CN107490692A - A kind of fluorescence immune chromatography method for quantitatively detecting hs-CRP and lipoprotein phospholipase A2 - Google Patents

A kind of fluorescence immune chromatography method for quantitatively detecting hs-CRP and lipoprotein phospholipase A2 Download PDF

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CN107490692A
CN107490692A CN201610401854.XA CN201610401854A CN107490692A CN 107490692 A CN107490692 A CN 107490692A CN 201610401854 A CN201610401854 A CN 201610401854A CN 107490692 A CN107490692 A CN 107490692A
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crp
antibody
fluorescent dye
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李亚星
刘凤鸣
刘冰
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CHANGZHOU BIOWIN BIOPHARM Co Ltd
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    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

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Abstract

The present invention relates to the technical field of immune detection analysis, especially a kind of fluorescence immune chromatography method for quantitatively detecting hs-CRP and lipoprotein phospholipase A2, includes blood, anti-hs-CRP antibody, anti-grease protein, phospholipid enzyme A2 antibody, fluorometric reagent and detector.Anti- hs-CRP antibody is one kind or the combination of anti-hs-CRP monoclonal antibody and anti-hs-CRP polyclonal antibody；Anti-grease protein, phospholipid enzyme A2 antibody is one kind or the combination of anti-grease protein, phospholipid enzyme A2 monoclonal antibodies and anti-grease protein, phospholipid enzyme A2 polyclonal antibodies；Fluorometric reagent is liquid phase and/or solid phase material containing the fluorescent material, and fluorescent material is one kind or the combination of organic fluorescent dye or rare earth element fluorescent dye；Detector is fluorescence detector.Using blood of human body as detection sample, it is reproducible can effectively to monitor hs-CRP and lipoprotein phospholipase A2, high specificity in blood, high sensitivity, it is simple to operate, special instruments and equipment is not required to, is not required to professional training, as a result clearly easily distinguish, it is easy to spread, it is suitable for Site Detection.

Description

A kind of fluorescence immunoassay for quantitatively detecting hs-CRP and lipoprotein phospholipase A2 Chromatography method
Technical field
The invention belongs to technical field of immune assay, more particularly to a kind of quantitatively detection hs-CRP and fat Protein, phospholipid enzyme A2 fluorescence immunoassay method.
Background technology
Cardiovascular and cerebrovascular disease is the serious threat mankind, particularly the common disease of the elderly's health, and the whole world is died from every year The number of cardiovascular and cerebrovascular diseases is up to million people, and it is the first to occupy the various causes of the death.Confirmed through research, cardiovascular and cerebrovascular disease occurrence and development are very big It is related with atherosclerosis (Atherosclerosis, AS) in degree.The assessment AS of qualitative, quantitative seriousness, contributes to It is effective to predict and judge cardiovascular and cerebrovascular embolism state, prevent the generation of cardiocerebrovasculaevents events.AS is a kind of inflammatory disease, inflammation Reaction take part in the links of its generation, be risen in AS starting, development and loss of stability and plaque rupture come off Important function.Lp-PLA2 is generally acknowledged a kind of new inflammatory reaction mark in the world at present, the Lp-PLA2 master in blood To be synthesized and be secreted by inflammatory cells such as monocytes/macrophages and lymphocytes.Lp-PLA2 contents are that prediction is cardiovascular in blood The risk profile factor of the diseases such as disease, coronary heart disease, cerebral apoplexy.Lp-PLA2 is also known as platelet-activating factor acetylhydrolase, is one Two acyl group ester bonds of glycerophosphatide that class is catalyzed on lipoprotein and cell membrane hydrolyze to form non-esterified fatty acid and lysophosphatide Enzyme race, belong to the phospholipase A2 superfamily of expansion, but it is different from other phospholipase A2 superfamily members, and its bioactivity is disobeyed Rely in calcium ion.Lp-PLA2 encoding genes (PLA2G7) are cloned first in nineteen ninety-five, are had 12 extrons, are positioned at No. 6 Chromosome p21.2~12, a kind of serine easterase being made up of 441 amino acid residues, molecular weight are 45 KDa.Lp-PLA2 It is divided to two classes, i.e., the Lp-PLA2 of secreting type in the circulating cycle and the Lp-PLA2 being present in atherosclerotic plaque.Circulate secreting type Lp-PLA2 about 70%~80% is combined with low-density lipoprotein LDL, 20%~30% with HDL or other lipoprotein knots Close.Lp-PLA2 is combined with LDL, generation lysolecithin (Lysophosphatidylcholine, Lyso-PC) and oxidized form trip From aliphatic acid (Oxidized Free Fatty Acids, Ox-FA), both are proinflammatory material, by stimulate generation stick because Son and cell factor, and promote monocyte to be assembled from lumen of vessels to endangium.Then, after monocyte is assembled under inner membrance Derivative is macrophage.Last macrophage phagocytosis oxidized low-density lipoprotein becomes foam cells, is finally gathered into AS spots Block.AS because its popularization it is wide, human body intrinsic incubation length, finally often with ischaemic, angina pectoris, miocardial infarction, apoplexy, The fatal sick form outburst such as coronary heart disease or heart failure.Basically the generation of cardiocerebrovasculaevents events is largely depended on In the stability of AS patches.Lp-PLA2 active reaction atherosclerotic lesion degree, it is relevant with the stability of vulnerable plaque, Relative to stability patch, the Lp-PLA2's of Vulnerable plaque is active significantly raised.Lp-PLA2 and other inflammatory factors such as C- Reactive protein etc. is compared, smaller because being influenceed by other hazards, and then detection has higher sensitivity and specificity.
CRP is a kind of Υ globulin, molecular weight 105KD, it is that plasma concentration is quick in infection and tissue damage, it is anxious Acute elevated main acute phase protein.CRP can play opsonic action with the phagocytosis of activating complement and reinforcement phagocyte, so as to Pathogenic microorganism and the damage of invasion body are removed, necrosis, the histocyte of apoptosis, is played during the innate immunity of body Important protective effect.Nineteen thirty, it was discovered by researchers that the serum of some acute infection patients can be with S. pneumoniae capsular C Precipitation reaction occurs for polysaccharide, and confirm to cause precipitation reaction is a kind of protein, and Acute and chronic inflammation exist it is such a anti- Should.This nonspecific inflammation albumen is referred to as C reactive protein（CRP）.CRP is by 5 same monomers containing 206 amino acid Formed with non-covalent bond, the calcium coupling collar containing crystalline in the homotaxis amyloid P.CRP structures of albumen, one sub- single Position and the calcium position of another subunit are connected to pentamer structure.CRP stimulates liver and epithelial cell by inflammatory lymphokine Synthesis, CRP congenital deficiencies are not yet found at present.CRP coordinates compound by end eventually（C5b-9）Participate in the formation of early stage AS. At the fragile position of plaque surface, monocyte（Macrophage）, T lymphocytes largely penetrate into, make patch unstable and rupture. In this process interleukins（IL）- 6 cause liver synthesis CRP increases.Atherosclerosis (atherosclerosis) patch Interior CRPmRNA is high 7~10 times compared with natural arterial and liver.The complement and CRP of activation, make patch unstable and broken in AS patches Split, ultimately result in thrombosis.Therefore, elevated CRP, which has, causes thrombotic danger.Clinically hs-CRP （hs-CRP）The significant rise in severe coronary artery disease sudden death, the intensity sum of hs-CRP and the immunohistochemical staining thin caps of AS Measure relevant.In a word, hs-CRP and AS degree is closely related, turns into the index of reflection AS inflammatory activities.
Both are combined, AS seriousness can more accurately and effectively be assessed by carrying out joint-detection, diagnose myocardial damage And its degree.
The detection methods of Lp-PLA2 clinically, mainly there is immunoturbidimetry, chemoluminescence method, ELISA and glue Body gold method.Hs-CRP detection method has simple immunodiffusion method, Latex Agglutination, enzyme-linked absorption method, rate nephelometry Deng.Colloidal gold method is one of the most commonly used quick detection means.Therefore, it is quick, easy, accurate using colloidal gold method, exploitation one The diagnostic kit of true and low-cost cardiovascular and cerebrovascular disease has important clinical meaning.
The content of the invention
The purpose of the present invention is to be directed to above-mentioned the deficiencies in the prior art, there is provided a kind of high sensitivity, accuracy be strong, Fluorescence immune chromatography detection method that is easy to operate, being capable of quick diagnosis hs-CRP and lipoprotein phospholipase A2.
In order to solve the above technical problems, the present invention uses fluorescence immune chromatography technology, specific technical scheme is：To solve Above-mentioned technical problem, the present invention use fluorescence immune chromatography technology, and specific technical scheme is：It is anti-that one kind quantitatively detects super quick C The fluorescence immune chromatography method of albumen and lipoprotein phospholipase A2 is answered, includes blood, anti-hs-CRP antibody, anti-grease Protein, phospholipid enzyme A2 antibody, fluorometric reagent and detector.The hs-CRP antibody is anti-hs-CRP Dan Ke One kind or combination of grand antibody and anti-hs-CRP polyclonal antibody；The anti-grease protein, phospholipid enzyme A2 antibody is anti-grease One kind or combination of protein, phospholipid enzyme A2 monoclonal antibodies and anti-grease protein, phospholipid enzyme A2 polyclonal antibodies；The fluorometric reagent is Liquid phase and/or solid phase material containing the fluorescent material, the fluorescent material are organic fluorescent dye or rare earth element fluorescence One kind of dyestuff or combination；The detector is fluorescence detector.
Further, described detection method comprises the following steps：
Step (1)：With specific effect between chemical crosslinking or biomolecule respectively by conjugated fluorescent dyes to monoclonal antibody, obtain To the monoclonal antibody of fluorescent dye modification, wave-length coverage is 300~1300nm；
Step (2)：The monoclonal antibody that the fluorescent dye that step (1) is obtained is modified is fixed in label pad；
Step (3)：One quantitative detection line and a nature controlling line are set respectively in chromatographic film, wherein, it is fixed with nature controlling line The goat anti-mouse igg that can be combined with monoclonal antibody specificity, reagent strip quantify and polyclonal antibody are fixed with detection line, and should The monoclonal antibody of fluorescent dye modification of the antigenic determinant that specific antibody is identified with being fixed in label pad is identified Antigenic determinant it is different；
Step (4)：Sample pad, label pad, chromatographic film, adsorptive pads and bottom plate are built into fluorescence immune chromatography test paper bar；
Step (5)：Fluorescence immune chromatography test paper bar is loaded.
Conjugated fluorescent dyes are arrived monoclonal antibody by the present invention using specific effect between chemical crosslinking or biomolecule Surface, obtain the monoclonal antibody of fluorescent dye modification.In the present invention, can when fluorescent dye surface has active group It is directly reacted with specific antibody, is not required to use chemical cross-linking agent；Conversely, then need to be coupled by chemical cross-linking agent.Wherein, change Learn crosslinking agent include 1- ethyls -3- (3- DimethylAminopropyls) carbodiimides (EDC), n-hydroxysuccinimide (NHS) and Glutaraldehyde etc..
In a preferred embodiment of the invention, monoclonal antibody, step are modified using the fluorescent dye of active group Suddenly it is：Fluorescent dye after purification is dissolved, then adds a certain amount of monoclonal antibody, using buffer solution as reaction medium, Reaction 2~4 hours, hydroxylamine hydrochloride terminating reaction is added, purified with modes such as chromatogram, chromatographic column or ultrafiltration centrifugations, obtain fluorescence The monoclonal antibody of dyestuff modification.
In order to improve the discrimination of signal and background, the wave-length coverage of fluorescent dye is 300~1300nm in the present invention, excellent Elect 550-800nm as.In addition, chromatographic film, bottom plate and buckle are typically extremely weak near infrared region (600~800nm) fluorescence intensity, It is therefore preferable that wave-length coverage is 550-800nm fluorescent dye further to improve sensitivity, more preferably fluorescent dye transmitted wave A length of 665nm.
Fluorescent dye includes organic fluorescent dye and rare earth element fluorescent dye.Fluorescent dye can be single compound Fluorescent dye or by several compound groups into composite fluorescent dye, but preferably single compound fluorescent dye and preferred tool There is the fluorescent dye of stronger photostability.
In order to reduce the influence to fluorescent dye fluorescence signal, the present invention uses chromatographic film, bottom plate and the button of hypofluorescence Card, its fluorescent noise be more than 550nm can be very weak, can good discrimination signal and the back of the body so as to ensure to obtain high fluorescence signal-to-background ratio Scape, and then improve detection sensitivity.It is preferred that bottom plate is white, surface has an adhesive sticker, and buckle, chromatographic film, bottom plate and does not do Glue does not contain fluorescer.
The fluorescence immune chromatography kit of the present invention is to the hs-CRP in blood sample and lipoprotein phospholipase A2 Carry out quantitative detection.During detection, blood sample is added in sample pad by well, sample is along chromatographic film to adsorptive pads side Moved to chromatography.The sample chromatography time is usually 8~25 minutes, preferably 15 minutes.After chromatography, with Wash buffer solution for cleaning layers Film is analysed, the time is 3-8 minutes, preferably 5 minutes, to reduce background, improves detection sensitivity.Wrapped in the Wash buffer solutions of the present invention Containing bovine serum albumin(BSA), sucrose and surfactant, pH value 7.0-8.0, wherein, the concentration of bovine serum albumin(BSA) for 0.05~ 2%, the concentration of sucrose is 1~15%, and the concentration of surfactant is 0.05~2%.The preferred polysorbas20 of surfactant, Qula Logical X-100, the preferred Tris-HCl buffer solutions of buffer solution, phosphate buffer.
After chromatography terminates, judge to detect the detection line and nature controlling line in window with fluorescent quantitation instrument, to obtain a result.
Brief description of the drawings
Fig. 1 is hs-CRP linearity test standard working curve figure；
Fig. 2 is lipoprotein phospholipase A2 linearity test standard working curve figure.
Embodiment
Embodiment 1：The preparation of hs-CRP and lipoprotein phospholipase A2 fluorescence immunoassay detection kit
1) coupling of fluorescent dye and hs-CRP antibody
The fluorescent dyes rhodamine that launch wavelength is 665nm is mixed with l mg/ml hs-CRP monoclonal antibody, 3h is reacted at room temperature, adds 1mol/L hydroxylamine hydrochloride terminating reactions, and with chromatographic column or chromatographs column separating purification, obtains fluorescent dye The hs-CRP monoclonal antibody of modification, the launch wavelength of fluorescent dye is 665nm；
2) coupling of fluorescent dye and lipoprotein phospholipase A2 antibody
The lipoprotein phospholipase A2 monoclonal antibody of fluorescent dyes rhodamine and l mg/ml that launch wavelength is 665nm is mixed Close, react at room temperature 3h, add 1mol/L hydroxylamine hydrochloride terminating reactions, and with chromatographic column or chromatograph column separating purification, obtain fluorescence The lipoprotein phospholipase A2 monoclonal antibody of dyestuff modification, fluorescence emission wavelengths 665nm；
3) structure of kit
Fluorochrome label thing is taken, bovine serum albumin(BSA) (content 1%), sucrose (content 10%) and surface is added and lives Property agent triton x-100 (content 0.8%), subsequent even application are sealed after 40 DEG C of dryings in label pad, protected at room temperature Deposit；
Hs-CRP fluorescence immunoassay detection reagent bar is assembled, is made up of sample pad, label pad, chromatographic film, adsorptive pads, it is suitable It is secondary to be pasted on white bottom plate.Wherein, sample pad is poroid barrier film, selects glass fibre, is testing sample collecting region；Pad In containing fluorescent dye modification hs-CRP monoclonal antibody；Quantitative detection line and nature controlling line are fixed with chromatographic film, is examined Survey line and nature controlling line at intervals of 5mm, and detection line is fixed with that to be different from goat-anti hs-CRP in label pad polyclonal Antibody, nature controlling line are fixed with sheep anti-mouse igg；
Lipoprotein phospholipase A2 fluorescence immunoassay detection reagent bar is assembled, is made up of sample pad, label pad, chromatographic film, adsorptive pads, it is suitable It is secondary to be pasted on white bottom plate.Wherein, sample pad is poroid barrier film, selects glass fibre, is testing sample collecting region；Pad In contain fluorescent dye modified lipoprotein phospholipase A2 monoclonal antibody；Quantitative detection line and nature controlling line are fixed with chromatographic film, Detection line and nature controlling line at intervals of 5mm, and detection line is fixed with that to be different from goat-anti human lipoprotein phospholipase A2 in label pad more Clonal antibody, nature controlling line are fixed with sheep anti-mouse igg；
After assembling, required width is cut into as requested, is placed in buckle, drier encapsulation is added, with Wash buffer solutions Hs-CRP and lipoprotein phospholipase A2 fluorescence immunoassay detection kit are configured to jointly.
Embodiment 2：The detection mode of hs-CRP and lipoprotein phospholipase A2 fluorescence immunoassay detection kit
1) blood sample to be detected is taken out；
2) 80ul is added dropwise in well, forward direction chromatography reaction 15min；
3) Wash buffer solutions, pH value 7.5 are prepared, buffer system is 20mM phosphate, and adding bovine serum albumin(BSA), (concentration is 1%), sucrose (concentration 10%) and surfactant triton x-100 (concentration 0.8%), 50 μ L are added in sample well, Stand 5min；
4) it is placed in fluorescent quantitation instrument and obtains fluorescence signal intensity, draws the concentration of institute's sample product.
Embodiment 3：Kit Performance Evaluation is tested
1) drafting of standard working curve：
Take hs-CRP antigen with negative serum be diluted to concentration for 30,20,10,5,0.5,0.1,0ng/mL sample, Take lipoprotein phospholipase A2 antigen with negative serum be diluted to concentration for 800,400,200,100,60,0ng/mL sample, then Its hs-CRP and lipoprotein phospholipase A2 are determined respectively, and to be made into value as X, fluorescence measured value is that Y draws standard work Curve, the expression formula through statistical fit standard working curve list regression equation and are：YHs-CRP=0.016423XHs-CRP+ 0.11849, fitting coefficient square is R2=0.983, as a result see accompanying drawing 1；YLipoprotein phospholipase A2=0.00070XLipoprotein phospholipase A2+ 0.05713, fitting coefficient square is R2=0.985 result is shown in accompanying drawing 2；
Fig. 1 is hs-CRP linearity test standard working curve, and measurement range is 0.1~30ng/ml；
Fig. 2 is lipoprotein phospholipase A2 linearity test standard working curve, lipoprotein phospholipase A2 measurement range is 60~ 800ng/ml；
2) accuracy testing
Same blood samples of patients sample is taken, detection 3 times is repeated with a batch of test strips, calculates relative deviation BHs-CRPValue For 5.23%, BLipoprotein phospholipase A2It is worth for 7.41%, meets product design requirement；
3) replica test
Same blood samples of patients sample is taken, is detected 20 times simultaneously with a batch of test strips, calculates coefficient of variation CVHs-CRP It is worth for 3.75%, CVLipoprotein phospholipase A2It is worth for 4.74%.Meet product design requirement.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.

Claims (4)

1. a kind of fluorescence immune chromatography method for quantitatively detecting hs-CRP and lipoprotein phospholipase A2, include blood, Anti- hs-CRP antibody, anti-grease protein, phospholipid enzyme A2 antibody, fluorometric reagent and detector, have following feature：
1）The hs-CRP antibody is that anti-hs-CRP monoclonal antibody and anti-hs-CRP are polyclonal One kind of antibody or combination；
2）The anti-grease protein, phospholipid enzyme A2 antibody is anti-grease protein, phospholipid enzyme A2 monoclonal antibodies and anti-grease protein, phospholipid enzyme A2 One kind of polyclonal antibody or combination；
3）The fluorometric reagent is liquid phase and/or solid phase material containing the fluorescent material, and the fluorescent material is organic glimmering One kind or combination of photoinitiator dye or rare earth element fluorescent dye；
4）The detector is fluorescence detector.
2. a kind of quantitatively detection hs-CRP according to claim 1 and the inspection of lipoprotein phospholipase A2 fluorescence immunoassay Survey method, it is characterised in that methods described comprises the following steps：
1）Conjugated fluorescent dyes are obtained glimmering to the surface of specific antibody 1 with specific effect between chemical crosslinking or biomolecule The specific antibody carrier of photoinitiator dye modification, the launch wavelength scope of fluorescent dye is 300~1300nm；
2）Prepare fluorescence immune chromatography test paper bar, the fluorescence immune chromatography test paper bar include label pad, chromatographic film, adsorptive pads and Bottom plate；
3）Cleaning buffer solution is configured, the cleaning buffer solution includes bovine serum albumin(BSA), sucrose and surfactant, and pH value is 7.0-8.0, wherein, the concentration of bovine serum albumin(BSA) is 0.05~2%, and the concentration of sucrose is 1~15%, surfactant it is dense Spend for 0.05~2%；
4）By step 1）The specific antibody 1 of obtained fluorescent dye modification is fixed in the label pad；
5）One detection line and a nature controlling line are set respectively in the chromatographic film, wherein, being fixed with nature controlling line can be with institute The biomolecule that the specific antibody carrier of fluorescent dye modification is combined is stated, specific antibody 2 is fixed with detection line；
6）By the label pad, chromatographic film, adsorptive pads and floor combination, fluorescence immune chromatography test paper bar is built into；
7）The blood of collection is loaded into the label pad, the contained antigen and the fluorescent dye in the blood The specific antibody of modification combines, and forms " fluorescent dye-specific antibody 1- antigens " compound, i.e. compound 1；
8）Liquid phase containing the compound 1 flows through chromatographic film, and " fluorescent dye-specific antibody 1- antigens-specificity is anti-for formation The compound of body 2 ", i.e. compound 2, and be captured and be fixed on the immobilon-p；
9）Fluorescent value caused by the fluorescent dye being indirectly fixed on the immobilon-p is detected with the fluorescence detector, with inspection Survey the content of antigen in the blood.
3. a kind of quantitatively detection hs-CRP according to claim 2 and the inspection of lipoprotein phospholipase A2 fluorescence immunoassay Test agent box, it is characterised in that：Fluorescent dye launch wavelength in described label pad is 550-800nm.
4. a kind of quantitatively detection hs-CRP according to claim 2 and the inspection of lipoprotein phospholipase A2 fluorescence immunoassay Test agent box, it is characterised in that：Fluorescence is very weak when more than 550nm or does not contain fluorescer for the chromatographic film and the bottom plate, It is preferred that bottom plate is white, surface has adhesive sticker, and both of which is free of fluorescer.
CN201610401854.XA 2016-06-09 2016-06-09 A kind of fluorescence immune chromatography method for quantitatively detecting hs-CRP and lipoprotein phospholipase A2 Pending CN107490692A (en)

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Publication number Priority date Publication date Assignee Title
CN108414754A (en) * 2018-03-08 2018-08-17 江苏瑞安生物技术有限公司 Detect the chromatography method of anti-grease protein, phospholipid enzyme A2 and the preparation method of kit
CN108872581A (en) * 2018-07-03 2018-11-23 江苏恒易生物科技有限公司 The fluorescence immune chromatography kit and preparation method of super quick CRP, lipoprotein phospholipase A2 and d-dimer are detected simultaneously

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Application publication date: 20171219