CN104280556A - Detection method and kit for simultaneously determining content of lipoprotein phospholipase A2 and C reactive protein in blood plasma - Google Patents
Detection method and kit for simultaneously determining content of lipoprotein phospholipase A2 and C reactive protein in blood plasma Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a detection method and a kit for simultaneously determining the content of lipoprotein phospholipase A2 and C reactive protein in blood plasma. The detection method and the kit are applicable to the two-index joint detection of evaluating the neurological severity and the infarction volume after the occurrence of the cerebral ischemic stroke. The kit is capable of realizing simultaneous analysis of two indexes by taking lipoprotein phospholipase A2 (Lp-PLA2) and the C reactive protein (CRP) in the blood plasma as detection indexes, using a double antibody sandwich method based on a porous plate, and fluorescent substances, such as a fluorescent dye, lanthanide or a quantum dot as a marker.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of detection kit adopting lipoprotein phospholipase A2 (Lp-PLA2) and c reactive protein (CRP) content in board-like luminescence method Simultaneously test blood plasma, thus the prevention of cerebral arterial thrombosis, condition assessment and/or prognosis are carried out to the kit of auxiliary judgment, wherein, the Monitoring lower-cut of CRP is 0.1 μ g/ml, be a kind of highly sensitive CRP analytical approach, relate to detection method and the kit of lipoprotein phospholipase A2 and c reactive protein content in a kind of Simultaneously test blood plasma particularly.
Background technology
Cerebral arterial thrombosis is a kind of common disease, frequently-occurring disease clinically, and its common cause of disease is that atherosclerotic causes luminal stenosis and cerebral thrombosis, causes local cerebral blood supply district blood flow ceases, and brain tissue ischemia, anoxic, softening necrosis occur.Large quantity research indicates that atherosclerotic itself is the process of an inflammation, and lasting low-level inflammation imply that the possibility that cardiovascular and cerebrovascular disease occurs.Lp-PLA2 is the marker of inflammation recently found, belongs to the non-calcium ionic dependent type phosphate in phospholipase A2 superfamily, primarily of inflammatory cell (as macrophage, lymphocyte etc.) secretion, and is subject to the adjustment of inflammatory mediator.CRP is the non-specific markers of a kind of systemic inflammatory reaction acute stage of being synthesized by liver, is one of the most strong predictor of cardiovascular and cerebrovascular disease, has best prediction and outcome to cardiovascular, the cerebrovascular and peripheral artery disease.
There are some researches show, after cerebral arterial thrombosis occurs, patient Lp-PLA2 and CRP level all obviously raise, and its level increases with neurological functional deficit and in increasing trend, difference has statistical significance.Serum CRP level is also significant to prediction cerebral infarction volume simultaneously.Detect the order of severity contributing to judging more all sidedly cerebral infarction while Lp-PLA2 and CRP and provide laboratory reference to its control and prognosis.
At present the diagnosis of cerebral arterial thrombosis not yet be there is to the detection method of being combined by Lp-PLA2 and CRP two indices.The present invention by realizing the judgement to cerebral infarction volume and neurological functional deficit to the detection of two indices, for evaluating the state of an illness and judging prognosis provides help.
Summary of the invention
Goal of the invention: the invention reside in detection method and kit that lipoprotein phospholipase A2 and c reactive protein content in a kind of Simultaneously test blood plasma are provided, by setting up a kind of immune analysis method of Two indices joint-detection, for the clinical analysis of cerebral arterial thrombosis, comprise the judgement of neurological functional deficit and Infarction volume size, provide a kind of highly sensitive, accuracy good and kit assay method easily and fast.
Technical scheme: for solving above-mentioned technical purpose, the present invention proposes the detection method of lipoprotein phospholipase A2 and c reactive protein content in a kind of Simultaneously test blood plasma, comprise the steps: to be coated with at the same time on the microwell plate of anti-Lp-PLA2 monoclonal antibody a and anti crp monoclonal antibody c, add Lp-PLA2 and CRP standard items or EDTA anticoagulated blood centrifugal after the serum sample that obtains in respective micropore, wash after oscillating reactions, add the anti-Lp-PLA2 monoclonal antibody b of fluorescent material mark and the anti crp monoclonal antibody d of fluorescent material mark, oscillating reactions, cleansing solution washs, measure its fluorescence intensity cps with luminoscope, fluorescence intensity, reference standard curve can determine Lp-PLA2 and CRP content in sample.
Wherein, described fluorescent material is any one in fluorescent dye, lanthanide chelate or quantum dot.
Described anti-Lp-PLA2 monoclonal antibody a identifies the different epi-position of Lp-PLA2 respectively from anti-Lp-PLA2 monoclonal antibody b and is independent of each other, wherein, described anti-Lp-PLA2 monoclonal antibody a and anti-Lp-PLA2 monoclonal antibody b is prepared by monoclonal hybridoma technology or is obtained by phage antibody technology screening.
Described anti crp monoclonal antibody c and anti crp monoclonal antibody d identifies the different epi-position of CRP respectively and is independent of each other, wherein, described anti crp monoclonal antibody c and anti crp monoclonal antibody d is prepared by monoclonal hybridoma technology or is obtained by phage antibody technology screening.
Described cleansing solution is the 0.01M phosphate buffer (1 × PBST) containing 0.05wt%TWEEN-20.
Particularly, anti-Lp-PLA2 monoclonal antibody a and anti crp monoclonal antibody c is coated in the optional position in plate hole after mixing, and anti-Lp-PLA2 monoclonal antibody b and the different fluorescent material of anti-Lp-PLA2 monoclonal antibody d mark; Or anti-Lp-PLA2 monoclonal antibody a and anti crp monoclonal antibody c is coated in the diverse location in plate hole respectively, and anti-Lp-PLA2 monoclonal antibody b marks with the identical fluorescent material of anti-Lp-PLA2 monoclonal antibody d.
The kit of lipoprotein phospholipase A2 and c reactive protein content in detection assay blood plasma while of invention also provides a kind of, it comprises as lower part: be coated with the anti crp monoclonal antibody d solution of the microwell plate of anti-Lp-PLA2 monoclonal antibody a and anti crp monoclonal antibody c, the anti-Lp-PLA2 monoclonal antibody b of mark fluorescent material and mark fluorescent material, Lp-PLA2 and CRP positive quality control serum, the negative quality controlled serum of Lp-PLA2 and CRP and concentrated washing lotion simultaneously.
Wherein, described anti-Lp-PLA2 monoclonal antibody a identifies the different epi-position of Lp-PLA2 respectively from anti-Lp-PLA2 monoclonal antibody b and is independent of each other, wherein, described anti-Lp-PLA2 monoclonal antibody a and anti-Lp-PLA2 monoclonal antibody b is prepared by monoclonal hybridoma technology or is obtained by phage antibody technology screening.
Described anti crp monoclonal antibody c and anti crp monoclonal antibody d identifies the different epi-position of CRP respectively and is independent of each other, wherein, described anti crp monoclonal antibody c and anti crp monoclonal antibody d is prepared by monoclonal hybridoma technology or is obtained by phage antibody technology screening.
Described Lp-PLA2 and CRP positive quality control serum adds Lp-PLA2 and CRP standard items in adding respectively in normal human serum to obtain, and addition is 3 times of the critical value of Lp-PLA2 and CRP content in normal serum; Wherein, normal serum Lp-PLA2Lp-PLA2 and CRP content critical value be respectively 175ng/ml and 1 μ g/ml; The negative quality controlled serum of described Lp-PLA2 and CRP is normal human serum, wherein, Lp-PLA2 content lower than 175ng/ml, CRP content lower than 1 μ g/ml.
Wherein, the concentrated washing lotion in described method or kit is the 0.2M phosphate buffer (20 × PBST) containing 1wt%TWEEN-20.Described cleansing solution is the 0.01M phosphate buffer (1 × PBST) containing 0.05wt%TWEEN-20.
Beneficial effect: compared with prior art, the present invention has advantage:
(1) board-like luminescence method detects Lp-PLA2 and CRP two indices and has high specificity, the feature that highly sensitive, accuracy is high, and comparing ELISA method can be quantitative, compares latex enhancing immune turbidimetry cost lower.
(2) Lp-PLA2 detects separately the defect degree can only pointing out nervous function to cerebral arterial thrombosis, can not reflect the size of Infarction volume.CRP is Acute reaction protein, close with injuring relation, and research shows the index that can be used as prediction cerebral infarction volume size and the order of severity.Therefore, the joint-detection of this two indices has more clinical meaning to ischemic cerebral stroke patients.
(3) the Two indices associated detecting method based on board-like luminescence method has unified the detection method of two indices, they can be detected in same reaction system, save the consumption of sample, also saved human and material resources and financial resources simultaneously.
Accompanying drawing explanation
Fig. 1 carries out joint-detection schematic diagram with different fluorescent materials.
Fig. 2 is for carry out joint-detection schematic diagram with position encoded in plate hole.
Embodiment
The preparation of embodiment 1 serum Lp-PLA2 and CRP two yoke plate formula luminescence method detection kit.
A kind of for while detection assay blood plasma in the kit of lipoprotein phospholipase A2 and c reactive protein content, comprise as lower component: be coated with simultaneously anti-Lp-PLA2 monoclonal antibody a (be hereinafter called for short monoclonal antibody a) with 96 orifice plates of anti crp monoclonal antibody c (being hereinafter called for short monoclonal antibody c), the anti-Lp-PLA2 monoclonal antibody b (being hereinafter called for short monoclonal antibody b) of mark fluorescent material and anti crp monoclonal antibody d (being hereinafter the called for short monoclonal antibody d) solution of mark fluorescent material, Lp-PLA2 and CRP positive quality control serum, the negative quality controlled serum of Lp-PLA2 and CRP and concentrated washing lotion.Wherein, Lp-PLA2 and CRP positive quality control serum adds Lp-PLA2 and CRP standard items in adding respectively in normal human serum to obtain, and addition is 3 times of the critical value of Lp-PLA2 and CRP content in normal serum; Wherein, normal serum Lp-PLA2Lp-PLA2 and CRP content critical value be respectively 175ng/ml and 1 μ g/ml; The negative quality controlled serum of Lp-PLA2 and CRP is normal human serum, wherein, Lp-PLA2 content lower than 175ng/ml, CRP content lower than 1 μ g/ml.
Wherein, various piece is prepared by the following method:
(1) the monoclonal antibody a of Lp-PLA2 different loci and the preparation of monoclonal antibody b is identified: two kinds of monoclonal antibodies all can be prepared by monoclonal hybridoma technology (increase preservation hybridoma cell strain by increment cultivation, then by the monoclonal antibody in sad-saturated ammonium sulfate method purifying nutrient solution) or obtained (expression vector by the high-affinity clone structure obtained after screening being obtained after transfection, expression, purification the monoclonal antibody of high-affinity) by phage antibody technology screening.In the present invention, Lp-PLA2 monoclonal antibody A selects anti-human lipoprotein phospholipase A2 (Lp-PLA 2) monoclonal antibody (article No. CLM0019006, buy from constant engineering in medicine company limited of Shenzhen), Lp-PLA2 monoclonal antibody B selects anti-human lipoprotein phospholipase A2 (Lp-PLA 2) monoclonal antibody (article No. CLM0019005, buy from constant engineering in medicine company limited of Shenzhen), two kinds of monoclonal antibodies identify the different epi-positions of Lp-PLA2 respectively.Certainly, two kinds of monoclonal antibodies can exchange use, as long as ensure the different epi-positions of two kinds of monoclonal antibody identification Lp-PLA2.
(2) the monoclonal antibody c of CRP different loci and the preparation of monoclonal antibody d is identified: two kinds of monoclonal antibodies can also be prepared by monoclonal hybridoma technology (increase preservation hybridoma cell strain by increment cultivation, then by the monoclonal antibody in sad-saturated ammonium sulfate method purifying nutrient solution) or obtained (expression vector by the high-affinity clone structure obtained after screening being obtained after transfection, expression, purification the monoclonal antibody of high-affinity) by phage antibody technology screening.Anti crp monoclonal antibody c (article No. CLM0032003 is selected respectively in the present invention, buy from constant engineering in medicine company limited of Shenzhen) and anti crp monoclonal antibody d (article No. CLM0032005, buy from constant engineering in medicine company limited of Shenzhen), two kinds of monoclonal antibodies identify the different epi-position of CRP respectively and are independent of each other.
The antibody bag quilt of (3) 96 microwell plates:
Method 1) two kinds of monoclonal antibodies are fixed on optional position in a micropore, with reference to Fig. 1.Become bag to be monoclonal antibody a and the monoclonal antibody c coating buffer of 1ng/mL by concentration with the MES buffer that 0.1M pH value is 6.0, and coating buffer is carried on microwell plate, 2-8 DEG C is wrapped by 18-24 hour.
Method 2) two kinds of monoclonal antibodies are fixed on fixed position in a micropore, with reference to Fig. 2.Become bag to be the monoclonal antibody a coating buffer of 1ng/mL by concentration with the MES buffer that 0.1M pH value is 6.0, and coating buffer is carried on the A place, position of microwell plate, same method for coating makes monoclonal antibody c coating buffer, is carried on the B place, position of microwell plate.2 ~ 8 DEG C of bags are by 18-24 hour.
(4) fluorescence labeling of monoclonal antibody b and monoclonal antibody d:
Corresponding to previous step (3), according to method 1), then monoclonal antibody b Cy3 dye marker, monoclonal antibody d Cy5 marks; According to method 2), then monoclonal antibody b and monoclonal antibody d can adopt Cy3 dyestuff to mark jointly.
Be labeled as example with the Cy3 dye marker of monoclonal antibody b, concrete operation method is: loaded in bag filter by monoclonal antibody b solution, and the 0.01M pH9.4 carbonate reaction be placed in containing Cy3 is spent the night.
(5) quality controlled serum of Lp-PLA2 and CRP: positive quality control serum is that (Lp-PLA2 is lower than 175ng/ml at normal human serum, CRP is lower than 1 μ g/ml) in add Lp-PLA2 and CRP standard items and obtain, addition is the critical value (being respectively 175ng/ml and 1 μ g/ml) of 3 times.Negative quality controlled serum is normal human serum (Lp-PLA2 lower than 175ng/ml, CRP lower than 1 μ g/ml).
(6) the 0.2M phosphate buffer (20 × PBST) of concentrated cleaning solution: 1wt%TWEEN-20
The course of work and principle:
The present invention adopts board-like luminescence method to utilize the content of mentioned reagent box joint-detection Lp-PLA2 and CRP, as shown in Figure 1, comprises the steps:
1) monoclonal antibody a and monoclonal antibody c is coated on simultaneously the optional position of micropore;
2) in 7 continuous plate holes, add standard items 1-5 and positive, negative quality controlled serum respectively, hatch 30min for 37 DEG C.In standard items 1-5, the content of Lp-PLA2 and CRP is respectively 1. 50ng/ml and 100ng/ml, 2. 300ng/ml and 400ng/ml, 3. 800ng/ml and 1000ng/ml, 4. 1500ng/ml and 2000ng/ml, 5. 3 μ g/ml and 3.5 μ g/ml;
3) add monoclonal antibody b and the monoclonal antibody d of different fluorescent material mark after washing plate, hatch 30min for 37 DEG C.Wherein, monoclonal antibody b Cy3 dye marker, monoclonal antibody d Cy5 dye marker;
4) reaction terminates rear washing, then reads the emitted luminescence intensity under different exciting light (554nm and 648nm) respectively, and draws the typical curve of Lp-PLA2 and CRP respectively.
The accuracy of embodiment 2 serum Lp-PLA2 and CRP two yoke plate formula luminescence method detection kit, degree of accuracy and stability test.
(1) accuracy and precision
Select the standard items of high, medium and low three concentration, respectively containing Lp-PLA2 and CRP concentration is 800ng/ml and 1000ng/ml, 300ng/ml and 450ng/ml, 50ng/ml and 100ng/ml.(Lp-PLA2 ELISA method is detected with the conventional sense kit on the board-like luminescence reagent box of the present invention's design and market, detect CRP latex enhancing immune turbidimetry) detect simultaneously, each Concentration Testing 10 times, acquired results is compared, determine accuracy and the precision of this kit, the testing result obtained is as following table:
Table 1.Lp-PLA2 testing result compares
This group data are done to the T inspection of paired sample, obtain P > 0.05, draw the difference not statistically significant between two groups of results, illustrate two kinds of methods no matter high, in or during the Lp-PLA2 of low concentration detects, all have good consistance, the accuracy of the detection method of this patent design is similar to commercially available detection kit.
Table 2.CRP testing result compares
This group data are done to the T inspection of paired sample, obtain P > 0.05, draw the difference not statistically significant between two groups of results, illustrate two kinds of methods no matter high, in or during the CRP of low concentration detects, all have good consistance, the accuracy of the detection method of this patent design is similar to commercially available detection kit.
Meanwhile, the coefficient of variation of this method duplicate detection result of two indexes under many concentration is all less than 10%, has better precision.
(2) stabilization of kit test:
Kit preservation condition is 2-8 DEG C, preserves after 6 months, measures the indices of kit, finds all within normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 6 days under 37 DEG C of conditions of preserving by kit, carry out accelerated aging tests, result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 DEG C of refrigerator freezings 5 days, measurement result also shows that kit indices is completely normal.Can show that kit at least can preserve more than 6 months at 2 ~ 8 DEG C from above result.
The above is full of the preferred embodiment of the present invention; it should be pointed out that for the person of ordinary skill of the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the detection method of lipoprotein phospholipase A2 and c reactive protein content in a Simultaneously test blood plasma, it is characterized in that, comprise the steps: to be coated with at the same time on the microwell plate of anti-Lp-PLA2 monoclonal antibody a and anti crp monoclonal antibody c, add Lp-PLA2 and CRP standard items or EDTA anticoagulated blood centrifugal after the serum sample that obtains in respective micropore, wash after oscillating reactions, add the anti-Lp-PLA2 monoclonal antibody b of fluorescent material mark and the anti crp monoclonal antibody d of fluorescent material mark, oscillating reactions, cleansing solution washs; Measure its fluorescence intensity cps with luminoscope, fluorescence intensity, reference standard curve can determine Lp-PLA2 and CRP content in sample.
2. detection method according to claim 1, is characterized in that, described fluorescent material is any one in fluorescent dye, lanthanide chelate or quantum dot.
3. detection method according to claim 1, it is characterized in that, described anti-Lp-PLA2 monoclonal antibody a identifies the different epi-position of Lp-PLA2 respectively from anti-Lp-PLA2 monoclonal antibody b and is independent of each other, wherein, described anti-Lp-PLA2 monoclonal antibody a and anti-Lp-PLA2 monoclonal antibody b is prepared by monoclonal hybridoma technology or is obtained by phage antibody technology screening.
4. detection method according to claim 1, it is characterized in that, described anti crp monoclonal antibody c and anti crp monoclonal antibody d identifies the different epi-position of CRP respectively and is independent of each other, wherein, described anti crp monoclonal antibody c and anti crp monoclonal antibody d is prepared by monoclonal hybridoma technology or is obtained by phage antibody technology screening.
5. detection method according to claim 1, is characterized in that, described cleansing solution is the 0.01M phosphate buffer (1 × PBST) containing 0.05wt%TWEEN-20.
6. detection method according to claim 1, it is characterized in that, anti-Lp-PLA2 monoclonal antibody a and anti crp monoclonal antibody c is coated in the optional position in plate hole after mixing, and anti-Lp-PLA2 monoclonal antibody b and the different fluorescent material of anti-Lp-PLA2 monoclonal antibody d mark; Or anti-Lp-PLA2 monoclonal antibody a and anti crp monoclonal antibody c is coated in the diverse location in plate hole respectively, and anti-Lp-PLA2 monoclonal antibody b marks with the identical fluorescent material of anti-Lp-PLA2 monoclonal antibody d.
7. the simultaneously kit of lipoprotein phospholipase A2 and c reactive protein content in detection assay blood plasma, it is characterized in that, comprise as lower part: be coated with the anti crp monoclonal antibody d solution of the microwell plate of anti-Lp-PLA2 monoclonal antibody a and anti crp monoclonal antibody c, the anti-Lp-PLA2 monoclonal antibody b of mark fluorescent material and mark fluorescent material, Lp-PLA2 and CRP positive quality control serum, the negative quality controlled serum of Lp-PLA2 and CRP and concentrated washing lotion simultaneously.
8. kit according to claim 6, it is characterized in that, described anti-Lp-PLA2 monoclonal antibody a identifies the different epi-position of Lp-PLA2 respectively from anti-Lp-PLA2 monoclonal antibody b and is independent of each other, wherein, described anti-Lp-PLA2 monoclonal antibody a and anti-Lp-PLA2 monoclonal antibody b is prepared by monoclonal hybridoma technology or is obtained by phage antibody technology screening.
9. kit according to claim 6, it is characterized in that, described anti crp monoclonal antibody c and anti crp monoclonal antibody d identifies the different epi-position of CRP respectively and is independent of each other, wherein, described anti crp monoclonal antibody c and anti crp monoclonal antibody d is prepared by monoclonal hybridoma technology or is obtained by phage antibody technology screening.
10. kit according to claim 6, it is characterized in that, described Lp-PLA2 and CRP positive quality control serum adds Lp-PLA2 and CRP standard items in adding respectively in normal human serum to obtain, and addition is 3 times of the critical value of Lp-PLA2 and CRP content in normal serum; Wherein, normal serum Lp-PLA2Lp-PLA2 and CRP content critical value be respectively 175ng/ml and 1 μ g/ml; The negative quality controlled serum of described Lp-PLA2 and CRP is normal human serum, wherein, Lp-PLA2 content lower than 175ng/ml, CRP content lower than 1 μ g/ml.
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| CN104849456A (en) * | 2015-05-18 | 2015-08-19 | 北京协和洛克生物技术有限责任公司 | Reagent kit for detecting concentration of Lp-PLA2 and preparing method thereof |
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| CN109521200A (en) * | 2018-12-29 | 2019-03-26 | 南京新耀医疗技术有限公司 | It is a kind of while detecting the kit of Multiple components content, method and its application in blood plasma |
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