CN102288766B - Carbohydrate antigen 15-3 (CA15-3) quantitative measurement kit and detection method thereof - Google Patents
Carbohydrate antigen 15-3 (CA15-3) quantitative measurement kit and detection method thereof Download PDFInfo
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Abstract
The invention discloses a carbohydrate antigen 15-3 (CA15-3) quantitative measurement kit. The CA15-3 quantitative measurement kit is characterized by containing a CA15-3 magnetic separation reagent, an enzyme reactant, a reaction reinforcing agent, diluent, a CA15-3 standard product, a CA15-3 quality control product, cleaning solution concentrated solution and substrate solution. The invention also discloses a preparation method of the kit. The kit combines a chemiluminescence technology with immune magnetic particles and provides a reaction system close to a homogeneous phase. Compared withthe prior art, the kit has higher detection sensitivity and specificity, shortens the time of showing the result by at least 10 minutes, achieves better performance parameters, and greatly reduces product cost.
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 sugar antigen 15-3 (CA15-3) content in the serum.
Background technology
CA15-3 is a kind of polymorphic epithelium glycoprotein, molecular weight 400KD.It is to detect breast cancer than important index that change of serum C A15-3 measures, 30%～50% patient with breast cancer's CA15-3 serum levels obviously raises, and the appearance of CA15-3 and histological grade, tumour size and axillary lymphatic metastasis is proportionate, and is very valuable to dynamic tracing, efficacy determination and the judgement relapse and metastasis of breast cancer.CA15-3 also can detect with other tumor markers, is used for the auxiliary diagnosis of related neoplasms case.When detecting with CA125, CA15-3 can be used as the index of the early stage recurrence of oophoroma; In liver, stomach, lung, pancreas and colon tumor patient, also may raise.In addition, the also phenomenon that raises of visible CA15-3 when nonmalignant diseases such as hepatitis, cirrhosis.The method of clinical detection CA15-3 has radiommunoassay (RIA), EIA enzyme immunoassay (ELISA), chemiluminescence immune assay (CLIA) and electrochemiluminescence immunoassay (ECLA) etc.
Past, to exempt from be that the sugar antigen 15-3 (CA15-3) of representative measures kit and is subjected to methodological restriction to put, its sensitivity and antijamming capability wretched insufficiency, there is very big drawback, withdraws from the market basically, use more be enzyme linked immunosorbent detection technology and chemiluminescence at present.It is continue Enzyme-multiplied immune technique and the emerging technology that grows up after putting immune technology the eighties that chemiluminescence rises in eighties of last century, because its high sensitivity, high specific, while method is easy, quick, the mark bond is stable, characteristics such as "dead" isotope damage and pollution have obtained develop rapidly in recent years.
Magnetic particle separation enzyme-linked immunoassay technology be a kind of be the solid phase carrier of separating with the magnetic particle, immune magnetic particle isolation technics is combined with the enzyme linked immunosorbent detection technology and a kind of novel immunologic detection method set 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 to have highly sensitively, detect few advantage of time spent.
Summary of the invention
The technical issues that need to address of the present invention are to provide a kind of sugar antigen 15-3 (CA15-3) quantitative determination reagent kit and detection method thereof, adopt this kit to carry out CA15-3 and detect time with higher sensitivity and specificity and shorter acquisition testing result and easier mode of operation.
The invention provides the quantitative determination reagent kit of a kind of sugar antigen 15-3 (CA15-3), its reagent that comprises has CA15-3 magnetic separation agent, enzyme reaction thing, increased response agent, dilution, standard items, quality-control product, cleaning fluid concentrate and substrate solution.
Described magnetic separation agent contains the magnetic microsphere that is marked with anti-CA15-3 monoclonal antibody.
Described enzyme reaction thing is the anti-CA15-3 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 standard items and quality-control product are the BSA protein solutions that contains a certain amount of CA15-3 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 sugar antigen 15-3 of the present invention (CA15-3) comprises the steps:
The first step: the preparation process of magnetic separation agent
One, magnetic bead buffer solution formulation operations step, preparation 1L:
1, takes by weighing TRIS 4.58g and NaCl6.81g in the 1L container, take by weighing 0.96g TWEEN-20 adds suitable quantity of water it is dissolved fully in the 20ml container after, pour in the said vesse;
2, with pipettor Proclin-300 is measured 0.2ml and in the beaker of 10ml purified water, fully after the dissolving, pour in the above-mentioned 1L container, in above-mentioned 1L container, add the 800ml purified water then, fully stir;
3, regulate its pH value of PH instrumentation amount, transfer PH, PH is between 7.95-8.05 in control;
4, taking by weighing BSA 3g pours in the above-mentioned 1L container;
5, be settled to 1000ml at last, filter with the 0.2um filter; Posting label after having filtered stores in 2-8 ℃ of freezer.
Two, the preparation process of magnetic separation agent
1, the 1.0mg disuccinimidyl suberate is dissolved among the 50ul DMSO, get in the 0.1mol/L PB damping fluid that the anti-CA15-3 monoclonal antibody of 2mg is dissolved in PH9.5 to cumulative volume be 1ml;
2, determine the input amount of disuccinimidyl suberate, draw disuccinimidyl suberate with liquid-transfering gun and join in the antibody-solutions of step 1, put room temperature 90min;
3, step 2 antibody-solutions is joined put into then in the Centricon-10 concentration tube in the high speed freezing centrifuge under 3000g, concentrate 30min to volume be 0.5ml;
4, get the 0.5ml magnetic bead, add in the 5ml reaction cup, put into special-purpose test tube rack, draw supernatant through magnet absorption after 2 minutes;
5, add 1.5ml PH9.5 0.1mol/L PB, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times at every turn; The antibody-solutions that step 4 is obtained joins in the above-mentioned magnetic bead, and room temperature reaction is 4 hours behind the mixing;
6,37 ℃ of the TRIS solution 15 minutes that add 0.3ml 1mol/L;
7, add 1.5ml PH 7.20.1mol/L PB at every turn clean the magnetic bead of mark, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times;
8, preserve liquid with the 100ml magnetic bead and change magnetic bead over to the 125ml vial, be 0.05% CA15-3 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, the magnetic separation agent that step 9 is obtained namely gets magnetic separation agent in the kit of the present invention with the ratio mixing of magnetic bead buffer solution according to 1: 1.
Second step: enzyme reaction thing preparation process
One, enzyme reaction thing diluent preparing operation steps, preparation 1L:
1, gets Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-3000.2ml and MgCl
20.05g in flask, in flask, add the 800ml purified water then, fully stir, reagent is dissolved fully;
2, transfer PH, PH is in the 7.35-7.45 scope in control;
3, taking by weighing BSA 3g pours in the above-mentioned beaker;
4, be settled to 1000ml at last, filter namely with the 0.2um filter.
Two, the coupling of alkaline phosphatase ALP and anti-CA15-3 monoclonal antibody
1, get 10mgALP and add in the 5ml physiological saline, join in the concentration tube, centrifugal 20 minutes of 3000RPM is concentrated into 1 milliliter;
2, obtain to add in the concentrate 0.1M NaIO of 0.2ml to step 1
4Solution, lucifuge stirred 20 minutes under the room temperature, and in the bag filter of packing into then, with the sodium-acetate buffer dialysis of 1mM PH4.4,4 ℃ are spent the night, and collect to keep liquid;
3, obtain to keep adding 0.2M PH9.5 carbonate buffer solution in the liquid to step 3, make PH be elevated to 9.0, add the anti-CA15-3 monoclonal antibody of 2.5mg then immediately, the room temperature lucifuge stirred 2 hours gently, added the 4mg/ml NaBH of 0.1ml again
4Solution, mixing, put 4 ℃ following 2 hours;
4, above-mentioned liquid is packed in the bag filter, to 0.15M PH7.4PBS dialysis, 4 ℃ are spent the night, and collect to keep liquid;
5, in step 4 to keep liquid, dropwise add the 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, sediment is dissolved among the PBS of 0.15M PH7.4 of 20ml at last;
6, the solution that step 5 is obtained is packed in the bag filter, removed ammonium ion in 5 hours with the dialysis of the PB buffer saline of 0.15M PH7.4, collect and keep behind the liquid 10,000rpm removed precipitation in centrifugal 30 minutes, collecting supernatant, is 1: 100 ratio interpolation 1M MgCl according to volume ratio
2Solution, namely get the conjugate of alkaline phosphatase (ALP) and CA15-3 monoclonal antibody, at last the alkaline phosphatase (ALP) the collected conjugate with the CA15-3 monoclonal antibody is mixed with the volume ratio of above-mentioned enzyme reaction thing dilution with 1: 1000, namely get the enzyme reaction thing.
The 3rd step:
Increased response agent preparation steps, preparation 1L:
1, takes by weighing TRIS1.56g and NaCl 4.23g in the 1L container; With pipettor with Proclin-300 measure 0.2ml in the beaker of 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;
2, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, transfer PH until dissolving fully, PH is between 7.35-7.45 in control;
3, take by weighing Mak330.9g in above-mentioned 1L container; Last constant volume 1000ml after the dissolving, filters with the 0.2um filter fully.
The 4th step:
The diluent preparing step, preparation 1L:
1, takes by weighing NaCl9.0g and BSA 60g in the container of 1L;
2, with pipettor Proclin-300 is measured 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml after the dissolving, filters with the 0.2um filter fully, posts label and stores in 2-8 ℃ of freezer, and the term of validity is 12 months.
The 5th step:
The preparation of CA15-3 calibration object and CA15-3 quality-control product:
The concentration of CA15-3 antigen is respectively 0.1,0.3,1,2.5,7.5U/ml U/ml in the CA15-3 calibration object; In the CA15-3 quality-control product concentration of CA15-3 antigen be respectively 0.3,2.5U/ml;
The 6th step:
The cleaning concentrate preparation steps, preparation 1L:
1, takes by weighing TRIS 12.54g and NaCl325.6g in the 1L container;
2, take by weighing 5g Tween-20 adds 20ml water it is dissolved fully in the 100ml container after, pour in the above-mentioned 1L container;
3, with pipettor with Proclin-300 measure 0.2ml in the beaker that fills the 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;
4, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolving fully;
5, transfer PH, control its scope between 7.35-7.45;
6, last constant volume 1000ml, filter namely with the 0.2um filter dissolving back fully.
The 7th step:
The substrate solution preparation steps, preparation 1L:
1, takes by weighing TRIS 2.35g, NaCl 6.41g, Na
2SO
30.002g and Proclin-3000.2ml is in the 1L beaker;
2, measure the 600ml purified water in the 1L beaker with graduated cylinder, fully stir, until dissolving fully, transfer PH, control its scope between 7.95-8.05;
3, add 250ml Lumi-Phos 480 after, filter with the 0.2um filter and to collect filtrate, be settled to 1000ml with purified water, behind the mixing namely.
Principle of work of the present invention: this product is a kind of detection method that the sandwich method chemiluminescence immunoassay combines with the magnetic particle isolation technics.In sample, calibration object and quality-control product, add quantitative enzyme mark CA15-3 monoclonal antibody, in conjunction with CA15-3 monoclonal antibody and the stabilizing reinforcer of magnetic particle.37 ℃ hatch after, the CA15-3 monoclonal antibody of combination on the magnetic particle is combined with the different epi-positions of CA15-3 molecule respectively with monoclonal antibody linked with peroxidase, forms " sandwich " structure.In externally-applied magnetic field, directly precipitate, do not need centrifugal namely separable.Remove supernatant, the compound of washing and precipitating adds enzyme-catalyzed chemical luminescence substrate then.Substrate under the enzyme effect by catalytic pyrolysis, form unsettled excited state intermediate, when the 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, CA15-3 content in the sample can be calculated according to typical curve.In sensing range, luminous intensity is directly proportional with CA15-3 concentration in the 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 near homogeneous phase is provided, compared with prior art, kit of the present invention has higher detection sensitivity and specificity, simultaneously prior art has shortened at least 10 minutes going out on the time as a result relatively, and has reached preferable 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 effectively sensitive, when enhancing product performance, and greatly reduces cost of products;
3, the magnetic separation agent in the kit, the enzyme reaction thing, stabilizing reinforcer, standard items, quality-control product, cleaning fluid concentrate, dilution and substrate solution all are 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: prescription sees Table 1, and 1L is example with preparation:
1, takes by weighing TRIS 4.58g and NaCl6.81g in the 1L container, take by weighing 0.96g TWEEN-20 adds suitable quantity of water it is dissolved fully in the 20ml container after, pour in the said vesse;
2, with pipettor with Proclin-300 measure 0.2ml in the beaker of a small amount of purified water fully the dissolving after, pour in the above-mentioned 1L container; In the 1L container, add the 800ml purified water then, fully stir, reagent is dissolved fully;
3, regulate its pH value of PH instrumentation amount; Transfer PH with HCl or NaOH, measure its PH and between 7.95-8.05, namely meet the requirements;
4, taking by weighing BSA (bovine serum albumin(BSA)) 3g pours in the said vesse;
5, be settled to 1000ml at last, survey pH value, scope namely meets the requirements between 7.95-8.05, filters with the 0.2um filter;
Post label after having filtered and store in 2-8 ℃ of freezer, 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 among the 50ul DMSO, namely concentration is 20mg/ml; Get in the 0.1mol/L PB damping fluid that the anti-CA15-3 monoclonal antibody of 2mg (day strong bio-pharmaceuticals (Tianjin) company limited, concentration is 2.55mg/ml) is dissolved in PH 9.5 to cumulative volume be 1ml;
2, calculate the input amount of DSS, calculate according to following formula: (antibody quality/16000) * 10 * 368/C
DSS), C wherein
DSSRefer to the amount of substance concentration mol/L of DSS;
3, the DSS with liquid-transfering gun absorption respective volume joins in the antibody-solutions of step 1, puts room temperature 90min;
4, step 3 antibody-solutions is joined put into then in the Centricon-10 concentration tube in the sigma2-16k high speed freezing centrifuge under the centrifugal force of 3000g, concentrate about 30min to volume be 0.5ml;
5, get the 0.5ml magnetic bead, magnetic bead concentration is 0.5g/ml, and described magnetic bead diameter adds in the 5ml reaction cup between 0.9 μ m, puts into special-purpose test tube rack, draws supernatant through magnet absorption after 2 minutes;
6, add 1.5ml PH9.50.1mol/L PB, mixing 30 seconds is put on the shelf, removes supernatant at every turn, repetitive operation 3 times; The antibody-solutions that step 4 is obtained joins in the magnetic bead, and room temperature reaction is 4 hours behind the mixing, keeps the mixing state;
7, the TRIS solution 37 that adds 0.3ml 1mol/L is spent 15 minutes, and wherein the application of sample amount of TRIS adds 0.15mlTRIS for 1mg antibody;
8, add 1.5ml PH 7.20.1mol/L PB at every turn clean the magnetic bead of mark, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times;
9, preserve liquid with the 100ml magnetic bead and change magnetic bead over to the 125ml vial, be 0.05% CA15-3 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, the magnetic separation agent that step 9 is obtained is with the ratio mixing of magnetic bead buffer solution according to 1: 1;
11, post label and store in 2-8 ℃ of freezer, the magnetic separation agent term of validity is 12 months;
Embodiment 2
One, enzyme reaction thing diluent preparing working specification: prescription sees Table 2, and 1L is example with preparation:
1, gets Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-3000.2ml and MgCl
20.05g in flask; In flask, add the 800ml purified water then, fully stir, reagent is dissolved fully;
2, transfer PH with HCl or NaOH, measurement makes PH in the 7.35-7.45 scope;
3, taking by weighing BSA 3g pours in the said vesse;
4, be settled to 1000ml at last, survey pH value, scope namely meets the requirements between 7.35-7.45, filters with the 0.2um filter; After having filtered, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months;
Enzyme reaction thing dilution table 2
Two, the coupling of alkaline phosphatase (ALP) and anti-CA15-3 monoclonal antibody
1, get 10mgALP and add in the 5ml physiological saline, join in the Centriprep YM10 concentration tube, centrifugal about 20 minutes of 3000RPM is concentrated into 1 milliliter.
2, obtain to add in the concentrate 0.1M NaIO of 0.2ml to step 1
4Solution, lucifuge stirred 20 minutes under the room temperature, and in the bag filter of packing into then, with the sodium-acetate buffer dialysis of 1mM PH4.4,4 ℃ are spent the night, and collect to keep liquid;
3, obtain to keep adding 0.2M PH9.5 carbonate buffer solution in the liquid to step 2, make PH be elevated to 9.0～9.5, add the anti-CA15-3 monoclonal antibody of 2.5mg (day strong bio-pharmaceuticals (Tianjin) company limited then immediately, concentration is 2.55mg/ml), the room temperature lucifuge stirred 2 hours gently, added the 4mg/ml NaBH of 0.1ml again
4(sodium borohydride) solution, mixing, put again 4 ℃ following 2 hours;
4, above-mentioned liquid is packed in the bag filter, to 0.15M PH7.4PBS dialysis, 4 ℃ are spent the night, and collect to keep liquid;
5, in step 4 to keep liquid, dropwise add the 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, sediment is dissolved among the PBS of a small amount of 0.15M PH7.4 at last;
6, the solution that step 5 is obtained is packed in the bag filter, dialyse with the PB buffer saline of 0.15M PH7.4 and to remove ammonium ion in about 5 hours, collect and keep behind the liquid 10,000rpm removed precipitation in centrifugal 30 minutes, collecting supernatant, is 1: 100 ratio interpolation 1M Mgcl according to volume ratio
2Solution, namely get the conjugate of alkaline phosphatase (ALP) and CA15-3 monoclonal antibody, at last the alkaline phosphatase (ALP) the collected conjugate with the CA15-3 monoclonal antibody is mixed with the volume ratio of above-mentioned enzyme reaction thing dilution with 1: 1000, namely get the enzyme reaction thing.
Embodiment 3
Increased response agent formulation operations rules: prescription is seen (table 3), and 1L is example with preparation:
1, take by weighing TRIS (trishydroxymethylaminomethane, molecular formula: (HOCH2) 3CNH2) 1.56g and NaCl 4.23g are in the 1L container; With pipettor with Proclin-300 measure 0.2ml in the beaker of a small amount of purified water fully the dissolving after, pour in the above-mentioned 1L container;
2, measure the 800ml purified water in the 1L container with graduated cylinder, fully stir, until dissolving fully; Transfer PH with HCL or NaOH, measure its scope between 7.35-7.45;
3, take by weighing Mak330.9g in above-mentioned 1L container;
4, last constant volume 1000ml after the dissolving, surveys pH value fully, and scope namely meets the requirements between 7.35-7.45, filters with the 0.2um filter; After having filtered, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months;
The preparation (table 3) of increased response agent
Embodiment 4
The dilution prescription is seen (table 4), and 1L is example with preparation:
1, takes by weighing NaCl9.0g and BSA 60g in the container of 1L;
2, with pipettor Proclin-300 is measured 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml after the dissolving, filters with the 0.2um filter fully, posts label and stores in 2-8 ℃ of freezer, 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, and impact point concentration is A, B, and C, D, E, F when preparing the solution of V volume, needs the volume of adding raw material for being respectively: table 5
| Concentration | Add standard items dilution volume | Add the 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, to be mixed with concentration point with dilution (concrete prescription see embodiment 4) be 0.1,0.3,1,2.5,7.5U/ml to sugar antigen 15-3 (CA15-3) quantitative determination reagent kit CA15-3 calibration object raw material (purchase the company in PPS, concentration is 13700U/ml); Quality-control product with the concentration point of diluent preparing be 0.3,2.5U/ml.
3, fully the dissolving after, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months.
Embodiment 6:
Cleaning concentrate formulation operations rules: prescription is seen (table 6), and 1L is example with preparation:
1, takes by weighing TRIS 12.54g and NaCl 325.6g in the 1L container;
2, take by weighing 5g Tween-20 adds suitable quantity of water it is dissolved fully in the 100ml container after, pour in the said vesse;
3, with pipettor with Proclin-300 measure 0.2ml in the beaker that fills the 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;
4, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolving fully;
5, transfer PH with HCL or NaOH, measure its scope between 7.35-7.45;
6, last constant volume 1000ml surveys pH value, and scope namely meets the requirements between 7.35-7.45, and filter with the 0.2um filter dissolving back fully; After having filtered, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months;
The preparation (table 6) of cleaning concentrate
Embodiment 7
Substrate solution formulation operations rules: prescription is seen (table 7), and 1L is example with preparation:
1, takes by weighing TRIS 2.35g, NaCl6.41g, Na
2SO
30.002g and Proclin-3000.2ml is in the 1L beaker;
2, measure the 600ml purified water in the 1L beaker with graduated cylinder, fully stir, until dissolving fully; Transfer PH with HCl or NaOH, measure its scope between 7.95-8.05;
3, behind the adding 250ml Lumi-Phos 480, filter collection filtrate with the 0.2um filter, be settled to 1000ml with purified water, behind the mixing, post label and store in 2-8 ℃ of freezer, 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, adds 15 μ l CA15-3 standard items, 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, cover test tube with plastic sheeting, the multitube vortex mixer behind the tube shaken frame 30s, is put 37 ℃ of water-baths 30 minutes gently.
6, the 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 the filter paper together with separation vessel, firmly bounces the separation vessel bottom to remove all drops that are bonded on the tube wall.
7, the cleaning fluid concentrate with 20 times of purified water dilutions after, add cleaning fluid after the 200 μ l dilution to each test tube, put the mixing 30s that vibrates gently on the multitube vortex mixer.Should avoid the application of sample dynamics excessive and cause magnetic bead to spill during application of sample.Mixing is wanted thoroughly.
8, repeating step 6,7,6 one times.
9, add 200 μ l substrate solutions mixing 3 seconds to the test tube, detect with ready luminous detector rapidly.
10, as running into high value HOOK sample, for fear of high value HOOK effect occurring, the suggestion clinician selects suitable extension rate that sample is diluted according to all the other test indexs.
Clinical testing:
1, detects data
1000 parts of normal human serum samples have been measured altogether.The serum sample physical examination result does not all have liver, brain, kidney, disease of digestive tract, does not have blood transfusion and major operation history in half a year, and the women is not in the gestational period and lactation.Measured value is carried out statistical analysis, draw normal serum term of reference:＜27.00U/ml.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 CA15-3 level also can be different, advises the range of normal value of each laboratory foundation oneself.
The CA15-3 value that can not only draw with this method is made diagnosis, should comprise patient's concrete condition and treatment situation in conjunction with clinical other analyses result.CA15-3 concentration value and this kit measurement result in the sample that obtains by additive method do not have direct comparability.The sample that exceeds the kit measurement scope, system can't provide definite numerical value.Measure its definite result as desire, measure again after the suggestion dilution.
2, kit performance index of the present invention
Sensitivity for analysis is defined as: to the mensuration of 20 zero standard product, get its mean deviation of 2 times, its corresponding concentration on typical curve is sensitivity for analysis; Sensitivity for analysis: 0.5U/ml; Accuracy: variation within batch CV%≤10.0%; Batch variation CV%≤15.0%; Linear coefficient: r 〉=0.9900; The range of linearity: 0.5-7.5U/ml; Specificity: measure the cross reacting material of high concentration, the result is as follows:
Claims (1)
1. the quantitative determination reagent kit of a sugar antigen CA15-3 is characterized in that, this kit comprises CA15-3 magnetic separation agent, enzyme reaction thing, increased response agent, dilution, CA15-3 standard items, CA15-3 quality-control product, cleaning fluid concentrate and substrate solution;
Each component makes according to the method for being prepared as follows in the described kit:
One, the preparation process of magnetic separation agent
One), magnetic bead buffer solution formulation operations step, the preparation 1L:
1, takes by weighing Tris 4.58g and NaCl6.81g in the 1L container, take by weighing 0.96g Tween-20 adds suitable quantity of water it is dissolved fully in the 20mL container after, pour in the above-mentioned 1L container;
2, with pipettor Proclin-300 is measured 0.2mL and in the beaker of 10mL purified water, fully after the dissolving, pour in the above-mentioned 1L container, in above-mentioned 1L container, add the 800mL purified water then, fully stir;
3, regulate pH, pH is between 7.95-8.05 in control;
4, taking by weighing BSA 3g pours in the above-mentioned 1L container;
5, be settled to 1000mL at last, filter namely with 0.2 μ m filter;
Two), the preparation process of magnetic separation agent
1, the 1.0mg disuccinimidyl suberate is dissolved among the 50 μ l DMSO, get in the 0.1mol/L PB damping fluid that the anti-CA15-3 monoclonal antibody of 2mg is dissolved in pH9.5 to cumulative volume be 1mL;
2, draw in the antibody-solutions that above-mentioned disuccinimidyl suberate joins step 1 with liquid-transfering gun, put room temperature 90min;
3, step 2 antibody-solutions is joined in the concentration tube, put into then in the high speed freezing centrifuge that centrifugal 30min is concentrated into 0.5mL under 3000g;
4, get the 0.5mL magnetic bead, add in the 5mL reaction cup, draw supernatant through magnet absorption after 2 minutes, add 1.5mL pH9.5 0.1mol/L PB then, mixing 30 seconds adds the antibody-solutions that step 3 obtains then, and room temperature reaction is 4 hours behind the mixing;
5, add 37 ℃ of the TRIS solution 15 minutes of 0.3mL 1mol/L, add 1.5mL pH 7.2 0.1mol/L PB then and clean the magnetic bead of mark, mixing 30 seconds;
6, preserve liquid with the 100mL magnetic bead and change magnetic bead over to vial;
7, the solution that step 6 is obtained and above-mentioned magnetic bead buffer solution namely get the magnetic separation agent according to 1: 1 volume ratio mixing;
Two, the preparation process of enzyme reaction thing
One), enzyme reaction thing diluent preparing operation steps, the preparation 1L:
1, gets Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-3000.2mL and MgCl
20.05g in flask, in flask, add the 800mL purified water then, fully stir, reagent is dissolved fully;
2, transfer pH, pH is in the 7.35-7.45 scope in control;
3, taking by weighing BSA 3g pours in the above-mentioned beaker;
4, be settled to 1000mL at last, filter namely with the 0.2um filter;
Two), the coupling of alkaline phosphatase ALP and anti-CA15-3 monoclonal antibody
1, get 10mg ALP and add in the 5mL physiological saline, join in the concentration tube, centrifugal 20 minutes of 3000rpm is concentrated into 1 milliliter;
2, obtain to add in the concentrate 0.1M NaIO of 0.2mL to step 1
4Solution, lucifuge stirred 20 minutes under the room temperature, and in the bag filter of packing into then, with the sodium-acetate buffer dialysis of 1mM pH4.4,4 ℃ are spent the night, and collect to keep liquid;
3, obtain to keep adding 0.2M pH9.5 carbonate buffer solution in the liquid to step 2, make pH be elevated to 9.0, add the anti-CA15-3 monoclonal antibody of 2.5mg then immediately, stirred 2 hours, add the 4mg/mL NaBH of 0.1mL again
4Solution, mixing, put 4 ℃ following 2 hours;
4, above-mentioned solution is packed in the bag filter, with 0.15M pH7.4 PBS dialysis, 4 ℃ are spent the night, and collect to keep liquid;
5, in step 4 to keep liquid, dropwise add the 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, sediment is dissolved among the PBS of 0.15M pH7.4 of 20mL at last;
6, the solution that step 5 is obtained is packed in the bag filter, removed ammonium ion in 5 hours with the dialysis of the PB damping fluid of 0.15M pH7.4, collect and keep behind the liquid 10,000rpm removed precipitation in centrifugal 30 minutes, collecting supernatant, is 1: 100 ratio interpolation 1M MgCl according to volume ratio
2Solution namely gets the conjugate of alkaline phosphatase ALP and anti-CA15-3 monoclonal antibody; With the alkaline phosphatase ALP that collects and the above-mentioned step 1 of conjugate of anti-CA15-3 monoclonal antibody) the enzyme reaction thing dilution that obtains mixes with 1: 1000 volume ratio, namely gets the enzyme reaction thing;
Three, the preparation process of increased response agent, preparation 1L:
1, get Tris 1.56g and NaCl 4.23g in the 1L container, get 0.2mL Proclin-300 in the beaker of 10mL purified water fully the dissolving after, pour in the above-mentioned 1L container;
2, get the 800mL purified water in above-mentioned 1L container, fully stir until dissolving fully, transfer pH between 7.35-7.45;
3, take by weighing Mak33 0.9g in above-mentioned 1L container; Last constant volume 1000mL after the dissolving, filters namely with 0.2 μ m filter fully;
Four, the preparation process of dilution, preparation 1L:
1, takes by weighing NaCl 9.0g 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 the dissolving, filters namely with the 0.2um filter fully;
Five, the preparation of CA15-3 calibration object and CA15-3 quality-control product:
The concentration of CA15-3 antigen is respectively 0.1,0.3,1,2.5,7.5U/mL in the CA15-3 calibration object;
In the CA15-3 quality-control product concentration of CA15-3 antigen be respectively 0.3,2.5U/mL;
Six, the preparation process of cleaning fluid concentrate, preparation 1L:
1, gets Tris 12.54g and NaCl 325.6g in the 1L container;
2, get 5g Tween-20 and in the 100mL container, add 20mL water it is dissolved fully after, pour in the above-mentioned 1L container;
3, get 0.2mL Proclin-300 in the beaker that fills the 10mL purified water fully the dissolving after, pour in the above-mentioned 1L container;
4, get the 800mL purified water in above-mentioned 1L container, fully stir, until dissolving fully;
5, transfer pH, control its scope between 7.35-7.45;
6, last constant volume 1000mL, filter namely with 0.2 μ m filter the dissolving back fully;
Seven, the preparation process of substrate solution, preparation 1L:
1, gets Tris 2.35g, NaCl6.41g, Na
2SO
30.002g and Proclin-3000.2mL is in the 1L beaker;
2, get the 600mL purified water in the 1L beaker, fully stir until dissolving fully, transfer pH between 7.95-8.05;
3, add 250mL Lumi-Phos 480, filter collection filtrate with 0.2 μ m filter, be settled to 1000mL with purified water, behind the mixing namely.
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| CN103293313A (en) * | 2012-02-24 | 2013-09-11 | 上海新波生物技术有限公司 | Carbohydrate antigen 15-3 time-resolved immunofluorescence assay and kit |
| CN103293300A (en) * | 2012-07-09 | 2013-09-11 | 深圳市艾瑞生物科技有限公司 | Time resolution fluorescence detection kit based on phosphorescence luminous technology, and preparation method and application of detection kit |
| CN103048461A (en) * | 2012-12-18 | 2013-04-17 | 苏州浩欧博生物医药有限公司 | Nanometer magnetic particle chemiluminescent assay kit for cancer antigen CA15-3, and preparation method and detection method thereof |
| CN103604925A (en) * | 2013-11-26 | 2014-02-26 | 卢氏实验室公司 | Quick immunodetection strip for monitoring cancers |
| CN103823064B (en) * | 2014-03-03 | 2016-05-25 | 中华人民共和国张家港出入境检验检疫局 | A kind of vomitoxin immue quantitative detection reagent box and using method thereof |
| CN106932583A (en) * | 2015-12-29 | 2017-07-07 | 北京大成生物工程有限公司 | Human epidermal growth factor acceptor Her-2/neu immue quantitative detection reagent boxes and preparation method and application |
| CN106124776A (en) * | 2016-06-30 | 2016-11-16 | 深圳市亚辉龙生物科技股份有限公司 | CA724 chemiluminescence immune detection reagent kit and preparation method thereof |
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