CN102721804A - Electrical chemiluminescence immunoassay based on gold magnetic particles - Google Patents
Electrical chemiluminescence immunoassay based on gold magnetic particles Download PDFInfo
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Abstract
The invention provides an electrical chemiluminescence immunoassay method based on gold magnetic particles, which realizes the quantitative detection on the content of antigen/antibody in human serum, blood plasma, cell culture supernatant or other related biological liquid so as to solve the problems of high cost, complicated steps, poor reaction controllability, no generality and the like in the prior art. The method mainly comprises the steps of 1) preparing immunomagnetic beads by gold magnetic particles; 2) labeling the antigen/antibody by Ru(bpy) <2+>3; 3) establishing a standard curve for quantitatively detecting the antigen/antibody; and 4) detecting a sample to be detected. A novel electrical chemiluminescence immunoassay technology is adopted, and the electrical chemiluminescence immunoassay method based on gold magnetic particles has the advantages that the operation is simple and convenient, the valence of antibody can not be affected in the process that the antibody is labeled by Ru(bpy) <2+>3, the labeling cost is low, and the universality is realized.
Description
Technical field
The invention belongs to field of medical examination, be specifically related to the electrochemiluminescence immuno analytical method.
Background technology
The concentration of certain antigen/antibody possibly have direct relation with its health status, physiological function, physiological period etc. in the human body, so the concentration of antigen/antibody is significant to human body in the detection by quantitative human body.Concentration like PSA in the human serum is significant at the aspects such as diagnosis, prognosis judgement and curative effect detection of prostate cancer.The detection technique of the antigen/antibody in human serum, blood plasma, cells and supernatant or other associated biomolecule liquid mainly contains at present: radiating immuning analysis technology (Radioimmunoassay; RIA), ELISA technology (Enzyme linked Immunosorbent Assay; ELISA), fluoroimmunoassay (Fluorescence Immunoassay; FIA), chemiluminescence epidemic disease analytical technology (Chemiluminescence Immunoassay, CLIA), immunochromatography technique (Lateral Flow Immunoassay, method such as LFIA); But these methods all have advantage and defect separately; And the electrochemiluminescence immunoassay (Electrochemiluminescence Immunoassay ECLIA) is a kind of deriving technology of chemiluminescence immune assay, is present state-of-the-art labelled immune analytical technology; It has inherited whole advantages of chemiluminescence immunoassay technology; Remedied the defective of chemiluminescence immunoassay technology, expanded the application space of chemiluminescence immunoassay technology, and enjoyed vast researcher, medical personnel and patient's favor with its special advantages.
The electrochemiluminescence immunoassay comprises two parts, is respectively immune response system and electrochemical analysis system.Immune response system is electricity consumption chemiluminescent labels labelled antigen or antibody, forms immune complex process to be checked through immune response and sequence of operations step then; Electrochemical analysis system be electrochemiluminescence material on the immune complex at working electrode surface generation electrochemiluminescence, utilize luminous surveying instrument to detect luminous intensity, thus the process that antigen or antibody are detected.
Compare the electrochemiluminescence immuno analytical method with other technologies and mainly contain following advantage: 1) highly sensitive, the range of linearity is wide, and the detection sensitivity and the range of linearity can meet or exceed radiommunoassay; 2) stable reagent, the harm of non-toxic and non-radioactive property; 3) good stability, automaticity is high; 4) detection speed is fast; 5) applied range.At present the electrochemiluminescence immuno analytical method has been widely used in the detection of hormone, tumor markers, hepatitis B each item index and other materials such as vitamin, folic acid, DNA, RNA, immunoglobulin (Ig) etc.But, relate to the biotinylation of antigen/antibody in the traditional electrical chemiluminescence immune analysis method, with processes such as functionalization base group modification magnetic microspheres, complex steps, cost is higher.
Summary of the invention
The invention provides a kind of electrochemiluminescence immune analysis method based on gold-magnetic particles; Realize the content of the antigen/antibody in detection by quantitative human serum, blood plasma, cells and supernatant or other associated biomolecule liquid, the prior art cost is higher to solve, complex steps, reaction poor controllability, do not have problem such as versatility.
For realizing above goal of the invention, the basic technology scheme that the present invention provides is following:
1, a kind of electrochemiluminescence immune analysis method based on gold-magnetic particles may further comprise the steps:
1) prepares immunomagnetic beads with gold-magnetic particles
Adopt coupling buffer dilution and the corresponding capture antibody/antigen of object to be measured, obtain capture antibody/antigenic solution; Pipette gold-magnetic particles (especially with mean grain size less than 5 μ m, saturation magnetization is greater than 40Am
2The gold-magnetic particles of/kg is good) in centrifuge tube, add coupling buffer jog mixing, magnetic resolution is abandoned supernatant; Getting said capture antibody/antigenic solution adding is equipped with in the centrifuge tube of said gold-magnetic particles; The jog mixing is fixed in constant temperature shaking table reaction with centrifuge tube, and the question response magnetic resolution that finishes is abandoned supernatant; Cleaning buffer solution is added jog mixing in this centrifuge tube, and magnetic resolution is abandoned supernatant; In this centrifuge tube, add sealer, and be placed on oscillating reactions in the constant temperature shaking table, reaction finishes the back magnetic resolution and abandons supernatant; Add at last and preserve damping fluid, the jog mixing makes immunomagnetic beads.
2) Ru (bpy)
3 2+The marker detection antibody/antigen
Other gets corresponding to said capture antibody/detection of antigens antibody/antigen, fully dialyses with the mark damping fluid; Get Ru (bpy)
3 2+-NHS ester is dissolved in it among DMSO (dimethyl sulfoxide); Adjustment detects antibody/antigen and Ru (bpy)
3 2+The mol ratio of-NHS ester makes it reach the optimum mark ratio; Both are mixed in the adding centrifuge tube lucifuge reaction in constant temperature oscillator; Question response finishes in above-mentioned centrifuge tube, to add the glycine solution of 100 μ L, 2M, continues lucifuge reaction 10min with cessation reaction; Reactant in the centrifuge tube is dialysed with preserving the damping fluid lucifuge; Promptly obtain Ru (bpy) after the dialysis
3 2+The antibody/antigen of mark is preserved subsequent use.
3) foundation of quantitative measurement antigen/antibody typical curve
Get many by-reaction pipe, in every by-reaction pipe, add quantitative said immunomagnetic beads, the antigen/antibody standard model of variable concentrations, said Ru (bpy) respectively
3 2+The antibody/antigen of mark is placed in the constant temperature shaking table and reacts, and forms the magnetic immuno compound:
Immunomagnetic beads-antigen-Ru (bpy)
3 2+The detection antibody mediated immunity compound of mark;
Immunomagnetic beads-antibody-Ru (bpy)
3 2+The antigen immune compound of mark;
Immunomagnetic beads-antigen-detection antibody-Ru (bpy)
3 2+Two anti-immune complexs of mark;
Clean and resuspended immune complex, and then it is sucked the sensing chamber of electrochemiluminescence immunity analysis instrument, introduce the ECL damping fluid that contains TPA simultaneously; Start the electrochemiluminescence reaction, record luminous value, production standard curve.
4) detect testing sample
Get reaction tube, add quantitative said immunomagnetic beads, testing sample, said Ru (bpy)
3 2+The antibody/antigen of mark is placed in the constant temperature shaking table and reacts, and forms said compound; Clean and resuspended this sandwich complex, and then it is sucked the sensing chamber of electrochemiluminescence immunity analysis instrument, introduce the ECL damping fluid that contains TPA simultaneously.
Start the electrochemiluminescence reaction, write down luminous value, calculate the concentration of antigen/antibody in the testing sample through said typical curve.
Above-mentioned steps 2) the preferred 0.005～0.1M of mark damping fluid in, the Na of pH 8～10
2CO
3/ NaHCO
3Damping fluid; Step 2) detects antibody/antigen and Ru (bpy) described in
3 2+The molecular number of-NHS ester is than 1:10～1:100; The concentration of detection antibody/antigen to be marked should transfer to 0.1～10mg/mL; Preferred 0.005～the 0.1M of the damping fluid of ECL described in the step 3), the PB damping fluid of pH 7～9 (contains 0.05%～0.1%TritonX-100,0.05%～0.1%Tween20,0.1%NaN
3, 100mM TPA).
Above-mentioned steps 1) coupling buffer described in is 0.005～0.1M; The Tris-HCl damping fluid of pH 6～8; The prescription of sealer is: 1%～10%BSA, 1%～5% calf serum, 0.1%～2%PEG20000.
Above-mentioned steps 1) and 2) described in to preserve damping fluid be 0.005～0.1M, the PB damping fluid of pH 6～8 (contains 0.1%～1%NaN
3).
Above-mentioned steps 1) and 2) in the revolution of constant temperature shaking table be 180rpm, temperature is 20～37 ℃.
Above-mentioned steps 1) and 3) described in cleaning buffer solution be 0.005～0.1M, the PBST of pH 6～8.
Luminous detection system of the present invention is that the luminescent substance on antigen or the antibody---tris (bipyridine) ruthenium is realized through being marked at.Its principle of luminosity is: luminous agent Ru (bpy)
3 2+Respectively lose an electronics and oxidation reaction takes place, Ru (bpy) at anode surface with electron donor TPA
3 2+Be oxidized to Ru (bpy)
3 3+, TPA is oxidized to TPA
+°; TPA
+° extremely instability can spontaneously lose a proton and form TPA °, and TPA ° is a kind of strong reductant, can give Ru (bpy) with an electron transport
3 3+Make it form Ru (bpy)
3 2+*, and TPA self is oxidized to di-n-propylamine and propionic aldehyde; Ru (bpy)
3 2+*Can launch the photon of a wavelength 620nm when getting back to ground state; This process is carried out at electrode surface circularly, produces many photons, and photomultiplier detects light intensity, light intensity and Ru (bpy)
3 2+Concentration linear, so can measure the content of determined antigen or antibody.
The present invention has the following advantages:
1, the present invention adopts a kind of mean grain size less than 5 μ m, and saturation magnetization is greater than 40Am
2The gold-magnetic particles of/kg prepares immunomagnetic beads as solid phase carrier, and this gold-magnetic particles has the performance of the efficient coupling biology/drug molecule of superparamagnetism and collaurum surface of magnetic nano-particle concurrently.The collaurum on this gold-magnetic particles surface and protein interaction can be protein adsorption to its gold surface; It between the two physisorption; No covalency absorption so the combination of the two does not influence activity of proteins, tightly is adsorbed on the colloidal gold particle surface on gold-magnetic particles surface like the Fc fragment of antibody protein; The Fab fragment of antibody protein is then outstanding outside, thus make the Fab fragment more easily and antigen react.
2, the present invention adopts a kind of novel with Ru (bpy)
3 2+The technology of labelled antigen/antibody has easy and simple to handlely, and labeling process can not influence antibody titer, and the mark cost is low, has advantages such as versatility.
3, based on the electrochemiluminescence immune analysis method of gold-magnetic particles; Except that the whole advantages that possess the traditional electrical chemiluminescence immunoassay technology; Also avoided biotinylated antigen/antibody in the traditional electrical chemiluminescence immune analysis method, with processes such as functionalization base group modification magnetic microspheres; Reduce cost, simplified experimental procedure.
Description of drawings
Fig. 1 is the structural representation of the used solid phase carrier-gold-magnetic particles of the present invention;
Fig. 2 is the present invention prepares immunomagnetic beads with gold-magnetic particles a operation chart;
Fig. 3, Fig. 4, Fig. 5 are that magnetic immuno compound involved in the present invention forms synoptic diagram.
Embodiment
Following examples are to the form example of scheme of the present invention with concrete experimental implementation, and experiment condition wherein and setup parameter should not be regarded as the limitation to basic technical scheme of the present invention.
Embodiment 1: use the concentration based on PSA in the electrochemiluminescence immune analysis method detection by quantitative human serum of gold-magnetic particles.
1) prepares immunomagnetic beads with gold-magnetic particles
1.1) with coupling buffer PSA monoclonal antibody (capture antibody) is made into 1mg/mL, take out a certain amount of antibody-solutions in a centrifuge tube, and be labeled as pre, give over to coupling efficiency and detect usefulness.
1.2) pipette
gold-magnetic particles (5mg/mL) 200 μ L in the 2mL centrifuge tube with pipettor; Add coupling buffer 400 μ L jog mixings; Magnetic resolution 2min abandons supernatant.
1.3) get in the centrifuge tube of 300 this kind of μ L antibody-solutions adding above-mentioned being equipped with
gold-magnetic particles; The jog mixing; Centrifuge tube is fixed in the constant temperature shaking table, 37 ℃, 180rpm oscillating reactions 1h, reaction finishes; Magnetic resolution 2min; Get supernatant in a centrifuge tube, and be labeled as post, give over to coupling efficiency and detect usefulness.
1.4) cleaning buffer solution 300 μ L are added jog mixing in the above-mentioned centrifuge tube, magnetic resolution is got supernatant in a centrifuge tube, and is labeled as wash
1, repeat this cleaning operation 1 time, get supernatant in another centrifuge tube, and be labeled as wash
2, give over to coupling efficiency and detect usefulness.
1.5) in above-mentioned centrifuge tube, adding the 1mL sealer, abundant mixed dissolution places 37 ℃ of constant temperature shaking tables, the 180rpm 1h that vibrates, and magnetic resolution is abandoned supernatant;
1.6) in above-mentioned centrifuge tube, add cleaning buffer solution 800 μ L at last, the jog mixing, magnetic resolution is abandoned supernatant, repeats this cleaning operation 3 times, and add 1mL and preserve damping fluid, the jog mixing, 4 ℃ of preservations are subsequent use.
1.7) with the coupling buffer the blank light absorption value of measuring pre, post at 280nm place respectively, be blank with the cleaning buffer solution, measure wash respectively
1, wash
2Light absorption value at the 280nm place; And calculating antibody exists
The coupling efficiency on gold-magnetic particles surface; Multiply by the amount that adds antibody with coupling efficiency, be and be coupled at
The amount of micron gold-magnetic particles surface antibody.
2) Ru (bpy)
3 2+Labelled antibody
2.1) PSA monoclonal antibody (detection antibody) is fully dialysed at 4 ℃ with the mark damping fluid.
2.2) treat that dialysis is accomplished after, measure AC with the lowry method, and the concentration of adjusting antibody is to 1mg/mL.
2.3) dissolving Ru (bpy)
3 2+-NHS ester is in DMSO, and making its concentration is 1mg/mL; The regulator solution consumption makes this antibody and Ru (bpy)
3 2+The mol ratio of-NHS ester reaches 1:50, and this ratio is Ru (bpy)
3 2+The mark ratio of the best of mark PSA antibody.
2.4) with this kind antibody and Ru (bpy)
3 2+-NHS ester mixes and adds in the centrifuge tube, under the room temperature, and 180rpm lucifuge vibration 4 hours.
2.5) its lucifuge is dialysed under 4 ℃ with preserving damping fluid, in dislysate, there is not the ECL signal.
2.6) after the dialysis, with lowry method mensuration AC; The comparison of tiring before and after making marks; At last that mark is good PSA monoclonal antibody is subsequent use 4 ℃ or-20 ℃ of preservations.
3) foundation of typical curve
3.1) take out above-mentioned immunomagnetic beads, balance 10min under room temperature;
3.2) in each reaction tube, add above-mentioned gold-magnetic particles 100 μ L, and then in each respective tube, add the PSA standard items of 100 μ L 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL respectively, 37 ℃, 180rpm places shaking table to react 30min.
3.3) take out each reaction tube and place on the magnetic separator, magnetic resolution is abandoned supernatant, cleans 3 times with cleaning buffer solution, and magnetic resolution is abandoned supernatant.
3.4) add Ru (bpy) to above-mentioned each reaction tube
3 2+The PSA monoclonal antibody 100 μ L of mark, blank does not add, and 37 ℃, 180rpm places shaking table to react 30min.
3.5) take out reaction tube and place on the magnetic separator, magnetic resolution is abandoned supernatant, cleans 3 times with cleaning buffer solution, and magnetic resolution is abandoned supernatant, and is resuspended with luminous damping fluid.
3.6) above-mentioned sandwich complex is sucked flow chamber, the ECL damping fluid that induces one simultaneously starts the electrochemiluminescence reaction, the record luminous value.
3.7) be horizontal ordinate with PSA concentration, luminous intensity is an ordinate drawing standard curve.
4) concentration of detection by quantitative PSA
4.1) take out above-mentioned immunomagnetic beads, balance 10min under room temperature;
4.2) in reaction tube, add above-mentioned gold-magnetic particles 100 μ L, and then to wherein adding the sample to be tested 100 μ L that contain PSA, 37 ℃, 180rpm places shaking table to react 30min.
4.3) take out reaction tube and place on the magnetic separator, magnetic resolution is abandoned supernatant, cleans 3 times with cleaning buffer solution, and magnetic resolution is abandoned supernatant.
4.4) add Ru (bpy) to above-mentioned each reaction tube
3 2+The PSA monoclonal antibody 100 μ L of mark, 37 ℃, 180rpm places shaking table to react 30min.
4.5) take out reaction tube and place on the magnetic separator, magnetic resolution is abandoned supernatant, cleans 3 times with cleaning buffer solution, and magnetic resolution is abandoned supernatant, and is resuspended with luminous damping fluid.
4.6) with the sensing chamber of above-mentioned solution suction electrochemiluminescence immunity analysis instrument, the ECL damping fluid that induces one simultaneously starts the electrochemiluminescence reaction, the record luminous value.
4.7) calculate the concentration of PSA in the sample to be tested according to above-mentioned typical curve.
Embodiment 2: use the concentration based on AFP in the electrochemiluminescence immune analysis method detection by quantitative human serum of gold-magnetic particles.
Except preparation during immunomagnetic beads with AFP antibody (capture antibody) encapsulate gold-magnetic particles, with Ru (bpy)
3 2+Mark AFP antibody (detection antibody), Ru (bpy)
3 2+Antibody and Ru (bpy) in the mark AFP antibody process
3 2+The mol ratio of-NHS ester and setting up outside the concentration of selected several AFP standard items in the typical curve process, other steps are with embodiment 1.
Embodiment 3: use the concentration based on TSH in the electrochemiluminescence immune analysis method detection by quantitative human serum of gold-magnetic particles.
Except preparation during immunomagnetic beads with TSH antibody (capture antibody) encapsulate gold-magnetic particles, with Ru (bpy)
3 2+Mark TSH antibody (detection antibody), Ru (bpy)
3 2+Antibody and Ru (bpy) in the mark TSH antibody process
3 2+The mol ratio of-NHS ester and setting up outside the concentration of several TSH standard items of selecting in the typical curve process, other steps are with embodiment 1.
Embodiment 4: use the concentration based on TP antibody in the electrochemiluminescence immune analysis method detection by quantitative human serum of gold-magnetic particles.
Except preparation during immunomagnetic beads with TP antigen (capture antigen) encapsulate gold-magnetic particles, with Ru (bpy)
3 2+Mark TP antigen (detection antigen), Ru (bpy)
3 2+Antigen and Ru (bpy) in the mark TP antigen process
3 2+The mol ratio of-NHSester and setting up outside the concentration of several TP antibody standard substance of selecting in the typical curve process, other steps are with embodiment 1.
When detecting the concentration of PSA in the serum, AFP, TSH, TP antibody according to above embodiment; With gold-magnetic particles as solid phase carrier; Because the collaurum on gold-magnetic particles surface and protein interaction can arrive its gold surface to protein adsorption, are physisorption between the two, no covalency absorption; So the combination of the two does not influence activity of proteins; Tightly be adsorbed on the colloidal gold particle surface on gold-magnetic particles surface like the Fc fragment of antibody protein, the Fab fragment of antibody protein is then outstanding outside, thus make the Fab fragment more easily and antigen react.Adopt Ru (bpy)
3 2+The technology of labelled antigen/antibody has easy and simple to handlely, and labeling process can not influence antibody titer, and the mark cost is low, has advantages such as versatility.Electrochemiluminescence immune analysis method based on gold-magnetic particles; Except that the whole advantages that possess the traditional electrical chemiluminescence immunoassay technology; Also avoided biotinylated antigen/antibody in the traditional electrical chemiluminescence immune analysis method, with processes such as functionalization base group modification magnetic microspheres; Reduce cost, simplified experimental procedure.
Claims (6)
1. electrochemiluminescence immune analysis method based on gold-magnetic particles may further comprise the steps:
1) prepares immunomagnetic beads with gold-magnetic particles
Adopt coupling buffer dilution and the corresponding capture antibody/antigen of object to be measured, obtain capture antibody/antigenic solution; Pipette gold-magnetic particles in centrifuge tube, add coupling buffer jog mixing, magnetic resolution is abandoned supernatant; Getting said antibody/antigen solution joins in the said centrifuge tube that gold-magnetic particles is housed; The jog mixing is fixed in oscillating reactions in the constant temperature shaking table with centrifuge tube, and magnetic resolution was abandoned supernatant after question response finished; Cleaning buffer solution is joined jog mixing in this centrifuge tube, and magnetic resolution is abandoned supernatant; In this centrifuge tube, add sealer, and be placed on oscillating reactions in the constant temperature shaking table, question response finishes the back magnetic resolution and abandons supernatant; Add at last and preserve damping fluid, the jog mixing makes immunomagnetic beads, preserves subsequent use;
2) with Ru (bpy)
3 2+Labelled antibody/antigen
Other gets corresponding to said capture antibody/detection of antigens antibody/antigen, fully dialyses with the mark damping fluid; Get Ru (bpy)
3 2+-NHS ester also is dissolved in it among DMSO; Adjustment detects antibody/antigen and Ru (bpy)
3 2+The mol ratio of-NHS ester makes it reach the optimum mark ratio; Both are mixed in the adding centrifuge tube lucifuge reaction in the constant temperature shaking table; Question response finishes in above-mentioned centrifuge tube, to add the glycine solution of 100 μ L, 2M, continues lucifuge reaction 10min with cessation reaction; Reactant in the centrifuge tube is dialysed with preserving the damping fluid lucifuge; Promptly obtain Ru (bpy) after the dialysis
3 2+The antibody/antigen of mark is preserved subsequent use;
3) foundation of the typical curve of quantitative measurement antigen/antibody
Get many by-reaction pipe, in every by-reaction pipe, add quantitative said immunomagnetic beads, the antigen/antibody standard items of variable concentrations, said Ru (bpy) respectively
3 2+The antibody/antigen of mark is placed in the constant temperature shaking table and reacts, and forms the magnetic immuno compound:
Immunomagnetic beads-antigen-Ru (bpy)
3 2+The antibody mediated immunity compound of mark;
Immunomagnetic beads-antibody-Ru (bpy)
3 2+The antigen immune compound of mark;
Immunomagnetic beads-Ag-Ab-Ru (bpy)
3 2+Two anti-immune complexs of mark;
Clean and resuspended immune complex, again it is joined the sensing chamber of electrochemiluminescence immunity analysis instrument, introduce the ECL damping fluid that contains TPA simultaneously, start the electrochemiluminescence reaction then, record luminous value, production standard curve;
4) detect testing sample
Get reaction tube, add quantitative said immunomagnetic beads, testing sample, said Ru (bpy)
3 2+The antibody/antigen of mark is placed in the constant temperature shaking table and reacts, and forms said immune complex; Clean and resuspended this immune complex, and then it is joined the sensing chamber of electrochemiluminescence immunity analysis instrument, introduce the ECL damping fluid that contains TPA simultaneously, start the electrochemiluminescence reaction; The record luminous value is through the concentration of antigen/antibody in the said typical curve calculating testing sample.
2. electrochemiluminescence immune analysis method according to claim 1 is characterized in that:
Step 2) the mark damping fluid in is 0.005～0.1M, the Na of pH 8～10
2CO
3/ NaHCO
3Damping fluid;
Step 2) detects antibody/antigen and Ru (bpy) described in
3 2+The mol ratio 1:10 of-NHS ester～1:100; The concentration of detection antibody/antigen to be marked should transfer to 0.1～10mg/mL;
The damping fluid of ECL described in the step 3) is 0.005～0.1M, and the PB damping fluid of pH 7～9 contains 0.05%～0.1%TritonX-100,0.05%～0.1%Tween20,0.1%NaN
3, 100mM TPA.
3. electrochemiluminescence immune analysis method according to claim 2 is characterized in that:
Coupling buffer described in the step 1) is 0.005～0.1M, the Tris-HCl damping fluid of pH 6～8; The prescription of sealer is: 1%～10%BSA, 1%～5% calf serum, 0.1%～2%PEG20000.
4. electrochemiluminescence immune analysis method according to claim 2 is characterized in that: preserving damping fluid step 1) and 2) is 0.005～0.1M, and the PB damping fluid of pH 6～8 contains 0.1%～1%NaN
3
5. electrochemiluminescence immune analysis method according to claim 2 is characterized in that: step 1) and 2) revolution of constant temperature shaking table is 100～180rpm, and temperature is 20～37 ℃.
6. electrochemiluminescence immune analysis method according to claim 2 is characterized in that: cleaning buffer solution step 1) and 3) is 0.005～0.1M, the PBST of pH 6～8.
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