CN108362687A - Chemiluminescent enhancement liquid and chemiluminescent substrate and its application - Google Patents
Chemiluminescent enhancement liquid and chemiluminescent substrate and its application Download PDFInfo
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Abstract
The present invention is provided to detect chemiluminescent enhancement liquid, chemiluminescent substrate, detection method and the kit of determinand in sample, improvements are that two kinds of chemiluminescence intensifiers are applied in combination：O-phenyl phenol and 4 imidazoles phenol.Both reinforcing agents play synergistic enhancing effect, significantly reduce background signal, and luminol luminous signal can be allowed to keep stable in a long time.Detection method and kit of the present invention can be matched with chemiluminescent analyzer, be can be used for tumor marker, infectious disease, steroids and other enzymes and exempted to promote the detection of epidemic disease chemiluminescence class product.
Description
Technical field
The present invention relates to the chemiluminescent enhancement liquid for detecting determinand in sample, chemiluminescent substrate, detection methods
And kit, it all refers to be applied in combination o-phenyl phenol and 4- imidazoles phenol both chemiluminescence intensifiers, belongs to immune skill
Art field.
Background technology
Since chemiluminescence immunoassay (chemiluminesent immunoassay, CLIA) is in eighties of last century seven
Since the eighties come out, because it uses nonradioactive labeling, and antigen-antibody immune response is mutually tied with chemiluminescence signal
It closes, thus with the excellent specific and sensitivity significantly improved, in Pharmaceutical Analysis, environmental analysis, food analysis and biology
The fields such as medicine, which are obtained for, to be widely applied, for detecting tumour class biomarker, such as first such as in biomedical sector
Fetoprotein (AFP), carcinomebryonic antigen (CEA), ferritin, prostate specific antigen (PSA), neuronspecific enolase
(NSE), CYFRA21-1, CA19-9, CA50, CA125, CA153, CA724 etc.；Sexual gland class biomarker, such as follicle-stimulating hormone (FSH)
(FSH), interstitialcellstimulating hormone (ICSH) (LH), prolactin, progesterone, testosterone, estradiol, estriol, β-human chorionic gonadtropin (β-
HCG)；First work(class biomarker, such as total triiodo thryonine (TT3), total thyroxin (TT4), free triiodo first gland are former
Propylhomoserin (FT3), free thyroxine (FT4), thyrotropic hormone (TSH), thyroglobulin, thyroglobulin antibody and
Thyroid peroxidase antibody etc.；Infectious disease class biomarker, as five indexes of hepatitis b (HBsAg, Anti-HBs antibody, anti-HBc,
HBeAg, anti-HBe), HCV antigens, HCV antibody, TP antigen, syphilis helicoid antibody, TORCH IgG/IgM, HIV
Antigen/antibody etc.；Diabetes class biomarker, such as insulin, C- peptides, insulin autoantibodies, islet cell antibodies, paddy ammonia
Acid decarboxylase antibody；Liver fibre heap class biomarker, such as laminin, hyaluronic acid, IV Collagen Type VIs and III procollagen type N
Hold peptide etc..
Currently, the CLIA technologies mainly utilized in market product can be divided into three categories：(1) immune point of enzyme-catalyzed chemical luminescence
Analysis method, chemiluminescent substrate shine through the degradation of enzyme, different according to enzyme type, are broadly divided into：A) horseradish peroxidase
Enzyme (HRP) chemiluminescence immunoassay, common chemiluminescent substrate can be selected from luminol, different luminol and its derivative
Object；B) alkaline phosphatase (ALP) chemiluminescence immunoassay, common chemiluminescent substrate are that 1,2- dichloroethane classes are spread out
Biology；(2) Electrochemiluminescence assay is exempted from as the Elecsys series and Cobas Series Electrochemicals of Roche companies shine
Epidemic disease analysis system is exactly in this way；(3) acridinium ester (acridinium ester) or acridine sulfonamide are based on
The chemiluminescence immunoassay of (acridinium sulfonylamide), chemical illuminating reagent are acridinium ester or acridine sulphur
Amide.
Luminol (luminol), different luminol (isoluminol) and its derivative are most early in chemiluminescence immunoassay point
The chemiluminescent substance used in analysis method.But, if not using reinforcing agent, Luminol shines as flash type, intensity
It is weak, the duration is short.By the way that reinforcing agent is added, fluorescent lifetime is obviously prolonged, when it being made to be improved to continue from of short duration flash type
Between long wide variety of glow-type, enhance luminous signal intensity and improve noise when sensitivity.Improve sensitivity most efficient method
It is that reinforcing agent is added in the reaction system to improve luminous signal intensity.The type of reinforcing agent is very extensive, initial reinforcing agent
Including fluorescein, substituted phenol, naphthols and hexahydroxy benzothiazole, it was found that other reinforcing agent substances, such as aromatic amine took again later
For boric acid, indophenols etc..
Chinese patent application CN1067257A refers to a kind of shielded compound using reinforcing agent o-phenyl phenol,
When detection, corresponding hydrolase is added, removes blocking group, generates the reinforcing agent o-phenyl phenol actually to play a role, carries
The luminous signal of high luminol.But this needs that a kind of hydrolase is additionally added, and is complicated so as to cause reaction system, but also
Testing cost can be improved.Furthermore the luminous signal of luminol although can be improved using only a kind of reinforcing agent of o-phenyl phenol, still
Still result in the problems such as HRP- luminol chemiluminescences system is higher poor with luminous signal stability there are background signal.This
Outside, o-phenyl phenol itself is because of certain unknown reason so that the luminol luminous signal of enhancing is difficult in a longer period of time
It keeps stablizing, luminous signal is decayed than very fast.This allows for many people and abandons using this reinforcing agent.
Invention content
In view of the deficiencies of the prior art, the present invention is in HRP- luminol chemiluminescence systems, while being added two kinds
Chemiluminescence intensifier：O-phenyl phenol (o-phenylphenol) and 4- imidazoles phenol (4- (imidazol-1-yl)
phenol).Making us unexpected is, both chemiluminescence intensifiers play synergistic enhancing effect.In addition, this is added simultaneously
Two kinds of chemiluminescence intensifiers, can significantly decrease the background signal in immunoassay, while allow the chemiluminescence signal of generation
It keeps stablizing in a long time.These advantages can improve detection accuracy and sensitivity.
The present invention provides a kind of chemiluminescent enhancement liquid, including o-phenyl phenol and 4- imidazoles phenol.The chemiluminescence
Enhancement solution is used to enhance the luminous signal of HRP- luminol chemiluminescence systems.
Further, a concentration of the 0.025 of a concentration of 0.008g/L~0.16g/L of o-phenyl phenol, 4- imidazoles phenol
~0.5g/L.
Further, a concentration of 0.008g/L~0.04g/L of o-phenyl phenol, 4- imidazoles phenol it is a concentration of
0.025g/L~0.125g/L.
Further, the chemiluminescent enhancement liquid further includes buffer solution, and the buffer solution is selected from Tris-HCl, borate
Buffer solution or carbonate buffer solution.
The present invention also provides a kind of composition enhancing HRP- luminol chemiluminescence systems luminous signal in application,
The composition includes the chemiluminescent enhancement liquid.
The present invention also provides a kind of chemiluminescent substrate, the chemiluminescent substrate includes A liquid and B liquid, and A liquid includes shining
Agent, B liquid include peroxide, wherein A liquid further includes the chemiluminescent enhancement liquid.
Further, a concentration of the 0.025 of a concentration of 0.008g/L~0.16g/L of o-phenyl phenol, 4- imidazoles phenol
~0.5g/L.
Further, a concentration of 0.008g/L~0.04g/L of o-phenyl phenol, 4- imidazoles phenol it is a concentration of
0.025g/L~0.125g/L.
Further, A liquid further includes buffer solution, and the buffer solution is selected from Tris-HCl, borate buffer solution or carbonate
Buffer solution.
Further, the luminous agent is selected from luminol, different luminol and derivative, a concentration of 0.2g/L~5g/
L, the peroxide are selected from hydrogen peroxide, urea peroxide or perborate, a concentration of 0.2g/L~2.0g/L.
Further, the luminous agent is luminol.
Further, the peroxide is urea peroxide.
Further, a concentration of 0.6g/L~1.0g/L of the luminous agent, a concentration of 0.2g/L of the peroxide
~0.3g/L.
The method of determinand, step include in a kind of detection sample：(1) it provides and is coated with the solid phase for capturing component；(2)
The determinand calibration object of various concentration is added or cleaning solution may be added containing the sample of determinand, after being incubated together with solid phase
Clean solid phase；(3) detected components of HRP labels are added, after being incubated, cleaning solution is added and cleans solid phase；(4) chemistry hair is added
After light substrate, luminous signal is detected；(5) luminous signal of various concentration determinand calibration object that detects and its corresponding is utilized
Determinand calibration object concentration draws standard curve；(6) according to the standard curve in the sample luminous signal and (5) detected, meter
The testing concentration in sample is calculated, wherein the capture component is specifically binding on determinand, the detection of the HRP labels
Component is specifically binding in determinand or the capture component, and the chemiluminescent substrate includes A liquid and B liquid, and A liquid contains
There are luminous agent, o-phenyl phenol and 4- imidazoles phenol, B liquid to contain peroxide.
Further, a concentration of the 0.025 of a concentration of 0.008g/L~0.16g/L of o-phenyl phenol, 4- imidazoles phenol
~0.5g/L.
Further, when determinand is antigen, the capture component and detected components are specifically in conjunction with described anti-
Former antibody.
Further, when determinand is antibody, the capture component and detected components are specifically in conjunction with described anti-
The antigen of body.
Further, when determinand is haptens, the capture group is divided into specifically in conjunction with the anti-of the haptens
The detected components of body, the HRP labels are the haptens or its analog of HRP labels.
Further, the solid phase is selected from microwell plate, plastic cement particle, magnetic bead, elastoplastic pipe or plastic bead.
Further, a concentration of 0.008g/L~0.04g/L of o-phenyl phenol, 4- imidazoles phenol it is a concentration of
0.025g/L~0.125g/L.
Further, A liquid further includes that the buffer solution is selected from Tris-HCl, borate buffer solution or carbonate buffer solution.
Further, the luminous agent is selected from luminol, different luminol and derivative, a concentration of 0.2g/L~5g/
L, the peroxide are selected from hydrogen peroxide, urea peroxide or perborate, a concentration of 0.2g/L~2.0g/L.
Further, the luminous agent is luminol.
Further, the peroxide is urea peroxide.
Further, a concentration of 0.6g/L~1.0g/L of the luminous agent, a concentration of 0.2g/L of the peroxide
~0.3g/L.
The kit of determinand in a kind of detection sample, the kit include capturing component, the detection group of HRP labels
Point, cleaning solution, chemiluminescent substrate and determinand calibration object, wherein the capture component is specifically binding on determinand,
The detected components of the HRP labels are specifically binding in determinand or the capture component, the chemiluminescent substrate
Including A liquid and B liquid, A liquid contains luminous agent, o-phenyl phenol and 4- imidazoles phenol, B liquid and contains peroxide.
Further, a concentration of the 0.025 of a concentration of 0.008g/L~0.16g/L of o-phenyl phenol, 4- imidazoles phenol
~0.5g/L.
Further, when determinand is antigen, the capture component and detected components are specifically in conjunction with described anti-
Former antibody.
Further, when determinand is antibody, the capture component and detected components are specifically in conjunction with described anti-
The antigen of body.
Further, when determinand is haptens, the capture group is divided into specifically in conjunction with the anti-of the haptens
The detected components of body, the HRP labels are the haptens or hapten derivant of HRP labels.
Further, A liquid further includes buffer solution, and the buffer solution is selected from Tris-HCl, borate buffer solution or carbonate
Buffer solution.
Further, a concentration of 0.008g/L~0.04g/L of o-phenyl phenol, 4- imidazoles phenol it is a concentration of
0.025g/L~0.125g/L.
Further, the luminous agent is selected from luminol, different luminol and derivative, a concentration of 0.2g/L~5g/
L, the peroxide are selected from hydrogen peroxide, urea peroxide or perborate, a concentration of 0.2g/L~2.0g/L.
Further, the luminous agent is luminol.
Further, the peroxide is urea peroxide.
Further, a concentration of 0.6g/L~1.0g/L of the luminous agent, a concentration of 0.2g/L of the peroxide
~0.3g/L.
Beneficial effects of the present invention：(1) present invention and without using the chemiluminescence intensifier that is protected, thus also with regard to nothing
Corresponding hydrolase need to be used to remove blocking group, so that reaction system is simplified, while reduce testing cost；(2)
Two kinds of chemiluminescence intensifiers are applied in combination in the present invention：O-phenyl phenol and 4- imidazoles phenol, have been surprisingly found that both reinforcing agents
Play synergistic enhancing effect, both not only together when the chemiluminescence signal that generates be more than the change generated in the presence of each
Learn the sum of luminous signal, and the two together when the signal-to-noise ratio that generates also be far longer than the noise generated in the presence of each
The sum of than；(3) two kinds of chemiluminescence intensifiers are applied in combination in the present invention：O-phenyl phenol and 4- imidazoles phenol, can effectively overcome
Using only luminous signal when o-phenyl phenol is unstable and the higher problem of background signal, and it can effectively overcome 4- imidazoles benzene
The more weaker problem of luminous signal intensity is changed in phenol enhancing, so that it is guaranteed that the luminous signal of the present invention is been significantly enhanced, protects
Relatively good luminous signal stability is held, while there is lower background signal；(4) chemiluminescent enhancement liquid of the invention and change
Learning luminous substrate has good storage stability, can at least be preserved at 37 DEG C 31 days, 18 are at least preserved at 2~8 DEG C
Month；(5) chemiluminescent enhancement liquid, chemiluminescent substrate, detection method and kit using the present invention, are remarkably improved detection
Accuracy, sensitivity and linear detection range；(6) present invention is made us by the way that o-phenyl phenol and 4- imidazoles phenol is applied in combination
Unexpectedly solve when o-phenyl phenol is only added, the luminol luminous signal of enhancing decay than be comparatively fast difficult to compared with
It keeps stablizing in for a long time so that the problem of many people abandon using o-phenyl phenol.
Description of the drawings
Fig. 1 detect the result of AFP500 calibration objects using the chemiluminescent substrate of the present invention.
It is measured when the o-phenyl phenol (being indicated with alphabetical D) of Fig. 2 various concentrations and 4- imidazoles phenol (being indicated with letter e)
Signal-to-noise ratio computation result figure.
Specific implementation mode
When using HRP- luminol chemiluminescence systems detection sample in determinand when, if determinand be antibody (IgE,
HCV antibody, HBeAb, HBsAb, HBcAb, HIV antibody etc.) when, dual-antigen sandwich method detection can be used, be at this moment attached to solid phase
On capture antigen and HRP label detection antigen binding to test antibodies on, chemiluminescent substrate is added, measures and shines per hole
Value, luminous signal intensity are directly proportional to the content of antibody in sample；It, can if determinand is antigen (such as AFP, CEA, PSA)
It is detected using double antibody sandwich method, the detection antibody that the capture antibody and HRP being at this moment attached in solid phase mark is attached to be measured
In the different loci of object, chemiluminescent substrate is added, measures per hole luminous value, the content of luminous signal intensity and antigen in sample
It is directly proportional；When determinand is haptens (such as estradiol, estriol, T3 and T4), A competitive inhibition method can be used and be detected,
At this moment haptens present in the haptens and sample of HRP labels can all go competitive binding to be fixed to this haptens in solid phase
Detection antibody, chemiluminescent substrate is added, measures per hole luminous value, the content of luminous signal intensity and haptens in sample at
Inverse ratio.
In the testing concentration in detecting the samples such as serum or blood plasma, it is bent standard should to be made first with determinand calibration object
Line.For this reason, it may be necessary to the high concentration determinand calibration object prepared in advance is subjected to a series of dilutions using calibration object dilution, to
The determinand calibration object of various concentration is obtained, or directly directly prepares difference using calibration object dilution and determinand calibration object
The determinand calibration object of concentration, in addition using the calibration object dilution without determinand as the determinand calibration object of zero-dose.Institute
It states calibration object dilution and can be selected from calf serum, newborn bovine serum or fetal calf serum, also selected from ddH2O, phosphate buffer
(PBS), physiological saline, HEPES buffer solution, PIPES buffer solutions, MOPS buffer solutions, Tricine buffer solutions, triethanolamine-hydrochloric acid
Buffer solution, Tris-HCl buffer solutions or barbital sodium-hydrochloride buffer etc..It, can be as needed in the calibration object dilution
The stabilizers such as suitable BSA, glutin or casein are added, suitable preservative can also be added as needed, as Sodium azide,
Thimerosal, Proclin-150, Proclin-200, Proclin-300 or Proclin-5000 etc..According to the determinand in sample
The measured value obtained in using HRP enzymatic chemical luminous immunoassays, and the standard curve that is obtained, so that it may calculate sample
Testing concentration in product.
In addition, in order to further increase the sensitivity of detection, it can be by albumin A, Protein G, biotin-avidin/strepto- parent
With element/neutral Avidin system, fluorescein isothiocynate (FITC)-anti-FITC antibody or fluorescein-anti-fluorescein antibody or its
Among his similar system combination to HRP- luminol chemiluminescence systems.
It is used herein for the capture antibody of determinand and detection when the determinand of detection is antigen or hapten
Antibody can be selected from anti-determinand monoclonal antibody (abbreviation monoclonal antibody) or polyclonal antibody (referred to as how anti-), be to utilize the to be measured of separation
Object or its segment (for antigen) or hapten-carrier protein conjugate (for haptens, by haptens with
What the carrier protein couplets such as BSA, KLH, BGG or OVA were formed together) caused by the animal of immunizing non-human.In addition, described catch
Obtain antibody and detection antibody can also be anti-determinand monoclonal antibody or it is how anti-after chemical reagent or enzymatic treatment still have determinand
In conjunction with active segment, the generated Fab segments such as after papain digestion, or generated after pepsin digestion
F(ab')2Or F (ab')2The Fab' segments generated after enzyme IdeS digestion.The non-human animal can be selected from mouse, big
The animals such as mouse, cavy, rabbit, chicken, pig, sheep, goat, horse, mule and camel.
When the determinand of detection is antibody, the capture antigen used herein for determinand and detection antigen be from
The either chemical means separated in sample synthesize or utilization genetic engineering means are in microbial cell or eukaryon
It recombinantly expresses and is generated (when capturing antigen and detection antigen is albumen) in biological cell.
No matter the capture antibody being bonded in solid phase, the capture antigen being also bonded in solid phase, the solid phase is selected from
Microwell plate, plastic cement particle, magnetic bead, elastoplastic pipe, plastic bead etc., preferably microwell plate.Microwell plate can be selected from 16 common holes
ELISA Plate, 48 hole elisa Plates and 96 hole elisa Plates etc., and plastic bead can be selected from Shenzhen bio tech ltd Niu Bang
PS02N/PS03N/PS04N/PS05N/PS06N/PS07N/PS08N model polystyrene microspheres etc..Magnetic bead is with four oxidations three
The paramagnets such as iron as kernel, surface by amino, carboxyl, hydroxyl, sulfydryl isoreactivity base group modification magnetic particle or
Magnetic microsphere.
When detecting the determinand in sample using HRP- luminol chemiluminescence systems, chemiluminescent substrate includes A liquid
With B liquid, A liquid contains luminous agent, and B liquid contains peroxide, wherein the luminous agent is selected from luminol (luminol), different Rumi
Promise (isoluminol) and its derivative, a concentration of 0.2g/L~5g/L, preferably 0.6g/L~1.0g/L；The peroxidating
Object is selected from hydrogen peroxide, urea peroxide and perborate, a concentration of 0.2g/L~2.0g/L, preferably 0.2g/L~0.3g/
L.In order to enhance luminous signal intensity and improve stability of photoluminescence, A liquid also contains o-phenyl phenol and 4- imidazoles phenol.It is molten in A
In liquid, a concentration of 0.008g/L~0.16g/L of o-phenyl phenol, a concentration of 0.025g/L~0.5g/L of 4- imidazoles phenol.
Preferably, A liquid also contains the buffer solution that pH is 9.0 ± 0.1, and the buffer solution is selected from Tris-HCl, borate buffer solution and carbon
Phthalate buffer, preferably phthalate buffer.
By largely studying and putting into practice discovery, two kinds of chemiluminescent enhancements of the present invention are added into chemiluminescent substrate
Agent unexpectedly plays synergistic enhancing effect, significantly enhances the luminous signal intensity of HRP- luminol chemiluminescence systems
And signal-to-noise ratio, while improving stability of photoluminescence.
Following embodiment further illustrates the present invention.These embodiments are not intended to limit the scope of the invention, and are to provide
A further understanding of the present invention.
1 chemical illuminating reagent of embodiment and its detection method
In the present invention, determinand is antigen, antibody or haptens, is illustrated by taking antigen A FP as an example here.Solid phase can
Selected from microwell plate, plastic cement particle, magnetic bead, elastoplastic pipe or plastic bead, illustrated by taking microwell plate as an example here.
1.HRP marker preparation methods
HRP markers refer to HRR label antigen, antibody or haptens, common labeling method have glutaraldehyde method and
Sodium periodate oxidation.Here it is illustrated by taking sodium periodate oxidation as an example.Its detailed step is：
(1) 5mg HRP are weighed, are dissolved in 1ml distilled water, the HRP solution of a concentration of 5mg/ml is formed；
(2) the 0.1M NaIO that 0.2ml newly matches are added into HRP solution4Solution is protected from light stirring 20min at room temperature；
(3) solution after stirring in (2) is fitted into bag filter, is carried out using the sodium-acetate buffer (pH4.4) of 1mM saturating
Analysis, at 4 DEG C overnight, the hydroformylation HRP solution after being dialysed；
(4) 20 μ l 0.2M sodium carbonate buffers (pH 9.5) are added, the pH of the hydroformylation HRP solution allowed in (3) rises to
9.0~9.5, then it is added the 1ml 0.01M carbonate buffer solutions of the AntiAFP antibody containing 10mg, room temperature is protected from light that be gently mixed 2 small
When；
(5) the 4mg/ml NaBH that 0.1ml newly matches are added4Solution, mixing are placed 2 hours at 4 DEG C；
(6) solution in (5) is fitted into bag filter, is dialysed at 4 DEG C using 0.15M PBS buffer solution (pH 7.4)
It is lower to stay overnight；
(7) solution after dialysis in (6) is purified with Sephedex G-75 gel chromatographies, it is anti-obtains HRP labels
AFP antibody-solutions.
2. chemiluminescent enhancement liquid and chemiluminescent substrate
The chemiluminescent substrate of the present invention includes A liquid and B liquid.
In one embodiment of the invention, a kind of chemiluminescent enhancement liquid, including o-phenyl phenol and 4- imidazoles benzene
Phenol, a concentration of 0.008g/L~0.16g/L of o-phenyl phenol, a concentration of 0.025~0.5g/L of 4- imidazoles phenol.Describedization
Learn the luminous signal that luminescence enhancement liquid is used to enhance HRP- Luminols.Preferably, the chemiluminescent enhancement liquid further includes
The buffer solution that pH is 9.0 ± 0.1, the buffer solution are selected from Tris-HCl, borate buffer solution and carbonate buffer solution, preferably
Carbonate buffer solution.
In another embodiment of the present invention, the formula of the A liquid and B liquid is as follows：A liquid：Luminol 0.2g/
L~5g/L, o-phenyl phenol 0.008g/L~0.16g/L, 4- imidazoles phenol 0.025g/L~0.5g/L, pH are 9.0 ± 0.1
Buffer solution, the buffer solution are selected from Tris-HCl, borate buffer solution and carbonate buffer solution, preferably carbonate buffer solution；B
Liquid：Urea peroxide 0.2g/L~2.0g/L, dilution are 0.2M phosphate buffers (pH is 7.4 ± 0.1).
In another embodiment of the present invention, the formula of the A liquid and B liquid is as follows：A liquid：Luminol 0.2g/
L~5g/L, o-phenyl phenol 0.008g/L~0.04g/L, 4- imidazoles phenol 0.025g/L~0.125g/L, pH are 9.0 ± 0.1
Buffer solution, the buffer solution is selected from Tris-HCl, borate buffer solution and carbonate buffer solution, preferably carbonate buffer
Liquid；B liquid：Urea peroxide 0.2g/L~2.0g/L, dilution are 0.2M phosphate buffers (pH is 7.4 ± 0.1).
In another embodiment of the present invention, the formula of the A liquid and B liquid is as follows：A liquid：Luminol 0.6g/L~
1.0g/L, o-phenyl phenol 0.008g/L~0.16g/L, 4- imidazoles phenol 0.025g/L~0.5g/L, pH are 9.0 ± 0.1
Buffer solution, the buffer solution are selected from Tris-HCl, borate buffer solution and carbonate buffer solution, preferably carbonate buffer solution；B
Liquid：Urea peroxide 0.2g/L~2.0g/L, dilution are 0.2M phosphate buffers (pH is 7.4 ± 0.1).
In another embodiment of the present invention, the formula of the A liquid and B liquid is as follows：A liquid：Luminol 0.6g/L~
1.0g/L, o-phenyl phenol 0.008g/L~0.04g/L, 4- imidazoles phenol 0.025g/L~0.125g/L, pH are 9.0 ± 0.1
Buffer solution, the buffer solution is selected from Tris-HCl, borate buffer solution and carbonate buffer solution, preferably carbonate buffer
Liquid；B liquid：Urea peroxide 0.2g/L~0.3g/L, dilution are 0.2M phosphate buffers (pH is 7.4 ± 0.1).
In another embodiment of the present invention, the formula of the A liquid and B liquid is as follows：A liquid：Luminol 0.8g/L, neighbour
The buffer solution that phenylphenol 0.04g/L, 4- imidazoles phenol 0.125g/L, pH are 9.0 ± 0.1, the buffer solution are selected from Tris-
HCl, borate buffer solution and carbonate buffer solution, preferably carbonate buffer solution；B liquid：Urea peroxide 0.3g/L, dilution are
0.2M phosphate buffers (pH is 7.4 ± 0.1).
In another embodiment of the present invention, the formula of the A liquid and B liquid is as follows：A liquid：Luminol 0.8g/L, neighbour
The carbonate buffer solution that phenylphenol 0.008g/L, 4- imidazoles phenol 0.025g/L, pH are 9.0 ± 0.1；B liquid：Urea peroxide
0.3g/L, dilution are 0.2M phosphate buffers (pH is 7.4 ± 0.1).
3. determinand calibration object is prepared
A certain amount of determinand (such as AFP, CEA, IgE, T4) is weighed, the PBS buffer solution containing 50% calf serum is utilized
(pH 7.2) carries out a series of dilutions, obtains the determinand calibration object of various concentration.For AFP, a concentration of 0ng/mL is obtained
AFP calibration objects (AFP0), the AFP calibration objects (AFP5) of a concentration of 5ng/mL, a concentration of 10ng/mL AFP calibration objects
(AFP10), the AFP calibration objects (AFP20) of a concentration of 20ng/mL, the AFP calibration objects (AFP120) of a concentration of 120ng/mL and dense
Degree is the AFP calibration objects (AFP500) of 500ng/mL.
4. object detecting method to be measured
When determinand be antigen or antibody, be detected frequently with sandwich method.Here dense to detect the antigen A FP in serum
It is illustrated for degree.Detailed detecting step is as follows：
(1) it prepares and is coated with the solid phase for capturing antibody
Anti- AFP capture antibody is dissolved in the carbonate buffer solution of pH 9.6, the anti-AFP of a concentration of 10 μ g/ml is obtained
Antibody coating buffer is captured, 50 μ l of coating buffer are added into ELISA Plate hole, are stood overnight at 4 DEG C；By the liquid in ELISA Plate hole
Dry or pump out, be added at room temperature into ELISA Plate hole cleaning solution (can be selected from the phosphate buffer of pH 7.2, pH 7~
8 Tris-HCl buffer solutions and the carbonate buffer solution etc. of pH 9.6) it washs 3~5 times；After having washed, by washing in enzyme mark hole
It washs liquid drying or pumps out, 5%BSA is added and was placed at 4 DEG C with closing binding site extra on ELISA Plate hole surface
Night；It after having closed, is washed 3~5 times using cleaning solution, to remove unbonded closed reagent；It, will be in enzyme mark hole after having washed
Cleaning solution is dried or is pumped out, and obtains the ELISA Plate for being coated with AntiAFP antibody, spare.
(2) calibration object or blood serum sample and detection antibody is added
By 50 μ l AFP calibration objects or may the blood serum sample containing AFP and 50 μ l HRP labels anti-AFP detections it is anti-
Body is added to being coated in the ELISA Plate hole of AntiAFP antibody of obtaining in (1), incubates 30min at 37 DEG C, washing is then added
Liquid washs 3~5 times.
(3) chemiluminescent substrate is added
ELISA Plate into (2) after washing per hole in be added 50 μ l A liquid and 50 μ l B liquid, after mixing, room temperature is protected from light
It after 5min, is put into chemiluminescent analyzer, measures each hole luminous signal intensity (RLU).
(4) standard curve is drawn
It is established using the luminous signal intensity of the various concentration AFP calibration objects of measurement and its corresponding AFP calibration objects concentration
Standard curve.
(5) the AFP concentration in serum is calculated
According to the standard curve in the blood serum sample luminous signal intensity of measurement and (4), the AFP calculated in blood serum sample is dense
Degree.
When determinand is antibody, the capture antibody in this detection method is replaced with detection antibody in conjunction with the antibody
Capture antigen (Ag1) and detection antigen (Ag2), coated in ELISA Plate hole in addition is Ag1.
As determinand haptens (hapten), the capture antibody in this detection method is replaced in conjunction with the haptens
Capture antibody (anti-hapten), the anti-AFP detection antibody of HRP labels replaces with the hapten of HRP labels or its is similar
Object, coated in ELISA Plate hole in addition is anti-hapten.
Embodiment 2：Chemiluminescent substrate is tested
(1) chemical intensifier is tested
Chemiluminescent substrate is made of A liquid and B liquid.In A liquid, buffer solution：The carbonate buffer solution that pH is 9.0 ± 0.1,
Luminol (LS)：0.6g/L；In B liquid, urea peroxide (UP)：0.3g/L, dilution are that (pH is 0.2M phosphate buffers
7.4±0.1).By changing o-phenyl phenol (D) concentration and 4- imidazoles phenol (E) concentration in A liquid, according in embodiment 1
Detect three parameters：Reaction system background signal, positive signal value and linear extent, wherein reaction system background signal, i.e., it is empty
White hair light value refers to not adding the reagents such as sample to be checked in reacting hole, chemiluminescent substrate is only added, and detects shining for generation
Signal；Positive signal value refers to that AFP500 is calibrated here by taking the AFP calibration objects (AFP500) of a concentration of 500ng/mL as an example
The luminous signal intensity (RLU) of product, while monitoring the stability of photoluminescence of different time；Linear extent refers to here by taking AFP as an example
Be AFP500 calibration objects luminous signal and a concentration of 0ng/mL AFP calibration objects (AFP0) luminous signal ratio.Detection
As a result as shown in table 1, table 2 and Fig. 1.
1. reaction system background signal of table
As shown in Table 1, when A liquid does not contain D and E, the background signal of luminol is relatively high.When D or E is only added, all may be used
Fall bigger when reducing the background signal of luminol, but only adding E.D and E is added simultaneously, can also reduce luminol
Background signal especially works as D：0.008g/L, E：0.025g/L or D：0.04g/L, E：When 0.125g/L, decline more
Significantly.
The positive signal value and reading stability of table 2.AFP500 calibration objects
By the 2nd~4 row and curve I, II, III and V of the 6th row and Fig. 1 of table 2 it is found that when A liquid does not contain D and E, Shandong
The luminous signal of minot is very low；When D is only added, the luminous signal of luminol can be enhanced, but after 35min, luminous signal
Drop to 57%, when arriving 50min, drops to 41%, i.e. stability of photoluminescence is poor, and luminous signal is decayed quickly；When E is only added
When, the luminous signal of luminol, and having good stability in 50min can be enhanced, but during 0~5 minute, E is to Shandong
The luminous signal of minot enhances degree less than D；When D and E is added simultaneously, the luminous signal of luminol can be further enhanced,
And synergistic enhancing effect is generated between D and E, i.e., the luminous signal generated when being added simultaneously is more than when being individually added into D and individually adds
Enter the sum of the luminous signal generated when E, and there is preferable stability of photoluminescence.
The positive signal value and reading stability of table 3.AFP500 calibration objects
Furthermore the concentration of reduction D and E is to 0.008g/L and 0.025g/L respectively, the luminous letter in 0min and 50min
Number, as a result as shown in the 2nd~4 row of the 5th row of table 2, the curve IV of Fig. 1 and table 3, it is known that when concentration reduces, while D is added
With the luminous signal that can further enhance luminol when E, synergistic enhancing effect is generated between D and E, and with preferable hair
Photostability；E enhances degree less than D to the luminous signal of luminol.In addition, increasing separately the concentration of D and E to 0.16g/L
And 0.5g/L, as a result as shown in the 5th~7 row of the 7th row of table 2, the curve VI of Fig. 1 and table 3, it is known that when concentration increases, together
When can further enhance the luminous signal of luminol when D and E is added, synergistic enhancing effect is generated between D and E, and have
Preferable stability of photoluminescence；E enhances degree less than D to the luminous signal of luminol.
By Tables 1 and 2 calculate AFP calibration objects linear extent and signal-to-noise ratio (here for for AFP500 calibration objects into
Row explanation, computational methods are the ratio of the luminous signal and reaction system background signal of AFP500 calibration objects), as a result such as 4 He of table
Shown in Fig. 2.
4. linear extent measured value of table
By table 4 and Fig. 2 it is found that working as D and E simultaneously in use, having relatively good linear extent, and D ought be added simultaneously
The sum of the linear extent for being greater than with the linear extent generated when E when D is only added and being generated when E only is added.In addition, working as D
With E simultaneously in use, also have relatively good signal-to-noise ratio, and have synergistic enhancing effect, i.e., when simultaneously D and E is added when produce
The sum of the signal-to-noise ratio that raw signal-to-noise ratio is greater than when D is only added and is generated when E only is added.
(2) luminol and urea peroxide experiment
Chemiluminescent substrate is made of A liquid and B liquid.In A liquid, o-phenyl phenol (D)：0.04g/L, 4- imidazoles phenol
(E)：0.125g/L, buffer solution：The carbonate buffer solution that pH is 9.0 ± 0.1, screens different luminols (LS) concentration；In B liquid
In：Dilution is 0.2M phosphate buffers (pH is 7.4 ± 0.1), screens different urea peroxides (UP) concentration.For this purpose, changing
Become LS and UP concentration, according to the detection method in embodiment 1, detects the AFP calibration objects of three kinds of various concentrations, i.e., a concentration of 0ng/
AFP calibration objects (AFP0), the AFP calibration objects (AFP5) of a concentration of 5ng/mL and the AFP calibration objects of a concentration of 500ng/mL of mL
(AFP500), luminous signal intensity (RLU) testing result under different time is as shown in table 5.In table 4, Sens.=
AFP5RLU/AFP0RLU refers to the ratio of the RLU of RLU and the AFP0 calibration object of AFP5 calibration objects, and this ratio is higher, table
Bright sensitivity is better, and when this ratio is less than 2.0, sensitivity is relatively low, cannot receive；Line.=AFP500RLU/
AFP5RLU refers to the ratio of the RLU of RLU and the AFP5 calibration object of AFP500 calibration objects, and this ratio is higher, shows linear
Width is bigger, and when this ratio is less than 20, linear extent is smaller, cannot receive.
The AFP calibration object testing results of 5. various concentration of table
As shown in Table 5, as a concentration of 0.2~5g/L of LS, when a concentration of 0.2~2g/L of UP, there is luminous signal, LS is most
Excellent concentration range is 0.6~1.0g/L, and the optimal concentration range of UP is 0.2~0.3g/L.
(3) chemiluminescent substrate buffer runs
Chemiluminescent substrate is made of A liquid and B liquid.In B liquid, urea peroxide (UP)：0.3g/L, dilution：0.2M phosphorus
Phthalate buffer (pH is 7.4 ± 0.1)；In A liquid, o-phenyl phenol (D)：0.04g/L, 4- imidazoles phenol (E)：0.125g/
L, luminol (LS)：0.8g/L changes the buffer solution in A liquid, detects the AFP calibration objects of various concentration, the results are shown in Table 6.
In table 6, Sens. and Line. meanings are identical as in table 5, and BB is borate buffer solution, and CB is carbonate buffer solution.
The different buffer solution selection results of table 6.
| Buffer solution | BB | CB | Tris-HCl |
| pH | 9.0±0.1 | 9.0±0.1 | 9.0±0.1 |
| Background | 941 | 676 | 103 |
| Sens. | 1.96 | 11.61 | 4.84 |
| Line. | 1947.85 | 394.44 | 437.31 |
As shown in Table 6, Sens.>=2.0, Line.>=20, so BB, CB and Tris-HCl buffering of pH 9.0 ± 0.1
Liquid is suitable in A liquid, wherein under the carbonate buffer solution of pH 9.0 ± 0.1, the Sens. highests measured have most
Good sensitivity, while also there is relatively high Line. values, in terms of comprehensive, effect is best.
Embodiment 3：Chemiluminescent substrate storage stability is tested
In order to test the stability of chemiluminescent substrate of the invention, A liquid is prepared：O-phenyl phenol (D) 0.04g/L, 4-
Imidazoles phenol (E) 0.125g/L, luminol 0.8g/L, buffer solution：0.1M carbonate buffer solutions (pH is 9.0 ± 0.1)；B liquid：It crosses
Urea (UP) 0.3g/L is aoxidized, dilution is 0.2M phosphate buffers (pH is 7.4 ± 0.1), according to the detection side in embodiment 1
Method, the AFP calibration objects for the 5 kinds of various concentrations prepared in detection embodiment 1 at 2~8 DEG C of reagent storage condition, in different time
Under luminous signal intensity (RLU), testing result is as shown in table 7.
Table 7.AFP calibration object testing results
| 2~8 DEG C (moons) | 0 | 3 | 6 | 9 | 12 | 15 | 18 |
| 0 | 3973 | 4390 | 3713 | 3765 | 2808 | 2220 | 1413 |
| 5 | 72250 | 79975 | 74758 | 61070 | 49698 | 80968 | 58543 |
| 10 | 194285 | 171315 | 195225 | 148948 | 119023 | 212373 | 177080 |
| 20 | 477720 | 420283 | 441938 | 309823 | 246605 | 441803 | 394983 |
| 500 | 14262238 | 14643470 | 14906343 | 12794303 | 13162728 | 13325253 | 14199725 |
| Related coefficient (r) | 1.0000 | 0.9999 | 1.0000 | 0.9999 | 0.9998 | 1.0000 | 0.9999 |
As shown in Table 7, chemiluminescent substrate of the invention at 18th month, still maintains extraordinary phase at 2~8 DEG C
Relationship number, luminous signal intensity keep stablizing, and have extraordinary storage stability.
Embodiment 4：Determinand is detected using chemiluminescent substrate
In order to detect the determinand in the samples such as serum using the chemiluminescent substrate of the present invention, A liquid is prepared：Adjacent phenyl benzene
Phenol (D) 0.04g/L, 4- imidazoles phenol (E) 0.125g/L, luminol 0.8g/L, buffer solution：(pH is 0.1M carbonate buffer solutions
9.0±0.1)；B liquid：0.3g/L urea peroxides (UP), dilution：0.2M phosphate buffers (pH is 7.4 ± 0.1), according to reality
The detection method in example 1 is applied, the AFP calibration objects, the CEA calibration objects of various concentration and the IgE of various concentration of various concentration are detected
Calibration object and AFP quality-control products Q1 (9.7ng/mL) and Q2 (118.4ng/mL), CEA quality-control products Q1 (11.3ng/mL) and Q2
(120.6ng/mL), IgE quality-control products Q1 (21.0IU/mL) and Q2 (113.7IU/mL), testing result is as shown in table 8.Wherein,
CEA and IgE calibration object preparation methods, as the AFP calibration objects in embodiment 1 are identical：For CEA calibration objects, weigh a certain amount of
CEA, carry out a series of dilutions using the PBS buffer solution (pH 7.2) containing 50% calf serum, obtain concentration be respectively 0,3,
5, the CEA calibration objects of 10,100 and 250ng/mL.For IgE calibration objects, a certain amount of IgE is weighed, using containing 50% calf
The PBS buffer solution (pH 7.2) of serum carries out a series of dilutions, and it is respectively 0,2.5,5,20,100 and 500IU/mL to obtain concentration
IgE calibration objects.
The different determinand calibration object of 8. 3 kinds of table and its quality-control product testing result
As shown in Table 8, chemiluminescent substrate of the invention also can detect other to be measured other than detecting antigen A FP
Object such as CEA and IgE, while having good detection accuracy, the range of linearity and detection sensitivity.This indicates that it is suitable for
Detect the different products under board-like chemiluminescence platform.
Claims (10)
1. a kind of chemiluminescent enhancement liquid, which is characterized in that including o-phenyl phenol and 4- imidazoles phenol.
2. chemiluminescent enhancement liquid as described in claim 1, which is characterized in that a concentration of 0.008g/L of o-phenyl phenol~
A concentration of 0.025~0.5g/L of 0.16g/L, 4- imidazoles phenol.
3. chemiluminescent enhancement liquid as claimed in claim 2, which is characterized in that a concentration of 0.008g/L of o-phenyl phenol~
A concentration of 0.025g/L~0.125g/L of 0.04g/L, 4- imidazoles phenol.
4. chemiluminescent enhancement liquid as described in claim 1, which is characterized in that further include buffer solution, the buffer solution is selected from
Tris-HCl, borate buffer solution or carbonate buffer solution.
5. a kind of application of composition in the luminous signal of enhancing HRP- luminol chemiluminescence systems, which is characterized in that institute
It includes the chemiluminescent enhancement liquid described in one of Claims 1 to 4 to state composition.
6. a kind of chemiluminescent substrate, the chemiluminescent substrate includes A liquid and B liquid, and A liquid includes luminous agent, and B liquid includes peroxide
Compound, which is characterized in that A liquid further includes the chemiluminescent enhancement liquid described in one of Claims 1 to 4.
7. chemiluminescent substrate as claimed in claim 6, which is characterized in that a concentration of 0.008g/L of o-phenyl phenol~
A concentration of 0.025~0.5g/L of 0.16g/L, 4- imidazoles phenol.
8. chemiluminescent substrate as claimed in claim 7, which is characterized in that a concentration of 0.008g/L of o-phenyl phenol~
A concentration of 0.025g/L~0.125g/L of 0.04g/L, 4- imidazoles phenol.
9. chemiluminescent substrate as claimed in claim 6, which is characterized in that A liquid further includes buffer solution, and the buffer solution is selected from
Tris-HCl, borate buffer solution or carbonate buffer solution.
10. the chemiluminescent substrate as described in one of claim 6~9, which is characterized in that the luminous agent of stating is selected from Rumi
Promise, different luminol and derivative, a concentration of 0.2g/L~5g/L, the peroxide are selected from hydrogen peroxide, urea peroxide
And perborate, a concentration of 0.2g/L~2.0g/L.
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