CN101363861A - Hepatitis b virus surface antigen chemiluminescence immune assay determination kit and method for preparing same - Google Patents
Hepatitis b virus surface antigen chemiluminescence immune assay determination kit and method for preparing same Download PDFInfo
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Abstract
The invention particularly relates to a kit for determining the surface antigen of the hepatitis B virus, and a preparation method thereof, which belongs to the medical field of immunological analysis. The kit comprises (1) a hepatits B virus surface antigen calibration material; (2) an avidin-coated carrier; (3) the polyclonal antibody or monoclonal antibody of a biotinylated hepatitis B virus surface antibody; (4) the polyclonal antibody or monoclonal antibody enzyme-labeled of the surface antibody; and (5) a chemiluminescent primer. Furthermore, the preparation method comprises the following steps of (1) preparing a calibration material from the pure surface antigen; (2) coating the carrier with avidin; (3) performing biotinylation of the polyclonal antibody or monoclonal antibody of the surface antibody; labeling the polyclonal antibody or monoclonal antibody of the surface body with an enzyme; (4) sub-packaging the calibration material and the chemiluminescent primer; and (5) assembling. The kit has the advantages of simpliness, rapidness, sensitiveness, stability and the like.
Description
Technical field
The present invention relates to the immunoassay medical domain, particularly, the invention provides a kind of hepatitis b virus s antigen (HBsAg) biotin-Avidin immunity amplifying technique and measure kit and preparation method thereof in conjunction with chemiluminescence immunoassay technology.
Background technology
Hepatitis type B virus (HBV) is the pathogen of hepatitis B, and hepatitis b virus s antigen is the important serologic marker thing of hepatitis type B virus.Exist hepatitis b virus s antigen to show person under inspection's hepatitis b virus infection in the serum (or other body fluid).Hepatitis b virus s antigen laboratory detection method is mainly immunoassay, and detection method commonly used is radiommunoassay (RIA) and EIA enzyme immunoassay (EIA), uses at present the widest be EIA enzyme immunoassay (EIA).
The research of labelled immune analytical technology and application development are rapid over past ten years, have been widely used in each field of biomedical fundamental research and clinical disease diagnosis.Wherein the technical matters maturation has advance and practical, and what be easy to promote mainly contains: four kinds of radiommunoassay, EIA enzyme immunoassay, time resolved fluoro-immunoassay and chemiluminescence immune assays etc.The basic theories of these ultramicron detection techniques is identical substantially, but used tracer agent and the signal that sent have nothing in common with each other.According to a large amount of test findings and clinical practice data,, be followed successively by: chemiluminescence immune assay, time resolved fluoro-immunoassay, radiommunoassay and EIA enzyme immunoassay from practicality, stability, accuracy and development prospect.There are many shortcomings in radio immunoassay, as complicated operation, measurement result instability, reagent holding time weak point, radioactive contamination, instrument costliness etc.
The sensitivity that biotin-avidin immunity amplifying technique can greatly improve detection method, and high specificity, stability is high, and applied widely, experimental cost is low.Biotin-avidin immunity amplifying technique has two kinds of fundamental types, one class is an intermediate with free Avidin, connect respectively and comprise macromolecular reaction system to be measured of biotin and mark biotin, after developed the avidin-biotin-complex technology on this basis; Another kind of is directly to connect biotinylation macromolecular reaction system with the mark Avidin to detect, and is called mark Avidin-biotin method.According to used in the reaction system to be measured be biotinylation first antibody or biotinylation second antibody, be divided into direct method and indirect method again.In indirect method, Avidin-biotin system can with the coupling of immunoassay detection architecture, amplify end reaction: will react simultaneously at the insolubilized antibodies of different antigenic determinants and biotinylated antibody and antigen (standard antigen and determined antigen) earlier, form the double-antibody sandwich immune complex at surface of solid phase carriers, adding Avidin again combines with biotin in the compound, reaction signal is amplified, thus the sensitivity that has improved immunoassay.
Chemiluminescence immunoassay is a kind of than elder generation and then effective method in conjunction with biotin-Avidin immunity amplifying technique.Chemiluminescence detection needs certain luminescence enhancer, and is furnished with superoxide enzyme system separately.Mainly be that biotin combines with affinity element, peroxidase (or alkaline phosphatase), pass through chemiluminescence and humidification again, light quantity is amplified the back detect.
At present, biotin-Avidin immunity amplifying technique is not used in the application facet of hepatitis b virus s antigen immunoassay product yet in conjunction with chemiluminescence immunoassay technology.On the one hand, the industry application need solves the high efficiency and the stability problem of finished product.On the other hand, chemical luminescence immune analysis reagent box of the prior art is the luminous measuring system of closed full-automatic chemical, need the expensive luminous measuring instrument of full-automatic chemical, promote the use of, can't be widely used in clinical diagnosis and research work thereby limited.Kit of the present invention adopts the microwell plate chemiluminescence immunoassay technology, both can be applicable to the open luminous measuring instrument of semi-automatic chemistry, also can be used for full automatic measuring system, can realize fast detection the in enormous quantities, and use cost is low, easier applying.
Summary of the invention
The present invention has solved the problems referred to above simultaneously, being about to biotin-Avidin immunity amplifying technique learns effectively with the immunoassay of hepatitis b virus s antigen in conjunction with chemiluminescence and combines, a kind of kit that can easy, quick, sensitive, stably detect hepatitis b virus s antigen is provided, and this kit is suitable for applying effectively on industry.
The purpose of this invention is to provide the kit of a kind of biotin-Avidin immunity amplifying technique in conjunction with the chemiluminscence immunoassay hepatitis b virus s antigen.
A further object of the present invention provides a kind of method for preparing the mentioned reagent box.
Kit according to the present invention comprises: 1) hepatitis b virus s antigen calibration object; 2) solid phase carrier of Avidinization; 3) polyclonal antibody of biotinylated anti-HBs or monoclonal antibody; 4) polyclonal antibody of the anti-HBs of enzyme labeling or monoclonal antibody; And 5) chemical luminous substrate that above-mentioned enzyme acted on.
According to kit of the present invention, wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle; Described marker enzyme is alkaline phosphatase or horseradish peroxidase; Described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol, wherein said 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes (AMPPD), CSPD or CDP-Star.
Further, the invention provides a kind of method for preparing the mentioned reagent box, may further comprise the steps:
1) with the pure product preparation of hepatitis b virus s antigen hepatitis b virus s antigen calibration object;
2) the solid phase carrier bag is by Avidin;
3) make the polyclonal antibody or the monoclonal antibody biotinylation of anti-HBs;
4) polyclonal antibody or the monoclonal antibody of usefulness enzyme labeling anti-HBs;
5) chemical luminous substrate that acted on of the polyclonal antibody of the anti-HBs of the monoclonal antibody of the above-mentioned hepatitis b virus s antigen calibration object of packing, biotinylated anti-HBs, enzyme labeling or monoclonal antibody and this enzyme; And
6) be assembled into finished product.
The method according to this invention, preferred, described solid phase carrier bag is by the step 2 of Avidin) may further comprise the steps:
1) bag quilt
With 0.050M pH value is the Avidin coating buffer that 9.6 carbonate buffer solution is mixed with desired concn, and coating buffer is carried on the solid phase carrier;
2) with physiological saline washing above-mentioned solid phase carrier; And
3) sealing
With containing 1%BSA, the pH value is 7.0～7.5, and concentration is that the phosphate buffer of 0.010M seals solid phase carrier after the above-mentioned washing as confining liquid.
Particularly, described method for coating can comprise:
1) bag quilt
The preparation coating buffer, comprise the natrium carbonicum calcinatum of 1.59g and the sodium bicarbonate of 2.94g based on the described coating buffer of 1000mL, the pH value of described coating buffer is 9.6, and the coating buffer with preparation mixes with the Avidin of debita spissitudo then, and the gained mixed liquor is carried on the solid phase carrier;
2) with physiological saline washing above-mentioned solid phase carrier; And
3) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10g BSA and 1mL biological preservative, the pH value of described confining liquid is 7.0～7.5, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
In said method, preferred, the solid phase carrier of described Avidin is microwell plate, plastic bead, plastic tube or magnetic-particle; Described enzyme is alkaline phosphatase or horseradish peroxidase; Described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol, wherein said 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
Concrete mentioned reagent box can comprise the microwell plate of hepatitis b virus s antigen calibration object, Avidinization, biotinylated antibody, enzyme labeling thing and chemical luminous substrate liquid, 20 times of concentrated cleaning solutions etc.Wherein, described hepatitis b virus s antigen calibration object is a standard level, and purity is not less than 90%, the microwell plate of Avidinization is the micropore lath in 48 or 96 holes, the enzyme of labelled antibody is coupling peroxidase (HRP), and chemical luminous substrate liquid is luminol, and concentrated cleaning solution is PBST.
The present invention's " hepatitis b virus surface antigen chemiluminescence immune assay determination kit " can detect the content of the hepatitis b virus s antigen in the human body behind the hepatitis b virus infection very single-mindedly, can be according to the variation of how much judging the result of treatment and the state of an illness thereof of hepatitis b virus s antigen content.It has advantages such as easy, quick, sensitive, stable.Every index of this hepatitis b virus s antigen mensuration kit (chemoluminescence method and the combination of immune amplifying technique) all reaches the analytic approach level of similar import reagent box.And, according to detection system of the present invention is open-sky technique, easy to be quick, does not need the expensive luminous measuring instrument of full-automatic chemical, be particularly suitable for vast middle and small hospital and promote the use of, for clinical diagnosis and research work provide a kind of very valuable detection means.According to kit of the present invention, the hepatitis b virus s antigen of the antibody of bag quilt and sample forms " double-antibody sandwich " structure on the antibody of enzyme labeling and the carrier, so " double-antibody sandwich single stage method " reaction pattern of the present invention's employing, both effectively utilize the chemiluminescence principle, guaranteed the sensitivity that detects again.In addition, this pattern also is convenient to operation and is produced.
What kit of the present invention was used is the enzymatic luminous substrate, by the chromogenic substrate in the light signal replacement EIA enzyme immunoassay that detects the luminous substrate generation, thereby have a specificity equal with EIA enzyme immunoassay, and sensitivity improves greatly, the sensitivity of ratio EIA enzyme immunoassay now improves about 10 times, and the diagnosis that can be hepatitis b virus s antigen provides more special, quick, reliable foundation.
In recent years studies confirm that in a large number that biotin-avidin system can combine with the multiple labelled reagent that comprises enzyme.Kit applicating biotin-avidin system of the present invention, with the polyclonal antibody of the anti-HBs of enzyme labeling or the strong bonded of monoclonal antibody high affinity, and produce multistage enlarge-effect, make biotin-avidin system immune labeled sensitive more, more efficiently the diagnosis of hepatitis b viral surface antigen with tracer analysis.
Description of drawings
Fig. 1 is the calibration object linear graph in the prepared kit of embodiment 1.
Embodiment
Embodiment 1 preparation hepatitis b virus surface antigen chemiluminescence immune assay determination kit of the present invention
One, the preparation of enzyme labelled antibody preparation and biotin coupling antibody
1. the polyclonal antibody of anti-HBs or monoclonal anti body and function glutaraldehyde method and horseradish peroxidase are fully dialysed to PBS, add equal-volume glycerine, preserve standby below-20 ℃.
2. biotin coupling anti-HBs polyclonal antibody or MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) with conventional method biotin (BNHS) is dissolved in N, dinethylformamide (DMF) is made into 1mg/mL;
(2) with 0.1mol/L pH value be 9.0 NaHCO
3With the polyclonal antibody or the monoclonal antibody dilution of the anti-HBs of purifying is 1～2mg/mL;
(3) be to mix about 1:8 or weight ratio 1:7 by BNHS with the antibody volumetric ratio, react 2～4h under the stirring at room;
(4) dialysis of packing into is 7.2 PBS to 0.05mol/LpH value, 4 ℃ of dialysed overnight, and bond adds equal-volume glycerine, packing in a small amount, preservation is standby below-20 ℃.
Two, the preparation of hepatitis b virus s antigen calibration object
With the pure product preparation of hepatitis b virus s antigen, totally 6 bottles of packing 0,0.3,1.5,6,30,150ng/mL.
Three, enzyme labelled antibody concentration is selected
Adopt the square formation method to select the working concentration of enzyme labelled antibody greater than 1:5000.
Four, the solid phase microwell plate of Avidinization preparation
(1) bag quilt
Natrium carbonicum calcinatum 0.795g
Sodium bicarbonate 1.47g
Distilled water 500mL
Behind the dissolving mixing, adjust pH to 9.6, add an amount of Avidin mixing, add then in each hole of microwell plate, every hole 130 μ L place 24h for 4 ℃.
(2) washing: it is inferior to give a baby a bath on the third day after its birth with physiological saline.
(3) sealing
NaH
2PO
4·2H
2O 0.2g
Na
2HPO
4·12H
2O 2.9g
BSA 10g
Proclin300 1mL
Distilled water is settled to 1000mL
The mentioned reagent weighing is put into clean container well, add the distilled water constant volume, the dissolving mixing, measuring the pH value is 7.0.
Every hole adds confining liquid 300 μ L respectively, and room temperature was placed 3 hours.Get rid of confining liquid, on thieving paper, pat dry.Room temperature removal moisture drying 24 hours.Carry out vacuum sealing bag immediately.Place behind the envelope and checked not have gas leakage in 15 minutes, if there is gas leakage to need envelope again.2～8 ℃ of preservations of labeling postposition.
Five, the many anti-or monoclonal antibody dilutions of enzyme mark
Tris 12.120g
BSA 5g
Proclin 1mL
Distilled water 1000mL
Six, chemical luminous substrate A liquid
Tris 53mM 3.29g
Borax 8.5mM 3.26g
BSA 0.1％ 1.0g
Luminol 10mM 1.772g
Benzofluoranthrene 0.05% (W/V) 0.5g
Proclin300 1mL
The fixed molten 1000mL of distilled water
pH 8.2～8.8
Seven, chemical luminous substrate B liquid
Na
2HPO
4·12H
2O 102.8mM 36.817g
Citric acid 174.7mM 10.21g
H
2O
2(30％) 0.03％ 1.0mL
Proclin300 1mL
The fixed molten 1000mL of distilled water
The pH value is 5.0～5.8
A liquid mixes with B liquid equal proportion during use.
Eight, 20 times of cleansing solutions
Na
2HPO
4·12H
2O 58g
NaH
2PO
4·2H
2O 2g
NaCl 160g
Tween-20 1mL
Proclin300 1mL
Deionized water 1000mL
Adjust pH to 7.2～7.4
Eight, semi-manufacture and finished product are formed
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract three parts of process specificitys, accuracy, sensitivity and stable assay approvals out and just can be assembled into hepatitis b virus s antigen mensuration kit (chemoluminescence method).Be assembled into also need inspect by random samples behind the kit and just can dispatch from the factory after qualified.
To sum up, in research process of the present invention, the present inventor has at first carried out shaker test and Quality Identification to used starting material, comprises the luminous intensity of activity, chemical luminous substrate of the absorption property of activity, Avidin carrier (as lighttight white microwell plate) of labelled antibody and biotinylated antibody and variation size, HRP and luminous duration etc.Then the method for Avidin carrier is studied, be cushioned liquid and confining liquid is tested, select optimal bag and be cushioned liquid and confining liquid, found best concentration conditions by the concentration test by the different bags of Avidin with different bags.For biotin coupling antibody with for the mark of HRP diverse ways can be arranged, by explore repeatedly and contrast test finally found easy, productive rate is high, cost is low, the reliable quality labeling method, and the dilution that biotin coupling antibody and enzyme labeling thing use carried out long-term investigation test, made and can make biotin coupling antibody and enzyme labeling thing keep active for a long time and stable dilution.Determined that by square formation cross matching repeatedly the proper proportion that biotinylated antibody mixes with the enzyme labeling thing is 1:5-1:10 once more.
The present inventor has also carried out experimental study to the reaction pattern and the reaction conditions of kit, finally determined double-antibody sandwich single stage method reaction pattern, and the influence of test findings is tested with regard to the different reaction time, determine the optimal reaction time.By the influence test to measured value shows to the mensuration of luminous duration of chemical luminous substrate liquid and different fluorescent lifetime: be measured as the best between 5～30 minutes after adding chemical luminous substrate liquid, its result is also comparatively accurate.
Embodiment 2～3 preparations hepatitis b virus surface antigen chemiluminescence immune assay determination kit of the present invention
Divided by the alkali phosphatase enzyme mark anti-HBs, with AMPPD as chemical luminous substrate, with plastic bead as the pH value of carrier, coating buffer be 4.5, the pH value of confining liquid is outside 7.5, all the other all prepare hepatitis b virus surface antigen chemiluminescence immune assay determination kit with the method identical with embodiment 1.
Embodiment 4 preparations hepatitis b virus surface antigen chemiluminescence immune assay determination kit of the present invention
As outside the carrier, all the other all prepare hepatitis b virus surface antigen chemiluminescence immune assay determination kit with the method identical with embodiment 1 divided by magnetic-particle.
The using method of embodiment 5 kits of the present invention
The concrete operations of the hepatitis b virus surface antigen chemiluminescence immune assay determination kit of above embodiment 1 preparation are as follows:
1) in 4 ℃ of refrigerators, takes out kit, equilibrium at room temperature 15 minutes.
2) take out coated slab, insert on the grillage.
3) add respectively in the reacting hole that each every hole of concentration calibration object adds 0,0.3,1.5,6,30, each 50 μ L of 150ng/ml, blank 1 hole is established in each test, each hole adds biotinylated antibody and enzyme labeling thing mixed volume 50 μ L except that blank well then, with the micro-oscillator mixing that fully vibrates, 37 ℃ of incubations 1 hour.
4) get rid of dereaction liquid, the cleansing solution after the dilution is filled it up with in every hole, washes plate 5 times, buckles on clean thieving paper at last and does.
5) each hole adds each 50 μ L of chemical luminous substrate liquid A, B, and fully vibrating with micro-oscillator mixes, room temperature (20～25 ℃) lucifuge reaction 5 minutes.
6) must measure in the 5th～30 minute after adding chemical luminous substrate liquid, on the chemiluminescence measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn, 1 second/hole of Measuring Time.
7) be horizontal ordinate with calibration object concentration, the RLU value is drawn typical curve for ordinate, finds the concentration of the hepatitis b virus s antigen of this serum on typical curve with each test serum RLU value.
The methodology of embodiment 6 kits of the present invention is identified
According to manufacturing conventional in this area and vertification regulation the kit of preparation among the embodiment 1 is identified, is seen the following form 1～3:
Table 1
| Interventions Requested | Test stone | Assay |
| Accuracy | Average recovery rate is at 90.0-110.0% | Conformance with standard |
| Specificity | Cross reacting rate≤0.01% with its analog | Conformance with standard |
| Accuracy CV (%) | ≤15％(n＝10) | Conformance with standard |
| Sensitivity | ≤0.1ng/ml | Conformance with standard |
| Stability | Each reagent set split 37 ℃ at least 3 days | Conformance with standard |
The national reference product testing result of table 2
| Country's reference product | Country's reference product test stone | Testing result |
| adr 0.2ng/ml adr 0.5ng/ml adr lng/ml | adr 0.5ng/ml | adr 0.2ng/ml |
| adw 0.5ng/ml adw lng/ml adw 2ng/ml | adw lng/ml | adw 0.5ng/ml |
| ay 0.5ng/ml ay lng/ml ay 2ng/ml | ay lng/ml | ay 0.5ng/ml |
| Stability | Each reagent set split 37 ℃ at least 3 days | Meet above-mentioned testing result |
Accuracy, specificity, accuracy, sensitivity and the stability that " hepatitis b virus surface antigen chemiluminescence immune assay determination kit " is described is fully qualified.
Table 3 calibration object concentration value and time calculation value
| Calibration object concentration value (ng/ml) | Concentration luminous value (RLU) | Concentration is returned calculation value (ng/ml) |
| 0 | 270 | 0.0 |
| 0.3 | 3119 | 0.3 |
| 1.5 | 15242 | 1.5 |
| 6 | 66029 | 6.0 |
| 30 | 367160 | 30.5 |
| 150 | 1994585 | 150.8 |
The test figure that embodiment 7 uses kit of the present invention to carry out clinical diagnosis
Reagent of the present invention and Luo Shi hepatitis b virus surface antigen chemiluminescence reagent reach 99.8% to 3540 parts of total coincidence rates of clinical blood serum sample testing result, and data are as follows:
From the data analysis of listed two kinds of reagent testing results, in the reagent testing result feminine gender of the present invention 6 parts of positives are arranged, there are 2 parts and Luo Shi reagent testing result not to be inconsistent in these 6 parts of positive findingses, this difference may be because the antigen/antibody material difference that reagent adopts, and the binding site of antigen/antibody difference causes the sensitivity of kit of the present invention, specificity, accuracy and similar import reagent box no significant difference; Easy to utilize, safe and reliable.
Claims (12)
1. a hepatitis b virus surface antigen chemiluminescence immune assay determination kit is characterized in that, described kit comprises:
1) hepatitis b virus s antigen calibration object;
2) solid phase carrier of Avidinization;
3) polyclonal antibody of biotinylated anti-HBs or monoclonal antibody;
4) polyclonal antibody of the anti-HBs of enzyme labeling or monoclonal antibody; And
5) chemical luminous substrate that above-mentioned enzyme acted on.
2. kit as claimed in claim 1 is characterized in that, the solid phase carrier of described Avidinization is microwell plate, plastic bead, plastic tube or magnetic-particle.
3. kit as claimed in claim 1 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
4. kit as claimed in claim 1 is characterized in that, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
5. kit as claimed in claim 4, it is characterized in that described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
6. kit as claimed in claim 1 is characterized in that, described chemical luminous substrate comprises A liquid and B liquid, wherein,
Based on the described chemical luminous substrate A of 1000mL liquid, comprise 3.29g Tris, 3.26g borax, 1.0g BSA, 1.772g luminol, 0.5g benzofluoranthrene, 1mL Proclin 300, its pH value is 8.2～8.8;
Based on the described chemical luminous substrate B of 100mL liquid, comprise 36.72g Na
2HPO
412H
2The H of O, 10.21g citric acid, 1.0mL30%
2O
2, 1mL Proclin 300, its pH value is 5.0～5.8.
7. method for preparing the described kit of claim 1 is characterized in that may further comprise the steps:
1) with the pure product preparation of hepatitis b virus s antigen hepatitis b virus s antigen calibration object;
2) the solid phase carrier bag is by Avidin;
3) make the polyclonal antibody or the monoclonal antibody biotinylation of anti-HBs;
4) polyclonal antibody or the monoclonal antibody of usefulness enzyme labeling anti-HBs;
5) chemical luminous substrate that acted on of the polyclonal antibody of the anti-HBs of the monoclonal antibody of the above-mentioned hepatitis b virus s antigen calibration object of packing, biotinylated anti-HBs, enzyme labeling or monoclonal antibody and this enzyme; And
6) be assembled into finished product.
8. method as claimed in claim 7 is characterized in that, described solid phase carrier bag is by the step 2 of Avidin) adopt following method:
1) bag quilt
With 0.050M pH value is the Avidin coating buffer that 9.6 carbonate buffer solution is mixed with desired concn, and coating buffer is carried on the solid phase carrier;
2) with physiological saline washing above-mentioned solid phase carrier; And
3) sealing
With containing 1%BSA, the pH value is 7.0～7.5, and concentration is that the phosphate buffer of 0.010M seals solid phase carrier after the above-mentioned washing as confining liquid.
9. as claim 7 or 8 described methods, it is characterized in that the solid phase carrier of described Avidinization is microwell plate, plastic bead, plastic tube or magnetic-particle.
10. method as claimed in claim 7 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
11. method as claimed in claim 7 is characterized in that, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
12. method as claimed in claim 11, it is characterized in that described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
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