CN110514844A - 3 magnetic microparticle chemiluminescence immune quantitative detection reagent boxes of a kind of poly- element of people five and preparation method thereof - Google Patents
3 magnetic microparticle chemiluminescence immune quantitative detection reagent boxes of a kind of poly- element of people five and preparation method thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention belongs to technical field of immune assay, are related to 3 (PTX3) magnetic microparticle chemiluminescence immune quantitative detection reagent boxes of a kind of poly- element of people five and preparation method thereof.The kit includes the coated magnetic particle suspension of Streptavidin, poly- plain 3 monoclonal antibodies of the people five of alkali phosphatase enzyme mark, poly- plain 3 monoclonal antibodies of the people five of biotin labeling, poly- plain 3 calibration objects of people five.For kit of the invention compared with existing ELISA kit currently on the market, detection range is wide, has preferably repeatability and sensitivity.
Description
Technical field
The invention belongs to technical field of immune assay, it is related to detecting people five in human serum or blood plasma and gathers 3 (PTX3) of element
Magnetic microparticle chemiluminescence immune assay kit of content and preparation method thereof.The kit includes that Streptavidin is coated
Magnetic particle suspension, poly- plain 3 monoclonal antibodies of the people five of alkali phosphatase enzyme mark, poly- plain 3 monoclonals of the people five of biotin labeling
Antibody, poly- plain 3 calibration objects of people five.Kit of the invention is compared with existing ELISA kit currently on the market, detection range
Width has preferably repeatability and sensitivity.
Background technique
People five gathers 3 (Pentraxin3, PTX3) of element and belongs to pentraxins family member, and also known as tumor necrosis factor stimulates base
It is a kind of typical acute phase protein because of 14 (TSG14).PTX3 can be combined as unique soluble pattern recognition receptors
A variety of soluble receptor ligands participate in immune defense, inflammation, Apoptosis, vascular remodeling, female reproduction and atherosclerosis
Etc. a variety of biological effects.Recently studies have shown that is compared with c reactive protein (CRP), PTX3 more can response organization part rapidly inflammation
Disease and damage are expected to become clinical more sensitive serologic marker and have attracted much attention.
Pentraxins include long-chain family and short chain family member.PTX3 is the long-chain pentraxins found earliest,
Containing 381 highly conserved amino acid long-chains, including 17 signal position peptides, wherein 203, N-terminal region amino acid and short chain
Family member CRP is homologous, with serum amyloid P component it is homologous be 178 amino acid peptide chain regions of C-terminal, molecular weight
About 40KD, peptide chain are longer than serum amyloid P component and CRP in N-terminal part.
PTX3 is a kind of multifunctional protein, and different effects is played in many diseases.In autoimmune disease and inflammation
In disease, such as the level and the activity of its disease and the reactive phase for the treatment of in rheumatoid arthritis and vasculitis patients serum
It closes.PTX3 is likely to become a kind of new biological markers of Clinical significance of detecting different from CRP, is used for clinical diagnosis, evaluates disease
Sick activity and seriousness, monitoring therapeuticing effect predict many aspects such as generation and the risk assessment of cardiovascular complication.Closely
Year document report: in certain autoimmune diseases and inflammatory injury model, PTX3 has played protectiveness effect, limitation damage
The occurrence and development of wound.
The quick diagnosis reagent kit for lacking the poly- element 3 of people five clinical at present.Existing basis and clinical research are to utilize life
The kit for being exclusively used in scientific research that object scientific & technical corporation provides detects (cannot be used for clinical diagnosis).These kits are mostly using enzyme
Join immunoadsorbent technics, detection time is relatively long (to be extracted microplate reader from sample and read absorbance value, then need to quantitative calculate
Want 5-8h), and it is with high costs.The marker of ideal reflection organ dysfunction should take into account sensibility and specificity, have accurate, letter
Just, the features such as inexpensive, noninvasive.Therefore, using modern science and technology, the clinical diagnosis examination of each index in Joint Index collection is developed
Agent box has great clinical meaning and economic significance, and has original creativity.
The PTX3 kit of invention utilizes chemiluminescence immunoassay point using magnetic microparticles as the solid phase of immune response
Analysis method and chemiluminescence class analyzer cooperate, for measuring poly- plain 3 contents of the people five in human serum/blood plasma, the kit
Cheap compared with the ELISA kit for being exclusively used in scientific research at present, high sensitivity is reproducible, and speed is fast.
Summary of the invention
In consideration of it, the present invention provides chemiluminescence immunoassay immue quantitative detection reagent box and its preparation side of a kind of poly- element 3 of people five
Method avoids the disadvantages of range of linearity existing for enzyme linked immunological kit is narrow, cumbersome, and it is low, stable to solve sensitivity
The defects of property is poor, at high cost.
In order to achieve the above objectives, the invention provides the following technical scheme:
The invention discloses a kind of preparation processes, and simple, low in cost, high sensitivity people five gathers plain 3 chemiluminescence immunoassays
Immue quantitative detection reagent box, including the coated magnetic particle suspension of Streptavidin, the poly- element 3 of the people five of alkali phosphatase enzyme mark are anti-
Body, poly- plain 3 antibody of the people five of biotin labeling, the poly- plain 3 antibody calibration objects of people five.
Preferably, the coated magnetic particle suspension of the Streptavidin is surface package with the super suitable of Streptavidin
Magnetic Nano material, partial size be 10~30nm, be suspended in magnetic particle coating object buffer in, concentration be 0.1mg/mL~
1.0mg/mL, preferred concentration are 0.5mg/mL；It is 20mM~200mM Tris (trihydroxy methyl that magnetic particle, which is coated with object buffer,
Aminomethane) buffer, PH range is 6.5~8.0, and preferred concentration is 100mM, and preferred PH is 7.0.
Preferably, poly- plain 3 antibody of the people five of the alkali phosphatase enzyme mark are alkaline phosphatase and poly- plain 3 antibody of people five
Conjugate, wherein poly- plain 3 antibody of people five are mouse monoclonal antibody.It is diluted in enzyme marker buffer, dilution ratio is
1:400~1:2000, preferred dilution ratio are 1:800；Enzyme marker buffer is 20mM~200mM Tris buffer,
PH range is 6.5~8.0, and preferred concentration is 20mM, and preferred PH is 7.4.
Preferably, poly- plain 3 antibody of the people five of the biotin labeling are the conjugate of biotin and poly- plain 3 antibody of people five,
Wherein poly- plain 3 antibody of people five are mouse monoclonal antibody.It is diluted in biotinylated derivative buffer, dilution ratio 1:200
~1:1000, preferred dilution ratio are 1:500；Biotinylated derivative buffer is 20mM~200mM Tris buffer, PH
Range is 6.5~8.0, and preferred concentration is 20mM, and preferred PH is 8.0.
Preferably, poly- plain 3 calibration objects of people five are the calibration object buffer containing 20% fetal calf serum, by poly- plain 3 antigens of people five
It is diluted to working concentration, respectively 0,0.50,5.00,25.00,50.00,100.00ng/mL；Calibration object buffer be 20mM~
200mM Tris buffer, PH range are 6.5~8.0, and preferred concentration is 20mM, and preferred PH is 7.4.
The invention also discloses the preparation methods of the poly- plain 3 chemiluminescence immunoassay immue quantitative detection reagent boxes of people five, including alkalinity
Poly- plain 3 preparation method for antibody of the people five of phosphatase enzyme mark, poly- plain 3 preparation method for antibody of the people five of biotin labeling, the poly- element 3 of people five
The preparation method of calibration object, assembling.
Preferably, poly- plain 3 preparation method for antibody of the people five of alkali phosphatase enzyme mark are that sodium carbonate is added in alkaline phosphatase
In buffer (pH8.0), it is added poly- plain 3 antibody of people five, 37 DEG C after reaction 4 hours, with ProteinG affinity column (GE company)
Enzyme labelled antibody is purified, the poly- plain 3 antibody enzyme conjugates of people five are obtained.It is diluted in enzyme marker buffer, dilution ratio 1:400
~1:2000, preferred dilution ratio are 1:1000.
Preferably, poly- plain 3 preparation method for antibody of the people five of biotin labeling are by people five poly- plain 3 antibody (mouse, Dan Ke
It is grand) it is added in sodium carbonate buffer (pH8.0), biotin derivative is added, it is affine with ProteinG after 37 DEG C are reacted 4 hours
Column (GE company) purifying biological element labelled antibody obtains the poly- plain 3 antibody biotin conjugates of people five.It is diluted in biotinylated derivative
In buffer, dilution ratio is 1:200~1:1000, and preferred dilution ratio is 1:500.
Preferably, the preparation method of poly- plain 3 calibration objects of people five is that people five is gathered plain 3 antigen diluents extremely with calibration object buffer
Working concentration, respectively 0,0.50,5.00,25.00,50.00,100.00ng/mL.
Preferably, the method for assembling is that mentioned reagent component is assembled into box, is saved under the conditions of 2~8 DEG C.
Kit Performance Evaluating Indexes of the present invention: accuracy, line are carried out to the kit using this method preparation
Property, precision, specificity and stability are measured.
Kit reaction principle of the present invention: using magnetic microparticles as the solid phase of immune response, chemiluminescence is utilized
Immunoassay method and chemiluminescence class analyzer cooperate, for measuring poly- plain 3 contents of the people five in human serum/blood plasma.Instead
The technical principle answered are as follows: sample to be tested, the poly- element 3 of people five of calibration object and the people five of alkali phosphatase enzyme mark gather plain 3, biotin mark
The poly- element 3 of the people five of note, which combines, forms compound, and the magnetic particle of coating Streptavidin is then added, passes through Streptavidin and life
Object element, which combines, is connected to antigen antibody complex on magnetic particle, the Direct precipitation in externally-applied magnetic field, and immune response is formed
Compound is separated with unbonded other materials.The compound of precipitating is cleaned, enzyme-catalyzed chemical luminescence substrate is added, substrate is made in enzyme
With lower by catalytic pyrolysis, unstable excitation state intermediate is formed, photon, shape are just issued when excitation state intermediate returns to ground state
At luminescence-producing reaction, the luminous intensity of light-emitting appearance measurement reaction is utilized.Within the measurement range, the people five in luminous intensity and sample is poly-
Plain 3 concentration are directly proportional, can quantitatively calculate the poly- element 3 of people five in sample to be tested using four parameter Logistic equation models of improvement
Concentration.
The present invention is different from the magnetic particle of routine techniques synthesis, the magnetic particle grain that routine techniques uses using super paramagnetic material
Diameter is generally 1~3um etc., and such partial size magnetic particle specific surface area is smaller, and detection resolution sensitivity is not high.Not with the prior art
With the magnetic particle that the present invention uses is made of superparamagnetic nanomaterial, and partial size is 10~30nm；Partial size is less than 30nm's
Superparamagnetic nano particle has big magnetic moment constant, and remanent magnetism and coercivity can be ignored, can make quickly to the magnetic field of application
Response；Large specific surface area simultaneously is easy dispersion, the ability for detecting single antigen molecule can be improved, improve the line of kit
Property range and detection sensitivity.
The poly- plain 3 magnetic microparticle chemiluminescence immunological quantitative determining kits of the people five of invention, preparation process is simple, at
This is cheap, stability is good, and performance is more than current ELISA kit.The novelty of invention is, at present the temporary nothing in market
Poly- plain 3 (PTX3) chemical luminescence reagent kits of people five, and prepare the poly- element 3 of the people five based on magnetic microparticle chemiluminescence technology platform
(PTX3) kit, high sensitivity is linear wide, and performance is more than the ELISA kit product of current scientific research.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Illustrate:
The poly- plain 3 magnetic microparticle chemiluminescence immunological quantitative determining principles of Fig. 1 behaviour five and flow chart.
Specific embodiment
The present invention is further explained in the light of specific embodiments, so that those skilled in the art can be better
Understand the present invention and can be practiced, illustrated embodiment is not as a limitation of the invention.
The preparation of embodiment 1, kit.
(1) magnetic particle coating object buffer preparation:
Above-mentioned material is added in 1000mL deionized water, dissolution is sufficiently stirred, adjusts PH to 7.00 ± 0.10.
(2) prepared by enzyme marker buffer:
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 1.00g |
| Bovine serum albumin(BSA) | 50.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized water, dissolution is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
(3) prepared by biotinylated derivative buffer:
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 4.50g |
| Tween-20 | 1.00g |
| Bovine serum albumin(BSA) | 10.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized water, dissolution is sufficiently stirred, adjusts PH to 8.00 ± 0.10.
(4) prepared by calibration object buffer:
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 1.00g |
| Fetal calf serum | 200mL |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized water, dissolution is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
(5) the coated magnetic particle suspension manufacturing methods of Streptavidin:
By the coated magnetic particle mother liquor of the Streptavidin of commercialization, (buying is in the limited public affairs of Nanjing Pan Gu's gene nano science and technology
Department) with magnetic bead coating object buffer diluted concentration be 0.5mg/mL.
(6) poly- plain 3 preparation method for antibody of the people five of alkali phosphatase enzyme mark:
100ug alkaline phosphatase is added in 1mL10mM sodium carbonate buffer (pH8.0), it is anti-that the poly- element 3 of 5ug people five is added
Body (it is company that buying is refined in Shenzhen) after 37 DEG C are reacted 4 hours, is resisted with ProteinG affinity column (GE company) purifying enzyme mark
Body obtains the poly- plain 3 antibody enzyme conjugates of people five.It is diluted in enzyme marker buffer, dilution ratio 1:1000.
(7) poly- plain 3 preparation method for antibody of the people five of biotin labeling:
It is slow that 1mL10mM sodium carbonate is added in poly- plain 3 antibody (mouse, monoclonal are purchased in Abcam company) of 20ug people five
In fliud flushing (pH8.0), 50ug biotin derivative is added, after 37 DEG C are reacted 4 hours, with ProteinG affinity column (GE company)
Purifying biological element labelled antibody obtains the poly- plain 3 antibody biotin conjugates of people five.It is diluted in biotinylated derivative buffer,
Dilution ratio is 1:500.
(8) preparation method of poly- plain 3 calibration objects of people five:
With calibration object buffer by poly- plain 3 antigen diluents of people five to working concentration, respectively 0,0.50,5.00,25.00,
50.00 100.00ng/mL.
(9) it assembles: mentioned reagent component being assembled into box, is saved under the conditions of 2~8 DEG C.
Embodiment 2, the test method of kit.
(1) it is loaded and is incubated for process: drawing poly- plain 3 calibration objects of people five or fresh patient's sample 50uL is added in reaction tube,
Then the poly- plain 3 antibody 50uL of the people five of the poly- 3 antibody 50uL of element of the people five of addition alkali phosphatase enzyme mark and biotin labeling, 37
DEG C incubation reaction 10 minutes；Then the coated magnetic particle suspension 50uL of Streptavidin is added, 37 DEG C of incubation reactions 10 are divided
Clock；
(2) Magneto separate cleaning process: the reaction tube after the completion of incubation reaction is placed on Magneto separate frame and stands 1 minute, is removed
Remove supernatant；Magnetic bead is added for the first time and is coated with object buffer 300uL, is placed on Magneto separate frame and stands 1 minute, remove supernatant；
Second of addition magnetic bead is coated with object buffer 300uL, is placed on Magneto separate frame and stands 1 minute, removes supernatant；Third time is added
Magnetic bead is coated with object buffer 300uL, is placed on Magneto separate frame and stands 1 minute, removes supernatant；
(3) luminescence process: addition 530 substrate solution of Lumi-Phos (buying is in Lumigen company, the U.S.) 200uL, 37 DEG C
It is protected from light incubation after five minutes, measures luminous value with the semi-automatic Chemiluminescence Apparatus of shore pine 9507.
Embodiment 3, the performance test results of kit.
By above result it can be seen that kit of the present invention is compared with ELISA kit, the performance test results
More excellent, the poly- plain 3 magnetic microparticle chemiluminescence immune quantitative detection reagent boxes of people five of the invention have good applicability and advanced
Property.
Claims (10)
1. a kind of chemiluminescence immunoassay immue quantitative detection reagent box of the poly- element 3 of people five, it is characterised in that: be coated with including Streptavidin
Magnetic particle suspension, poly- plain 3 antibody of the people five of alkali phosphatase enzyme mark, poly- plain 3 antibody of the people five of biotin labeling, people five is poly-
Plain 3 calibration objects.
2. a kind of kit for detecting Drugs for Cardiovascular Diseases gene according to claim 1, it is characterised in that: the strepto-
The coated magnetic particle suspension of Avidin is the superparamagnetic nanomaterial that surface package has Streptavidin, and it is micro- to be suspended in magnetic
In grain coating object buffer, concentration is 0.1mg/mL~1.0mg/mL；It is 20mM~200mM that magnetic particle, which is coated with object buffer,
Tris (trishydroxymethylaminomethane) buffer, PH are 6.5~8.0.
3. a kind of kit for detecting Drugs for Cardiovascular Diseases gene according to claim 1, it is characterised in that: the alkalinity
Poly- plain 3 antibody of the people five of phosphatase enzyme mark are the conjugate of alkaline phosphatase and poly- plain 3 antibody of people five, and wherein the poly- element 3 of people five is anti-
Body is mouse monoclonal antibody.It is diluted in enzyme marker buffer, dilution ratio is 1:400~1:2000；Enzyme marker is slow
Fliud flushing is 20mM~200mM Tris buffer, and PH is 6.5~8.0.
4. a kind of kit for detecting Drugs for Cardiovascular Diseases gene according to claim 1, it is characterised in that: the biology
Poly- plain 3 antibody of the people five of element label are the conjugate of biotin and poly- plain 3 antibody of people five, and wherein poly- plain 3 antibody of people five are mouse
Monoclonal antibody.It is diluted in biotinylated derivative buffer, dilution ratio is 1:200~1:1000；Biotinylated derivative is slow
Fliud flushing is 20mM~200mM Tris buffer, and PH is 6.5~8.0.
5. a kind of kit for detecting Drugs for Cardiovascular Diseases gene according to claim 1, it is characterised in that: the people five
3 calibration objects of poly- element are the calibration object buffer containing 20% fetal calf serum, by poly- plain 3 antigen diluents of people five to working concentration, respectively
It is 0,0.50,5.00,25.00,50.00,100.00ng/mL；Calibration object buffer is 20mM~200mM Tris buffer, PH
It is 6.5~8.0.
6. the preparation method that any one of Claims 1 to 5 kit is used to detect the poly- element 3 of people five, which is characterized in that including
Following steps:
1) preparation of poly- plain 3 antibody of the people five of alkali phosphatase enzyme mark；
2) preparation of poly- plain 3 antibody of the people five of biotin labeling；
3) preparation of poly- plain 3 calibration objects of people five；
4) it assembles.
7. according to the method described in claim 6, it is characterized by: the people five of the alkali phosphatase enzyme mark is poly- in step 1)
The preparation method of plain 3 antibody is that alkaline phosphatase is added in sodium carbonate buffer (pH8.0), and poly- plain 3 antibody of people five are added,
After 37 DEG C are reacted 4 hours, enzyme labelled antibody is purified with ProteinG affinity column (GE company), obtains the poly- plain 3 abzyme knots of people five
Close object.It is diluted in enzyme marker buffer, dilution ratio is 1:400~1:2000.
8. according to the method described in claim 6, it is characterized by: the poly- element 3 of the people five of the biotin labeling is anti-in step 2)
The preparation method of body is that poly- plain 3 antibody (mouse, monoclonal) of people five are added in sodium carbonate buffer (pH8.0), biology is added
Plain derivative, with ProteinG affinity column (GE company) purifying biological element labelled antibody, obtains people five after 37 DEG C are reacted 4 hours
3 antibody biotin conjugates of poly- element.It is diluted in biotinylated derivative buffer, dilution ratio is 1:200~1:1000.
9. according to the method described in claim 6, it is characterized by: the people five gathers the preparation side of plain 3 calibration objects in step 3)
Method is, with calibration object buffer by poly- plain 3 antigen diluents of people five to working concentration, respectively 0,0.50,5.00,25.00,
50.00 100.00ng/mL.
10. according to the method described in claim 6, it is characterized by: the assembling steps are, by mentioned reagent in step 4)
Component is assembled into box, saves under the conditions of 2~8 DEG C.
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| CN111929440A (en) * | 2020-07-29 | 2020-11-13 | 西南医科大学附属中医医院 | Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay |
| CN112730826A (en) * | 2020-12-23 | 2021-04-30 | 中南大学湘雅三医院 | Human phosphorylated vasodilator-stimulated phosphoprotein magnetic particle chemiluminescence immune quantitative detection kit, and detection method and application thereof |
| CN114152740A (en) * | 2021-11-16 | 2022-03-08 | 珠海科域生物工程股份有限公司 | Enzymatic chemiluminescence kit, preparation method and detection method |
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