CN103018464B - 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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- CN103018464B CN103018464B CN201210536453.7A CN201210536453A CN103018464B CN 103018464 B CN103018464 B CN 103018464B CN 201210536453 A CN201210536453 A CN 201210536453A CN 103018464 B CN103018464 B CN 103018464B
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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 and a kind ofly measure the reagent of Procalcitonin and the preparation method of this reagent, particularly adopt reagent and the preparation method of the detection Procalcitonin in latex enhancing immune turbidimetry for Determination serum or blood plasma, medical science and technological field of biochemistry can be widely used in.
Background technology
Levels of Serum Procalcitonin (Procalcitionin, PCT) is made up 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, lower than 0.5ng/ml in normal human blood.When serious bacterial, fungi, parasitic infection and pyemia and MOFE, its level in blood plasma raises; And the immunity of self, allergy and when diseases such as local infection, virus infections, chronic nonspecific inflammation, cancer heating, the reactions of graft host rejection PCT level can not raise; This just determines the high degree of specificity of PCT, therefore also can be used for the antidiastole of various clinical setting.PCT concentration becomes positive correlation with severity of inflammation, and is reduced to normal level along with the control of inflammation and the alleviation of the state of an illness, and thus PCT can be used as again the reliability index judging the state of an illness and prognosis and observation of curative effect.PCT can detect after 2 hours after infection, significant to clinical early diagnosis, within 12 ~ 24 hours, peaks after infection.PCT has extensive and important using value clinically; And provide standard to the mortality ratio of patient ICU and hospital stays.
The laboratory method that current detection PCT is conventional has radioimmunology analytic approach (RIA), chemiluminescence immunoassay (CLIA), collaurum colourimetry (GICA) and euzymelinked immunosorbent assay (ELISA) (ELISA), and radioimmunology analytic approach utilizes the polyclonal antibody of Prof. Du Yucang to identify specifically and is connected synthesizing amino acid Procalcitonin.The method can detect the blood-serum P CT of normal person, and reliable susceptibility is 4pm/mL, detection be the potpourri of free PCT, mating type PCT and CGRP precursor, and above-mentioned three kinds of materials can not be distinguished.This method detects length consuming time (19 ~ 22h), and has the pollution of radioelement, label poor stability, and discarded object is difficult to process and makes it be restricted.Chemiluminescence immunoassay (CLIA) utilizes chemiluminescent substance through catalyst and oxidizing, forms the intermediate of an excited state; When the intermediate of this excited state gets back to stable ground state, send photon, utilize luminous signal measuring instrument measurement of photon number, thus the concentration of indirect determination PCT.Have tubular type and board-like two kinds of kits in the market, but this method exist acridinium ester, luminol, the direct labelled antibody of different luminol luminescence efficiency low, the shortcoming of label instability; And this direct labelling method belongs to moment light emitting-type, be difficult to the stability and the repeatability that ensure test result, also need special detecting instrument.Be not easy to Routine Test Lab carry out.And utilize biotin-avidin system to detect the electrochemiluminescence immune reagent kit of PCT, need the fully automatic electric chemiluminescence detector of supporting costliness, Routine Test Lab cannot be carried out, and brings unnecessary expense to patient, increases patient burden.Euzymelinked immunosorbent assay (ELISA) detects by the PCT monoclonal antibody of Prof. Du Yucang, though method is special, but detects and be minimumly limited to 10 μ g/L, cannot detect the PCT concentration in normal human serum.Chinese patent (application number: 201110210250.4) also disclose a kind of kit detecting Procalcitonin, utilize magnetic particle coupled antibody as fixedly separated phase, the multistage amplification system of biotin-Streptavidin, be catalyzing enzyme catalytic luminescence substrate with horseradish peroxidase, although improve the sensitivity of detection, but whole reaction time consumption is longer (at least needing 40 minutes) also, and sample needs magnetic resolution pre-service.This method needs washing reaction pipe, if hand washing can bring personal error, if washed with instrument, then needs another standby washing instrument, increases the cost in laboratory.
Summary of the invention
Technical matters to be solved by this invention is the deficiency overcoming above-mentioned background technology, provides a kind of Procalcitonin to detect reagent and preparation method; The reagent provided should have the feature that accuracy is high, reproducible, simple to operate, minute is short, be applicable to various types of automatic clinical chemistry analyzer; The preparation method of reagent thereof provided should have easy to make and that cost is not high feature.
Technical scheme provided by the invention is:
Measure a reagent for Procalcitonin, comprise 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 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 the 5 parts of reference calibrations product forming 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; Or, be prepared into the Procalcitonin reference calibrations product of single-point concentration; Such as 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 acid (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.
In described Procalcitonin reagent 1, the concentration of damping fluid is 20-200mmol/L, 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), sodium chloride.
Described electrolyte is anion electrolyte or cationic polyelectrolyte.
Described electrolyte is the sodium chloride (NaCl) in 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, polyoxyethylene laurel ether serial, polyoxyethylene phenyl ether, polyoxethylene octylphenyl phenylate, polyoxyethylene alkyl phenyl ether, ethylene nonyl phenyl 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 goat-anti HCT original antibody or rabbit anti-human Procalcitonin antibody or the anti-human Procalcitonin antibody of chicken or mouse-anti HCT original antibody.
Measure a preparation method for 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 pH value to 7.0-8.0;
2) Procalcitonin reagent 2:
(1) suspending liquid that carboxylated latex microballoon damping fluid is diluted to 0.01mg/ml is got;
(2) ratio of the N-hydroxy-succinamide sulfonate sodium (NHS) of 20mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDAC carboxyl activator) and 40mg is added 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 is reacted 15-30 minute after 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 the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, make its concentration be 0.01mg/ml;
(4) anti-human Procalcitonin antibody is dissolved in damping fluid, makes the protein concentration of anti-human Procalcitonin antibody be 0.25mg/ml;
(5) the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the anti-human Procalcitonin antibody 1ml in step (4) immediately; The concentration of carboxylated latex microballoon in this mixed liquor, anti-human Procalcitonin antibody and damping fluid is made to be respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
(6) potpourri is reacted 3 hours at 37 DEG C, constantly stir;
(7) ratio adding the monoethanolamine of 2.5 μ l in 1ml reaction mixture adds monoethanolamine, and reaction 10-30 minute, constantly stirs;
(8) centrifugal treating removes unconjugated protein and monoethanolamine, and with damping fluid dilution, the concentration making anti-human PCT antibody latex particle is 0.002-0.01ml/ml, then adds stabilizing agent, namely antiseptic obtains Procalcitonin reagent 2 after dissolving mixing;
3) liquid-type Procalcitonin reference calibrations product:
First by formula ratio antiseptic, stabilizing agent and damping fluid mixed and be divided into 5 portions of mixed liquors, then Procalcitonin reference calibrations product concentration as required, the Procalcitonin sterling of the 1000ng/ml of respective amount is added in above-mentioned mixed liquor respectively, obtains the reference calibrations product that 5 parts 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; Or,
Obtain mixed liquor after antiseptic, stabilizing agent and damping fluid being mixed by formula ratio, in mixed liquor, then add the Procalcitonin sterling of a certain amount of 1000ng/ml, obtain the Procalcitonin reference calibrations product of single-point high concentration.Such as, the Procalcitonin content in these reference calibrations product is 100.0ng/ml or 500.0ng/ml; Become the reference calibrations product of multiple variable concentrations again with normal saline dilution during use; Here have no particular limits, as long as obtained Procalcitonin reference calibrations product can compare with sample, can the content of Procalcitonin in working sample.
Described centrifugal treating adopts hydro-extractor, and centrifuge speed is 15000 revs/min.
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 sample and reagent, reactant liquor turbidity is increased, in reactant liquor turbidity and sample, the amount of antigen is linear within the specific limits, Biochemical Analyzer or other optical detecting instrument can be used at 520-600nm wavelength place assaying reaction liquid absorbance, and reactant liquor absorbance is directly proportional to surveyed PCT concentration.
The invention has the beneficial effects as follows: the Procalcitonin of the employing Immunoturbidimetry provided detects reagent, by CG antibody is combined with carboxylated latex microballoon, be exaggerated the surface area of CG antigen and antibody response, there is accuracy high (with the correlativity R of chemiluminescence immunoassay
2for 0.9991-0.9998), reproducible, the feature that detects fast (at most only needing 10 minutes to obtaining a result from mensuration, even shorter), sample mensuration in enormous quantities can be carried out on routine biochemistry instrument, substantially increase testing efficiency.The preparation method of this reagent is easy and simple to handle, and raw material is easy to get (all selecting outsourcing raw material), and cost is not high, is applicable to all kinds of medical research unit and Routine Test Lab application.
Accompanying drawing explanation
Fig. 1 shows the mutual relationship by the measured value of the inventive method gained PCT reagent and the PCT measured value of chemiluminescence immunoassay gained in embodiment 1.Through regretional analysis: y=1.0062x+0.6087; R
2=0.9991, demonstrate the present embodiment 1, with chemiluminescence immunoassay, there is good correlativity.
Fig. 2 shows the mutual relationship by the measured value of the inventive method gained PCT reagent and the PCT measured value of chemiluminescence immunoassay gained in embodiment 2.Through regretional analysis: y=1.0096x+0.7981; R
2=0.9996, demonstrate the present embodiment 2, with chemiluminescence immunoassay, there is good correlativity.
Fig. 3 shows the mutual relationship by the measured value of the inventive method gained PCT reagent and the PCT measured value of chemiluminescence immunoassay gained in embodiment 3.Through regretional analysis: y=1.0092x+0.7507; R
2=0.9998, demonstrate the present embodiment 3, with chemiluminescence immunoassay, there is good correlativity.
Fig. 4 shows the mutual relationship by the measured value of the inventive method gained PCT reagent and the PCT measured value of chemiluminescence immunoassay gained in embodiment 4.Through regretional analysis: y=1.0056x+2.8301; R
2=0.9998 demonstrates the present embodiment 4 has good correlativity with chemiluminescence immunoassay.
Embodiment
The present invention detects primary raw material needed for reagent and calibration object:
1, anti-human Procalcitonin antibody, has much commercial anti-human Procalcitonin antibody available, as DaKo company of Finland, Japanese UNF company; The only former reaction with HCT of this antibody, with other antigen without immunological cross-reaction, can be polyclonal antibody or monoclonal antibody, be not particularly limited here, if can with latex microsphere coupling.Its anti-human Procalcitonin antibody can be goat-anti (i.e. 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 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, as German Merck company, Japanese UNF company, Japanese JSR company and Thermo company; Its basic material is polystyrene or co polystyrene, and surperficial carboxylic group (COOH) is modified, and also can be not used as any modification, will select different microballoons here according to selecting antibody different with the associated methods of microballoon.As selected chemical crosslink technique (also can adopt physisorphtion), then with the microballoon that surface is modified through carboxyl, amino, aldehyde radical etc.The latex microsphere that the preferred carboxylic group of this law is modified, the preferred 20nm-500nm of particle diameter of carboxylated latex microballoon, further preferred 80nm-300nm.
3, Procalcitonin: purchased from DaKo company of Finland, need reference calibrations product for the preparation of this reagent place can be that natural calcitonin is former, also can be genetic recombination Procalcitonin.
Describe reagent of the present invention in detail below in conjunction with embodiment, but these embodiments should not thought to limit definition of the present invention.
Embodiment one
One) Procalcitonin reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust PH to 7.2 with hydrochloric acid;
Two) Procalcitonin reagent 2
1. the MES damping fluid getting carboxylated latex microballoon 50mmol/L, pH6.0 of 80nm is diluted to the suspending liquid of 0.01mg/ml;
2. the ratio adding 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 is reacted 15-30 minute after mixing immediately, constantly stir;
3., with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, PH6.0, remove unreacted NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
4. goat-anti HCT original antibody is dissolved in the borate buffer solution of 50mmol/L, PH8.5, makes the protein concentration of goat-anti HCT original antibody be 0.25mg/ml;
5. the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the goat-anti HCT original antibody 1ml in step (4) immediately; Now in this mixed liquor, the concentration of carboxylated latex microballoon, goat-anti HCT original antibody and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
6. potpourri is reacted 3 hours at 37 DEG C, constantly stir;
7. the ratio adding 2.5 μ l monoethanolamines in 1ml reaction mixture adds monoethanolamine, reacts 15 minutes, constantly stirs;
8. with 15000 revs/min of unconjugated protein of centrifugal removing and monoethanolamine, dilute with the MOPSO damping fluid of 50mmol/L, PH7.2, the concentration making goat-anti people PCT antibody latex particle is 0.01ml/ml, then adds after bovine serum albumin(BSA) (stabilizing agent) 3mmol/L, 3mmol/L Sodium azide (antiseptic) dissolve mixing and namely obtains Procalcitonin reagent 2;
Three) liquid-type PCT reference calibrations product
Corresponding PCT sterling 1000ng/ml adds in above-mentioned mixed liquor by PCT reference calibrations product concentration as required, is prepared into 5 the reference calibrations product forming series concentration; In 5 reference calibrations product, PCT content is respectively 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml from low to high.
Pattern detection (contrasting detection 30 samples respectively with Chemiluminescence immunoassay):
According to laboratory operating procedures, in 10 μ L samples, add the Procalcitonin reagent 1 of 240 μ L, hatch 3 minutes at 37 DEG C, add 60 μ L Procalcitonin reagent 2,37 DEG C and hatch after 10 seconds in wavelength 600nm reading absorbance A
1, react and read absorbance A to when 5 minutes
2, calculate twice absorbance changing value △ A=A
2-A
1.By comparing with the same calibration solution processed, calculate the PCT concentration in sample.Can find out from table 1 and Fig. 1 (Fig. 1 is according to table 1 Plotting data) according to result, although the inventive method is simple to operate, required time, only within 10 minutes, also shows very good correlativity (correlativity R with Chemiluminescence immunoassay
2reach 0.9991).
Table 1
Embodiment two
One) Procalcitonin reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust PH to 8.0 with NaOH;
Two) Procalcitonin reagent 2
1. the MES damping fluid getting carboxylated latex microballoon 50mmol/L, PH6.0 of 150nm is diluted to the suspending liquid of 0.01mg/ml;
2. the ratio adding the NHS of 20mg EDAC and 40mg in the carboxylated latex microsphere suspension liquid of 1ml adds EDAC and NHS, in room temperature, potpourri is reacted 15-30 minute immediately, constantly stir after mixing;
3., with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, PH6.0, remove unreacted NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
4. be dissolved in the borate buffer solution of 50mmol/L, PH8.5 by anti-human for rabbit Procalcitonin antibody, the protein concentration making the anti-human Procalcitonin antibody of rabbit is 0.25mg/ml;
5. the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the rabbit anti-human Procalcitonin antibody 1ml in step (4) immediately; Now in this mixed liquor, 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;
6. potpourri is reacted 3 hours at 37 DEG C, constantly stir;
7. the ratio adding 2.5 μ l monoethanolamines in 1ml reaction mixture adds monoethanolamine, reacts 30 minutes, constantly stirs;
8. with 15000 revs/min of unconjugated protein of centrifugal removing and monoethanolamine, dilute with the phosphate buffer of 50mmol/L, PH7.4, the concentration making rabbit anti-human PCT antibody latex particle is 0.006ml/ml, then adds after bovine serum albumin(BSA) (stabilizing agent) 3mmol/L, 3mmol/L Sodium azide (antiseptic) dissolve mixing and namely obtains Procalcitonin reagent 2;
Three) liquid-type PCT reference calibrations product
Corresponding PCT sterling 1000ng/ml adds in above-mentioned mixed liquor by PCT reference calibrations product concentration as required, be prepared into the reference calibrations product of 5 series concentration, in 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.
Pattern detection (contrasting detection 30 samples respectively with Chemiluminescence immunoassay):
According to laboratory operating procedures, in 10 μ L samples, add the Procalcitonin reagent 1 of 240 μ L, hatch 3 minutes at 37 DEG C, add 60 μ L Procalcitonin reagent 2,37 DEG C and hatch after 10 seconds in wavelength 600nm reading absorbance A
1, react and read absorbance A to when 5 minutes
2, calculate twice absorbance changing value △ A=A
2-A
1.By comparing with the same calibration solution processed, calculate the PCT concentration in sample.From table 2 and Fig. 2 (Fig. 2 is according to table 2 Plotting data), result can be found out, although the inventive method is simple to operate, required time, only within 10 minutes, also shows very good correlativity (correlativity R with Chemiluminescence immunoassay
2reach 0.9996).
Table 2
Embodiment three
One) Procalcitonin reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust PH to 8.0 with NaOH;
Two) Procalcitonin reagent 2
1. the MES damping fluid getting carboxylated latex microballoon 50mmol/L, PH6.0 of 300nm is diluted to the suspending liquid of 0.01mg/ml;
2. the ratio adding the NHS of 20mg EDAC and 40mg in the carboxylated latex microsphere suspension liquid of 1ml adds EDAC and NHS, immediately after mixing in room temperature by this potpourri reaction 15-30 minute, constantly stir;
3., with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, PH6.0, remove unreacted NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
4. mouse-anti HCT original antibody is dissolved in the borate buffer solution of 50mmol/L, PH8.5, makes the protein concentration of mouse-anti HCT original antibody be 0.25mg/ml;
5. the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the mouse-anti HCT original antibody 1ml in step (4) immediately; Now in this mixed liquor, the concentration of carboxylated latex microballoon, mouse-anti HCT original antibody and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
6. potpourri is reacted 3 hours at 37 DEG C, constantly stir;
7. the ratio adding 2.5 μ l monoethanolamines in 1ml reaction mixture adds monoethanolamine, reacts 10 minutes, constantly stirs;
8. with 15000 revs/min of unconjugated protein of centrifugal removing and monoethanolamine, dilute with the PIPES damping fluid of 50mmol/L, PH8.0, the concentration making mouse-anti people PCT antibody latex particle is 0.002ml/ml, then adds after bovine serum albumin(BSA) (stabilizing agent) 3mmol/L, 3mmol/L Sodium azide (antiseptic) dissolve mixing and namely obtains Procalcitonin reagent 2.
Three) liquid-type PCT reference calibrations product
Corresponding PCT sterling 1000ng/ml adds in above-mentioned mixed liquor by PCT reference calibrations product concentration as required, be prepared into the reference calibrations product of 5 series concentration, in 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.
Pattern detection (contrasting detection 30 samples respectively with Chemiluminescence immunoassay):
According to laboratory operating procedures, in 10 μ L samples, add the Procalcitonin reagent 1 of 240 μ L, hatch 3 minutes at 37 DEG C, add 60 μ L Procalcitonin reagent 2,37 DEG C and hatch after 10 seconds in wavelength 600nm reading absorbance A
1, react and read absorbance A to when 5 minutes
2, calculate twice absorbance changing value △ A=A
2-A
1.By comparing with the same calibration solution processed, calculate the PCT concentration in sample.From table 3 and Fig. 3 (Fig. 3 is according to table 3 Plotting data), result can be found out, although the inventive method is simple to operate, required time, only within 10 minutes, also shows very good correlativity (correlativity R with Chemiluminescence immunoassay
2reach 0.9998).
Table 3
Embodiment four
One) Procalcitonin reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust PH to 7.6 with hydrochloric acid or NaOH;
Two) Procalcitonin reagent 2
1. the MES damping fluid getting carboxylated latex microballoon 50mmol/L, PH6.0 of 220nm is diluted to the suspending liquid of 0.01mg/ml;
2. the ratio adding the NHS of 20 mg EDAC and 40 mg in the carboxylated latex microsphere suspension liquid of 1ml adds EDAC and NHS, in room temperature, this potpourri is reacted 15-30 minute immediately, constantly stir after mixing;
3., with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, PH6.0, remove unreacted NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
4. be dissolved in the borate buffer solution of 50mmol/L, PH8.5 by anti-human for rabbit Procalcitonin antibody, the protein concentration making the anti-human Procalcitonin antibody of rabbit is 0.25mg/ml;
5. the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the rabbit anti-human Procalcitonin antibody 1ml in step (4) immediately; Now in this mixed liquor, 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;
6. potpourri is reacted 3 hours at 37 DEG C, constantly stir;
7. the ratio adding 2.5 μ l monoethanolamines in 1ml reaction mixture adds monoethanolamine, reacts 20 minutes, constantly stirs;
8. with 15000 revs/min of unconjugated protein of centrifugal removing and monoethanolamine, dilute with the HEPES damping fluid of 50mmol/L, PH7.6, the concentration making rabbit anti-human PCT antibody latex particle is 0.004ml/ml, then adds bovine serum albumin(BSA) (stabilizing agent) 4mmol/L, 2mmol/L Sodium azide (antiseptic) and dissolves the rear i.e. Procalcitonin reagent 2 of mixing;
Three) liquid-type PCT reference calibrations product
Corresponding PCT sterling 1000ng/ml adds in above-mentioned mixed liquor by PCT reference calibrations product concentration as required, be prepared into the reference calibrations product of 5 series concentration, in 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.
Pattern detection (contrasting detection 30 samples respectively with Chemiluminescence immunoassay):
According to laboratory operating procedures, in 10 μ L samples, add the Procalcitonin reagent 1 of 240 μ L, hatch 3 minutes at 37 DEG C, add 60 μ L Procalcitonin reagent 2,37 DEG C and hatch after 10 seconds in wavelength 600nm reading absorbance A
1, react and read absorbance A to when 5 minutes
2, calculate twice absorbance changing value △ A=A
2-A
1.By comparing with the same calibration solution processed, calculate the PCT concentration in sample.From table 4 and Fig. 4 (Fig. 4 is according to table 4 Plotting data), result can be found out, although the inventive method is simple to operate, required time, only within 10 minutes, also shows very good correlativity (correlativity R with Chemiluminescence immunoassay
2reach 0.9998).
Table 4
Be below Procalcitonin reagent measurement operation step of the present invention and make a concrete analysis of parameter on automatic clinical chemistry analyzer:
Determination step
Analytical parameters (parameter for Hitachi 7080 biochemical instruments)
Method: Two point end assay, temperature of reaction: 37 DEG C
Predominant wavelength: 520-600nm commplementary wave length: 800nm(can not select)
Sample size: 10 μ L, reagent 1 amount/reagent 2 amount: 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
Can carry out the mensuration of Procalcitonin after instrument completes calibration automatically, instrument is by automatically calculating corresponding PCT concentration.
The present invention, by being combined with carboxylated latex microballoon by PCT antibody, being exaggerated the surface area of PCT antigen and antibody response, achieving the object of carrying out sample mensuration in enormous quantities on routine biochemistry instrument.Be applicable to the mensuration that Routine Test Lab is used for PCT concentration.What demand was understood is; above-described is only the preferred embodiments of the invention, for the those of ordinary skill in this technology neck city, otherwise under departing from the prerequisite of the principle of the invention; can also as some improvement and adjustment, these adjustment improved also should be considered as protection scope of the present invention.
Claims (4)
1. measure a reagent for Procalcitonin, comprise following composition and content:
1) Procalcitonin reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust pH to 7.2 with hydrochloric acid;
2) Procalcitonin reagent 2
(1) the MES damping fluid getting carboxylated latex microballoon 50mmol/L, pH6.0 of 80nm is diluted to the suspending liquid of 0.01mg/ml;
(2) ratio of 20mg 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and 40mg N-hydroxy-succinamide sulfonate sodium is added in 1ml carboxylated latex microsphere suspension liquid, in room temperature, this potpourri is reacted 15-30 minute after being mixed immediately by reagent, constantly stir;
(3) with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, pH6.0, remove unreacted Sulfo-NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
(4) goat-anti HCT original antibody is dissolved in the borate buffer solution of 50mmol/L, pH8.5, makes the protein concentration of goat-anti HCT original antibody be 0.25mg/ml;
(5) the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the goat-anti HCT original antibody 1ml in step (4) immediately; Now in this mixed liquor, the concentration of carboxylated latex microballoon, goat-anti HCT original antibody and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
(6) potpourri is reacted 3 hours at 37 DEG C, constantly stir;
(7) ratio adding 2.5 μ l monoethanolamines in 1ml reaction mixture adds monoethanolamine, reacts 15 minutes, constantly stirs;
(8) with 15000 revs/min of unconjugated protein of centrifugal removing and monoethanolamine, dilute with the MOPSO damping fluid of 50mmol/L, pH7.2, the concentration making goat-anti people PCT antibody latex particle is 0.01ml/ml, then adds after bovine serum albumin(BSA) 3mmol/L, 3mmol/L Sodium azide dissolves mixing and namely obtains Procalcitonin reagent 2;
3) liquid-type PCT reference calibrations product
Corresponding PCT sterling 1000ng/ml adds in above-mentioned mixed liquor by PCT reference calibrations product concentration as required, is prepared into 5 the reference calibrations product forming series concentration; In 5 reference calibrations product, PCT content is respectively 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml from low to high.
2. measure a reagent for Procalcitonin, comprise following composition and content:
1) Procalcitonin reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust pH to 8.0 with NaOH;
2) Procalcitonin reagent 2
(1) the MES damping fluid getting carboxylated latex microballoon 50mmol/L, pH6.0 of 150nm is diluted to the suspending liquid of 0.01mg/ml;
(2) add the ratio of the Sulfo-NHS of 20mg EDAC and 40mg in the carboxylated latex microsphere suspension liquid of 1ml, in room temperature, potpourri is reacted 15-30 minute after being mixed immediately by reagent, constantly stir;
(3) with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, pH6.0, remove unreacted Sulfo-NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
(4) be dissolved in the borate buffer solution of 50mmol/L, pH8.5 by anti-human for rabbit Procalcitonin antibody, the protein concentration making the anti-human Procalcitonin antibody of rabbit is 0.25mg/ml;
(5) the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the rabbit anti-human Procalcitonin antibody 1ml in step (4) immediately; Now in this mixed liquor, 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;
(6) potpourri is reacted 3 hours at 37 DEG C, constantly stir;
(7) ratio adding 2.5 μ l monoethanolamines in 1ml reaction mixture adds monoethanolamine, reacts 30 minutes, constantly stirs;
(8) with 15000 revs/min of unconjugated protein of centrifugal removing and monoethanolamine, dilute with the phosphate buffer of 50mmol/L, pH7.4, the concentration making rabbit anti-human PCT antibody latex particle is 0.006ml/ml, then adds after bovine serum albumin(BSA) 3mmol/L, 3mmol/L Sodium azide dissolves mixing and namely obtains Procalcitonin reagent 2;
3) liquid-type PCT reference calibrations product
Corresponding PCT sterling 1000ng/ml adds in above-mentioned mixed liquor by PCT reference calibrations product concentration as required, be prepared into the reference calibrations product of 5 series concentration, in 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.
3. measure a reagent for Procalcitonin, comprise following composition and content:
1) Procalcitonin reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust pH to 8.0 with NaOH;
2) Procalcitonin reagent 2
(1) the MES damping fluid getting carboxylated latex microballoon 50mmol/L, pH6.0 of 300nm is diluted to the suspending liquid of 0.01mg/ml;
(2) add the ratio of the Sulfo-NHS of 20mg EDAC and 40mg in the carboxylated latex microsphere suspension liquid of 1ml, in room temperature, this potpourri is reacted 15-30 minute after being mixed immediately by reagent, constantly stir;
(3) with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, pH6.0, remove unreacted Sulfo-NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
(4) mouse-anti HCT original antibody is dissolved in the borate buffer solution of 50mmol/L, pH8.5, makes the protein concentration of mouse-anti HCT original antibody be 0.25mg/ml;
(5) the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the mouse-anti HCT original antibody 1ml in step (4) immediately; Now in this mixed liquor, the concentration of carboxylated latex microballoon, mouse-anti HCT original antibody and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
(6) potpourri is reacted 3 hours at 37 DEG C, constantly stir;
(7) ratio adding 2.5 μ l monoethanolamines in 1ml reaction mixture adds monoethanolamine, reacts 10 minutes, constantly stirs;
(8) with 15000 revs/min of unconjugated protein of centrifugal removing and monoethanolamine, dilute with the PIPES damping fluid of 50mmol/L, pH8.0, the concentration making mouse-anti people PCT antibody latex particle is 0.002ml/ml, then adds after bovine serum albumin 3mmol/L, 3mmol/L Sodium azide dissolves mixing and namely obtains Procalcitonin reagent 2;
3) liquid-type PCT reference calibrations product
Corresponding PCT sterling 1000ng/ml adds in above-mentioned mixed liquor by PCT reference calibrations product concentration as required, be prepared into the reference calibrations product of 5 series concentration, in 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.
4. measure a reagent for Procalcitonin, comprise following composition and content:
1) Procalcitonin reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust pH to 7.6 with hydrochloric acid or NaOH;
2) Procalcitonin reagent 2
(1) the MES damping fluid getting carboxylated latex microballoon 50mmol/L, pH6.0 of 220nm is diluted to the suspending liquid of 0.01mg/ml;
(2) add the ratio of the Sulfo-NHS of 20mg EDAC and 40mg in the carboxylated latex microsphere suspension liquid of 1ml, in room temperature, this potpourri is reacted 15-30 minute after being mixed immediately by reagent, constantly stir;
(3) with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, pH6.0, remove unreacted Sulfo-NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
(4) be dissolved in the borate buffer solution of 50mmol/L, pH8.5 by anti-human for rabbit Procalcitonin antibody, the protein concentration making the anti-human Procalcitonin antibody of rabbit is 0.25mg/ml;
(5) the carboxylated latex microsphere suspension liquid 1ml got in step (3) adds the rabbit anti-human Procalcitonin antibody 1ml in step (4) immediately; Now in this mixed liquor, 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;
(6) potpourri is reacted 3 hours at 37 DEG C, constantly stir;
(7) ratio adding 2.5 μ l monoethanolamines in 1ml reaction mixture adds monoethanolamine, reacts 20 minutes, constantly stirs;
(8) with 15000 revs/min of unconjugated protein of centrifugal removing and monoethanolamine, dilute with the HEPES damping fluid of 50mmol/L, pH7.6, the concentration making rabbit anti-human PCT antibody latex particle is 0.004ml/ml, then adds bovine serum albumin(BSA) 4mmol/L, 2mmol/L Sodium azide and dissolves the rear i.e. Procalcitonin reagent 2 of mixing;
3) liquid-type PCT reference calibrations product
Corresponding PCT sterling 1000ng/ml adds in above-mentioned mixed liquor by PCT reference calibrations product concentration as required, be prepared into the reference calibrations product of 5 series concentration, in 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.
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| CN103558400A (en) * | 2013-11-25 | 2014-02-05 | 重庆中元生物技术有限公司 | Procalcitonin latex enhanced immunoturbidimetry detection kit |
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