CN104950111A - Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit - Google Patents
Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit Download PDFInfo
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention belongs to the technical field of in-vitro immune detection and particularly relates to a kit for detecting the concentration of myeloperoxidase (MPO) in a sample and a preparation method of the kit. The kit comprises microspheres coated with monoclonal antibodies of MPO, a platform carrier, an MPO concentration gradient standard, an MPO quantity control material, a fluorescent labeling reagent labeled MPO antibody, a sample buffer solution and washing liquor. A detector disclosed by the invention is Luminex MAGPIX, can be used for measuring the concentration of MPO of multiple individuals accurately, is capable of increasing types of detection labels and performing accurate diagnosis on cardiovascular diseases, and has important application prospects.
Description
Technical field
The invention belongs to ion vitro immunization detection technique field, be specifically related to a kind ofly detect kit of myeloperoxidase concentration determination in sample and preparation method thereof.
Background technology
Add up according to world Heart Federation, the death toll that angiocardiopathy causes accounts for 1/3 of the total death toll in the world, the morbidity rate of cardiovascular and cerebrovascular disease, M & M, in ascendant trend year by year, have become " number one killer " of human health, the life of the serious threat mankind.Accurate Diagnosis prediction for angiocardiopathy has great significance.
The leucoprotease of myeloperoxidase (MPO) to be a kind of relative molecular mass be 144KD is glycosylated hemoprotein.Under given conditions, its can induced oxidation stress and tissue damage, as the mark of system inflammation and oxidative stress.Increasing with coronary heart disease of epidemiological studies display MPO concentration is closely related, and the concentration therefore detecting MPO in patient's blood plasma has very important reference value for patients with coronary heart disease.
At present for the immune diagnostic method of angiocardiopathy, can only detect a kind of single index, often there is high false negative rate in the sensitivity existed due to different index, degree of accuracy and specific difference in clinical diagnosis.
Summary of the invention
The present invention, relative to traditional immunoassay, aims to provide high flux, the technology platform that simultaneously can detect multiple cardiovascular mark fast and accurately and kit, and this kit can also reduce false negative rate simultaneously.
A liquid chip kit for myeloperoxidase enzyme concentration in quantitative detection sample, comprises myeloperoxidase enzyme antibody, sample buffer, the washing lotion of the microballoon of the monoclonal antibody being coated with anti-myeloperoxidase, platform carrier with holes, the concentration gradient standard items of myeloperoxidase, myeloperoxidase quality-control product, fluorescent labeling reagent mark.
Preferably, described microballoon be surface with iridescent, fluorescence-encoded polystyrene magnetic microsphere can be realized, be coated with 1000-5000 microballoon inside the every hole of platform carrier with holes, and each microballoon contains 2*10
-6-5*10
-5the monoclonal antibody of the anti-myeloperoxidase of g.
Preferably, described platform carrier with holes is micro reaction plate.
Preferably, the concentration gradient standard items of described myeloperoxidase and myeloperoxidase quality-control product are freeze-dried powder, the concentration gradient standard items of described myeloperoxidase be by 6, concentration is respectively 0,50,100,200,400, the myeloperoxidase standard items of 800ng/ml make freeze-dried powder; Myeloperoxidase quality-control product is by 2, and concentration is 80, the myeloperoxidase quality-control product of 500ng/ml makes freeze-dried powder.
Preferably, the concentration gradient standard items of described myeloperoxidase and myeloperoxidase quality-control product are all to dilute according to 1:200000-1:1000 with myeloperoxidase sterling and homemade myeloperoxidase standard dilutions to form, described myeloperoxidase standard items are no less than 2, described myeloperoxidase quality-control product is different with the concentration of described myeloperoxidase standard items, the effect of described myeloperoxidase quality-control product is in kit use procedure, play the effect checking and approving check and correction, namely whether within the scope of Quality Control, approval check and correction is carried out to myeloperoxidase standard items matching curve out by myeloperoxidase quality-control product measured value, if myeloperoxidase quality-control product measured value is qualified, typical curve then can be utilized to simulate the concentration value of the myeloperoxidase in sample.
Preferably; described myeloperoxidase standard dilutions; be be in 10-100mM phosphate buffer in PH7.0-7.4, concentration, add the animal blood serum of 1-10% massfraction respectively, the antiseptic of 0.01-0.5% massfraction, the protein protective agent of 1-10% massfraction, the surfactant of 0.05-1% massfraction mix.
Preferably, the myeloperoxidase enzyme antibody of described fluorescent labeling reagent mark is monoclonal antibody.
Preferably, described fluorescent labeling reagent is phycoerythrin.
Preferably, described sample buffer is the phosphate buffer containing protein protective agent, is in 10-100mM phosphate buffer, adds the protein protective agent of 1-10% massfraction respectively, the antiseptic of 0.01-0.5% massfraction mixes.
Preferably, described surfactant is Qu Datong X-100, polysorbas20, Brij-35, polysorbate40, polysorbate60, Tween 80, sodium dodecylsulphonate, this dish 20, alkylphenol-polyethenoxy 4 ether, Macrogol 2000, Macrogol 4000.
Preferably, described antiseptic is Proclin 300, Sodium azide, thimerosal, gentamicin sulphate.
Preferably, described protein protective agent is bovine serum albumin(BSA), ox γ albumen, trehalose, glycerine, sucrose.
Prepare the method for described kit, comprise following preparation process, described preparation process in no particular order:
Be coated with the preparation of the microballoon of the monoclonal antibody of anti-myeloperoxidase: activate adding phosphate sodium dihydrogen buffer solution, N-hydroxy thiosuccinimide solution and Carbodiimide solution in the microballoon not wrapping quilt; Then the monoclonal antibody 1-125 μ g adding specific anti-myeloperoxidase carries out bag quilt at 4-8 DEG C; Washing; The albuminised confining liquid of animal blood added again containing 1-10% massfraction spends the night closed; Finally in 10-100mM phosphate buffer, lucifuge, 4-8 DEG C deposit;
The concentration gradient standard items of myeloperoxidase and the preparation of myeloperoxidase quality-control product: be first in 10-100mM phosphate buffer in PH 7.0-7.4, concentration, add the animal blood serum of 1-10% massfraction respectively, the antiseptic of 0.01-0.5% massfraction, the protein protective agent of 1-10% massfraction, the surfactant of 0.05-1% massfraction mix standard dilutions; Again sterling and homemade standard dilutions are diluted according to 1:200000-1:1000 and form that to indicate concentration be the concentration gradient standard items of 0-800ng/ml and the myeloperoxidase quality-control product of 60-250ng/ml;
The myeloperoxidase enzyme antibody of fluorescent labeling reagent mark: fluorescent labeling reagent is carried out derivatization by coupling agent, then covalent cross-linking is carried out with myeloperoxidase enzyme antibody, terminator cessation reaction is used after reaction, the antibody of fluorescent labeling reagent mark can be obtained, the myeloperoxidase enzyme antibody that fluorescent labeling reagent is marked in containing in protectant phosphate buffer, 4-8 DEG C of preservation;
The preparation of sample buffer: in 10-200mM phosphate buffer, adds the protein protective agent of 1-10% massfraction, and the antiseptic of 0.01-0.5% massfraction mixes;
The preparation of washing lotion: in 10-100mM phosphate buffer, the surfactant adding 1% massfraction mixes.
Preferably, described N-hydroxy thiosuccinimide solution and Carbodiimide solution are bought in Pierce company, and described animal blood serum albumen is bovine serum albumin(BSA).
The present invention is based on Luminex xMAP technology platform, be that the method for the polystyrene little magnetic ball fluorescent dye of 6.5um is encoded diameter, obtain by regulating the different ratio of two kinds of fluorescent dyes and can reach at most the microballoon that 100 kinds have different characteristic fluorescence Spectra, then by capture molecules such as the antigen for particular detection thing on often kind of coding microball covalent cross-linking, antibody or nucleic acid probes.During detection microballoon by micro liquid transfer system defiled by two bundle laser, the color of a branch of judgement particle thus determine the specificity (qualitative test) of measured object; Fluorescence labeling intensity that another bundle measures on particulate is quantitative to detected survey thing, and the data of gained directly can be used for judged result after computer process.Luminex xMAP technology has independent assortment, high flux, high speed, low cost, accuracy is high, reproducible, highly sensitive, the range of linearity wide, without the need to washing, advantage easy and simple to handle.In clinical diagnosis, introduce liquid microarrays technology and product, greatly will improve detection efficiency and reduce testing cost.
The present invention is on Luminex xMAP technology platform, by double-antibody method, accurate quantitative analysis is carried out to the concentration of MPO, independent assortment can be carried out from different marks simultaneously, detect while reaching multiple markers, improve the accuracy that angiocardiopathy detects, reduce the false negative rate in clinical detection.
Accompanying drawing explanation
Fig. 1 is embodiment 4 canonical plotting.
Fig. 2 is embodiment 5 canonical plotting.
Fig. 3 is embodiment 6 canonical plotting.
Fig. 4 is embodiment 7 canonical plotting.
Fig. 5 is embodiment 8 canonical plotting.
Fig. 6 is embodiment 9 canonical plotting.
Fig. 7 is embodiment 10 canonical plotting.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment 1:
Prepare the myeloperoxidase enzyme antibody of the fluorescent labeling reagent mark in this kit by the following method, the myeloperoxidase enzyme antibody of wherein said fluorescent labeling reagent mark is the myeloperoxidase monoclonal antibody of phycoerythrin mark:
1) derivatization of phycoerythrin
Getting 4.5mg phycoerythrin is dissolved in 1.0mL phosphate buffer, add 10 μ L 3-(2-pyridine dimercapto) propionic acid N-hydroxy-succinamide ester (SPDP) absolute methanol solution (4mg/mL), SPDP and phycoerythrin mol ratio is made to be about 10, normal-temperature reaction 50min, through sephadex chromatography desalination, phosphate buffer balance and wash-out, collect phycoerythrin solution peak.
2) sulfhydrylation of monoclonal antibody
Get the antibody of 0.5mg, add above-mentioned SPDP absolute methanol liquid, make the mol ratio of SPDP and antibody be about 10, normal-temperature reaction 40min, adds dithiothreitol (DTT) (DTT) and makes its final concentration be 25 μMs, normal-temperature reaction 25min, the same sephadex chromatography excessively, collects protein peak.
3) phycoerythrin and monoclonal antibody is crosslinked
Get the fluorescence phycoerythrin of 0.75mg/mL derivatization and the monoclonal antibody mixed in equal amounts of 0.25mg/mL sulfhydrylation, shaking table low-speed oscillation, 4 DEG C of reactions are spent the night.Phycoerythrin mark goods are finally dissolved in phosphate buffer, 4 DEG C of preservations.
Embodiment 2:
Prepare the myeloperoxidase enzyme antibody of the fluorescent labeling reagent mark in this kit by the following method, the myeloperoxidase enzyme antibody of wherein said fluorescent labeling reagent mark is the myeloperoxidase monoclonal antibody of phycoerythrin mark:
1) derivatization of phycoerythrin
Getting 4.5mg phycoerythrin is dissolved in 1.0mL phosphate buffer, add 5 μ L 3-(2-pyridine dimercapto) propionic acid N-hydroxy-succinamide ester (SPDP) absolute methanol solution (4mg/mL), SPDP and phycoerythrin mol ratio is made to be about 5, normal-temperature reaction 50min, through sephadex chromatography desalination, phosphate buffer balance and wash-out, collect phycoerythrin solution peak.
2) sulfhydrylation of monoclonal antibody
Get the antibody of 0.5mg, add above-mentioned SPDP absolute methanol liquid, make the mol ratio of SPDP and antibody be about 5, normal-temperature reaction 40min, adds dithiothreitol (DTT) (DTT) and makes its final concentration be 25 μMs, normal-temperature reaction 25min, the same sephadex chromatography excessively, collects protein peak.
3) phycoerythrin and monoclonal antibody is crosslinked
Get the fluorescence phycoerythrin of 0.75mg/mL derivatization and the monoclonal antibody mixed in equal amounts of 0.25mg/mL sulfhydrylation, shaking table low-speed oscillation, 4 DEG C of reactions are spent the night.Phycoerythrin mark goods are finally dissolved in phosphate buffer, 4 DEG C of preservations.
Embodiment 3:
The operation using method of myeloperoxidase of the present invention (MPO) concentration liquid chip inspecting reagent unit is as follows:
1. standard items and quality-control product are redissolved with deionized water respectively and mix, respectively to adding standard items 50ul/ hole, quality-control product 10ul/ hole, sample buffer 40ul/ hole in microwell plate;
2. the magnetic microsphere of coated antibody is diluted 25 times, then respectively to adding 50ul magnetic microsphere in microwell plate, normal temperature oscillating reactions 120min;
3. wash plate twice with washing trigger;
4. in microwell plate, add the antibody 100ul/ hole of phycoerythrin mark, be placed on room temperature reaction 60min on shaking table;
5. wash plate twice with washing trigger;
6. in microwell plate, add the sheath fluid in 100ul/ hole, normal temperature vibration 10min;
7. utilize Luminex MAGPIX to read the fluorescent value of each reacting hole;
8. the concentration value of standard items and fluorescent value are carried out quafric curve regression fit and go out typical curve, then the fluorescent value of sample is brought in typical curve, the concentration value of MPO in sample can be drawn.
Embodiment 4:
Prepare this kit by the following method:
Be coated with the preparation of the microballoon of the monoclonal antibody of anti-myeloperoxidase respectively: get the microballoon 5.0 × 10 not wrapping quilt
6individual, the pH value adding 60 μ L is the 100mM phosphate sodium dihydrogen buffer solution of 6.2, the 25mg/mL N-hydroxy thiosuccinimide solution of 20 μ L and the 25mg/mL Carbodiimide solution of 20 μ L activate, and then adds specific monoclonal antibody 25 μ g and carries out bag quilt; The confining liquid added again containing the bovine serum albumin(BSA) of 1% massfraction is closed; Last in the phosphate buffer of 10mM lucifuge, 4 DEG C deposit;
The preparation method of the special anti-myeloperoxidase monoclonal antibody that phycoerythrin (PE) marks is identical with embodiment 1;
The concentration gradient standard items of myeloperoxidase and the preparation of myeloperoxidase quality-control product: with the NBCS containing 1% massfraction, the Proclin300 of 0.01% massfraction, the bovine serum albumin(BSA) of 1% massfraction, the sterling of mark is mixed with and indicates concentration by the 10mM buffer solution of sodium phosphate (PH7.0) of the Triton X-100 of 0.05% massfraction is respectively 0, 50, 100, 200, 400, the concentration gradient standard items of 800ng/ml and 60, the quality-control product of 250ng/ml, then freeze-dried powder is made, 4 DEG C of preservations,
The preparation of sample buffer: add respectively in 10mM phosphate buffer (PH7.4): the bovine serum albumin(BSA) of 1% massfraction and the Proclin 300 of 0.01% massfraction, fully mix;
The preparation of washing lotion: in 10mM phosphate buffer, the Tween-20 adding 1% massfraction mixes.
Be the canonical plotting of the present embodiment as shown in Figure 1, the testing result of table 1 for adopting the method described in embodiment 3 to detect the present embodiment quality-control product, sample, wherein sample 1,2 derives from human plasma, the detected value of quality-control product 1 and quality-control product 2 is within the scope of the target value of correspondence as can be seen from Table 1, so can determine that quality-control product is controlled, credible result; And the detected value deviation of sample 1,2 is compared with its corresponding target value, deviation is less than 10%, illustrates that this kit Detection accuracy is high.
Table 1 quality-control product, pattern detection result
Embodiment 5:
Prepare this kit by the following method:
Be coated with the preparation of the microballoon of the monoclonal antibody of anti-myeloperoxidase respectively: get the microballoon 5.0 × 10 not wrapping quilt
6individual, the pH value adding 60 μ L is the 100mM phosphate sodium dihydrogen buffer solution of 6.2, the 25mg/mL N-hydroxy thiosuccinimide solution of 20 μ L and the 25mg/mL Carbodiimide solution of 20 μ L activate, and then adds specific monoclonal antibody 25 μ g and carries out bag quilt; The confining liquid added again containing the bovine serum albumin(BSA) of 10% massfraction is closed; Last in the phosphate buffer of 100mM lucifuge, 8 DEG C deposit;
The concentration gradient standard items of myeloperoxidase and the preparation of myeloperoxidase quality-control product: with the NBCS containing 10% massfraction, the Proclin300 of 0.5% massfraction, the bovine serum albumin(BSA) of 10% massfraction, the sterling of mark is mixed with and indicates concentration by the 100mM buffer solution of sodium phosphate (PH8.0) of the Triton X-100 of 1% massfraction is respectively 0, 50, 100, 200, 400, the concentration gradient standard items of 800ng/ml and 60, the quality-control product of 250ng/ml, then freeze-dried powder is made, 4 DEG C of preservations,
The preparation method of the special anti-myeloperoxidase monoclonal antibody that phycoerythrin (PE) marks is identical with embodiment 2;
The preparation of sample buffer: add respectively in 100mM phosphate buffer (PH7.4): the bovine serum albumin(BSA) of 10% massfraction and the Proclin 300 of 0.5% massfraction, fully mix;
The preparation of washing lotion: in 100mM phosphate buffer (PH8.0), the Tween-20 adding 10% massfraction mixes.
Be the canonical plotting of the present embodiment as shown in Figure 2, the testing result of table 2 for adopting the method described in embodiment 3 to detect the present embodiment quality-control product, sample, wherein sample 1,2 derives from human plasma, the detected value of quality-control product 1 and quality-control product 2 is within the scope of the target value of correspondence as can be seen from Table 2, so can determine that quality-control product is controlled, credible result; And the detected value deviation of sample 1,2 is compared with its corresponding target value, deviation is less than 10%, illustrates that this kit Detection accuracy is high.
Table 2 quality-control product, pattern detection result
Embodiment 6:
Substantially the same manner as Example 4, unlike:
The concentration gradient standard items of myeloperoxidase and the preparation of myeloperoxidase quality-control product: with the NBCS containing 2% massfraction, the Proclin300 of 0.05% massfraction, the bovine serum albumin(BSA) of 5% massfraction, the sterling of mark is mixed with and indicates concentration by the 20mM buffer solution of sodium phosphate (PH7.4) of the Triton X-100 of 0.15% massfraction is respectively 0, 50, 100, 200, 400, the one concentration gradient standard items of 800ng/ml and 60, the quality-control product of 250ng/ml, then freeze-dried powder is made, 4 DEG C of preservations,
The preparation of sample buffer: add respectively in 100mM phosphate buffer (PH7.4): the bovine serum albumin(BSA) of 1% massfraction and the Proclin 300 of 0.05% massfraction, fully mix.
Be the canonical plotting of the present embodiment as shown in Figure 3, table 3 is the present embodiment quality-control product, pattern detection result, as can be seen from Table 3, the detected value of quality-control product 1 and quality-control product 2 within the scope of the target value of correspondence, so can determine that quality-control product is controlled, credible result; And the detected value deviation of sample 1,2 is compared with its corresponding target value, deviation is less than 10%, and illustrate that this kit Detection accuracy is high, wherein sample 1,2 comes from human plasma.
Table 3 quality-control product, pattern detection result
Embodiment 7:
The present embodiment is substantially the same manner as Example 5, unlike:
Prepare the microballoon of the monoclonal antibody of the anti-myeloperoxidase in this kit by the following method:
Get the microballoon 5.0 × 10 not wrapping quilt
6individual, the pH value adding 60 μ L is the 100mM phosphate sodium dihydrogen buffer solution of 6.2, the 25mg/mL N-hydroxy thiosuccinimide solution of 20 μ L and the 125mg/mL Carbodiimide solution of 20 μ L activate, and then adds specific monoclonal antibody 50 μ g and carries out bag quilt; The confining liquid added again containing 1% bovine serum albumin(BSA) is closed; Last in phosphate buffer lucifuge, 4 DEG C deposit;
The concentration gradient standard items of myeloperoxidase and the preparation of quality-control product: with the 20mM buffer solution of sodium phosphate (PH7.4) containing 10% NBCS, 0.5%Proclin300,10% bovine serum albumin(BSA), 1%Triton X-100 respectively the sterling of mark is mixed with indicate concentration be 0,50,100,200,400, a series of concentration gradient standard items of 800ng/ml and 60, the quality-control product of 250ng/ml; Then freeze-dried powder is made, 4 DEG C of preservations;
The preparation of sample buffer: add respectively in 100mM phosphate buffer (PH7.4): 10% bovine serum albumin(BSA) and 1%Proclin 300, fully mix.
Be the canonical plotting of the present embodiment as shown in Figure 4, table 4 is the present embodiment quality-control product, pattern detection result, as can be seen from Table 4, the detected value of quality-control product 1 and quality-control product 2 within the scope of the target value of correspondence, so can determine that quality-control product is controlled, credible result; And the detected value deviation of sample 1,2 is compared with its corresponding target value, deviation is less than 10%, and illustrate that this kit Detection accuracy is high, wherein sample 1,2 comes from human plasma.
Table 4 quality-control product, pattern detection result
Embodiment 8:
Substantially the same manner as Example 6, unlike:
Prepare the microballoon of the monoclonal antibody of the anti-myeloperoxidase in this kit by the following method:
Get the microballoon 5.0 × 10 not wrapping quilt
6individual, the pH value adding 60 μ L is the 100mM phosphate sodium dihydrogen buffer solution of 6.2, the 25mg/mL N-hydroxy thiosuccinimide solution of 20 μ L and the 125mg/mL Carbodiimide solution of 20 μ L activate, and then adds specific monoclonal antibody 50 μ g and carries out bag quilt; The confining liquid added again containing 1% bovine serum albumin(BSA) is closed; Last in phosphate buffer lucifuge, 4 DEG C deposit;
Be the canonical plotting of the present embodiment as shown in Figure 5, table 5 is the present embodiment quality-control product, pattern detection result.
Table 5 quality-control product, pattern detection result
Embodiment 9:
Substantially the same manner as Example 6, unlike:
Prepare the microballoon of the monoclonal antibody of the anti-myeloperoxidase in this kit by the following method:
Get the microballoon 5.0 × 10 not wrapping quilt
6individual, the pH value adding 60 μ L is the 100mM phosphate sodium dihydrogen buffer solution of 6.2, the 25mg/mL N-hydroxy thiosuccinimide solution of 20 μ L and the 125mg/mL Carbodiimide solution of 20 μ L activate, and then adds specific monoclonal antibody 100 μ g and carries out bag quilt; The confining liquid added again containing 1% bovine serum albumin(BSA) is closed; Last in phosphate buffer lucifuge, 4 DEG C deposit;
Be the canonical plotting of the present embodiment as shown in Figure 6, table 6 is the present embodiment quality-control product, pattern detection result.
Table 6 quality-control product, pattern detection result
Embodiment 10:
Substantially the same manner as Example 6, unlike:
Prepare the microballoon of the monoclonal antibody of the anti-myeloperoxidase in this kit by the following method:
Get the microballoon 5.0 × 10 not wrapping quilt
6individual, the pH value adding 60 μ L is the 100mM phosphate sodium dihydrogen buffer solution of 6.2, the 25mg/mL N-hydroxy thiosuccinimide solution of 20 μ L and the 125mg/mL Carbodiimide solution of 20 μ L activate, and then adds specific monoclonal antibody 125 μ g and carries out bag quilt; The confining liquid added again containing 1% bovine serum albumin(BSA) is closed; Last in phosphate buffer lucifuge, 4 DEG C deposit;
Be the canonical plotting of the present embodiment as shown in Figure 7, table 7 is the present embodiment quality-control product, pattern detection result.
Table 7 quality-control product, pattern detection result
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. quantitatively detect a liquid chip kit for myeloperoxidase enzyme concentration in sample, comprise myeloperoxidase enzyme antibody, sample buffer, the washing lotion of the microballoon of the monoclonal antibody being coated with anti-myeloperoxidase, platform carrier with holes, the concentration gradient standard items of myeloperoxidase, myeloperoxidase quality-control product, fluorescent labeling reagent mark.
2. microballoon described in be surface with iridescent, fluorescence-encoded polystyrene magnetic microsphere can be realized, be coated with 1000-5000 microballoon inside the every hole of platform carrier with holes, and each microballoon contains 2*10
-6-5*10
-5the monoclonal antibody of the anti-myeloperoxidase of g.
3. platform carrier with holes described in is micro reaction plate.
4. the concentration gradient standard items of myeloperoxidase described in and myeloperoxidase quality-control product are all to dilute according to 1:200000-1:1000 with myeloperoxidase sterling and homemade myeloperoxidase standard dilutions to form, described myeloperoxidase standard items are no less than 2, described myeloperoxidase quality-control product is different with the concentration of described myeloperoxidase standard items, the effect of described myeloperoxidase quality-control product is in kit use procedure, play the effect checking and approving check and correction, namely whether within the scope of Quality Control, approval check and correction is carried out to myeloperoxidase standard items matching curve out by myeloperoxidase quality-control product measured value, if myeloperoxidase quality-control product measured value is qualified, typical curve then can be utilized to simulate the concentration value of the myeloperoxidase in sample.
5. the myeloperoxidase standard dilutions described in; be be in 10-100mM phosphate buffer in PH 7.0-7.4, concentration, add the animal blood serum of 1-10% massfraction respectively, the antiseptic of 0.01-0.5% massfraction, the protein protective agent of 1-10% massfraction, the surfactant of 0.05-1% massfraction mix.
6. described in, the myeloperoxidase enzyme antibody of fluorescent labeling reagent mark is monoclonal antibody.
7. fluorescent labeling reagent described in is phycoerythrin.
8. sample buffer described in is the phosphate buffer containing protein protective agent, and be in 10-100mM phosphate buffer, add the protein protective agent of 1-10% massfraction, the antiseptic of 0.01-0.5% massfraction mixes.
9. prepare the method for described kit, comprise following preparation process, described preparation process in no particular order:
Be coated with the preparation of the microballoon of the monoclonal antibody of anti-myeloperoxidase: activate adding phosphate sodium dihydrogen buffer solution, N-hydroxy thiosuccinimide solution and Carbodiimide solution in the microballoon not wrapping quilt; Then the monoclonal antibody 1-125 μ g adding specific anti-myeloperoxidase carries out bag quilt at 4-8 DEG C; Washing; The albuminised confining liquid of animal blood added again containing 1-10% massfraction spends the night closed; Finally in 10-100mM phosphate buffer, lucifuge, 4-8 DEG C deposit;
The concentration gradient standard items of myeloperoxidase and the preparation of myeloperoxidase quality-control product: be first in 10-100mM phosphate buffer in PH 7.0-7.4, concentration, add the animal blood serum of 1-10% massfraction respectively, the antiseptic of 0.01-0.5% massfraction, the protein protective agent of 1-10% massfraction, the surfactant of 0.05-1% massfraction mix standard dilutions; Again sterling and homemade standard dilutions are diluted according to 1:200000-1:1000 and form that to indicate concentration be the concentration gradient standard items of 0-800ng/ml and the myeloperoxidase quality-control product of 60-250ng/ml;
The myeloperoxidase enzyme antibody of fluorescent labeling reagent mark: fluorescent labeling reagent is carried out derivatization by coupling agent, then covalent cross-linking is carried out with myeloperoxidase enzyme antibody, terminator cessation reaction is used after reaction, the antibody of fluorescent labeling reagent mark can be obtained, the myeloperoxidase enzyme antibody that fluorescent labeling reagent is marked in containing in protectant phosphate buffer, 4-8 DEG C of preservation;
The preparation of sample buffer: in 10-200mM phosphate buffer, adds the protein protective agent of 1-10% massfraction, and the antiseptic of 0.01-0.5% massfraction mixes;
The preparation of washing lotion: in 10-100mM phosphate buffer, the surfactant adding 1% massfraction mixes.
10. N-hydroxy thiosuccinimide solution and Carbodiimide solution described in are bought in Pierce company, and described animal blood serum albumen is bovine serum albumin(BSA).
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| CN201510264522.7A CN104950111A (en) | 2015-05-22 | 2015-05-22 | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit |
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| CN201510264522.7A CN104950111A (en) | 2015-05-22 | 2015-05-22 | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit |
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Cited By (5)
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| CN105498874A (en) * | 2016-01-29 | 2016-04-20 | 中国农业大学 | Chip, system and method for detecting peroxidase concentration, as well as chip production method |
| CN106290190A (en) * | 2016-08-05 | 2017-01-04 | 长春恒晓生物科技有限责任公司 | The method measuring MPO halogenase activity |
| CN107991494A (en) * | 2017-11-22 | 2018-05-04 | 贾赟 | A kind of liquid phase protein chip kit of the external and new hair animal epidemic of while four kinds of detection and preparation method thereof |
| CN108445215A (en) * | 2018-02-01 | 2018-08-24 | 浙江艾明德生物科技有限公司 | A kind of kit and preparation method quantitatively detecting myeloperoxidase |
| CN110716052A (en) * | 2019-08-14 | 2020-01-21 | 殷丽 | Human eosinophil cationic protein and myeloperoxidase detection kit and application thereof |
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| CN105498874A (en) * | 2016-01-29 | 2016-04-20 | 中国农业大学 | Chip, system and method for detecting peroxidase concentration, as well as chip production method |
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| CN106290190B (en) * | 2016-08-05 | 2019-01-25 | 长春恒晓生物科技有限责任公司 | The method for measuring MPO halogenase activity |
| CN107991494A (en) * | 2017-11-22 | 2018-05-04 | 贾赟 | A kind of liquid phase protein chip kit of the external and new hair animal epidemic of while four kinds of detection and preparation method thereof |
| CN108445215A (en) * | 2018-02-01 | 2018-08-24 | 浙江艾明德生物科技有限公司 | A kind of kit and preparation method quantitatively detecting myeloperoxidase |
| CN110716052A (en) * | 2019-08-14 | 2020-01-21 | 殷丽 | Human eosinophil cationic protein and myeloperoxidase detection kit and application thereof |
| CN110716052B (en) * | 2019-08-14 | 2023-04-25 | 武汉大白小白科技有限公司 | Kit for detecting human eosinophil cationic protein and myeloperoxidase |
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Application publication date: 20150930 |