CN104820097A - Liquid-chip kit for quantitatively detecting concentration of lipoprotein phospholipase A2 in sample and preparation method thereof - Google Patents
Liquid-chip kit for quantitatively detecting concentration of lipoprotein phospholipase A2 in sample and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of in-vitro immunodetection and particularly relates to a kit for detecting the concentration of lipoprotein phospholipase A2 in a sample and a preparation method thereof. The kit comprises microspheres coated with lipoprotein phospholipase A2, a micropore reaction plate, a standard product and a quality control product of a concentration gradient of lipoprotein phospholipase A2, a biotin-marked lipoprotein phospholipase A2 antibody, streptavidin-marked phycoerythrin, and a phosphate buffer solution containing a protein protection agent. Detection instruments used for the detection is MAGPIX of Luminex and Luminex 200, 3D; on the platform, measurement for the concentration of lipoprotein phospholipase A2 of human plasma by high flux, high speed, low cost, high accuracy and good repeatability can be realized, and simultaneously, detection markers of different types can be freely combined and added, so that simultaneous detection of multiple markers is realized, the accuracy rate for diagnosis of cardiovascular diseases is increased and the kit has important application prospect.
Description
Technical field
The invention belongs to ion vitro immunization detection technique field, be specifically related to a kind ofly detect kit of lipoprotein phospholipase A2 concentration determination in sample and preparation method thereof.
Background technology
Lp-PLA2 belongs to phospholipase A2 superfamily, and a kind of serine lipase be made up of 441 amino acid residues, relative molecular weight is 45kD.With all the other members of phospholipase A2 family unlike, Lp-PLA2 does not need calcium ion to maintain its catalytic activity, it has the activity of degraded platelet activating factor, therefore platelet-activating factor acetylhydrolase (platelet-activating factor acetylhydrolase, PAF-AH) is also referred to as.In blood, Lp-PLA2 is primarily of the macrophage of maturation and lymphocyte synthesis and secretion, exists in a large number, and can be regulated by inflammatory mediator at atherosclerotic position.The activated Lp-PLA2 of tool can ester bond on selective hydrolysis oxidized phospholipids sn-2 position in short acyl chain and generate two kinds of strong pro-inflammatory mediators---oxidation free fatty acid and lysolecithin.These two kinds of inflammatory mediators can be used as MCF, the activation of induced monocyte, and in atherosclerotic generation, development, tool has certain effect.Epidemiological studies display Lp-PLA2 is a kind of enzyme can predicting the atherosclerotic relevant with coronary events and headstroke danger, and it is as one independently inflammatory factor, has and promotes atherosclerotic effect.
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.The present invention is on new technology platform, by double-antibody method, accurate quantitative analysis is carried out to the concentration of LP-PLA2, 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.
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.
A liquid chip kit for lipoprotein phospholipase A2 concentration in quantitative detection sample, comprises gradient concentration standard items, the quality-control product of lipoprotein phospholipase A2, biotin labeled lipotropism protein, phospholipid enzyme A2 antibody, the phycoerythrin (SAPE) of streptavidin, the sample buffer of the microballoon of the monoclonal antibody being coated with lipotropism protein, phospholipid enzyme A2, platform carrier, lipoprotein phospholipase A2.
Preferably, described microballoon is the polystyrene magnetic microsphere of surface with iridescent, be coated with 1000-5000 microballoon/hole, and each microballoon contains 2*10 inside platform carrier with holes
-6-5*10
-5the monoclonal antibody of the lipotropism protein, phospholipid enzyme A2 of μ g.
Preferably, described platform carrier with holes is micro reaction plate.
Preferably, the gradient concentration standard items of described lipoprotein phospholipase A2 and the quality-control product of lipoprotein phospholipase A2 are all to dilute according to 1:200000-1:1000 with lipoprotein phospholipase A2 sterling and homemade standard dilutions to form, described standard items are no less than 2, described quality-control product and the difference of described standard items are that concentration is different, the effect of described 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 standard items matching curve out by quality-control product measured value, if quality-control product measured value is qualified, typical curve then can be utilized to simulate the concentration value of the lipoprotein phospholipase A2 in sample.
Preferably, described lipoprotein phospholipase A2 sterling is the natural lipoproteins phospholipase A2 albumen of human plasma purification or the restructuring lipoprotein phospholipase A2 albumen of Bacillus coli expression.
Preferably; described standard dilutions; 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.
Preferably, described sample buffer is in the phosphate buffer of 10-100mM, adds the protein protective agent of 1-10% massfraction respectively, the antiseptic of 0.01-0.5% massfraction mixes.
Preferably, described biotin labeled lipotropism protein, phospholipid enzyme A2 antibody is any one in monoclonal antibody or polyclonal antibody.
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 lipotropism protein, phospholipid enzyme A2: activate adding phosphate sodium dihydrogen buffer solution, N-hydroxy thiosuccinimide solution and Carbodiimide solution in the microballoon not wrapping quilt, the monoclonal antibody 1-125 μ g then adding specific lipotropism protein, phospholipid enzyme A2 carries out bag quilt at 4-8 DEG C; Wash three times; The albuminised confining liquid of animal blood added again containing 1-10% massfraction is closed; Finally in 10-100mM phosphate buffer, lucifuge, 4-8 DEG C deposit;
The preparation of lipoprotein phospholipase A2 gradient concentration standard items and lipoprotein phospholipase A2 quality-control product: be first in 10-100mM phosphate buffer in PH 7.0-7.4, concentration, adds 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 mixes standard dilutions; Again sterling and homemade standard dilutions are diluted the lipoprotein phospholipase A2 quality-control product forming and indicate gradient concentration standard items that concentration is 0-800ng/ml and 80-300ng/ml according to 1:200000-1:1000;
Biotin labeling lipotropism protein, phospholipid enzyme antibody: lipotropism protein, phospholipid enzyme antibody is mixed according to mol ratio 1:20 with biotin, then obtain biotin labeled lipotropism protein, phospholipid enzyme antibody by purifying;
The Streptavidin of streptavidin phycoerythrin: 0.5mg, add heterobifunctional agent absolute methanol liquid, the mol ratio of heterobifunctional agent and Streptavidin is made to be 20, normal-temperature reaction 60min, adding dithiothreitol (DTT) (DTT) makes its final concentration be 25mM, normal-temperature reaction 30min, the same sephadex chromatography excessively, collect protein peak, obtain the Streptavidin of sulfhydrylation;
Getting the fluorescence phycoerythrin of 0.75mg/mL derivatization and the Streptavidin of 0.25mg/mL sulfhydrylation, to be that 3:1 mixes according to mass concentration ratio crosslinked, shaking table low-speed oscillation, and 4-8 DEG C of reaction is spent the night;
The preparation of sample buffer: in 10-100mM phosphate buffer, adds the protein protective agent of 1-10% massfraction, and the antiseptic of 0.01-0.5% 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).
Preferably, described heterobifunctional agent is one in succinimide-4-thiacyclohexane-1-carbonic ester or 3-(2-pyridine dimercapto) propionic acid N-hydroxy-succinamide ester or both potpourris.
The present invention is based on Luminex xMAP technology platform, be that the method for the polystyrene little magnetic ball fluorescent dye of 6.5 μm 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) 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.This 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's detecting instrument used is MAGPIX and Luminex200 of Luminex, 3D, this platform can realize the mensuration that high flux, high speed, low cost, accuracy carry out human plasma lipoprotein phospholipase A2 concentration high, reproduciblely, simultaneously can increase the kind of certification mark thing by independent assortment, thus realize multiple label and detect simultaneously, increase the accuracy rate to cardiovascular disease diagnosis, have important application prospect.
Accompanying drawing explanation
Fig. 1 is embodiment 3 canonical plotting.
Fig. 2 is embodiment 4 canonical plotting.
Fig. 3 is embodiment 5 canonical plotting.
Fig. 4 is embodiment 6 canonical plotting.
Fig. 5 is embodiment 7 canonical plotting.
Fig. 6 is embodiment 8 canonical plotting.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail.
Embodiment 1:
The operation using method of lipoprotein phospholipase A2 (LP-PLA2) concentration liquid chip inspecting reagent unit of the present invention is as follows:
1. by standard items and quality-control product deionized water redissolution mixing, respectively to adding each 10 μ l of standard items 50 μ l/ hole, Quality Control and sample and 40 μ l sample buffer/holes in microwell plate;
2. the magnetic particle of coated antibody is diluted 25 times, then respectively to adding 50 μ l magnetic particles in microwell plate, normal temperature oscillating reactions 120min;
3. wash plate twice with washing trigger;
4. in microwell plate, add biotin labeled monoclonal antibody 25 μ l/ hole, be placed on room temperature reaction 60min on shaking table;
5. in microwell plate, add the phycoerythrin 25 μ l/ hole of streptavidin, be placed on room temperature reaction 30min on shaking table;
6. wash plate twice with washing trigger;
7. in microwell plate, add the sheath fluid in 150 μ l/ holes, normal temperature vibration 10min;
8. utilize Luminex 200 to read the fluorescent value of each reacting hole;
9. 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 LP-PLA2 in sample can be drawn.
Embodiment 2:
Prepare the phycoerythrin of this kit streptavidin by the following method:
The streptavidin of phycoerythrin:
1) desalting processing of phycoerythrin
The R-PE of purifying is generally kept in the sulfate of ammoniac damping fluid of 60% saturation degree, palpus centrifugal segregation supernatant before using, and is resuspended in 50mmol/L PBS, uses desalting column to remove sulfate of ammoniac.
2) 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 120min, through sephadex chromatography desalination, phosphate buffer balance and wash-out, collect phycoerythrin solution peak.
3) sulfhydrylation of Streptavidin
Get the Streptavidin of 0.5mg, add above-mentioned SMCC absolute methanol liquid, the mol ratio of SMCC and Streptavidin is made to be about 20, normal-temperature reaction 60min, adding dithiothreitol (DTT) (DTT) makes its final concentration be 25mM, normal-temperature reaction 30min, the same sephadex chromatography excessively, collects protein peak.
4) phycoerythrin and Streptavidin is crosslinked
Get the fluorescence phycoerythrin of 0.75mg/mL derivatization and the Streptavidin mixed in equal amounts of 0.25mg/mL sulfhydrylation, shaking table low-speed oscillation, 4 DEG C of reactions are spent the night.
5) termination of cross-linking reaction
By the sodium iodoacetate 60 μ l adding 20mg/ml be cross-linked, reaction 30min, cessation reaction.Finally phycoerythrin being marked goods is dissolved in phosphate buffer, 4 DEG C of preservations.
Embodiment 3:
Prepare this kit by the following method:
Be coated with the preparation of the microballoon of the monoclonal antibody of lipotropism protein, phospholipid enzyme A2 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 concentration gradient standard items of lipoprotein phospholipase A2 and the preparation of quality-control product: with the 10mM buffer solution of sodium phosphate (PH7.0) containing the NBCS of 1% massfraction, 0.01%Proclin300,1% bovine serum albumin(BSA), 0.05%Triton X-100 respectively the sterling of mark is mixed with indicate concentration be 0,50,100,200,400, the concentration gradient standard items of 800ng/ml and 80, the quality-control product of 300ng/ml; Then freeze-dried powder is made, 4 DEG C of preservations;
Biotin labeling lipotropism protein, phospholipid enzyme antibody: lipotropism protein, phospholipid enzyme antibody is mixed according to mol ratio 1:20 with biotin, then obtain biotin labeled lipotropism protein, phospholipid enzyme antibody by purifying;
The preparation of the phycoerythrin of streptavidin: identical with embodiment 1;
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.
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 1 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 4:
Prepare this kit by the following method:
Be coated with the preparation of the microballoon of the monoclonal antibody of lipotropism protein, phospholipid enzyme A2 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 lipoprotein phospholipase A2 and the preparation of quality-control product: with the 100mM buffer solution of sodium phosphate (PH7.4) of the Triton X-100 of the Proclin300 containing the NBCS of 10% massfraction, 0.5% massfraction, 10% bovine serum albumin(BSA), 1% massfraction respectively the sterling of mark is mixed with indicate concentration be 0,50,100,200,400, the concentration gradient standard items of 800ng/ml and 80, the quality-control product of 300ng/ml; Then freeze-dried powder is made, 4 DEG C of preservations;
Biotin labeling lipotropism protein, phospholipid enzyme antibody: identical with embodiment 3;
The preparation of the phycoerythrin of streptavidin: 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.
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 1 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 5:
Substantially the same manner as Example 3, unlike:
The concentration gradient standard items of lipoprotein phospholipase A2 and the preparation of quality-control product: with the 20mM buffer solution of sodium phosphate (PH7.4) containing 2% NBCS, 0.05%Proclin300,5% bovine serum albumin(BSA), 0.15%Triton X-100 respectively the sterling of mark is mixed with indicate concentration be 0,50,100,200,400, the concentration gradient standard items of 800ng/ml and 80, the quality-control product of 300ng/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): 1% bovine serum albumin(BSA) and 0.05% Proclin 300, fully mix.
Be the canonical plotting of the present embodiment as shown in Figure 3, the testing result of table 3 for adopting the method described in embodiment 1 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 3, 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 3 quality-control product, pattern detection result
Embodiment 6:
Substantially the same manner as Example 3, unlike:
Be coated with the preparation of the magnetic bead of the monoclonal antibody of lipotropism protein, phospholipid enzyme A2 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 50 μ g and carries out bag quilt; The confining liquid added again containing bovine serum albumin(BSA) is closed; Last in phosphate buffer lucifuge, 4 DEG C deposit;
The concentration gradient standard items of lipoprotein phospholipase A2 and the preparation of quality-control product: with the 20mM buffer solution of sodium phosphate (PH7.4) containing 2% NBCS, 0.05%Proclin300,5% bovine serum albumin(BSA), 0.10%Triton X-100 respectively the sterling of mark is mixed with indicate concentration be 0,50,100,200,400, the concentration gradient standard items of 800ng/ml and 80, the quality-control product of 300ng/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): 1% bovine serum albumin(BSA) and 0.05% Proclin 300, fully mix.
Be the canonical plotting of the present embodiment as shown in Figure 4, the testing result of table 4 for adopting the method described in embodiment 1 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 4, 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 4 quality-control product, pattern detection result
Embodiment 7:
Substantially the same manner as Example 6, unlike:
Be coated with the preparation of the magnetic bead of the monoclonal antibody of lipotropism protein, phospholipid enzyme A2 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 100 μ g and carries out bag quilt; The confining liquid added again containing bovine serum albumin(BSA) is closed; Last in phosphate buffer lucifuge, 4 DEG C deposit;
The concentration gradient standard items of lipoprotein phospholipase A2 and the preparation of quality-control product: with the 20mM buffer solution of sodium phosphate (PH7.4) containing 2% NBCS, 0.05%Proclin300,5% bovine serum albumin(BSA), 0.10%Triton X-100 respectively the sterling of mark is mixed with indicate concentration be 0,50,100,200,400, the concentration gradient standard items of 800ng/ml and 80, the quality-control product of 300ng/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): 1% bovine serum albumin(BSA) and 0.05% Proclin 300, fully mix.
Be the canonical plotting of the present embodiment as shown in Figure 5, the testing result of table 5 for adopting the method described in embodiment 1 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 5, 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 5 quality-control product, pattern detection result
Embodiment 8:
Substantially the same manner as Example 6, unlike:
Be coated with the preparation of the magnetic bead of the monoclonal antibody of lipotropism protein, phospholipid enzyme A2 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 125 μ g and carries out bag quilt; The confining liquid added again containing bovine serum albumin(BSA) is closed; Last in phosphate buffer lucifuge, 4 DEG C deposit;
The concentration gradient standard items of lipoprotein phospholipase A2 and the preparation of quality-control product: with the 20mM buffer solution of sodium phosphate (PH7.4) containing 2% NBCS, 0.05%Proclin300,5% bovine serum albumin(BSA), 0.10%Triton X-100 respectively the sterling of mark is mixed with indicate concentration be 0,50,100,200,400, the concentration gradient standard items of 800ng/ml and 80, the quality-control product of 300ng/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): 1% bovine serum albumin(BSA) and 0.05% Proclin 300, fully mix.
Be the canonical plotting of the present embodiment as shown in Figure 6, the testing result of table 6 for adopting the method described in embodiment 1 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 6, 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 6 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 lipoprotein phospholipase A2 concentration in sample, it is characterized in that: the gradient concentration standard items, the quality-control product of lipoprotein phospholipase A2, biotin labeled lipotropism protein, phospholipid enzyme A2 antibody, the phycoerythrin of streptavidin, the sample buffer that comprise the microballoon of the monoclonal antibody being coated with lipotropism protein, phospholipid enzyme A2, platform carrier with holes, lipoprotein phospholipase A2.
2. kit according to claim 1, is characterized in that: described microballoon is the polystyrene magnetic microsphere of surface with iridescent, be coated with 1000-5000 microballoon/hole, and each microballoon contains 2*10 inside platform carrier with holes
-6-5*10
-5the monoclonal antibody of the lipotropism protein, phospholipid enzyme A2 of μ.
3. kit according to claim 2, is characterized in that: described platform carrier with holes is micro reaction plate.
4. kit according to claim 3, is characterized in that: the gradient concentration standard items of described lipoprotein phospholipase A2 and the quality-control product of lipoprotein phospholipase A2 are all to dilute according to 1:200000-1:1000 with lipoprotein phospholipase A2 sterling and homemade standard dilutions to form.
5. the kit according to claims 4, is characterized in that: described lipoprotein phospholipase A2 sterling is the natural lipoproteins phospholipase A2 albumen of human plasma purification or the restructuring lipoprotein phospholipase A2 albumen of Bacillus coli expression.
6. kit according to claim 4; it is characterized in that: described standard dilutions; 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.
7. kit according to claim 6, is characterized in that: described biotin labeled lipotropism protein, phospholipid enzyme A2 antibody is any one in monoclonal antibody or polyclonal antibody.
8. prepare the method for kit as claimed in claim 1, it is characterized in that: comprise the following steps, following steps are regardless of tandem:
Be coated with the preparation of the microballoon of the monoclonal antibody of lipotropism protein, phospholipid enzyme A2: 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 lipotropism protein, phospholipid enzyme A2 carries out bag quilt at 4-8 DEG C; Washing; The albuminised confining liquid of animal blood added again containing 1-10% massfraction is closed; Finally in 10-100mM phosphate buffer, lucifuge, 4-8 DEG C deposit;
The preparation of the gradient concentration standard items of lipoprotein phospholipase A2 and the quality-control product of lipoprotein phospholipase A2: be first in 10-100mM phosphate buffer in PH 7.0-7.4, concentration, adds 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 mixes standard dilutions; Again sterling and homemade standard dilutions are diluted the lipoprotein phospholipase A2 quality-control product forming and indicate gradient concentration standard items that concentration is 0-800ng/ml and 80-300ng/ml according to 1:200000-1:1000;
Biotin labeling lipotropism protein, phospholipid enzyme A2 antibody: lipotropism protein, phospholipid enzyme A2 antibody is mixed according to mol ratio 1:20 with biotin, then obtain biotin labeled lipotropism protein, phospholipid enzyme A2 antibody by purifying;
The Streptavidin of streptavidin phycoerythrin: 0.5mg, add heterobifunctional agent absolute methanol liquid, the mol ratio of heterobifunctional agent and Streptavidin is made to be 20, normal-temperature reaction 60min, adding dithiothreitol (DTT) (DTT) makes its final concentration be 25mM, normal-temperature reaction 30min, the same sephadex chromatography excessively, collect protein peak, obtain the Streptavidin of sulfhydrylation;
Getting the fluorescence phycoerythrin of 0.75mg/mL derivatization and the Streptavidin of 0.25mg/mL sulfhydrylation, to be that 3:1 mixes according to mass concentration ratio crosslinked, shaking table low-speed oscillation, and 4-8 DEG C of reaction is spent the night;
The preparation of sample buffer: in 10-100mM phosphate buffer, adds the protein protective agent of 1-10% massfraction, and the antiseptic of 0.01-0.5% massfraction mixes.
9. the preparation method of kit as claimed in claim 8, it is characterized in that: described N-hydroxy thiosuccinimide solution and Carbodiimide solution are bought in Pierce company, described animal blood serum albumen is bovine serum albumin(BSA).
10. the preparation method of kit as claimed in claim 8, is characterized in that: described heterobifunctional agent is one in succinimide-4-thiacyclohexane-1-carbonic ester or 3-(2-pyridine dimercapto) propionic acid N-hydroxy-succinamide ester or both potpourris.
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