CN102360014A - Quantitative determination kit for alpha-fetoprotein (AFP), and detection method thereof - Google Patents
Quantitative determination kit for alpha-fetoprotein (AFP), and detection method thereof Download PDFInfo
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Abstract
The present invention discloses a quantitative determination kit for alpha-fetoprotein (AFP). The kit is characterized in that: the kit comprises an AFP magnetic separation reagent, an enzyme reactant, a reaction reinforcing agent, a dilution, an AFP standard material, an AFP quality control material, a cleaning solution, a concentration solution and a substrate solution. The invention further discloses a preparation method for the kit. According to the kit of the present invention, the chemiluminescence technology and the immune magnetic particles are combined, a reaction system approaching the homogeneous phase is provided; compared to the prior art, the kit of the present invention has higher detection sensitivity and detection specificity, the time for obtaining the result is reduced by at least 10 minutes than the time in the prior art, the excellent performance parameters of the kit of the present invention are achieved, and the product cost of the kit of the present invention is substantially reduced.
Description
Technical field
The present invention relates to measure the kit and the method for testing thereof of serum, especially relate to kit and the method for testing thereof of measuring alpha-fetoprotein (AFP) content in the serum.
Background technology
Alpha-fetoprotein (AFP) is that molecular weight is the glycoprotein of 70KD.Under the normal condition, synthetic by liver and yolk bag in early days in development of fetus, synthetic by fetus vitellin cell and fetus intestines and stomach in a small amount.AFP is topmost fetal serum protein, and the content of birth back in serum descends rapidly, reduces to minimum the year end second until growing up.Find that clinically HCC and system genitale tumour cell can synthesize AFP, serum concentration of AFP is raise, so Serum AFP content is the important indicator of this type of early diagnosis tumour.The situation that the HAS AFP of nonmalignant disease raises mainly appears at 6-9 month of pregnant female, and some hepatopaths person that especially liver cell lesion thes occur, like cirrhosis, acute, chronic hepatitis and hepatonecrosis.The possibility of the unusual indication generation Down Syndrome (Down syndrome) of pregnant female AFP value.The method that detects at present AFP clinically mainly contains the method for exempting from of putting, ELISA method, chemoluminescence method etc.
Past, to exempt from be that the alpha-fetoprotein (AFP) of representative is measured kit and received 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.Chemiluminescence rise in eighties of last century be the eighties continue Enzyme-multiplied immune technique with put immune technology after the emerging technology that grows up; Because its high sensitivity, high specific; While method is easy, quick; The mark bond is stable, and 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 alpha-fetoprotein (AFP) quantitative determination reagent kit and detection method thereof; Adopt this kit to carry out AFP 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 alpha-fetoprotein (AFP), its reagent that comprises has AFP 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-AFP monoclonal antibody.
Described enzyme reaction thing is the anti-AFP monoclonal antibody that contains alkali phosphatase enzyme mark.
Described increased response agent is the damping fluid that contains Tris.
Said dilution is to contain BSA solution.
Described standard items and quality-control product are the BSA protein solutions that contains a certain amount of AFP antigen.
Said cleaning fluid concentrate is the damping fluid that contains TWEEN-20 and Proclin-300.
Described substrate solution is an enzyme-catalyzed chemical luminescence substrate solution.
The preparation method of the quantitative determination reagent kit of alpha-fetoprotein of the present invention (AFP) 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-AFP monoclonal antibody of 2mg is dissolved in PH 9.5 to cumulative volume be 1ml;
2, confirm 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, with step 2 antibody-solutions join 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 after 2 minutes through magnet absorption;
5, add 1.5mlPH9.50.1mol/LPB, 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 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% AFP 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 promptly 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: the enzyme reaction thing prepares 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, promptly get with the filtration of 0.2um filter.
Two, the coupling of alkaline phosphatase ALP and anti-AFP 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-AFP 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 BS of 0.15M PH7.4, collect and keep behind the liquid 10,000rpm removed deposition in centrifugal 30 minutes; Collecting supernatant, is 1: 100 ratio interpolation 1M MgCl according to volume ratio
2Solution; Promptly get the conjugate of alkaline phosphatase (ALP) and AFP monoclonal antibody; At last the conjugate of the alkaline phosphatase of collecting (ALP) with the AFP monoclonal antibody mixed with the volume ratio of above-mentioned enzyme reaction thing dilution with 1: 1000, promptly 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 calibration object and quality-control product:
Calibration object concentration is respectively 2,10,50,200,500ng/ml; Quality-control product concentration is respectively 10,200ng/ml.
The 6th step:
The cleaning concentrate preparation steps, preparation 1L:
1, takes by weighing TRIS 12.54g and NaCl 325.6g 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, the dissolving back is filtered with the 0.2um filter and is promptly got 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,, transfer PH, control its scope between 7.95-8.05 until dissolving fully;
3, behind the adding 250ml Lumi-Phos 480, filter collection filtrating, be settled to 1000ml, promptly get behind the mixing with purified water with the 0.2um filter.
Principle of work of the present invention: a kind of detection method that these article combine with the magnetic particle isolation technics for the sandwich method chemiluminescence immunoassay.In sample, calibration object and quality-control product, add AFP monoclonal antibody and the stabilizing reinforcer that quantitative enzyme is marked the AFP monoclonal antibody, combined magnetic particle.37 ℃ hatch after, the AFP monoclonal antibody that combines on the magnetic particle combines with the different epi-positions of AFP molecule respectively with monoclonal antibody linked with peroxidase, forms " sandwich " structure.In externally-applied magnetic field, directly precipitate, do not need centrifugal promptly 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 intermedium, when the excited state intermedium 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, the AFP content in the sample can be calculated according to typical curve.In sensing range, luminous intensity is directly proportional with AFP 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, and 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 is seen table 1, and 1L is an 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, promptly 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 promptly 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, promptly concentration is 20mg/ml; Get in the 0.1mol/L PB damping fluid that the anti-AFP monoclonal antibody of 2mg (day strong bio-pharmaceuticals (Tianjin) company limited, concentration is 3.29mg/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, with step 3 antibody-solutions join 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% AFP 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 is seen table 2, and 1L is an 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 promptly 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-AFP 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-AFP monoclonal antibody of 2.5mg (day strong bio-pharmaceuticals (Tianjin) company limited then immediately; Concentration is 3.29mg/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 BS of 0.15M PH7.4 and to remove ammonium ion in about 5 hours, collect and keep behind the liquid 10,000rpm removed deposition in centrifugal 30 minutes; Collecting supernatant, is 1: 100 ratio interpolation 1M Mgcl according to volume ratio
2Solution; Promptly get the conjugate of alkaline phosphatase (ALP) and AFP monoclonal antibody; At last the conjugate of the alkaline phosphatase of collecting (ALP) with the AFP monoclonal antibody mixed with the volume ratio of above-mentioned enzyme reaction thing dilution with 1: 1000, promptly get the enzyme reaction thing.
Embodiment 3
Increased response agent formulation operations rules: prescription is seen (table 3), and 1L is an 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 promptly 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 an 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, the volume that needs the adding raw material is 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, alpha-fetoprotein (AFP) quantitative determination reagent kit AFP calibration object raw material (purchase in the sky strong bio-pharmaceuticals (Tianjin) company limited, concentration is 2.5mg/ml) is mixed with dilution (specifically fill a prescription and see embodiment 4) that concentration point is 2,10,50,200,500ng/ml; Quality-control product uses that the concentration point of diluent preparing is 10,200ng/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 an 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 promptly 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 an example with preparation:
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 with HCl or NaOH, measure its scope between 7.95-8.05;
3, behind the adding 250ml Lumi-Phos 480, filter collection filtrating 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 following
1, adds 15 μ l AFP standard items, quality-control product, sample to be measured to corresponding test tube bottom.
2, add 45 μ l enzyme reaction things to each test tube.
3, add 45 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, is placed on the test tube of reversing 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 is 6,7,6 one times.
9, added in 200 μ l substrate solution to the test tubes mixing 3 seconds, detect with ready luminous detection appearance 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
Sample picks up from the normal health check-up of 1500 examples, blood donor.The 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:＜25.00ng/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 AFP level also can be different, advises the range of normal value of each laboratory foundation oneself.The AFP value that can not only draw with this method is made diagnosis, only as the intermediate data reference role, should combine clinical other analyses result, comprises patient's concrete condition and treatment situation.AFP concentration value and this kit measurement result in the sample that obtains through 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 like desire, measure again after the suggestion dilution.The testing result of this kit only supplies clinical reference, can not be separately as the foundation of making a definite diagnosis or get rid of case, and for reaching diagnostic purpose, this testing result will be used in combination with clinical examination, medical history and other inspection.These article can be used for the mensuration of serum sample, and the reliability that is used for other body fluid samples AFP concentration determination is fully confirmed.
2, kit performance index of the present invention
Sensitivity for analysis is defined as: to the mensuration of 20 zero standard article, get its mean deviation of 2 times, its pairing concentration on typical curve is sensitivity for analysis;
Sensitivity for analysis: 0.30ng/ml
Accuracy: variation within batch CV%≤10.0%; Batch variation CV%≤13.0%
Linear coefficient: r >=0.9900
The range of linearity: 0.30-300ng/ml
Specificity: measure the AFP of 1000 μ g/L, degree of disturbance＜0.03%.
Claims (5)
1. the quantitative determination reagent kit of an alpha-fetoprotein AFP is characterized in that, this kit comprises AFP magnetic separation agent, enzyme reaction thing, increased response agent, dilution, AFP standard items, AFP quality-control product, cleaning fluid concentrate and substrate solution.
2. kit as claimed in claim 1 is characterized in that, described AFP magnetic separation agent contains the magnetic microsphere that is marked with anti-AFP monoclonal antibody;
Described enzyme reaction thing is the anti-AFP monoclonal antibody that contains alkali phosphatase enzyme mark;
Described increased response agent is the damping fluid that contains Tris;
Said dilution is the solution that contains BSA;
Described standard items and quality-control product are the BSA solution that contains a certain amount of AFP antigen;
Said cleaning fluid concentrate is the damping fluid that contains TWEEN-20 and Proclin-300;
Described substrate solution is an enzyme-catalyzed chemical luminescence substrate solution.
3. let alone a described kit like claim 1-2, it is characterized in that, each component makes according to the method for being prepared as follows in the said 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, promptly get with the filtration of 0.2um filter;
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-AFP monoclonal antibody of 2mg is dissolved in PH 9.5 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 PH9.50.1mol/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 7.20.1mol/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, solution that step 6 is obtained and above-mentioned magnetic bead buffer solution promptly get claim 1 or 2 according to 1: 1 volume ratio mixing
Magnetic separation agent in the said kit;
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, promptly get with the filtration of 0.2um filter;
Two), the coupling of alkaline phosphatase ALP and anti-AFP 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 2, make PH be elevated to 9.0, add the anti-AFP 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 PH7.4PBS 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 damping fluid of 0.15M PH7.4, collect and keep behind the liquid 10,000rpm removed deposition in centrifugal 30 minutes; Collecting supernatant, is 1: 100 ratio interpolation 1M MgCl according to volume ratio
2Solution promptly gets the conjugate of alkaline phosphatase ALP and anti-AFP monoclonal antibody; The conjugate of alkaline phosphatase ALP that collects and anti-AFP monoclonal antibody is used above-mentioned step 1) the enzyme reaction thing dilution that obtains mixes with 1: 1000 volume ratio, promptly gets the enzyme reaction thing.
Three, the preparation process of increased response agent, preparation 1L:
1, get TRIS1.56g and NaCl4.23g 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, transfer PH between 7.35-7.45 until dissolving fully;
3, take by weighing Mak330.9g in above-mentioned 1L container; Last constant volume 1000ml after the dissolving, promptly gets with the filtration of 0.2um filter fully;
Four, the preparation process of dilution, preparation 1L:
1, takes by weighing 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 the dissolving, promptly gets with the filtration of 0.2um filter fully;
Five, the preparation of AFP calibration object and AFP quality-control product:
In the AFP calibration object concentration of AFP antigen be respectively 2,10,50,200,500ng/ml; In the AFP quality-control product concentration of AFP antigen be respectively 10,200ng/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, the dissolving back is filtered with the 0.2um filter and is promptly got fully;
Seven, the preparation process of substrate solution, preparation 1L:
1, gets TRIS 2.35g, NaCl 6.41g, 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, transfer PH between 7.95-8.05 until dissolving fully;
3, add 250ml Lumi-Phos 480, filter with the 0.2um filter and collect filtrating, be settled to 1000ml, promptly get behind the mixing with purified water.
4. let alone a described kit like claim 1-3, it is characterized in that, the method for application of said kit is following:
1), adds 15 μ l AFP standard items, AFP quality-control product, sample to be measured to corresponding test tube bottom;
2), add 45 μ l enzyme reaction things to each test tube;
3), add 45 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) put to magnetic separator, then, guarantee that every test tube all contacts with separator surface, precipitate 2 minutes, the reversing separation vessel is poured out supernatant;
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;
8), added in 200 μ l substrate solution to the test tubes mixing 3 seconds, the luminous detection appearance detects.
5. kit as claimed in claim 4 is characterized in that, when sample to be measured is high value HOOK sample, uses dilution that sample is diluted as required.
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| CN103969453A (en) * | 2014-05-30 | 2014-08-06 | 上海度微医学技术有限公司 | Application of alpha fetoprotein autoantibody singly or jointly with alpha fetoprotein acting as tumor diagnosis, therapeutic effect evaluation and relapse monitoring marker |
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| CN107022030A (en) * | 2017-03-23 | 2017-08-08 | 广东谷盈生物科技产业研究院有限公司 | Detect monoclonal antibody and kit and the application of alpha-fetoprotein |
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| CN114544978A (en) * | 2022-03-04 | 2022-05-27 | 美康生物科技股份有限公司 | IGF-1 detection kit, preparation method and detection method thereof |
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Application publication date: 20120222 |