CN105467113A - Immunoassay method based on fluorescein and luciferase bioluminescent reaction and application thereof - Google Patents
Immunoassay method based on fluorescein and luciferase bioluminescent reaction and application thereof Download PDFInfo
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- 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
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- 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
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Abstract
The invention provides an immunoassay method based on fluorescein and luciferase bioluminescent reaction and application thereof. The method comprises the steps of: (a) coating a coated material on a solid carrier, incubating and washing with a buffer; (2) adding a determinand standard solution, incubating and washing with a buffer; (3) adding ALP labeled secondary antibody which is an anti-determinand antibody, incubating and washing with a buffer; (4) adding an ATP solution, reacting, adding a bio-luminescent substrate, reacting, and finally testing a luminescence value to obtained a result I; (5) replacing the determinand standard solution in the step (2) with a determinand sample solution, repeating the operations in the step (1) to (4), detecting a luminescence value to obtain a result II, comparing the result II with the result I, and conversing to obtain the concentration of the determinand in the sample solution. The immunoassay method has the advantages of high sensitivity, simple signal readout mode, wide linear range, and fast analysis.
Description
Technical field
The invention belongs to technical field of immune assay, be specifically related to a kind of immune analysis method based on fluorescein and luciferase bioluminescence reaction and application thereof.
Background technology
Build High Sensitive Analysis method is one of target of analytical chemistry field pursuit always, and High Sensitive Analysis method has promoted the development of analytical chemistry and the application in fields such as clinical diagnosis, environmental monitoring, food security, bio-imagings thereof.In serious infectious diseases, bacterium or the early diagnosis of the disease such as virus infections, cancer and the examination of food and Environmental Trace noxious material, High Sensitive Analysis method seems particularly important.Immune biomarker analysis method based on antibody-antigene specific recognition is a kind of analytical approach high sensitivity of biomarker technology and immunoreactive high degree of specificity combined, have highly sensitive, specificity good, analyze the advantages such as quick.Enzyme linked immunological is representative wherein, and the method has high specificity, reproducible, simple to operate, cost is low, be easy to the advantage such as commercialization and robotization.But traditional enzyme-linked immune analytic method is all generally amplify based on time enzymatic signal of between immune labeled enzyme and substrate, and its sensitivity can only reach ng/mL.At the highly sensitive analysis field of needs, traditional enzyme-linked immune analytic method is difficult to satisfy the demand, and therefore, how to realize simple and convenient high-sensitivity immunity analysis and is still one of the significant challenge in analytical chemistry field.
At present, the realization of high-sensitive immune analysis method mainly relies on: 1) effective enrichment of object and amplification in sample; 2) effective signal amplifies strategy.Immune magnetic enrichment method based on nanometer magnetic bead can effective object in enriched sample, has the advantages such as simple to operate, velocity of separation is fast, bioaccumulation efficiency is high, is widely used in immunoassay.Meanwhile, suitable signal is adopted to amplify strategy most important for the raising of immune analysis method sensitivity.In the last few years, researchers have developed a series of method for amplifying signal, mainly comprise: (1) is based on the signal amplifying system of nano material: novel nano-material (nanometer magnetic bead, collaurum, quantum dot, Graphene, Si-based nanometer material etc.) to have specific surface area large, stability and the advantage such as bio-compatibility is good, can a large amount of signal reporter group of coupling as fluorescence molecule or enzyme, reach the object that signal amplifies.(2) based on the signal amplifying system of click reaction: click reaction refers to that a class has stereoselectivity, reaction conditions is simple, react chemical reaction fast, be widely applied in fields such as Bioconjugation, clinical diagnosis, cell imaging, bio-sensings.In immune analysis method, antibody by more enzyme or other signal reporter molecules in the coupling of click controllable reaction, thus can improve the sensitivity of method.(3) based on the signal amplifying system of nucleic acid amplification: by nucleic acid amplification technologies as PCR and ring mediated isothermal amplification combine with immune analysis method, the signal of analysans is converted to nucleic acid signal analysis, thus improve existing methodical sensitivity, but the method operation more complicated, have impact on its practical application.Although above-mentioned 3 class amplification systems can improve the sensitivity (improving 1-2 the order of magnitude) of analytical approach, its amplification effect still can be subject to many-sided restriction.Such as, in immunoassay, the multistep signal amplifying system based on click reaction can increase experimental procedure, have impact on speed and the accuracy of analysis.Based on the signal amplifying system experimental implementation more complicated of nucleic acid amplification, have impact on its practical application.Therefore, be necessary that research and development sensitivity is higher, analysis speed is faster, signal exports the more simple immune analysis method of reading manner.
Summary of the invention
The object of the invention is the situation that can not meet practical application request for current immune analysis method in detection sensitivity, analysis speed and signal playback mode etc., and a kind of immune analysis method based on fluorescein and luciferase bioluminescence reaction and application thereof are provided.Immune analysis method of the present invention carries out based on " ATP-fluorescein-luciferase " bioluminescence reaction, has the advantages such as highly sensitive, signal playback mode simple, detect linear wide ranges, analysis speed is fast.
For achieving the above object, the invention provides following technical proposals.
One of technical scheme provided by the invention is: a kind of immune analysis method based on bioluminescence reaction, it comprises the steps:
(1) encrusting substance matter is coated on solid phase carrier, hatches rear damping fluid and rinse;
(2) add the determinand standard solution with variable concentrations gradient, hatch rear damping fluid and rinse;
(3) add with ALP mark two resist, hatch rear damping fluid and rinse, described two resist the antibody for anti-determinand;
(4) after adding ATP solution reaction, then add bioluminescence substrate and react, finally detect luminous value, obtain result I;
(5) the determinand standard solution of the variable concentrations gradient described in step (2) is replaced to testing sample solution, repeat the operation of step (1) ~ (4), finally detect luminous value, obtain result II, by result II reference result I, judge the concentration of determinand in testing sample solution;
Wherein, when determinand is comlete antigen, described encrusting substance matter is to combine from described comlete antigen is corresponding and resists the antibody with different antigen binding site with described two, when determinand is antibody, described encrusting substance matter is the artificial antigen that the comlete antigen corresponding with described antibody or hapten molecule and protein carrier coupling are formed;
Described bioluminescence substrate is the solution containing fluorescein and luciferase.
In the present invention, step (1) is: be coated on solid phase carrier by encrusting substance matter, hatches rear damping fluid and rinses.
As this area is conventional, described encrusting substance matter, for catching material, is generally antibody, if hapten molecule, then carries out bag quilt again by after hapten molecule and protein carrier coupling.The enough determinands described in specific binding step (2) of described encrusting substance mass-energy.
Described solid phase carrier is all kinds of solid phase carriers described in field of immunodetection routine, and it special requirement for concrete material and specification, as long as it is suitable for bag quilt and carries out other routine immunization operations.Preferably, described solid phase carrier is ELISA Plate, and the ELISA Plate of the various conventional material in this area and specification is all applicable to the present invention.
Described routine operation of hatching as this area, be generally by bag by after solid phase carrier be placed in suitable temperature conditions under place a few hours, treat that encrusting substance matter is firmly adsorbed in solid phase carrier.Preferably, described in hatch for by bag by after solid phase carrier be placed in 4 ~ 40 DEG C place 0.5 ~ 12 hour, more preferably in 37 DEG C place 2 hours.
Described damping fluid is that this area is conventional, and all kinds of damping fluid for rinsing excessive encrusting substance matter is all applicable to the present invention, specifically can select depending on different detection reaction systems.Preferably, described damping fluid is PBS damping fluid, and the PBS damping fluid more preferably containing 1 ‰ Tween-20s, described permillage refers to volume permillage.
Described flushing is the routine operation of this area, is generally to add damping fluid on solid phase carrier, leaves standstill to get rid of after several minutes only to pat dry, and generally rinses for several times.
In the present invention, step (2) is: add the determinand standard solution with variable concentrations gradient, hatches rear damping fluid and rinses;
Wherein, described determinand is material to be detected, and specific combination enough occurs for itself and the encrusting substance mass-energy described in step (1), and its attribute is generally comlete antigen or antibody.The source of described antibody is not particularly limited, as being the antibody encrusting substance matter immunity mammalian species described in step (1) obtained, as mouse-anti or rabbit resist, its type does not have particular/special requirement yet, both can be monoclonal antibody, also can be polyclonal antibody.
Describedly hatch same step (1), it is the routine operation of this area, is generally to place a few hours under solid phase carrier being placed in suitable temperature conditions, and thing to be added is firmly adsorbed on solid phase carrier.Preferably, hatch described in and place 0.5 ~ 12 hour for solid phase carrier being placed in 4 ~ 40 DEG C, more preferably in 37 DEG C of placements 2 hours.
The same step of described damping fluid (1), it is that this area is conventional, and all kinds of damping fluid for rinsing excessive additive is all applicable to the present invention, specifically can select depending on different detection reaction systems.Preferably, described damping fluid is PBS damping fluid, and the PBS damping fluid more preferably containing 1 ‰ Tween-20s, described permillage refers to volume permillage.
The same step of described flushing (1), it is the routine operation of this area, is generally to add damping fluid on solid phase carrier, leaves standstill to get rid of after several minutes only to pat dry, and generally rinses for several times.
In the present invention, step (3) is: add and resist with two of ALP mark, hatch rear damping fluid and rinse, described two resist the antibody for anti-determinand.
Wherein, described ALP is the English abbreviation of alkaline phosphatase (alkalinephosphatase), can generate AMP (AMP) by enzymolysis ATP (atriphos) efficiently.
Described two sources resisted are not particularly limited, as being by the determinand immunity mammalian species described in step (2) antibody that obtains, as anti-in goat-anti, horse etc., its type does not have particular/special requirement yet, both can be monoclonal antibody, also can be polyclonal antibody.
Describedly hatch same step (1), it is the routine operation of this area, is generally to place a few hours under solid phase carrier being placed in suitable temperature conditions, and thing to be added is firmly adsorbed on solid phase carrier.Preferably, hatch described in and place 0.5 ~ 12 hour for solid phase carrier being placed in 4 ~ 40 DEG C, more preferably in 37 DEG C of placements 2 hours.
The same step of described damping fluid (1), it is that this area is conventional, and all kinds of damping fluid for rinsing excessive additive is all applicable to the present invention, specifically can select depending on different detection reaction systems.Preferably, described damping fluid is PBS damping fluid, and the PBS damping fluid more preferably containing 1 ‰ Tween-20s, described permillage refers to volume permillage.
The same step of described flushing (1), it is the routine operation of this area, is generally to add damping fluid on solid phase carrier, leaves standstill to get rid of after several minutes only to pat dry, and generally rinses for several times.
In the present invention, described step (3) can also substitute with step (3 '), and described step (3 ') comprises sub-step (3 ') a and sub-step (3 ') b;
Described step (3 ') a is: add and resist with two of biotin or marked by streptavidin, hatch rear damping fluid and rinse, described two resist the antibody for anti-determinand;
Described sub-step (3 ') b is: add Streptavidin or biotin labeled ALP, hatches rear damping fluid and rinses;
Wherein, when add in sub-step (3 ') a with biotin labeled two anti-time, then add the ALP of marked by streptavidin in sub-step (3 ') b; When adding anti-with two of marked by streptavidin in sub-step (3 ') a, then add biotin labeled ALP in sub-step (3 ') b.
Same step (1) is hatched described in sub-step (3 ') a and (3 ') b, it is the routine operation of this area, be generally place a few hours under solid phase carrier being placed in suitable temperature conditions, thing to be added is firmly adsorbed on solid phase carrier.Preferably, hatch described in and place 0.5 ~ 12 hour for solid phase carrier being placed in 4 ~ 40 DEG C, more preferably in 37 DEG C of placements 2 hours.
Sub-step (3 ') a and the same step of damping fluid (1) described in (3 ') b, it is that this area is conventional, all kinds of damping fluid for rinsing excessive additive is all applicable to the present invention, specifically can select depending on different detection reaction systems.Preferably, described damping fluid is PBS damping fluid, and the PBS damping fluid more preferably containing 1 ‰ Tween-20s, described permillage refers to volume permillage.
Sub-step (3 ') a and the same step of flushing (1) described in (3 ') b, it is the routine operation of this area, is generally to add damping fluid on solid phase carrier, leaves standstill to get rid of after several minutes only to pat dry, and generally rinses for several times.
After sub-step (3 ') a and (3 ') b terminates, as usual carry out the operation of step (4).
In the present invention, step (4) is: after adding ATP solution reaction, then adds bioluminescence substrate and react, and finally detects luminous value, obtains result I.
Described bioluminescence substrate is the solution containing fluorescein and luciferase.
The method of described detection luminous value is that this area is conventional, as the corresponding detecting instrument of collection of biological luminescence-producing reaction luminous value can be adopted to carry out, preferably detects bioluminescence value with ATP detector.
The mode of described acquisition result I is that this area is conventional, as carried out by means of supporting ATP detector, to obtain accurate data, or further, can also after acquisition precise information, make the typical curve of reflection determinand standard concentration, thus provide reference foundation for subsequent detection testing sample, reach and detect fast and the object of accurate quantification.
In the present invention, step (5) for: the determinand standard solution of the variable concentrations gradient described in step (2) is replaced to testing sample solution, repeat the operation of step (1) ~ (4), finally detect luminous value, obtain result II, by result II reference result I, judge the concentration of determinand in testing sample solution.
As this area is conventional, generally speaking, the substance of carrying out testing sample after the reference data obtaining contrast detects.In the present invention, general operation is the operation repeating step (1) ~ (4), just the determinand standard solution of the variable concentrations gradient described in step (2) is replaced to testing sample solution, obtains result II.The mode of described acquisition result II is identical with the mode obtaining result I, as being carry out by means of supporting ATP detector, to obtain accurate data, similarly, reference result I (can be preferably reference standard curve), reaches the object that accurate quantification detects sample to be tested.
In concrete operations, when the material detected is the material without standard items such as mycoplasma pulmonis antibody, method of the present invention preferably comprises the steps:
(1) encrusting substance matter is coated on solid phase carrier, hatches rear damping fluid and rinse;
(2) add determinand, hatch rear damping fluid and rinse;
(3) add with ALP mark two resist, hatch rear damping fluid and rinse, described two resist the antibody for anti-determinand;
(4) after adding ATP solution reaction, then add bioluminescence substrate and react, finally detect luminous value, read result;
Wherein, when determinand is comlete antigen, described encrusting substance matter is to combine from described comlete antigen is corresponding and resists the antibody with different antigen binding site with described two, when determinand is antibody, described encrusting substance matter is the artificial antigen that the comlete antigen corresponding with described antibody or hapten molecule and protein carrier coupling are formed;
Described bioluminescence substrate is the solution containing fluorescein and luciferase.
Above technical scheme, eliminates the step reading standard items result, directly reads the result of testing sample, carries out qualitative detection.
The principle of the method for the invention is: provide the condition of energy at ATP under, and luciferase can be oxidized to oxidized fluorescence element by catalytic fluorometry element, can produce bioluminescence, and the content of luminous intensity and ATP is directly proportional in the process of oxidation.And ALP enzyme can generate AMP (AMP) by enzymolysis ATP efficiently, AMP can not provide energy for the bioluminescence reaction of Luciferase catalyses, thus inhibits luminous generation.The present invention is with alkaline phosphatase (alkalinephosphatase, ALP) as the marker enzyme in immune response, ATP is as its catalytic substrate, pass through immune response, the content of object in sample is converted into the content of ALP, and ALP can enzymolysis ATP, makes it to suppress bioluminescence, thus the most at last in sample the content of object be converted into bioluminescence signal, thus construct a kind of novel bioluminescence immunoassay method.
The schematic diagram of the inventive method as shown in Figure 1.In enzyme-linked immuno assay, by the specific recognition between antibody-antigene, the content of object in sample can be converted into the content of alkaline phosphatase, and alkaline phosphatase can decompose ATP efficiently, and the content of ATP and bioluminescence intensity are proportional, so in the immune analysis method of " double antibodies sandwich ", the content of object is higher, alkaline phosphatase is adsorbed more in enzyme mark hole, the ATP consumed is more, the knots modification of the bioluminescence reaction caused also is larger, and the intensity of bioluminescence reaction can be able to be collected by portable ATP detector, measure.Therefore, the content of object can by this information response of bioluminescence intensity out.
Two of technical scheme provided by the invention is: preceding method is in the application of field of immunodetection.
Of the present inventionly to be of wide application, such as to may be used for the detection of mycoplasma pneumoniae, Procalcitonin etc. in drugs Small molecular and serum in urine.
Should be noted that; above-mentioned application may relate to medical diagnosis on disease and the treatment of medical domain indication; also the situation of the Diagnosis and Treat of non-diseases may be related to; the content etc. of objectionable impurities in microorganism in such as testing environment, detection food; claim corresponding to the present invention is only limitted to the scope of the Diagnosis and Treat method of non-diseases, relates to the technical scheme of the Diagnosis and Treat method of disease not in the scope that the present invention is claimed.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can combination in any, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
Compare traditional immune analysis method signal read-out system, this bioluminescence reaction in the present invention, " fluorescein-luciferase-ATP " bioluminescence reaction had following advantage as the signal read-out system in immune response: the sensitivity 1) with superelevation, because can reach 10 to the detection sensitivity of ATP itself
-15m, this ATP can as the enzymatic substrate of the ALP enzyme in immunoassay simultaneously, and the method can realize two enzyme signal cascade and amplify, and result can be quantitative; 2) range of linearity is wide, can reach 5 orders of magnitude to the range of linearity that ATP detects, therefore also very wide to the range of linearity of other objects; 3) enzymatic luminescence-producing reaction speed is fast, only needs 10s clock namely to can read result; 4) signal-obtaining mode is simple, and available portable detecting instrument reads.Owing to being archebiosis light, the optical texture that readout equipment does not need external excitation light source and filter disc etc. complicated, good portability, handled easily.
The present invention is that the development of immuno analytical method provides new approaches, and can be applicable to trace poisonous and harmful substances in the early diagnosis of serious infectious diseases or environment and food highly sensitive, detect fast, control and prevention of disease and guarantee environment and food security are had important practical significance.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the inventive method.
Fig. 2 is the typical curve reflecting goat-anti people lgG content and bioluminescence value knots modification in embodiment 1, and wherein, horizontal ordinate is the concentration (ng/mL) of goat anti-human igg, and ordinate is the knots modification (a.u.) of biological luminous value.
Fig. 3 is the typical curve reflecting meth content and bioluminescence value knots modification in embodiment 2, and wherein, horizontal ordinate is the concentration (ng/mL) of meth, and ordinate is the knots modification (a.u.) of biological luminous value.
Fig. 4 is the typical curve reflecting Procalcitonin content and bioluminescence value knots modification in embodiment 3, and wherein, horizontal ordinate is the concentration (pg/mL) of Procalcitonin, and ordinate is the knots modification (a.u.) of biological luminous value.
Fig. 5 is the result figure detecting mycoplasma pneumoniae antibody in actual serum in embodiment 4, sample 1-3 is negative sample, and 4-15 is positive.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Except specified otherwise, equipment used in each embodiment and the equal conventional commercial of reagent can obtain.
In each embodiment, the source of portion of material and reagent is as follows:
Portable ATP detector is purchased from Xi'an Tianlong Science & Technology Co., Ltd.;
Alkaline phosphatase (150000U/L), the sheep anti-Mouse two of biotin and marked by streptavidin resists, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) available from Sigma;
Identify that the antibody of methyl A Feitaming drugs is purchased from Mo Zhidong bio tech ltd, Beijing;
Identify that the antibody of Procalcitonin (PCT) is purchased from Beijing Abzymo Biosciences Technology Co., Ltd.;
Anti-(Ab2-ALP) (1mg/mL) of horse anti-mouse two of alkali phosphatase enzyme mark and the anti-sheep two of rabbit of alkali phosphatase enzyme mark resist purchased from Beijing Bo Aosen Bioisystech Co., Ltd;
Mycoplasma pneumoniae recombinant protein: grand bio tech ltd of Xiamen Wanke;
Goat anti-human igg antibody: Beijing Suo Laibao Bioisystech Co., Ltd;
The comlete antigen that methyl A Feitaming coupling BSA is formed: purchased from Mo Zhidong bio tech ltd, Beijing;
Bioluminescence substrate (solution containing fluorescein and luciferase): available from Sigma.
Part Experiment solution preparation method is as follows:
The preparation of biotinylated antibody
The biotin that 4.52mg/mL identifies the monoclonal antibody of PCT and 5mg/mL in molar ratio 1:20 mixes and is dissolved in 300 μ LPBS buffer solution, shaken at room temperature 1h, react ultra-filtration centrifuge tube centrifugal 10min under 1000r/g condition of rear 10kD, then clean once with PBS, finally the reagent in super filter tube is dissolved in 300 μ LPBS, is finally placed on 4 DEG C and saves backup.
In the following embodiments, based on fluorescein-luciferase-ATP " bio-luminescence system is to the response of the hypersensitive of ATP concentration change and ultrafast enzyme-catalyzed change speed; utilize the alkaline phosphatase (AlkalinePhosphatase, ALP) that this bio-luminescence system comes in detection reaction system.The method is using the substrate of ATP as ALP, and in the basic conditions, ATP can be degraded to AMP by ALP efficiently, and AMP can not provide energy for bioluminescence reaction, thus inhibits luminous generation.Bioluminescence value after suppressed is set to B2, and bioluminescence value when ALP concentration is zero is set to B1, then the knots modification of bioluminescence value is Δ B=B1-B2, and the knots modification of bioluminescence value and the content of ALP are proportionate.ALP enzyme and luciferase all can keep high catalytic activity in the buffer solution of pH=8.0, and therefore these two kinds of enzymes can play efficient catalytic effect in same system.
Embodiment 1 is based on " fluorescein-luciferase-ATP " bio-luminescence system detecting pattern albumen (goat-anti people lgG)
One, drawing standard curve
(1) by people lgG carbonate buffer solution (pH=9.6,0.01M) dilute 2000 times, concentration is 10 μ g/mL, is coated on elisa plate, 4 DEG C are spent the night, and then clean 3 times with PBST washing lotion (the PBS damping fluids containing 1 ‰ Tween-20s); 150 μ L3%BSA confining liquids are closed reaction plate, and react 2h at 37 DEG C, PBST washing lotion cleans 3 times; By goat-anti people lgG titer dilution variable concentrations, respectively add 150 μ L, at 37 DEG C, react 1h; PBST washing lotion cleans 3 times, reacts 1h at 37 DEG C; PBST washing lotion cleans 3 times; The anti-sheep two anti-(1mg/mL) of rabbit of ALP mark dilutes 1000 times, reaction plate respectively adds 150 μ L, reacts 1h at 37 DEG C; PBST washing lotion cleans 1 time, washes 2 times.
(2) Tris-HCl of certain density ATP pH8.0 dissolves, and above-mentioned 96 orifice plates respectively adds 150 μ L, reacts 45min at 37 DEG C; Add ATP bioluminescence substrate (fluorescein and luciferase) subsequently to react with reactant liquor, by Portable Test Instrument for ATP or multi-functional microplate reader, bioluminescence value is measured.Acquired results drawing standard curve as shown in Figure 2.
Two, the mensuration of sample to be tested
Detecting step is with the process of drawing standard curve manipulation, and difference is to change goat-anti people lgG titer into each sample to be tested solution, after reading result, result is contrasted with the typical curve of Fig. 2, obtains the content of goat-anti people lgG in each sample to be tested.
Embodiment 2 detects the methyl A Feitaming in urine based on " fluorescein-luciferase-ATP " bio-luminescence system
One, drawing standard curve
(1) comlete antigen (concentration: 2mg/mL) carbonate buffer solution (pH=9.6 methyl A Feitaming coupling BSA formed, 0.01M) dilute 4000 times, in 96 hole elisa plates, every hole adds the solution of the above-mentioned dilution of 100 μ L, 4 DEG C of bags are spent the night, PBST cleans 3 times, with 3%BSA100 μ L in 37 DEG C of closed 2h.
Methyl A Feitaming standard solution is diluted to concentration is: 500,200,100,50,20,10,5,1,0.5,0.1,0.05,0ng/mL, join on above-mentioned elisa plate respectively, every hole 50 μ L, every hole adds antibody (1mg/mL the dilutes 1000 times) solution that 50 μ L identify methyl A Feitaming drugs simultaneously, after 37 DEG C of reaction 1.5h.
Clean 4 times with PBST, then the horse anti-mouse two anti-(Ab2-ALP) marked by ALP dilutes 2000 times, every hole adds 100 μ L, and 37 DEG C of reactions 45min, PBST clean 2 times, wash 2 times.
(2) 100 μ L1 μM ATP (Tris-HCl, pH8.5) join on above-mentioned elisa plate, 37 DEG C of reaction 45min, finally add the fluorescein of 50 μ L and the solution of luciferase, with portable ATP detector collection of biological luminous signal, result drawing standard curve as shown in Figure 3.
Two, the mensuration of urine specimen
Detecting step is with the process of drawing standard curve manipulation, difference is to change methyl A Feitaming standard solution into each urine to be checked, after reading result, result is contrasted with the typical curve of Fig. 3, obtains the content of methyl A Feitaming in each urine specimen.
Embodiment 3 detects the Procalcitonin (procalcitonin, PCT) in serum based on " fluorescein-luciferase-ATP " bio-luminescence system
One, drawing standard curve
(1) enzyme linked immunoassay capture antigen
The capture antibody (PCT-83) of 5.15mg/mL dilutes 1000 times with the carbonate coating buffer of pH9.6, and on 96 orifice plates, each hole adds the dilution of 150 μ L, and 4 DEG C of reactions are spent the night; PBST washing lotion cleans 3 times; 150 μ L3%BSA confining liquids are closed reaction plate, react 2h at 37 DEG C; PBST washing lotion cleans 3 times; By PCT titer dilution variable concentrations, respectively add 150 μ L, at 37 DEG C, react 1h; PBST washing lotion cleans 3 times; PCT-79-Biotin (the PCT-79 antibody of biotin coupling) dilutes 500 times with PBS, reaction plate respectively adds 150 μ L, reacts 1h at 37 DEG C; PBST washing lotion cleans 3 times; ALP-SA (alkaline phosphatase of Streptavidin coupling) dilutes 1000 times, reaction plate respectively adds 150 μ L, reacts 1h at 37 DEG C; PBST washing lotion cleans 1 time, washes 2 times.
(2) ATP biloluminescence method carries out quantitatively antigen
The Tris-HCl of certain density ATP pH8.0 dissolves, and above-mentioned 96 orifice plates respectively adds 150 μ L, reacts 45min at 37 DEG C; Add ATP bioluminescence substrate (fluorescein and luciferase) subsequently to react with reactant liquor, measured bioluminescence value by portable ATP detector, result is depicted as typical curve as shown in Figure 4.
Two, the mensuration of serum sample
Detecting step is with the process of drawing standard curve manipulation, and difference is to change PCT titer into each serum sample to be checked, after reading result, result is contrasted with the typical curve of Fig. 4, obtains the content of Procalcitonin in each serum sample.
Embodiment 4 detects the mycoplasma pneumoniae antibody in serum based on " fluorescein-luciferase-ATP " bio-luminescence system
1, immunoreactive step
(1) mycoplasma pneumoniae recombinant protein stoste dilution, bag quilt: get 1 μ L mycoplasma pneumoniae recombinant protein and add in carbonate buffer solution (pH9.6), be diluted to best bag by concentration, 4 DEG C of refrigerator overnight.3 times are washed with PBST buffer solution.
(2) close: the BSA solution of 150 μ L3% is joined in the elisa plate in 96 holes, at 37 DEG C of reaction 2h, then wash 3 times with PBST buffer solution.
(3) blood serum sample will collected, dilutes 100 times with PBS buffer solution (pH=7.4,0.01M), then joins in ELISA hole above respectively, at 37 DEG C of reaction 2h, washs three times with PBST;
(4) the goat-anti people lgG conjugate of alkali phosphatase enzyme mark dilutes 1000 times, reaction plate respectively adds 150 μ L, reacts 1h at 37 DEG C; PBST washing lotion cleans 1 time, washes 2 times.
2, the ATP biloluminescence method signal collection stage
The Tris-HCl of certain density ATP pH8.0 dissolves, and above-mentioned 96 orifice plates respectively adds 150 μ L, reacts 45min at 37 DEG C; Add ATP bioluminescence substrate (fluorescein and luciferase) subsequently to react with reactant liquor, by portable ATP detector, bioluminescence value is measured.Result as shown in Figure 5.As can be seen from Figure 5,1-3 sample is negative sample, and its bioluminescence value is all very little, 4-15 is positive, so its bioluminescence value is higher than 1-3 sample significantly, the result of Fig. 5 shows, method of the present invention can be applied to the detection of mycoplasma pneumoniae antibody in actual blood serum sample.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. based on an immune analysis method for bioluminescence reaction, it is characterized in that, it comprises the steps:
(1) encrusting substance matter is coated on solid phase carrier, hatches rear damping fluid and rinse;
(2) add the determinand standard solution with variable concentrations gradient, hatch rear damping fluid and rinse;
(3) add with ALP mark two resist, hatch rear damping fluid and rinse, described two resist the antibody for anti-determinand;
(4) after adding ATP solution reaction, then add bioluminescence substrate and react, finally detect luminous value, obtain result I;
(5) the determinand standard solution of the variable concentrations gradient described in step (2) is replaced to testing sample solution, repeat the operation of step (1) ~ (4), finally detect luminous value, obtain result II, by result II reference result I, judge the concentration of determinand in testing sample solution;
Wherein, when determinand is comlete antigen, described encrusting substance matter is to combine from described comlete antigen is corresponding and resists the antibody with different antigen binding site with described two, when determinand is antibody, described encrusting substance matter is the artificial antigen that the comlete antigen corresponding with described antibody or hapten molecule and protein carrier coupling are formed;
Described bioluminescence substrate is the solution containing fluorescein and luciferase.
2. method according to claim 1, is characterized in that, in step (1), described solid phase carrier is ELISA Plate.
3. method according to claim 1, is characterized in that, in step (1), described in hatch for by bag by after solid phase carrier be placed in 4 ~ 40 DEG C place 0.5 ~ 12 hour, more preferably in 37 DEG C place 2 hours; And/or,
Described damping fluid is PBS damping fluid, and the PBS damping fluid more preferably containing 1 ‰ Tween-20s, described permillage refers to volume permillage.
4. method according to claim 1, is characterized in that, in step (2),
Described hatching is placed 0.5 ~ 12 hour for solid phase carrier being placed in 4 ~ 40 DEG C, more preferably places 2 hours in 37 DEG C; And/or,
Described damping fluid is PBS damping fluid, and the PBS damping fluid more preferably containing 1 ‰ Tween-20s, described permillage refers to volume permillage.
5. method according to claim 1, is characterized in that, in step (3),
Described hatching is placed 0.5 ~ 12 hour for solid phase carrier being placed in 4 ~ 40 DEG C, more preferably places 2 hours in 37 DEG C; And/or,
Described damping fluid is PBS damping fluid, and the PBS damping fluid more preferably containing 1 ‰ Tween-20s, described permillage refers to volume permillage.
6. method according to claim 1, is characterized in that, described step (3) substitutes with step (3 '), and described step (3 ') comprises sub-step (3 ') a and sub-step (3 ') b;
Described step (3 ') a is: add and resist with two of biotin or marked by streptavidin, hatch rear damping fluid and rinse, described two resist the antibody for anti-determinand;
Described sub-step (3 ') b is: add Streptavidin or biotin labeled ALP, hatches rear damping fluid and rinses;
Wherein, when add in sub-step (3 ') a with biotin labeled two anti-time, then add the ALP of marked by streptavidin in sub-step (3 ') b; When adding anti-with two of marked by streptavidin in sub-step (3 ') a, then add biotin labeled ALP in sub-step (3 ') b.
7. method according to claim 1, is characterized in that, hatching described in sub-step (3 ') a and sub-step (3 ') b is placed 0.5 ~ 12 hour for solid phase carrier being placed in 4 ~ 40 DEG C, more preferably places 2 hours in 37 DEG C;
Sub-step (3 ') a and the damping fluid described in sub-step (3 ') b are PBS damping fluid, and the PBS damping fluid more preferably containing 1 ‰ Tween-20s, described permillage refers to volume permillage.
8. method according to claim 1, is characterized in that, in step (4), described bioluminescence substrate is the solution containing fluorescein and luciferase.
9. method according to claim 1, is characterized in that, in step (4), described detection luminous value detects bioluminescence value with ATP detector.
10. method described in any one of claim 1 ~ 9 is in the application of field of immunodetection.
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