CN103901192A - Enzyme-labeled immunoassay kit and application of thereof to serum detection - Google Patents
Enzyme-labeled immunoassay kit and application of thereof to serum detection Download PDFInfo
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- CN103901192A CN103901192A CN201410108594.8A CN201410108594A CN103901192A CN 103901192 A CN103901192 A CN 103901192A CN 201410108594 A CN201410108594 A CN 201410108594A CN 103901192 A CN103901192 A CN 103901192A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
Abstract
The invention discloses an enzyme-labeled immunoassay kit which comprises an antibody pre-coating reaction plate, an enzyme-labeled antibody and luminous substrate liquid, wherein the enzyme-labeled antibody takes SBP (soybean protease) as marker enzyme; the luminous substrate liquid comprises H2O2, luminol and an enhancer; the enhancer consists of MORPH (4-marlene pyridine) and SPTZ (3-(10'-phenothiazinyl) propyl-1-sulfonate). According to the enzyme-labeled immunoassay kit, the SBP is introduced to serve as the marker enzyme; on one hand, the thermal stability of the enzyme is higher than that of HRP (hypothalamic regulatory peptide), and the enzyme has the advantages of wide substrate working range, high heat resistance, high acid-alkaline stability, wide pH application range and the like; on the other hand, when the enhancers MORPH and SPTZ are used for enhancing chemical illumination, a chemical luminous signal is enhanced by nearly 100 times, and the detection sensitivity is greatly improved.
Description
Technical field
The present invention relates to immune reagent kit field, be specifically related to a kind of enzyme-labeled immunity kit and the application in serum detects thereof.
Background technology
ELISA method is a new technology in immunodiagnosis, is now successfully applied to the immunodiagnosis of the aspects such as the caused infectious disease of multiple pathogenic microorganisms, parasitic disease and non-infective disease.Also be applied to the quantitative measurement of large molecular antigen and little molecular antigen, had sensitive, special, simple, quick, stable and be easy to the features such as automation mechanized operation.Be not only applicable to the inspection of clinical samples, and can check even thousands of parts of samples of hundreds of within one day, therefore, be suitable for seroepidemiological survey yet.Not only can be used for measuring antibody, and can be used for measuring the circulating antigen in body fluid, so be also a kind of good method of early diagnosis.Therefore ELISA method is at the range of application expanding day in the each field of biomedicine.
In ELISA method, the activity of peroxidase is normally by Catalyzed Synthesis By Peroxidase hydrogen peroxide (H
2o
2) coloured product of obtaining of oxidation substrates (as tetramethyl benzidine TMB), with colorimetric method for determining coloured product.Found afterwards based at H
2o
2under the existence of reinforcing agent, horseradish peroxidase (HRP) can catalytic oxidation luminol, occurs " enhanced chemiluminescence reaction ", and the immuno-enzymatic kit that various chemiluminescence methods are measured enzymes work arises at the historic moment.Why these enhanced chemiluminescence reaction immuno-enzymatic kits are used widely, and be because method is high compared with the sensitivity of colorimetric detection immunization for this reason, and under the condition that there is no reinforcing agent, the chemiluminescence signal that HRP catalysis produces are lower.At present, in HRP catalytic luminescence system, the reinforcing agent the most widely of use is to iodophenol (PIP).But reaction kinetics research shows, the H of HRP catalysis
2o
2the chemiluminescence intensity that oxidation luminol reaction detection arrives is unstable.Just start, within very short time, luminous intensity strengthens rapidly and reaches maximal value, then reduces very soon.This decay be by substrate oxidation reaction free radical product with make the inactivation of HRP being connected of HRP due to.Therefore the chemiluminescence signal that the luminol oxidation reaction that, HRP catalysis causes produces is unstable.
Summary of the invention
The object of this invention is to provide a kind of enzyme-labeled immunity kit, adopting soybean peroxidase (SBP) is marker enzyme, MORPH and SPTZ are reinforcing agent, have solved the unsettled problem of chemiluminescence signal that in prior art, HRP catalysis causes luminol oxidation reaction to produce.The present invention also provides the application of this enzyme-labeled immunity kit in serum detects.
For addressing the above problem, the present invention by the following technical solutions:
A kind of enzyme-labeled immunity kit, comprises the pre-coated reaction plate of antibody, enzyme labelled antibody and luminous substrate liquid, and described enzyme labelled antibody is take soybean peroxidase as marker enzyme; Described luminous substrate liquid comprises H
2o
2, luminol and reinforcing agent; Described reinforcing agent is MORPH(4-horse beautiful jade pyridine) and SPTZ(3-(10 '-phenothiazinyl) propyl group-1-sulfonate).
The pre-coated reaction plate of described antibody prepares through following steps:
The modification of step 1, orifice plate
Select complete 96 black hole ELISA Plate, modified antibodies damping fluid is selected the carbonate of pH9.6,100 μ L concentration is that 5 μ g/mL Ab1 antibody add in described 96 hole ELISA Plate coated, 4 ℃ of night incubation; PBST fully washs three times, and PBS washs three times again, pats dry gently 4 ℃ of preservations;
Join in the 96 hole ELISA Plate that are fixed with Ab1 antibody that step 1 prepares with 200 μ L2%BSA, room temperature sealing 2h, PBS washing three times, pats dry gently, 4 ℃ of preservations, wherein, described 2%BSA is PBS preparation;
Wherein, the PBS that step 1 and step 2 relate to is 10mM pH7.4, and PBST is the 0.1%Tween of PBS preparation.
Described enzyme labelled antibody adopts improvement sodium periodate method to prepare:
Step 1, get 1mg soybean peroxidase and be dissolved in 100 μ L PBS, add the 12.8mg/mLNaIO of new preparation
4solution 50 μ L, mix, and put room temperature 15min;
In step 3, the solution that obtains to step 2, add the antibody containing 2mg purifying Ab2, mix, and pack bag filter into, in 4 ℃ of dialysis 18h of 50mM pH9.6 carbonate buffer solution, make soybean peroxidase and the combination of Ab2 antibody;
In step 4, the solution that obtains to step 3, add 5mg/ml NaBH
4solution 20 μ L, mix, and put 4 ℃ of reductase 12 h;
Step 5, slowly add the isopyknic saturated ammonium sulfate solution of the solution obtaining with step 4, the jog 0.5h that saltouts, the centrifugal 15min of 132000g, removes supernatant, and precipitation is dissolved with a little PBS, packs bag filter into, 4 ℃ of dialysed overnight in a large amount of PBS;
Step 6, next day take out centrifugal, and to remove insolubles, the upper strata stillness of night obtains soybean peroxidase-Ab2 antibody conjugates, adds to 500 μ L with PBS;
After step 7, titration are qualified, add equivalent glycerine, packing bottle, low temperature is preserved;
Wherein, the PBS that step 1 to seven relates to is 10mM pH7.4.
Described luminous substrate liquid prepares through following steps: 1.2mM SPTZ, 0.6mM MORPH, 0.4mMLuminol and 0.4mM H
2o
2in the Tris-HCl of 50mM pH8.5 damping fluid.
The application of the enzyme-labeled immunity kit of above-mentioned preparation in serum detects.
Principle of the present invention: research shows there is no under reinforcing agent H
2o
2the reaction of oxidation luminol just can be effectively by SBP catalysis.Another feature of SBP is: luminol oxidation product can produce long chemiluminescence signal of a duration.And SBP stability is higher than HRP, the advantage such as the substrate-function scope of enzyme is wide, and heat resistance is high, and ph stability is good, and the pH scope of application is wide, these are also the important parameters that stores enzyme immunoreagent.In the time adding reinforcing agent SPTZ and MMORPH, chemiluminescence intensity can continue to strengthen nearly a hundred times, and detection signal is further amplified, and detection sensitivity improves greatly.
Beneficial effect of the present invention:
The advantages such as 1, enzyme-labeled immunity kit of the present invention is introduced the SBP enzyme that serves as a mark, and the thermal stability of this enzyme is higher than HRP on the one hand, has substrate-function scope wide, and heat resistance is high, and ph stability is good, and the pH scope of application is wide.On the other hand, in the time using reinforcing agent MORPH and SPTZ enhanced chemiluminescence, chemiluminescence intensity is by original 3.5 × 10
5bring up to 1.5 × 10
7, chemiluminescence signal can strengthen nearly two orders of magnitude, and detection sensitivity can be greatly improved.
2, use enzyme-labeled immunity kit of the present invention not only to realize high flux, the fast detecting of the many indexs of a blood sample, and operation steps same as the prior art and detecting instrument, directly can use, without the redevelopment of professional operating personnel's retraining and instrument, software.Therefore its study on the industrialization is with a wide range of applications and important meaning.
Accompanying drawing explanation
Fig. 1 is the implementation process schematic diagram of enzyme-labeled immunity kit of the present invention.
Embodiment
Below in conjunction with drawings and Examples, the present invention is done further and explained.
A kind of enzyme-labeled immunity kit, comprises the pre-coated reaction plate of antibody, enzyme labelled antibody and luminous substrate liquid, and described enzyme labelled antibody is take soybean peroxidase as marker enzyme; Described luminous substrate liquid comprises H
2o
2, luminol and reinforcing agent; Described reinforcing agent is MORPH and SPTZ, and following examples MORPH and SPTZ are purchased from Sigma Aldrich.
Embodiment 1
Enzyme-labeled immunity kit implementation process of the present invention as shown in Figure 1.
The PBS that the present embodiment relates to is 10mM pH7.4, and PBST is the 0.1%Tween of 10mM pH7.4PBS preparation.The SBP using is purchased from Bio-Research Products company of the U.S..
The first step, the preparation of enzyme labelled antibody
Adopt improvement sodium periodate method: 1, get 1mg SBP and be dissolved in 100 μ LPBS, add the 12.8mg/mLNaIO of new preparation
4solution 50 μ L, mix, and put room temperature 15min; 2, after taking-up, add 9 μ L/mL ethylene glycol PBS solution 50 μ L, room temperature is placed 30min; 3, add containing 2mg purifying HCG human chorionic promoting sexual gland hormone antibody (β-HCG), mix, and pack bag filter into, in 4 ℃ of dialysis 18h of 50mM pH9.6 carbonate buffer solution, make SBP and the combination of Ab2 antibody; 4, add 5mg/ml NaBH
4solution 20 μ L, mix, and put 4 ℃ of reductase 12 h; 5, in above solution, slowly add isopyknic saturated ammonium sulfate solution, the jog 0.5h that saltouts, the centrifugal 15min of 132000g, removes supernatant, and precipitation is dissolved with a little PBS, packs bag filter into, 4 ℃ of dialysed overnight in a large amount of PBS; 6, next day, taking-up was centrifugal, and to remove insolubles, the upper strata stillness of night obtains SBP-Ab2 antibody conjugates, adds to 500 μ L with PBS; 7, titration qualified after, add equivalent high-quality glycerine, packing bottle, low temperature preserve.
Second step, the modification of orifice plate
Orifice plate is selected 96 hole ELISA Plate of black, high-bond entirely, and modified antibodies damping fluid is selected the carbonate into pH9.6.100 μ L concentration are that 5 μ g/mL α-HCG antibody add in orifice plate coated, 4 ℃ of night incubation.PBST fully washs three times, and PBS washs three times again, pats dry gently 4 ℃ of preservations.
The 3rd step, seals non-specific avtive spot
With 200 μ L2%BSA(PBS preparations) add in the orifice plate of the above-mentioned Ab1 of being fixed with antibody room temperature sealing 2h.PBS washing three times, pats dry 4 ℃ of preservations gently.
The 4th step, the detection of serum
The detection of test serum: 100 μ L test serums are added in the orifice plate that above-mentioned sealing is good, incubated at room 1h, PBST washing three times, PBS washs three times again, pats dry gently.
The 5th step, the catching of enzyme labelled antibody
Catching of enzyme labelled antibody: add the enzyme labelled antibody of above-mentioned preparation of the certain dilutability of 100 μ L (2000 ×) in above-mentioned orifice plate, incubated at room 1h, PBST washing three times, PBS washs three times again, pats dry gently.
The 6th step, luminous substrate liquid adds, and luminous signal detects
First, optimize luminol, H
2o
2, the concentration of MORPH, SPTZ and the pH value of luminous substrate liquid damping fluid used, make luminous intensity maximum.In each hole, add the luminous substrate liquid after optimization: 1.2mM SPTZ, 0.6mMMORPH, 0.4mM Luminol and 0.4mM H simultaneously
2o
2in the Tris-HCl of 50mM pH8.5 damping fluid, utilize CCD imaging to gather immediately the chemiluminescence signal in each hole.Because of each hole corresponding be each index in serum test, thereby, simultaneously can detect each index content in same serum.
The foregoing is only preferred embodiments of the present invention; protection scope of the present invention is not limited with above-mentioned embodiment; in every case the equivalence that those of ordinary skills do according to disclosed content is modified or is changed, and all should include in the protection domain of recording in claims.
Claims (5)
1. an enzyme-labeled immunity kit, comprises the pre-coated reaction plate of antibody, enzyme labelled antibody and luminous substrate liquid, it is characterized in that, described enzyme labelled antibody is take soybean peroxidase as marker enzyme; Described luminous substrate liquid comprises H
2o
2, luminol and reinforcing agent; Described reinforcing agent is MORPH and SPTZ.
2. enzyme-labeled immunity kit according to claim 1, is characterized in that, the pre-coated reaction plate of described antibody prepares through following steps:
The modification of step 1, orifice plate
Select complete 96 black hole ELISA Plate, modified antibodies damping fluid is selected the carbonate of pH9.6,100 μ L concentration is that 5 μ g/mL Ab1 antibody add in described 96 hole ELISA Plate coated, 4 ℃ of night incubation; PBST fully washs three times, and PBS washs three times again, pats dry gently 4 ℃ of preservations;
Step 2, seal non-specific avtive spot
Join in the 96 hole ELISA Plate that are fixed with Ab1 antibody that step 1 prepares with 200 μ L2%BSA, room temperature sealing 2h, PBS washing three times, pats dry gently, 4 ℃ of preservations, wherein, described 2%BSA is PBS preparation;
Wherein, the PBS that step 1 and step 2 relate to is 10mM pH7.4, and PBST is the 0.1%Tween of PBS preparation.
3. enzyme-labeled immunity kit according to claim 1, is characterized in that, described enzyme labelled antibody adopts improvement sodium periodate method to prepare:
Step 1, get 1mg soybean peroxidase and be dissolved in 100 μ L PBS, add the 12.8mg/mLNaIO of new preparation
4solution 50 μ L, mix, and put room temperature 15min;
Step 2, take out the solution that obtains of step 1, after add 9 μ L/mL ethylene glycol PBS solution 50 μ L, room temperature is placed 30min;
In step 3, the solution that obtains to step 2, add the antibody containing 2mg purifying Ab2, mix, and pack bag filter into, in 4 ℃ of dialysis 18h of 50mM pH9.6 carbonate buffer solution, make soybean peroxidase and the combination of Ab2 antibody;
In step 4, the solution that obtains to step 3, add 5mg/ml NaBH
4solution 20 μ L, mix, and put 4 ℃ of reductase 12 h;
Step 5, slowly add the isopyknic saturated ammonium sulfate solution of the solution obtaining with step 4, the jog 0.5h that saltouts, the centrifugal 15min of 132000g, removes supernatant, and precipitation is dissolved with a little PBS, packs bag filter into, 4 ℃ of dialysed overnight in a large amount of PBS;
Step 6, next day take out centrifugal, and to remove insolubles, the upper strata stillness of night obtains soybean peroxidase-Ab2 antibody conjugates, adds to 500 μ L with PBS;
After step 7, titration are qualified, add equivalent glycerine, packing bottle, low temperature is preserved;
Wherein, the PBS that step 1 to seven relates to is 10mM pH7.4.
4. enzyme-labeled immunity kit according to claim 1, is characterized in that, described luminous substrate liquid prepares through following steps: 1.2mM SPTZ, 0.6mM MORPH, 0.4mM Luminol and 0.4mM H
2o
2in the Tris-HCl of 50mM pH8.5 damping fluid.
5. the application of the enzyme-labeled immunity kit that prepared by the arbitrary claim of claim 1-4 in serum detects.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104597253A (en) * | 2015-02-02 | 2015-05-06 | 东南大学 | Method for preparing biological probes by coupling soybean peroxidase and protein molecules |
| CN104614519A (en) * | 2015-02-02 | 2015-05-13 | 东南大学 | Soybean peroxidase labelled chemiluminescence immunoassay kit, as well as use method and application thereof |
| CN106198504A (en) * | 2016-07-25 | 2016-12-07 | 武汉赛维尔生物科技有限公司 | A kind of enhanced chemical luminescence detection kit and method |
| CN107561261A (en) * | 2017-08-18 | 2018-01-09 | 江苏凯基生物技术股份有限公司 | A kind of enhanced chemical luminescence reagent box and preparation method thereof |
| CN110554183A (en) * | 2019-09-17 | 2019-12-10 | 沈阳万类生物科技有限公司 | Hypersensitive ECL chemiluminescent reagent |
| WO2023225776A1 (en) * | 2022-05-23 | 2023-11-30 | 孙嘉怡 | Method for preparing highly sensitive and stable chemiluminescent substrate liquid and use thereof in immunoassay |
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| CN101661042A (en) * | 2009-09-25 | 2010-03-03 | 中国农业大学 | Porcine reproductive and respiratory syndrome virus (PRRSV) double-antibody sandwich ELISA kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104597253A (en) * | 2015-02-02 | 2015-05-06 | 东南大学 | Method for preparing biological probes by coupling soybean peroxidase and protein molecules |
| CN104614519A (en) * | 2015-02-02 | 2015-05-13 | 东南大学 | Soybean peroxidase labelled chemiluminescence immunoassay kit, as well as use method and application thereof |
| CN106198504A (en) * | 2016-07-25 | 2016-12-07 | 武汉赛维尔生物科技有限公司 | A kind of enhanced chemical luminescence detection kit and method |
| CN107561261A (en) * | 2017-08-18 | 2018-01-09 | 江苏凯基生物技术股份有限公司 | A kind of enhanced chemical luminescence reagent box and preparation method thereof |
| CN110554183A (en) * | 2019-09-17 | 2019-12-10 | 沈阳万类生物科技有限公司 | Hypersensitive ECL chemiluminescent reagent |
| WO2023225776A1 (en) * | 2022-05-23 | 2023-11-30 | 孙嘉怡 | Method for preparing highly sensitive and stable chemiluminescent substrate liquid and use thereof in immunoassay |
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| CN103901192B (en) | 2016-02-17 |
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