CN103018464A - Reagent for determining procalcitonin and preparation method of reagent - Google Patents
Reagent for determining procalcitonin and preparation method of reagent Download PDFInfo
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Abstract
The invention relates to a reagent for determining procalcitonin and a preparation method of the reagent, in order to provide the reagent which has the characteristic of high accuracy degree, and the provided preparation method has the characteristic of convenience in preparation. The technical scheme is that the reagent for determining procalcitonin comprises the following components: a. a procalcitonin reagent 1, b. a procalcitonin reagent 2, and c. a liquid-type procalcitonin reference calibration item. The preparation method of the reagent for determining procalcitonin comprises the following steps of: (1) uniformly mixing the procalcitonin reagent 1; (2) removing the procalcitonin reagent 2, the removing process specifically comprises the following steps of: a. taking a suspension, b. carrying out reaction on a mixture, c. acquiring a carboxylic latex microsphere suspension, d. regulating the concentration, e. taking the suspension in step (3), and adding into the step (4), f. reacting, g. adding cholamine, and h. carrying out centrifugal treatment; and (3) treating the liquid-type procalcitonin reference calibration product: mixing according to the amount of a formula, and ranking according to the content of the procalcitonin or adding a procalcitonin pure product in the mixed liquid.
Description
Technical field
The present invention relates to a kind of preparation method who measures reagent and this reagent of Procalcitonin, particularly adopt reagent and the preparation method of the detection Procalcitonin in latex enhancing immune turbidimetry for Determination serum or the blood plasma, can be widely used in medical science and technological field of biochemistry.
Background technology
Levels of Serum Procalcitonin (Procalcitionin, PCT) is comprised of 116 amino acid, and molecular weight is the glycoprotein of 13KD.Its half life period is 25-30 hour.Human normal plasma PCT content is extremely low, is lower than 0.5ng/ml in normal human blood.Its level in blood plasma raises when serious bacterium, fungi, parasitic infection and pyemia and MOFE; And the immunity of self, allergy and when the diseases such as local infection, virus infections, chronic nonspecific inflammation, cancer heating, graft host rejection the PCT level can not raise; This has just determined the high degree of specificity of PCT, therefore also can be used for the antidiastole of various clinical settings.PCT concentration becomes positive correlation with the inflammation order of severity, and is reduced to normal level along with the alleviation of the control of inflammation and the state of an illness, thereby PCT can be used as again the reliability index of judging the state of an illness and prognosis and observation of curative effect.PCT can detect after rear 2 hours in infection, and is significant to clinical early diagnosis, peaks in 12～24 hours after infection.PCT has extensive and important using value clinically; And mortality ratio and hospital stays to patient ICU provide standard.
Detecting at present PCT laboratory method commonly used has radioimmunology analytic approach (RIA), chemiluminescence immunoassay (CLIA), collaurum colourimetry (GICA) and euzymelinked immunosorbent assay (ELISA) (ELISA), and the radioimmunology analytic approach is to utilize the polyclonal antibody that manually synthesizes to identify specifically and be connected the synthesizing amino acid Procalcitonin.The method can detect normal person's blood-serum P CT, and reliable susceptibility is 4pm/mL, detection be the potpourri of free PCT, mating type PCT and CGRP precursor, and can not distinguish above-mentioned three kinds of materials.This method detects length consuming time (19～22h), and have the pollution of radioelement, label poor stability, discarded object to be difficult to process and it is restricted.Chemiluminescence immunoassay (CLIA) is to utilize chemiluminescent substance through catalyst and oxygenant oxidation, forms the intermediate of an excited state; The intermediate of this excited state is got back to when stablizing ground state, sends photon, utilizes luminous signal measuring instrument measurement of photon number, thus the concentration of indirect determination PCT.Tubular type and board-like two kinds of kits are arranged in the market, but this method exists the luminescence efficiency of acridinium ester, luminol, the direct labelled antibody of different luminol low, the unsettled shortcoming of label; And this direct labelling method belongs to the moment light emitting-type, is difficult to guarantee the stability of test result and repeated, also needs special detecting instrument.Being not easy to Routine Test Lab carries out.And utilize biotin-avidin system to detect the electrochemiluminescence immune reagent kit of PCT, and needing the fully automatic electric chemiluminescence detector of supporting costliness, Routine Test Lab can't be carried out, and brings unnecessary expense to the patient, increases the patient burden.Euzymelinked immunosorbent assay (ELISA) is to detect with artificial synthetic PCT monoclonal antibody, though method is special, but detects the minimum 10 μ g/L that are limited to, and can't detect the PCT concentration in the normal human serum.Chinese patent (application number: 201110210250.4) also disclose a kind of kit that detects Procalcitonin, utilize magnetic particle coupling antibody as fixedly separated phase, the multistage amplification system of biotin-Streptavidin, be catalyzing enzyme catalytic luminescence substrate with horseradish peroxidase, although improved the sensitivity that detects, but whole reaction time consumption is also grown (needing at least 40 minutes), and sample needs the magnetic resolution pre-service.This method needs the washing reaction pipe, if hand washing can bring personal error, if wash with instrument, then needs in addition standby washing instrument, increases the cost in laboratory.
Summary of the invention
Technical matters to be solved by this invention is the deficiency that overcomes the above-mentioned background technology, provides a kind of Procalcitonin to detect reagent and preparation method; The reagent that provides should have accuracy height, good reproducibility, simple to operate, minute short, be applicable to the characteristics of various types of automatic clinical chemistry analyzers; The preparation method of reagent thereof that provides should have characteristics easy to make and that cost is not high.
Technical scheme provided by the invention is:
A kind of reagent of measuring Procalcitonin comprises following composition and content:
A, Procalcitonin reagent 1:
The pH value of this reagent 1 is 7.0-8.0;
B, Procalcitonin reagent 2:
The particle diameter of the carboxylated latex microballoon in the described anti-human PCT antibody latex particle is 20nm-500nm;
C, liquid-type Procalcitonin reference calibrations product:
Described liquid-type Procalcitonin reference calibrations product comprise 5 parts of reference calibrations product that form series concentration; Procalcitonin content in 5 parts of reference calibrations product is respectively 1.25ng/ml, 2.5ng/ml, 5.0ng/ml, 10.0ng/ml, 20.0ng/ml from low to high; Perhaps, be prepared into the Procalcitonin reference calibrations product of single-point concentration; For example concentration is the Procalcitonin reference calibrations product of 100ng/ml or 500ng/ml.
Described damping fluid is the mixing of one or more arbitrary proportions in phosphate buffer, trishydroxymethylaminomethane (Tris) damping fluid, Isosorbide-5-Nitrae-piperazine two ethyl sulfonic acids (PIPES) damping fluid, 4-(2-hydroxyethyl) piperazine-1-1 ethane sulfonic acid (HEPES) damping fluid, Pehanorm base propane sulfonic acid (TAPS) damping fluid, glycine buffer, borate buffer, 3-morpholine propane sulfonic acid (MOPS) damping fluid, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid (MOPSO) damping fluid, MES (MES) damping fluid.
The concentration of damping fluid is 20-200mmol/L in the described Procalcitonin reagent 1, pH value 7.0-8.0.
Described reaction accelerator is polyglycol or dextran sulfate (DS-50).
The preferred 0.05-30g/L of described reaction accelerator concentration.
Described stabilizing agent is the mixing of one or more arbitrary proportions in disodium ethylene diamine tetraacetate, bovine serum albumin(BSA), the sodium chloride.
Described electrolyte is anion electrolyte or cationic polyelectrolyte.
Described electrolyte is the sodium chloride (NaCl) in the kation, the preferred 50-200mmol/L of concentration.
Described glass or plastic containers, cationic surfactant, anionic surfactant or zwitterionic surfactant.
Described non-ionic surfactant is: the mixing of one or more arbitrary proportions in Theist, Tween series, serial, the polyoxyethylene phenyl ether of polyoxyethylene laurel ether, polyoxyethylene octyl group phenylate, polyoxyethylene alkyl phenyl ether, the polyoxyethylene nonylplenyl ether.
The preferred 0.5-1g/L of described surfactant concentration.
The preferred 1.0-10mg/ml of concentration of described anti-human PCT antibody latex particle.
Described anti-human Procalcitonin antibody is the anti-human Procalcitonin antibody of goat-anti HCT original antibody or rabbit or the anti-human Procalcitonin antibody of chicken or mouse-anti HCT original antibody.
A kind of preparation method who measures Procalcitonin reagent, carry out as follows:
1) Procalcitonin reagent 1:
By formula ratio damping fluid, electrolyte, surfactant, reaction accelerator, antiseptic, stabilizing agent and purified water are mixed, set up the pH value to 7.0-8.0;
2) Procalcitonin reagent 2:
(1) gets the carboxylated latex microballoon is diluted to 0.01mg/ml with damping fluid suspending liquid;
(2) add the ratio of the N-hydroxy-succinamide sulfonate sodium (NHS) of 20mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDAC carboxyl activator) and 40mg in the carboxylated latex microsphere suspension liquid of 1ml, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide sulfonate sodium, in room temperature this potpourri was reacted 15-30 minute behind the mixing immediately, constantly stir;
(3) with damping fluid or purified water washing carboxylated latex microballoon, remove unreacted 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide sulfonate sodium, and with the latex microsphere carboxylated latex microsphere suspension liquid that suspends in purified water to get, making its concentration is 0.01mg/ml;
(4) anti-human Procalcitonin antibody is dissolved in the damping fluid, the protein concentration that makes anti-human Procalcitonin antibody is 0.25mg/ml;
(5) get carboxylated latex microsphere suspension liquid 1ml in the step (3) and add immediately anti-human Procalcitonin antibody 1ml in the step (4); Make the concentration of carboxylated latex microballoon in this mixed liquor, anti-human Procalcitonin antibody and damping fluid be respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
(6) potpourri was reacted 3 hours at 37 ℃, constantly stir;
(7) ratio that adds the monoethanolamine of 2.5 μ l in the 1ml reaction mixture adds monoethanolamine, react 10-30 minute, constantly stirring;
(8) centrifugal treating is removed unconjugated protein and monoethanolamine, and with damping fluid dilution, the concentration that makes anti-human PCT antibody latex particle is 0.002-0.01ml/ml, namely gets Procalcitonin reagent 2 after then adding stabilizing agent, antiseptic dissolving mixing;
3) liquid-type Procalcitonin reference calibrations product:
By formula ratio antiseptic, stabilizing agent and damping fluid are mixed and are divided into 5 portions of mixed liquors first, then as required Procalcitonin reference calibrations product concentration, the Procalcitonin sterling of the 1000ng/ml of respective amount is added respectively in the above-mentioned mixed liquor, obtain 5 parts of reference calibrations product that form series concentration; Procalcitonin content in 5 parts of reference calibrations product is respectively 1.25ng/ml, 2.5ng/ml, 5.0ng/ml, 10.0ng/ml, 20.0ng/ml from low to high; Perhaps,
Press and get mixed liquor after formula ratio mixes antiseptic, stabilizing agent and damping fluid, then in mixed liquor, add the Procalcitonin sterling of a certain amount of 1000ng/ml, obtain the Procalcitonin reference calibrations product of single-point high concentration.For example, the Procalcitonin content in these reference calibrations product is 100.0ng/ml or 500.0ng/ml; Become again the reference calibrations product of a plurality of variable concentrations during use with normal saline dilution; Here have no particular limits, if the Procalcitonin reference calibrations product that make can with sample relatively, can working sample in the content of Procalcitonin get final product.
Described centrifugal treating adopts hydro-extractor, and centrifuge speed is 15000 rev/mins.
The reaction principle that PCT provided by the present invention measures reagent is: the anti-human PCT antibody latex particle generation antigen-antibody reaction in the PCT in the sample and the reagent, the reactant liquor turbidity is increased, the amount of antigen is linear in reactant liquor turbidity and the sample within the specific limits, can use Biochemical Analyzer or other optical detecting instrument at 520-600nm wavelength place assaying reaction liquid absorbance, the reactant liquor absorbance is directly proportional with survey PCT concentration.
The invention has the beneficial effects as follows: the Procalcitonin of the employing Immunoturbidimetry that provides detects reagent, by CG antibody is combined with the carboxylated latex microballoon, amplify the surface area of CG antigen and antibody response, had the accuracy height (with the correlativity R of chemiluminescence immunoassay
2Be 0.9991-0.9998), the quick characteristics of (begin at most only to need 10 minutes to obtaining a result from mensuration, even shorter) of good reproducibility, detection, can carry out sample in enormous quantities at the routine biochemistry instrument and measure, greatly improved testing efficient.The preparation method of this reagent is easy and simple to handle, and raw material is easy to get (all selecting the outsourcing raw material), and cost is not high, is fit to all kinds of medical research units and Routine Test Lab and uses.
Description of drawings
Fig. 1 shows among the embodiment 1 mutual relationship by the PCT measured value of the measured value of the inventive method gained PCT reagent and chemiluminescence immunoassay gained.Through regretional analysis: y=1.0062x+0.6087; R
2=0.9991, demonstrate present embodiment 1 and have good correlativity with chemiluminescence immunoassay.
Fig. 2 shows among the embodiment 2 mutual relationship by the PCT measured value of the measured value of the inventive method gained PCT reagent and chemiluminescence immunoassay gained.Through regretional analysis: y=1.0096x+0.7981; R
2=0.9996, demonstrate present embodiment 2 and have good correlativity with chemiluminescence immunoassay.
Fig. 3 shows among the embodiment 3 mutual relationship by the PCT measured value of the measured value of the inventive method gained PCT reagent and chemiluminescence immunoassay gained.Through regretional analysis: y=1.0092x+0.7507; R
2=0.9998, demonstrate present embodiment 3 and have good correlativity with chemiluminescence immunoassay.
Fig. 4 shows among the embodiment 4 mutual relationship by the PCT measured value of the measured value of the inventive method gained PCT reagent and chemiluminescence immunoassay gained.Through regretional analysis: y=1.0056x+2.8301; R
2=0.9998 demonstrates present embodiment 4 has good correlativity with chemiluminescence immunoassay.
Embodiment
The present invention detects reagent and the required primary raw material of calibration object:
1, anti-human Procalcitonin antibody has many commercial anti-human Procalcitonin antibody available, such as Finland DaKo company, Japanese UNF company; This antibody only with the former reaction of HCT, with other antigen without immunological cross-reaction, can be polyclonal antibody or monoclonal antibody, be not particularly limited here, as long as can with the latex microsphere coupling.Its anti-human Procalcitonin antibody can be that goat-anti (is goat-anti HCT original antibody; As follows), also can be that rabbit is anti-, chicken anti-or mouse-anti.As long as adopt known immunoelectrophoresis to measure, this antibody only and between the people PCT presents single heavy alignment, and the titre of anti-human Procalcitonin antibody is more than or equal to 0.5mg/ml.
2, latex microsphere has many commercial Nano microspheres available, such as German Merck company, Japanese UNF company, Japanese JSR company and Thermo company; Its basic material is polystyrene or co polystyrene, and the surface is modified with carboxylic group (COOH), also can be not used as any modification, will select different microballoons according to the associated methods of selecting antibody and microballoon is different here.As select chemical crosslink technique (also can adopt physisorphtion), then with the microballoon of surface through modifications such as carboxyl, amino, aldehyde radicals.The latex microsphere that the preferred carboxylic group of this law is modified, the preferred 20nm-500nm of the particle diameter of carboxylated latex microballoon, further preferred 80nm-300nm.
3, Procalcitonin: available from Finland DaKo company, needing the reference calibrations product for the preparation of this reagent place, can be that natural calcitonin is former, also can be the genetic recombination Procalcitonin.
Describe reagent of the present invention in detail below in conjunction with embodiment, but these embodiment should not think to limit definition of the present invention.
Embodiment one
One) Procalcitonin reagent 1
, add again purified water and be settled to 1000ml after the above-mentioned raw materials dissolving with purified water 800ml, then transfer PH to 7.2 with hydrochloric acid;
Two) Procalcitonin reagent 2
1. the carboxylated latex microballoon of getting 80nm is diluted to the suspending liquid of 0.01mg/ml with the MES damping fluid of 50mmol/L, pH6.0;
2. the ratio that adds 20mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDAC) and 40 mg N-hydroxy-succinamide sulfonate sodiums (NHS) in 1ml carboxylated latex microsphere suspension liquid adds 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide sulfonate sodium, in room temperature this potpourri was reacted 15-30 minute behind the mixing immediately, constantly stir;
3. with the MES damping fluid of 50mmol/L, PH6.0 or purified water washing carboxylated latex microballoon, remove unreacted NHS and EDAC, and with the latex microsphere carboxylated latex microsphere suspension liquid that suspends to get, wherein the content of carboxylated latex microballoon is 0.01mg/ml in purified water;
4. goat-anti HCT original antibody is dissolved in the borate buffer solution of 50mmol/L, PH8.5, the protein concentration that makes goat-anti HCT original antibody is 0.25mg/ml;
5. get carboxylated latex microsphere suspension liquid 1ml in the step (3) and add immediately goat-anti HCT original antibody 1ml in the step (4); The concentration of carboxylated latex microballoon, goat-anti HCT original antibody and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L in this mixed liquor at this moment;
6. potpourri was reacted 3 hours at 37 ℃, constantly stir;
7. add the ratio adding monoethanolamine of 2.5 μ l monoethanolamines in the 1ml reaction mixture, reacted 15 minutes, constantly stir;
8. with 15000 rev/mins of centrifugal unconjugated protein and monoethanolamines removed, MOPSO damping fluid dilution with 50mmol/L, PH7.2, making the concentration of goat-anti people PCT antibody latex particle is 0.01ml/ml, namely gets Procalcitonin reagent 2 after then adding bovine serum albumin(BSA) (stabilizing agent) 3mmol/L, 3mmol/L Sodium azide (antiseptic) dissolving mixing;
Three) liquid-type PCT reference calibrations product
PCT reference calibrations product concentration as required adds corresponding PCT sterling 1000ng/ml in the above-mentioned mixed liquor, is prepared into 5 reference calibrations product that form series concentration; PCT content is respectively 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml from low to high in 5 reference calibrations product.
Pattern detection (contrasting respectively 30 samples of detection with the chemiluminescence immunoassay method):
According to the experimental implementation step, add the Procalcitonin reagent 1 of 240 μ L in the 10 μ L samples, hatched 3 minutes at 37 ℃, add 2,37 ℃ of 60 μ L Procalcitonin reagent and hatch after 10 seconds and read absorbance A at wavelength 600nm
1, read absorbance A when reacting to 5 minutes
2, calculate absorbance changing value △ A=A twice
2-A
1Compare by the calibration solution with same processing, calculate the PCT concentration in the sample.Can find out according to the result that from table 1 and Fig. 1 (Fig. 1 draws according to table 1 data) although the inventive method is simple to operate, required time only in 10 minutes, also shows very good correlativity (correlativity R with the chemiluminescence immunoassay method
2Reach 0.9991).
Table 1
Embodiment two
One) Procalcitonin reagent 1
, add again purified water and be settled to 1000ml after the above-mentioned raw materials dissolving with purified water 800ml, then transfer PH to 8.0 with NaOH;
Two) Procalcitonin reagent 2
1. the carboxylated latex microballoon of getting 150nm is diluted to the suspending liquid of 0.01mg/ml with the MES damping fluid of 50mmol/L, PH6.0;
2. add ratio adding EDAC and the NHS of the NHS of 20mg EDAC and 40mg in the carboxylated latex microsphere suspension liquid of 1ml, in room temperature potpourri was reacted 15-30 minute behind the mixing immediately, constantly stir;
3. with the MES damping fluid of 50mmol/L, PH6.0 or purified water washing carboxylated latex microballoon, remove unreacted NHS and EDAC, and with the latex microsphere carboxylated latex microsphere suspension liquid that suspends to get, wherein the content of carboxylated latex microballoon is 0.01mg/ml in purified water;
4. the anti-human Procalcitonin antibody of rabbit is dissolved in the borate buffer solution of 50mmol/L, PH8.5, the protein concentration that makes the anti-human Procalcitonin antibody of rabbit is 0.25mg/ml;
5. get carboxylated latex microsphere suspension liquid 1ml in the step (3) and add immediately the anti-human Procalcitonin antibody of rabbit 1ml in the step (4); The concentration of carboxylated latex microballoon, the anti-human Procalcitonin antibody of rabbit and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L in this mixed liquor at this moment;
6. potpourri was reacted 3 hours at 37 ℃, constantly stir;
7. add the ratio adding monoethanolamine of 2.5 μ l monoethanolamines in the 1ml reaction mixture, reacted 30 minutes, constantly stir;
8. with 15000 rev/mins of centrifugal unconjugated protein and monoethanolamines removed, phosphate buffer dilution with 50mmol/L, PH7.4, making the concentration of the anti-human PCT antibody latex of rabbit particle is 0.006ml/ml, namely gets Procalcitonin reagent 2 after then adding bovine serum albumin(BSA) (stabilizing agent) 3mmol/L, 3mmol/L Sodium azide (antiseptic) dissolving mixing;
Three) liquid-type PCT reference calibrations product
PCT reference calibrations product concentration as required adds corresponding PCT sterling 1000ng/ml in the above-mentioned mixed liquor, be prepared into the reference calibrations product of 5 series concentration, PCT content is respectively 7.5ng/ml, 15ng/ml, 30ng/ml, 60ng/ml, 120ng/ml from low to high in the reference calibrations product of 5 series concentration.
Pattern detection (contrasting respectively 30 samples of detection with the chemiluminescence immunoassay method):
According to the experimental implementation step, add the Procalcitonin reagent 1 of 240 μ L in the 10 μ L samples, hatched 3 minutes at 37 ℃, add 2,37 ℃ of 60 μ L Procalcitonin reagent and hatch after 10 seconds and read absorbance A at wavelength 600nm
1, read absorbance A when reacting to 5 minutes
2, calculate absorbance changing value △ A=A twice
2-A
1Compare by the calibration solution with same processing, calculate the PCT concentration in the sample.The result can find out from table 2 and Fig. 2 (Fig. 2 draws according to table 2 data), although the inventive method is simple to operate, required time only in 10 minutes, also shows very good correlativity (correlativity R with the chemiluminescence immunoassay method
2Reach 0.9996).
Table 2
Embodiment three
One) Procalcitonin reagent 1
, add again purified water and be settled to 1000ml after the above-mentioned raw materials dissolving with purified water 800ml, then transfer PH to 8.0 with NaOH;
Two) Procalcitonin reagent 2
1. the carboxylated latex microballoon of getting 300nm is diluted to the suspending liquid of 0.01mg/ml with the MES damping fluid of 50mmol/L, PH6.0;
2. add ratio adding EDAC and the NHS of the NHS of 20mg EDAC and 40mg in the carboxylated latex microsphere suspension liquid of 1ml, in room temperature this potpourri was reacted 15-30 minute behind the mixing immediately, constantly stir;
3. with the MES damping fluid of 50mmol/L, PH6.0 or purified water washing carboxylated latex microballoon, remove unreacted NHS and EDAC, and with the latex microsphere carboxylated latex microsphere suspension liquid that suspends to get, wherein the content of carboxylated latex microballoon is 0.01mg/ml in purified water;
4. mouse-anti HCT original antibody is dissolved in the borate buffer solution of 50mmol/L, PH8.5, the protein concentration that makes mouse-anti HCT original antibody is 0.25mg/ml;
5. get carboxylated latex microsphere suspension liquid 1ml in the step (3) and add immediately mouse-anti HCT original antibody 1ml in the step (4); The concentration of carboxylated latex microballoon, mouse-anti HCT original antibody and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L in this mixed liquor at this moment;
6. potpourri was reacted 3 hours at 37 ℃, constantly stir;
7. add the ratio adding monoethanolamine of 2.5 μ l monoethanolamines in the 1ml reaction mixture, reacted 10 minutes, constantly stir;
8. with 15000 rev/mins of centrifugal unconjugated protein and monoethanolamines removed, PIPES damping fluid dilution with 50mmol/L, PH8.0, making the concentration of mouse-anti people PCT antibody latex particle is 0.002ml/ml, namely gets Procalcitonin reagent 2 after then adding bovine serum albumin(BSA) (stabilizing agent) 3mmol/L, 3mmol/L Sodium azide (antiseptic) dissolving mixing.
Three) liquid-type PCT reference calibrations product
PCT reference calibrations product concentration as required adds corresponding PCT sterling 1000ng/ml in the above-mentioned mixed liquor, be prepared into the reference calibrations product of 5 series concentration, PCT content is respectively 10ng/ml, 40ng/ml, 80ng/ml, 160ng/ml, 320ng/ml from low to high in the reference calibrations product of 5 series concentration.
Pattern detection (contrasting respectively 30 samples of detection with the chemiluminescence immunoassay method):
According to the experimental implementation step, add the Procalcitonin reagent 1 of 240 μ L in the 10 μ L samples, hatched 3 minutes at 37 ℃, add 2,37 ℃ of 60 μ L Procalcitonin reagent and hatch after 10 seconds and read absorbance A at wavelength 600nm
1, read absorbance A when reacting to 5 minutes
2, calculate absorbance changing value △ A=A twice
2-A
1Compare by the calibration solution with same processing, calculate the PCT concentration in the sample.The result can find out from table 3 and Fig. 3 (Fig. 3 draws according to table 3 data), although the inventive method is simple to operate, required time only in 10 minutes, also shows very good correlativity (correlativity R with the chemiluminescence immunoassay method
2Reach 0.9998).
Table 3
Embodiment four
One) Procalcitonin reagent 1
, add again purified water and be settled to 1000ml after the above-mentioned raw materials dissolving with purified water 800ml, then transfer PH to 7.6 with hydrochloric acid or NaOH;
Two) Procalcitonin reagent 2
1. the carboxylated latex microballoon of getting 220nm is diluted to the suspending liquid of 0.01mg/ml with the MES damping fluid of 50mmol/L, PH6.0;
2. add ratio adding EDAC and the NHS of the NHS of 20 mg EDAC and 40 mg in the carboxylated latex microsphere suspension liquid of 1ml, in room temperature this potpourri was reacted 15-30 minute behind the mixing immediately, constantly stir;
3. with the MES damping fluid of 50mmol/L, PH6.0 or purified water washing carboxylated latex microballoon, remove unreacted NHS and EDAC, and with the latex microsphere carboxylated latex microsphere suspension liquid that suspends to get, wherein the content of carboxylated latex microballoon is 0.01mg/ml in purified water;
4. the anti-human Procalcitonin antibody of rabbit is dissolved in the borate buffer solution of 50mmol/L, PH8.5, the protein concentration that makes the anti-human Procalcitonin antibody of rabbit is 0.25mg/ml;
5. get carboxylated latex microsphere suspension liquid 1ml in the step (3) and add immediately the anti-human Procalcitonin antibody of rabbit 1ml in the step (4); The concentration of carboxylated latex microballoon, the anti-human Procalcitonin antibody of rabbit and damping fluid is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L in this mixed liquor at this moment;
6. potpourri was reacted 3 hours at 37 ℃, constantly stir;
7. add the ratio adding monoethanolamine of 2.5 μ l monoethanolamines in the 1ml reaction mixture, reacted 20 minutes, constantly stir;
8. with 15000 rev/mins of centrifugal unconjugated protein and monoethanolamines removed, HEPES damping fluid dilution with 50mmol/L, PH7.6, making the concentration of the anti-human PCT antibody latex of rabbit particle is 0.004ml/ml, and then adding behind bovine serum albumin(BSA) (stabilizing agent) 4mmol/L, 2mmol/L Sodium azide (antiseptic) the dissolving mixing is Procalcitonin reagent 2;
Three) liquid-type PCT reference calibrations product
PCT reference calibrations product concentration as required adds corresponding PCT sterling 1000ng/ml in the above-mentioned mixed liquor, be prepared into the reference calibrations product of 5 series concentration, PCT content is respectively 45ng/ml, 90ng/ml, 180ng/ml, 320ng/ml, 640ng/ml from low to high in the reference calibrations product of 5 series concentration.
Pattern detection (contrasting respectively 30 samples of detection with the chemiluminescence immunoassay method):
According to the experimental implementation step, add the Procalcitonin reagent 1 of 240 μ L in the 10 μ L samples, hatched 3 minutes at 37 ℃, add 2,37 ℃ of 60 μ L Procalcitonin reagent and hatch after 10 seconds and read absorbance A at wavelength 600nm
1, read absorbance A when reacting to 5 minutes
2, calculate absorbance changing value △ A=A twice
2-A
1Compare by the calibration solution with same processing, calculate the PCT concentration in the sample.The result can find out from table 4 and Fig. 4 (Fig. 4 draws according to table 4 data), although the inventive method is simple to operate, required time only in 10 minutes, also shows very good correlativity (correlativity R with the chemiluminescence immunoassay method
2Reach 0.9998).
Table 4
Below be Procalcitonin reagent measurement operation step of the present invention and make a concrete analysis of parameter at automatic clinical chemistry analyzer:
Determination step
Analytical parameters (take the parameter of Hitachi's 7080 biochemical instruments as example)
Method: Two point end assay, temperature of reaction: 37 ℃
Predominant wavelength: 520-600nm commplementary wave length: 800nm(can not select)
Sample size: 10 μ L, reagent 1 amount/reagent 2 amounts: 240 μ L/60 μ L
The Direction of Reaction: forward (rising), calibration mode: multiple spot is non-linear
The read point time: be respectively and 21 points (suitable 420 seconds of total reaction time) at 5
Instrument is finished the mensuration that can carry out Procalcitonin after the calibration automatically, and instrument is by automatically calculating corresponding PCT concentration.
The present invention has amplified the surface area of PCT antigen and antibody response by PCT antibody is combined with the carboxylated latex microballoon, has realized carrying out the purpose that sample in enormous quantities is measured at the routine biochemistry instrument.Be fit to the mensuration that Routine Test Lab is used for PCT concentration.Demand is understood that; above-described only is the preferred embodiments of the invention, for the those of ordinary skill in present technique neck city, otherwise breaks away under the prerequisite of the principle of the invention; can also be as some improvement and adjustment, these improved adjustment also should be considered as protection scope of the present invention.
Claims (10)
1. reagent of measuring Procalcitonin comprises following composition and content:
A, Procalcitonin reagent 1:
The pH value of this reagent 1 is 7.0-8.0;
B, Procalcitonin reagent 2:
The particle diameter of the carboxylated latex microballoon in the described anti-human PCT antibody latex particle is 20nm-500nm;
C, liquid-type Procalcitonin reference calibrations product:
Described liquid-type Procalcitonin reference calibrations product comprise 5 parts of reference calibrations product that form series concentration; Procalcitonin content in 5 parts of reference calibrations product is respectively 1.25ng/ml, 2.5ng/ml, 5.0ng/ml, 10.0ng/ml, 20.0ng/ml from low to high; Perhaps,
Be prepared into the Procalcitonin reference calibrations product of single-point concentration.
2. a kind of reagent of measuring Procalcitonin according to claim 1, it is characterized in that described damping fluid is phosphate buffer, trishydroxymethylaminomethane (Tris) damping fluid, Isosorbide-5-Nitrae-piperazine two ethyl sulfonic acids (PIPES) damping fluid, 4-(2-hydroxyethyl) piperazine-1-1 ethane sulfonic acid (HEPES) damping fluid, Pehanorm base propane sulfonic acid (TAPS) damping fluid, glycine buffer, borate buffer, 3-morpholine propane sulfonic acid (MOPS) damping fluid, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid (MOPSO) damping fluid, the mixing of one or more arbitrary proportions in MES (MES) damping fluid.
3. a kind of reagent of measuring Procalcitonin according to claim 2, the concentration that it is characterized in that damping fluid in the described Procalcitonin reagent 1 is 20-200mmol/L, pH value 7.0-8.0.
4. a kind of reagent of measuring Procalcitonin according to claim 3 is characterized in that described reaction accelerator is polyglycol or dextran sulfate.
5. a kind of reagent of measuring Procalcitonin according to claim 4 is characterized in that described electrolyte is anion electrolyte or cationic polyelectrolyte.
6. a kind of reagent of measuring Procalcitonin according to claim 5 is characterized in that described glass or plastic containers, cationic surfactant, anionic surfactant or zwitterionic surfactant; Described non-ionic surfactant is: the mixing of one or more arbitrary proportions in Theist, Tween series, serial, the polyoxyethylene phenyl ether of polyoxyethylene laurel ether, polyoxyethylene octyl group phenylate, polyoxyethylene alkyl phenyl ether, the polyoxyethylene nonylplenyl ether.
7. a kind of reagent of measuring Procalcitonin according to claim 6 is characterized in that described stabilizing agent is the mixing of one or more arbitrary proportions in disodium ethylene diamine tetraacetate, bovine serum albumin(BSA), the sodium chloride.
8. a kind of reagent of measuring Procalcitonin according to claim 7 is characterized in that described anti-human Procalcitonin antibody is the anti-human Procalcitonin antibody of goat-anti HCT original antibody or rabbit or the anti-human Procalcitonin antibody of chicken or mouse-anti HCT original antibody.
9. the described a kind of preparation method who measures the reagent of Procalcitonin of claim 1, carry out as follows: 1) Procalcitonin reagent 1:
By formula ratio damping fluid, electrolyte, surfactant, reaction accelerator, antiseptic, stabilizing agent and purified water are mixed, set up the pH value to 7.0-8.0;
2) Procalcitonin reagent 2:
(1) gets the carboxylated latex microballoon is diluted to 0.01mg/ml with damping fluid suspending liquid;
(2) add the ratio of the N-hydroxy-succinamide sulfonate sodium (NHS) of 20mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDAC carboxyl activator) and 40mg in the carboxylated latex microsphere suspension liquid of 1ml, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide sulfonate sodium, in room temperature this potpourri was reacted 15-30 minute behind the mixing immediately, constantly stir;
(3) with damping fluid or purified water washing carboxylated latex microballoon, remove unreacted 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide sulfonate sodium, and with the latex microsphere carboxylated latex microsphere suspension liquid that suspends in purified water to get, making its concentration is 0.01mg/ml;
(4) anti-human Procalcitonin antibody is dissolved in the damping fluid, the protein concentration that makes anti-human Procalcitonin antibody is 0.25mg/ml;
(5) get carboxylated latex microsphere suspension liquid 1ml in the step (3) and add immediately anti-human Procalcitonin antibody 1ml in the step (4); Make the concentration of carboxylated latex microballoon in this mixed liquor, anti-human Procalcitonin antibody and damping fluid be respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
(6) potpourri was reacted 3 hours at 37 ℃, constantly stir;
(7) ratio that adds the monoethanolamine of 2.5 μ l in the 1ml reaction mixture adds monoethanolamine, react 10-30 minute, constantly stirring;
(8) centrifugal treating is removed unconjugated protein and monoethanolamine, and with damping fluid dilution, the concentration that makes anti-human PCT antibody latex particle is 0.002-0.01ml/ml, namely gets Procalcitonin reagent 2 after then adding stabilizing agent, antiseptic dissolving mixing;
3), liquid-type Procalcitonin reference calibrations product:
By formula ratio antiseptic, stabilizing agent and damping fluid are mixed and are divided into 5 portions of mixed liquors first, then as required Procalcitonin reference calibrations product concentration, the Procalcitonin sterling of the 1000ng/ml of respective amount is added respectively in the above-mentioned mixed liquor, obtain 5 parts of reference calibrations product that form series concentration; Procalcitonin content in 5 parts of reference calibrations product is respectively 1.25ng/ml, 2.5ng/ml, 5.0ng/ml, 10.0ng/ml, 20.0ng/ml from low to high; Perhaps,
Press and get mixed liquor after formula ratio mixes antiseptic, stabilizing agent and damping fluid, then the Procalcitonin sterling that in mixed liquor, adds a certain amount of 1000ng/ml, obtain the Procalcitonin reference calibrations product of single-point high concentration, its Procalcitonin content is 50.0ng/ml or 100.0ng/ml.
10. a kind of preparation method who measures the reagent of Procalcitonin according to claim 9 is characterized in that described centrifugal treating adopts hydro-extractor, and centrifuge speed is 15000 rev/mins.
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