CN102809648A - High-sensitivity immunoassay method for enzyme signal cyclic amplification - Google Patents
High-sensitivity immunoassay method for enzyme signal cyclic amplification Download PDFInfo
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- CN102809648A CN102809648A CN2011101444913A CN201110144491A CN102809648A CN 102809648 A CN102809648 A CN 102809648A CN 2011101444913 A CN2011101444913 A CN 2011101444913A CN 201110144491 A CN201110144491 A CN 201110144491A CN 102809648 A CN102809648 A CN 102809648A
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Abstract
The invention relates to the field of enzyme-linked immunosorbent assay (ELISA), in particular to an anti-horseradish peroxidase (HRP) polyclonal antibody-HRP compound which is applicable to an ELISA technology for enzyme signal cyclic amplification. An immunoassay method based on the enzyme signal cyclic amplification is applicable to various types of immunoassay systems based on enzyme signal amplification. The immunoassay systems have the advantage that the immunoassay sensitivity can be improved to the greatest extent.
Description
Technical field
The present invention relates to technical field of immunoassay, particularly a kind of based on enzyme signal cycle amplification hypersensitive immunologic detection method, belong to enzyme linked immunological (ELISA) technical field.
Background technology
Enzyme linked immunological (ELISA) technology is a technology of outbalance in the modern immunoassay, and especially the various types of preliminary screening agent boxes based on elisa technique are widely used in clinical examination, food security field.The reagent that 3 necessity are arranged in this detection technique: the 1. antigen of solid phase or antibody; 2. the antigen of enzyme labeling or antibody; 3. the substrate of enzyme effect.According to source and the proterties of sample and the satisfying the requirements of detection of reagent, can design various dissimilar detection methods, survey antigen, double antigens sandwich survey antibody, indirect method survey antibody, competition law isotype like double-antibody sandwich.Elisa technique detects Projects with Different; Its detection sensitivity and corresponding antigen and antibody are closely connected; And along with inspection and quarantine require more and more stricter, to method sensitivity require increasingly high, and based on traditional elisa technique in that often to improve the space aspect the sensitivity limited; Also can only rely on the affinity that improves antigen-antibody, thereby improve its detection sensitivity indirectly.
Summary of the invention
Demand to current elisa technique development trend: how its detection sensitivity effectively is provided, and technical matters to be solved by this invention is exactly on original ELISA detection architecture basis, carries out innovative design to obtain higher detection sensitivity.Innovation of the present invention mainly is in original ELISA detection architecture, to introduce " antienzyme antibody-enzyme labeling compound " like the anti-horseradish peroxidase of rabbit (Horseradish peroxidase; HRP) resist-the HRP compound more; Anti-HRP-alkaline phosphatase (the Alkaline phosohatase of rabbit; AP) compound etc. is to reach higher detection sensitivity.
The object of the present invention is to provide a kind of ELISA detection system of amplifying based on enzyme signal cycle signal.
Another object of the present invention is to provide one type of " antienzyme antibody-enzyme labeling thing " preparation method and be applied in the traditional E LISA technology.
A purpose more of the present invention is to utilize one type " antienzyme antibody-enzyme labeling thing " to realize the circulation amplification of enzyme signal among the traditional E LISA, thereby improves detection sensitivity.
The object of the invention is realized through following technical scheme: the preparation of one type of antienzyme antibody-enzyme labeling thing also is applied in the traditional E LISA technology.To the HRP color development system, prepare anti-HRP and resist-the HRP compound more; To the AP color development system, prepare anti-AP and resist-the AP compound more; The compound that also possibly prepare other types colour developing enzyme antibody enzyme mark compound and dissimilar colour developing enzyme antibody thereof-enzyme intersection preparation; This type of compound can be realized the circulation amplification of enzyme signal; Traditional E LISA is provided the sensitivity of detection architecture, and this instructions is that example is described of the present invention designing a model with HRP.
Technical scheme of the present invention is that how anti-at first obtain anti-HRP through how anti-technology of preparing, and carries out compatible reaction with certain amount of H RP and form how anti--HRP compound of anti-HRP.Choose a certain ELISA detection model such as double antibodies sandwich and detect the new E LISA detection architecture that HBsAg foundation is amplified based on the enzyme signal cycle.
Above-mentioned anti-HRP how preparation flow and the detection system of anti--HRP compound makes up and specifically can comprise the steps:
(1), with the HRP immunize rabbit of purifying, obtain containing the rabbit anteserum of anti-HRP antibody; Earlier with (NH
4)
2SO
4The precipitation method are the preliminary purification Immunoglobulin IgG from the serum of rabbit, is further purified the IgG of anti-HRP again with the ProteinA-Sepharose affinity column, and SDS-PAGE and Western blotting identify how anti-the anti-HRP of separation and purification is; (2), how anti-the anti-HRP that gets behind the above-mentioned purifying is, adds certain amount of H RP and form how anti--HRP compound of undersaturated anti-HRP, this compound not only has the HRP enzymatic activity, and still has the avtive spot with the HRP enzyme reaction; (3), applicating example 1: the double antibodies sandwich method detects on the basis of hepatitis B surface antigen (HBsAg); Introduce anti-HRP resists-the HRP compound more; Add before the ELISA development step above-mentioned anti-HRP how anti--wash behind HRP compound reaction 30min, carry out the enzyme development step again; (4), applicating example 2: indirect method is surveyed on foot and mouth disease virus (FMDV) the antibody ELISA kit basis; Introduce anti-HRP resists-the HRP compound more; Add before the ELISA development step above-mentioned anti-HRP how anti--wash behind HRP compound reaction 30min, carry out the enzyme development step again.
The invention provides the how preparation method of anti--HRP compound of anti-HRP, this compound can be used in the ELISA detection architecture, forms based on the enzyme signal cycle and amplifies immune detection system, can improve the sensitivity of detection from far away.
Description of drawings
Fig. 1 enzyme signal cycle is amplified application example 1-double-antibody sandwich elisa pattern.
Fig. 2 enzyme signal cycle is amplified application example 2-indirect method and is surveyed the antibody ELISA pattern.
Embodiment
Embodiment be to anti-HRP provided by the present invention how anti--HRP compound preparation and be applied to further specifying of enzyme signal cycle amplification system applicating example; But the working of an invention mode is not limited thereto; For the ELISA of other modes, this invention strategy is same suitable.
Embodiment 1 anti-HRP is the preparation of anti--HRP compound how
(1) preparation of Water-In-Oil antigen emulsifying agent
Mix 2 mg purifying HRP with Freund's complete adjuvant or incomplete Freund 1.2mL; Mixed 2 hours with homogenizer; The emulsifying agent that makes is splashed in the beaker that fills cold water; If an emulsifying agent state intactly rests on the water surface, and indiffusion shows to form stable Water-In-Oil antigen emulsifying agent.
(2) immune new zealand white rabbit
It is big to choose for 4 weeks, 2 of the healthy new zealand white rabbits of the about 1.5Kg of body weight.Hair with the new zealand white rabbit back carefully cuts off earlier; Get the Water-In-Oil antigen emulsifying agent that 600 μ L prepare then, carry out hypodermic injection with micro syringe multidigit point ground, back part of animal two side injection sums are about 8～10 points; Antigen can slowly be spread; Every whether at a distance from 1 ~ 2 all immunity once it is red and swollen to observe injection point, if rotten to the corn available gentian violet is received does.After immune 3-4 time, from the about 1mL of the auricular vein of rabbit blood drawing, behind the centrifugal 10min, the evaluation of must serum can tiring.Carry out 6 immunity altogether, adopt the arteria carotis depletion method to get blood after back 5 days in the last immunity.
(3) arteria carotis depletion method
Do skin incision in the White Rabbit neck outside, draw back the nutator of visible diagonal behind the skin, this flesh passivity is separated and pushed the rear to, can see the resilient total artery of pale red.With this artery free gently (together with the vagus nerve of going together with it), with the distal end ligation, proximal part is clamped with hemostatic forceps with silk thread, and another hemostatic forceps is clamped the artery vagus nerve, in order to fixing.Cut off blood vessel along ligation place, with fixing hemostatic forceps the broken ends of fractured bone is vised, insert the blood transfusion needle tubing, slowly open the hemostatic forceps of clamping, arterial blood flows in the bottle through the blood transfusion needle tubing immediately.Adopt the room temperature natural coagulation to separate antiserum, place then 37 ℃ 1 hour, 4 ℃ are spent the night again, treat clot retracts, centrifugal 15 minutes of 4000rpm collects supernatant.
(4) sero-fast purifying
Saturated ammonium sulfate solution: get 500ml distilled water and be heated to 70～80 ℃, 400g ammonium sulfate is dissolved in wherein, stir 20min, cooling.At the bottom of treating that ammonium sulfate crystallization is sunken to bottle, its supernatant is saturated ammonium sulfate.Transfer pH7.0 with 28% ammoniacal liquor before use.Extract with 55% saturated ammonium sulfate: serum adds 1 part of mixing of physiological saline for 1 part, dropwise adds then in 2 parts of the saturated ammonium sulfates, and the limit edged stirs, and prevents to form agglomerate and reduces sedimentary specificity. and leave standstill 30min behind the mixing or put 4 ℃ of refrigerator overnight.The centrifugal 10min of low-temperature and high-speed 10 000/min discards supernatant (containing albumin), and taking precipitate (containing globulin) is dissolved in a small amount of physiological saline.
Extract with 33% saturated ammonium sulfate: 2 parts of said extracted thing normal saline solutions are added 1 part of saturated ammonium sulfate.And then 10 000/min centrifugal, all the other operations are the same.Repeating above-mentioned steps with 33% saturated ammonium sulfate extracts once.
With the extract bag filter of packing into, in physiological saline, dialyse, to remove wherein contained ammonium sulfate.Putting-20 ℃ of refrigerators preserves.Carry out being further purified of antibody according to ProteinA-Sepharose affinity column purification kit operational manual.And adopt SDS-PAGE and Western blotting to identify how anti-the anti-HRP of separation and purification is.
(5) preparation of anti-HRP antibody-HRP compound
Get the good HRP antibody 10mg of purifying, add certain amount of H RP, the two is affine to form unsaturated anti-HRP antibody-HRP compound, and the bag filter of packing into is dialysed in physiological saline, puts-20 ℃ of refrigerators and preserves.
Embodiment 2 enzyme signal cycle are amplified high-sensitivity immunity and are detected hepatitis B surface antigen system (double-antibody sandwich survey antigen)
(1) material is prepared
HBsAg encapsulates plate: how anti-get anti-HBsAg, and with PH 9.6 carbonate buffer solutions dilution final concentration 5-8 ug/ml, encapsulating volume is 100～150ul/ hole.Encapsulating 4 ° of C of condition spends the night.1%BSA, 0.01M PBS, PH7.4,37 ° of C sealing 2h dry subsequent use.
Anti-HBsAg enzyme conjugates: 1. get 5 mg HRP and be dissolved in that 0.5ml is two to be heated up in a steamer in the water, add 0.06 Mol/L NaIO of new preparation
4The WS 0.5 ml, mixing is put 4 ℃ of 30 min; 2. take out the back and add 0.16 Mol/L glycol water, 0.5 ml, room temperature is placed 30 min; 3. add the WS 1ml that contains 5mg Purification of HBsAg antibody, mixing, and the bag filter of packing into slowly stir dialysis 6 h to 0.05 Mol/L pH, 9.5 carbonate buffer solutions, make it to combine; 4. add NaBH
4Solution (5 mg/ml) 0.2 ml, mixing is put 4 ℃ of 2 h; 5. in above solution, slowly add isopyknic saturated ammonium sulfate solution, mixing, 4 ℃ of 30 min, centrifugal, remove supernatant, deposition is with a little 0.02 Mol/L pH7.4 PBS liquid dissolving, and the bag filter of packing into spends the night at 4 ℃ of dialysis desalinations with same liquid; 6. next day, taking-up was centrifugal, to remove insolubles, promptly got the anti-HBsAg enzyme conjugates, added to 5 ml with 0.02 Mol/L pH, 7.4 PBS liquid; 7. titration qualified after, add equivalent high-quality glycerine, packing bottle, cryopreservation.
Developer A liquid: sodium acetate 13.6g, citric acid 1.6g, H2O2 (30%) 0.3ml, dH2O 500ml;
Developer B liquid: EDTA-Na 0.2g, citric acid 0.95g, glycerine 50ml, TMB (tetramethyl benzidine) 0.2g
Stop buffer: 2M H
2SO4;
20 * concentrated cleaning solution: 8 mM sodium phosphates, the 2mM potassium phosphate, 0.14 M NaCl, 100 mM KCl, 0.05% Tween-20, pH 7.4.
(2) operation steps
Take out the antibody sandwich plate, reserve blank well 1 hole, negative control 3 holes, positive control 2 holes, all the other every holes, each hole add 50 μ L samples to be tested.Yin and yang attribute contrasts every hole and adds 50 μ L control serums.Every then hole adds 50 μ L anti-HBsAg enzyme conjugates (not comprising blank well), and blank well is vacant.Behind the mixing, 37 ℃ of incubations 30 minutes.Cleansing solution with dilution is good is washed plate 1 time, dries the back and adds anti-HRP antibody-HRP compound 50 μ L/ holes (dilution ratio 1000-10000), 37 ℃ of incubations 30 minutes.Cleansing solution with dilution is good is washed plate 4 times, and cleansing solution should be filled with plate hole when washing plate, but should not overflow, and should soak 15 seconds after injecting cleansing solution at every turn.Every hole adds 50 μ L developer A liquid earlier, adds 50 μ L developer B liquid again, behind the mixing, and 37 ℃ of lucifuge incubations 30 minutes.Every hole adds 100 μ L stop buffers, mixing.Selecting ELIASA to measure wavelength is 450nm, and reference wavelength 630nm measures each hole absorption value.
Embodiment 3 enzyme signal cycle are amplified high-sensitivity immunity and are detected Schweineseuche antiviral antibody system (indirect method survey antibody)
(1) material is prepared
Envelope antigen: get O type foot and mouth disease virus VP1 albumen, with PH 9.6 carbonate buffer solutions dilution final concentration 3-10 ug/ml, encapsulating volume is the 150ul/ hole.Encapsulating 4 ° of C of condition spends the night.5% skimmed milk power, 0.01M PBS, 0.05% Tween20, PH7.4,37 ° of C sealing 2h dry subsequent use.
ELIAS secondary antibody: 1. get 5 mg HRP and be dissolved in that 0.5ml is two to be heated up in a steamer in the water, add 0.06 Mol/L NaIO of new preparation
4The WS 0.5 ml, mixing is put 4 ℃ of 30 min; 2. take out the back and add 0.16 Mol/L glycol water, 0.5 ml, room temperature is placed 30 min; 3. add and contain the WS 1ml that 5mg two resists, mixing, and the bag filter of packing into slowly stir dialysis 6 h to 0.05 Mol/L pH, 9.5 carbonate buffer solutions, make it to combine; 4. add NaBH
4Solution (5 mg/ml) 0.2 ml, mixing is put 4 ℃ of 2 h; 5. in above solution, slowly add isopyknic saturated ammonium sulfate solution, mixing, 4 ℃ of 30 min, centrifugal, remove supernatant, deposition is with a little 0.02 Mol/L pH7.4 PBS liquid dissolving, and the bag filter of packing into spends the night at 4 ℃ of dialysis desalinations with same liquid; 6. next day, taking-up was centrifugal, to remove insolubles, promptly got the anti-HBsAg enzyme conjugates, added to 5 ml with 0.02 Mol/L pH, 7.4 PBS liquid; 7. titration qualified after, add equivalent high-quality glycerine, packing bottle, cryopreservation.
Developer A liquid: sodium acetate 13.6g, citric acid 1.6g, H2O2 (30%) 0.3ml, dH2O 500mL;
Developer B liquid: EDTA-Na 0.2g, citric acid 0.95g, glycerine 50mL, TMB (tetramethyl benzidine) 0.2g
Stop buffer: 1M HCl;
20 * concentrated cleaning solution: 8 mM sodium phosphates, the 2mM potassium phosphate, 0.14 M NaCl, 100 mM KCl, 0.05% Tween-20, pH 7.4.
(2) operation steps
Get the PV1 antigen coated microplate, reserve blank well 1 hole, negative control 3 holes, positive control 2 holes, all the other every holes, each hole add 50 μ L samples to be tested.Every then hole adds 50 μ L ELIAS secondary antibodies (not comprising blank well), and blank well is vacant.Behind the mixing, 37 ℃ of incubations 30 minutes.Cleansing solution with dilution is good is washed plate 1 time, dries the back and adds anti-HRP antibody-HRP compound 50 μ L/ holes (dilution ratio 1000-10000), 37 ℃ of incubations 30 minutes.Cleansing solution with dilution is good is washed plate 4 times, and cleansing solution should be filled with plate hole when washing plate, but should not overflow, and should soak 15 seconds after injecting cleansing solution at every turn.Every hole adds 50 μ L developer A liquid earlier, adds 50 μ L developer B liquid again, behind the mixing, and 37 ℃ of lucifuge incubations 30 minutes.Every hole adds 100 μ L stop buffers, mixing.Selecting ELIASA to measure wavelength is 450nm, and reference wavelength 630nm measures each hole absorption value.
The foregoing description is an embodiment of the present invention part, but embodiment of the present invention is not restricted to the described embodiments, and other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplification.All should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. one kind is amplified the structure of immunologic detection method based on the enzyme signal cycle, it is characterized in that: adopt anti-HRP how the circulation of carrying out the enzyme signal of anti--HRP compound amplify, and be not limited only to HRP, also the enzyme of other types such as alkaline phosphatase AP etc.
2. enzyme signal cycle according to claim 1 is amplified immunologic detection method; Be that the circulation of on traditional E LISA detecting pattern, carrying out the enzyme signal is again amplified, traditional common detecting pattern have double-antibody sandwich survey antigen, double antigens sandwich survey antibody, indirect method survey antibody, competition law survey antibody dissimilar ELISA detecting pattern such as antigen, Avidin-biotin-ELISA or the like.
3. one kind like claim 1; 2 described enzyme signal cycle are amplified immunologic detection method; It is characterized in that using a kind of anti-HRP to resist-HRP compound (or other types enzyme corresponding compound thing of equivalence), what it adopted is the how anti-of anti-HRP, and forms unsaturated type complex with HRP more; This compound has the HRP enzymatic activity, can also combine with HRP simultaneously.
4. anti-HRP according to claim 1 is the preparation method of anti--HRP compound how, it is characterized in that the HRP immunize rabbit with purifying, obtains containing the rabbit anteserum of anti-HRP antibody; Earlier with (NH
4)
2SO
4The precipitation method are the preliminary purification Immunoglobulin IgG from the serum of rabbit, is further purified the IgG of anti-HRP again with the ProteinA-Sepharose affinity column, and SDS-PAGE and Western blotting identify how anti-the anti-HRP of separation and purification is.
5. how anti-the anti-HRP that gets behind the above-mentioned purifying is, adds certain amount of H RP and form how anti--HRP compound of undersaturated anti-HRP.
6. according to claim 1,2 described enzyme signal cycle are amplified immunologic detection method, it is characterized in that realizing the circulation amplification of enzyme signal, improve its detection sensitivity greatly, adopt which kind of ELISA detecting pattern and be not restricted to.
7. one kind like claim 1, and 2 described enzyme signal cycle amplification methods are applicable to the development of all kinds of ELISA kits, to require be a kind of new E LISA detection system of amplifying based on the enzyme signal cycle.
8. how anti-how anti-an anti-HRP antibody as claimed in claim 1 belong to, and be not limited to which kind of type, and how anti-promptly can be rabbit, can be also how anti-mouse is, sheep is how anti-etc.
9. the described enzyme signal cycle of claim 1 is amplified the application of immune detection system in association areas such as clinical examination, food security, animal epidemics.
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Cited By (2)
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| CN104101711A (en) * | 2013-04-07 | 2014-10-15 | 广州瑞博奥生物科技有限公司 | Improved enzyme-linked immunoassay kit and detection method thereof |
| CN114636820A (en) * | 2022-05-07 | 2022-06-17 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114636820A (en) * | 2022-05-07 | 2022-06-17 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
| CN114636820B (en) * | 2022-05-07 | 2022-10-21 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
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