of magnetic separation agent
One), magnetic bead buffer solution formulation operations step, preparation 1L:
1, take TRIS 4.58g and NaCl6.81g in 1L container, after taking 0.96g Tween-20 and adding suitable quantity of water in 20ml container it is dissolved completely, pour in above-mentioned 1L container;
2, with pipettor, Proclin-300 is measured after 0.2ml dissolves completely in the beaker of 10ml purified water, pour in above-mentioned 1L container, then in above-mentioned 1L container, add 800ml purified water, fully stir;
3, regulate pH, control pH between 7.95-8.05;
4, taking BSA 3g pours in above-mentioned 1L container;
5, be finally settled to 1000ml, with 0.2 μ m filter, filter and get final product;
Two), the preparation process of magnetic separation agent
1,1.0mg disuccinimidyl suberate is dissolved in 50 μ l DMSO, get in the 0.1mol/L PB damping fluid that the anti-PGI monoclonal antibody of 2mg is dissolved in pH 9.5 to cumulative volume be 1ml;
2, with liquid-transfering gun, draw in the antibody-solutions that above-mentioned disuccinimidyl suberate joins step 1, put room temperature 90min;
3, the solution of step 2 is joined in concentration tube, then put in high speed freezing centrifuge centrifugal 30min under 3000g and be concentrated into 0.5ml;
4, get 0.5ml magnetic bead, add in 5ml reaction cup, through magnet adsorption, after 2 minutes, draw supernatant, then add 1.5ml pH9.50.1mol/L PB, mix 30 seconds, the antibody-solutions that then adds step 3 to obtain, mixes rear room temperature reaction 4 hours;
5,37 ℃ of the Tris solution 15 minutes that add 0.3ml 1mol/L, then add 1.5ml pH 7.2 0.1mol/L PB to clean the magnetic bead of mark, mix 30 seconds;
6, with 100ml magnetic bead, preserve liquid magnetic bead is proceeded to vial;
7, solution step 6 being obtained and above-mentioned magnetic bead buffer solution mix according to the volume ratio of 1: 1, obtain magnetic separation agent;
Two, the preparation process of enzyme reaction thing
One), enzyme reaction thing diluent preparing operation steps, preparation 1L:
1, get Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-300 0.2ml and MgCl
20.05g, in flask, then adds 800ml purified water in flask, fully stirs, and reagent is dissolved completely;
2, adjust pH, control pH within the scope of 7.35-7.45;
3, taking BSA 3g pours in above-mentioned beaker;
4, be finally settled to 1000ml, with 0.2 μ m filter, filter and get final product;
Two), the coupling of alkaline phosphatase and anti-PGI monoclonal antibody
1, get 10mgALP and add in 5ml physiological saline, join in concentration tube, centrifugal 20 minutes of 3000rpm, is concentrated into 1 milliliter;
2, to step 1, obtain the 0.1M NaIO that adds 0.2ml in concentrate
4solution, under room temperature, lucifuge stirs 20 minutes, then packs in bag filter, and with the sodium-acetate buffer dialysis of 1mM pH4.4,4 ℃ are spent the night, and collect and retain liquid;
3, to step 2, obtain in reservation liquid and add 0.2M PH9.5 carbonate buffer solution, make pH be elevated to 9.0, then add immediately the anti-PGI monoclonal antibody of 2.5mg, stir 2 hours, then add the 4mg/ml NaBH of 0.1ml
4solution, mixes, and puts at 4 ℃ 2 hours;
4, above-mentioned solution is packed in bag filter, with 0.15M pH7.4PBS dialysis, 4 ℃ are spent the night, and collect and retain liquid;
5, in step 4 to retain liquid, dropwise add equal-volume saturated ammonium sulfate, put 4 ℃ 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment is washed secondary with semi-saturation ammonium sulfate, finally sediment is dissolved in the PBS of 0.15M pH7.4 of 20ml;
6, solution step 5 being obtained packs in bag filter, with the PB damping fluid dialysis of 0.15M pH7.4, within 5 hours, remove ammonium ion, collect and retain after liquid 10,000rpm removes precipitation for centrifugal 30 minutes, collect supernatant, the ratio that is 1: 100 according to volume ratio is added 1M MgCl
2solution, obtains the conjugate of alkaline phosphatase and anti-PGI monoclonal antibody; By the alkaline phosphatase of collecting and the above-mentioned step 1 of the conjugate of anti-PGI monoclonal antibody) the enzyme reaction thing dilution that obtains mixes with the volume ratio of 1: 1000, obtains enzyme reaction thing;
Three, the preparation process of increased response agent, preparation 1L:
1, get TRIS1.56g and NaCl 4.23g in 1L container, after getting 0.2ml Proclin-300 and dissolving completely in the beaker of 10ml purified water, pour in above-mentioned 1L container;
2, get 800ml purified water in above-mentioned 1L container, fully stir until dissolving completely adjusts pH between 7.35-7.45;
3, take Mak33 0.9g in above-mentioned 1L container; Last constant volume 1000ml, after dissolving completely, filters and get final product with 0.2 μ m filter;
Four, the preparation process of dilution, preparation 1L:
1, take NaCl9.0g and BSA 60g in the container of 1L;
2, get 0.5ml Proclin-300 and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml, after dissolving completely, filters and get final product with 0.2 μ m filter;
Five, the preparation of PGI calibration object and PGI quality-control product:
In PGI calibration object the concentration of PGI antigen be respectively 5,15,30,80,160ng/ml; In PGI quality-control product the concentration of PGI antigen be respectively 15,80ng/ml;
Six, the preparation process of cleaning fluid concentrate, preparation 1L:
1, get TRIS 12.54g and NaCl 325.6g in 1L container;
2, after getting 5g Tween-20 and adding 20ml water in 100ml container it is dissolved completely, pour in above-mentioned 1L container;
3, after getting 0.2ml Proclin-300 and dissolving completely in the beaker that fills 10ml purified water, pour in above-mentioned 1L container;
4, get 800ml purified water in above-mentioned 1L container, fully stir, until dissolve completely;
5, adjust pH, control its scope between 7.35-7.45;
6, last constant volume 1000ml, filters and get final product with 0.2 μ m filter after dissolving completely;
Seven, the preparation process of substrate solution, preparation 1L:
1, get TRIS 2.35g, NaCl 6.41g, Na
2sO
30.002g and Proclin-300 0.2ml are in 1L beaker;
2, get 600ml purified water in 1L beaker, fully stir until dissolving completely adjusts pH between 7.95-8.05;
3, add 250ml Lumi-Phos 480, with 0.2 μ m filter, filter to collect filtrate, by purified water, be settled to 1000ml, after mixing and get final product.
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| CN102654499A (en) * | 2012-05-16 | 2012-09-05 | 江苏省原子医学研究所 | Method for detecting optically-excited chemiluminiscence of pepsinogen 1 and reagent kit |
| CN104597251A (en) * | 2015-01-23 | 2015-05-06 | 河南美凯生物科技有限公司 | Chemiluminescent quantitative detection kit of pepsinogen I and preparation method thereof |
| CN106198986A (en) * | 2016-08-31 | 2016-12-07 | 潍坊市康华生物技术有限公司 | The chemiluminescence detection kit of combined diagnosis of gastrosis and preparation, using method |
| CN109521004A (en) * | 2018-11-21 | 2019-03-26 | 北京利德曼生化股份有限公司 | For detecting the magnetic microparticle separating chemiluminescence immunoassay of glial fibrillary acid protein (GFAP) |
| CN112098654B (en) * | 2020-09-15 | 2023-07-18 | 武汉生之源生物科技股份有限公司 | Pepsinogen I/II assay kit and preparation method thereof |
| CN113109325A (en) * | 2021-03-16 | 2021-07-13 | 华南理工大学 | Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof |
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Denomination of invention: Kit for the quantitative determination of pepsinogen I (PGI) and its detection method Effective date of registration: 20180830 Granted publication date: 20140122 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: WUHAN JINGHONG WANFANGTANG PHARMACEUTICAL TECHNOLOGY CO.,LTD. Registration number: 2018420000042 |
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Denomination of invention: Quantitative determination kit of pepsinogen I (PGI) and its detection method Effective date of registration: 20190909 Granted publication date: 20140122 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: WUHAN JINGHONG WANFANGTANG PHARMACEUTICAL TECHNOLOGY CO.,LTD. Registration number: Y2019420000013 |
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Address after: 430074 No.18, East bio Garden Road, Donghu New Technology Development Zone, Wuhan City, Hubei Province Patentee after: Wuhan Jinghong Technology Co.,Ltd. Address before: 430000, building 7-5, 6 Industrial Park, Kanto New Technology Development Zone, Wuhan, Hubei, East Lake Patentee before: WUHAN JINGHONG WANFANGTANG PHARMACEUTICAL TECHNOLOGY CO.,LTD. |
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Denomination of invention: Kit for the quantitative determination of pepsinogen I (PGI) and its detection method Effective date of registration: 20201016 Granted publication date: 20140122 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: Wuhan Jinghong Technology Co.,Ltd. Registration number: Y2020420000070 |
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Denomination of invention: Pepsinogen I (PGI) quantitative determination kit and its detection method Effective date of registration: 20211027 Granted publication date: 20140122 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: Wuhan Jinghong Technology Co.,Ltd. Registration number: Y2021420000117 |
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Denomination of invention: Pepsinogen I (PGI) quantitative assay kit and its detection method Granted publication date: 20140122 Pledgee: China Construction Bank Corporation Wuhan Provincial Sub-branch Pledgor: Wuhan Jinghong Technology Co.,Ltd. Registration number: Y2024980015122 |