CN101609090A - A kind of employing acridinium ester labelled antigen or antibody analysis method - Google Patents
A kind of employing acridinium ester labelled antigen or antibody analysis method Download PDFInfo
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Abstract
The present invention relates to a kind of chemiluminescent immunoassay(CLIA) method, be particularly related to a kind of employing acridinium ester labelled antigen or antibody analysis method and prepare a kind of immunoassay kit with this method, the acridinium ester label has the luminous specific groups of generation on chemical constitution, in the luminescence immunoassay process, participate in luminescence-producing reaction directly, usually this class material does not have background luminescence, in reaction, can be used to detect the sample of low concentration or micro-concentrations, it is the very high luminous agent of a class luminescence efficiency, acridinium ester I, II molecule and acridinium carboxamide III all can combine with antibody (or antigen), generate to have the chemiluminescence activity by force, the labelled antibody that the immune response specificity is high.
Description
Technical field:
The present invention relates to a kind of chemiluminescent immunoassay(CLIA) method, particularly a kind of employing acridinium ester labelled antigen or antibody analysis method and prepare a kind of immunoassay kit with this method.
Background technology:
Chemiluminescent immunoassay(CLIA) (Chemiluminesent Immunoassay, CLIA) be immunoassay continue enzyme immune technology (EIA), put immune technology (RIA), immunofluorence technic (FIA) and time-resolved fluoroimmunoassay technology (TRFIA) after the development an emerging determination techniques.
Chemiluminescence is a kind of mensuration mode that grows up over nearly two, 30 years, its free energy that utilizes chemical reaction to discharge excites intermediate (common alkali acid phosphatase-amantadine), make it get back to ground state from excited state, when intermediate such as can discharge at the photon of energy level when excited state is got back to ground state, photon is measured and carried out quantitative test.
Chemiluminescence has the specificity of fluorescence, do not need simultaneously exciting light, the influence of exciting light parasitic light has very high sensitivity in the fluorescence analysis with regard to having avoided, and has strong environmental pollution and health hazard unlike radiometric analysis, is a kind of very outstanding quantitative analysis method.Though chemiluminescence possesses very high specificity and very little interference, the not specificity of chemical analysis itself has restricted the use of entire method.Therefore, utilize the signal specificity of immunoreactive specificity and chemiluminescence itself to form said chemiluminescent immunoassay(CLIA) (CLIA) technology as RIA, FIA etc.
The chemiluminescent immunoassay(CLIA) technology is a kind of extremely sensitive microdetermination technology, and all have antigenic material (comprising haptens) and can use the chemiluminescent immunoassay(CLIA) technical measurement.It possesses following characteristics:
1. extremely sensitive, the limit reaches 10-17-10-19M/L, and is far above RIA, EIA, suitable but more cheap than TRFIA with TRFIA.
2. high specificity, good reproducibility C.V.＜5%.
3. measurement range is wide, can reach 7 orders of magnitude.
4. reagent stability is good, the pollution-free term of validity 6-12 month.
5. simple to operate, be easy to robotization.
Chemiluminescence immune assay is divided into: two kinds of chemiluminescence enzyme immunoassay and flash type chemiluminescence immune assays.Wherein chemiluminescence enzyme immunoassay belongs to a type of enzyme immunoassay (EIA), and just the used substrate of final step enzymatic reaction is a luminous agent, measures on specific instrument by the light of luminescence-producing reaction.Aspect mark: marker enzyme commonly used have horseradish peroxidase (horseradish peroxidase, HRP) and alkaline phosphatase (alkaline phosphatse, AP), and the flash type chemiluminescence immune assay is to adopt acridinium ester labelled antigen or antibody.
Horseradish peroxidase is that a kind of molecular weight reaches 44000 glycoprotein, by colourless zymoprotein and dark-brown iron porphyrin be combined into.The luminous substrate of horseradish peroxidase is luminol (Lumimol).Luminol belongs to the compound of phenylhydrazine class.Under alkali condition, peroxidase plays catalytic action to luminol and hydroperoxidation.
Alkaline phosphatase is a kind of phosphate hydrolytic enzyme, and the AP molecular weight that extracts from Escherichia coli is 80kDa, and optimum pH is 8.0; The AP molecular weight that extracts from mucous membrane of small intestine is 100kDa, and optimum pH is that 9.6.AMPPD is the luminous substrate of alkaline phosphatase.
In the molecular structure of AMPPD by two piths: one is the dioxy ring that connects phenyl ring and diamantane, and it can rupture and ballistic phonon: another is a phosphate group, and it keeps the stable of whole molecular structure.Under the effect of alkaline phosphatase, the rapid dephosphorylate group of this substrate generates unsettled intermediate A MPD.AMPD is very fast to be decomposed voluntarily, gets back to the low energy stable state from high energy excited state, sends photon simultaneously.
HRP and AP are used for labelled antibody (antigen) and form enzymic-labelled antibody (antigen) bond in chemiluminescence, and this bond had both kept the immunocompetence of antibody (antigen), and the enzyme of Bao Liuing is to the catalytic activity of substrate again.After the immune response of enzyme labelled antibody (antigen) and antigen (antibody) is carried out, utilize it to the luminous substrate catalytic action and directly luminous, directly carry out quantitatively by the mensuration of luminous intensity.
The enzyme-catalyzed chemical luminescence diagnostic products all rests on semi-automatic level at present, and the running time is longer, testing result poor repeatability, false drop rate height.Went out the result at least 1 hour as T4 enzyme-catalyzed chemical luminescence diagnostic reagent, just and detect after often needing to gather certain sample size in order to reduce cost, the patient of health check-up so the earliest waits for 2 days at least, at most first quarter moon.
The purpose of carrying out of the present invention makes the user can obtain to detect data easier, fast, accurately exactly.The present invention mainly utilizes the specificity and the affinity of antigen and antibody mediated immunity reaction, by what can be used for spike at antigen or antibody labeling a kind of micro substance diagnostic techniques that chemiluminescent organic molecule is determined the concentration of determined antigen can take place.
In addition, the present invention also reaches the purpose of rapid automatized diagnosis in conjunction with advanced at present magnetic particle technology.
Summary of the invention:
The object of the present invention is to provide a kind of flash type chemiluminescence immune analysis method, particularly adopt acridinium ester labelled antigen or antibody analysis method.
The acridinium ester label has the luminous specific groups of generation on chemical constitution, participate in luminescence-producing reaction directly in the luminescence immunoassay process.Usually this class material does not have background luminescence, can be used to detect the sample of low concentration or micro-concentrations in reaction, is the very high luminous agent of a class luminescence efficiency.Acridinium ester I, II molecule and acridinium carboxamide III all can combine with antibody (or antigen), generate to have the labelled antibody that chemiluminescence is active by force, the immune response specificity is high.
Acridinium ester is marked on the amino of antigen (antibody), and with antigen reactive variable region, mainly by being made up of several amino, these several amino have determined immunoreactive specificity and compatibility on the antibody.Owing to the possibility with the shielding of the amino on the variable region is arranged during the acridinium ester labelled antibody, will cause antibody to lose biologically active, lose specificity like this.We adopt a kind of special process, can avoid the variable region, acridinium ester are marked on the fixed area of antibody, make it both keep due characteristic, are difficult for inactivation again.
The present invention adopts the flash type chemiluminescent substance acridinium ester thing that serves as a mark, and acridinium ester is an organic molecule, and luminous signal is corresponding one by one with the molecule number, and is not affected by environment, so the favorable reproducibility of testing result.
Another object of the present invention is to provide a kind of coating technique.
The present invention adopts at present advanced in the world polystyrene coating technique, can be so that participating in immunoreactive antibody concentration increase greatly, and then improve detection sensitivity and reaction rate.Because carrying out immune response at polystyrene surface is similar to liquid phase reactor, compare with present state endoperidium plate technique, the microreaction process does not need reactant to carry out molecular collision by the liquid phase main diffusion again to solid-liquid interface, therefore significantly reduce the probability that the interfering material in the liquid environment disturbs whole immunoreaction process, made testing result more accurate.
In order to improve the susceptibility of chemiluminscence immunoassay, numerous mensuration amplification systems has appearred, and be one of them based on the amplification system of biotin-avidin reaction.Aspect coating technique, the amplification system on the biotin-avidin reaction basis that we utilize.
Biotin can be used for mark range protein (comprising antigen, antibody, enzyme etc.), formation biotinylated protein matter derivant by after the carboxyl on the thiphene ring valeric acid side chain and the coupled activation of multiple big molecule.A protein molecule can combine with a plurality of biotin molecules, thereby has higher specific activity.The macromolecular polyvalency of biotinylation is the material base of the multistage amplification of biotin-avidin system.
The application of Avidin-biotin system in chemiluminescence has various ways, also can be used for wrapping indirectly quilt.Can on solid phase, wrap in advance, formerly be combined with biotin by the antibody of solid phase or antigen, biotinylated antibody or antigen be combined with Avidin by Avidin-biotin reaction with the absorption method bag by Avidin.This bag not only can be increased the antibody or the antigen amount of absorption by method, and its binding site is fully exposed.
Biotin (biotin, B) molecular weight is 244.3kDa, and isoelectric point is that the imidazolone ring in the pH value 3.5. biotin structure is the main position that combines with Avidin, and thiphene ring valeric acid side chain terminal carboxyl is the main position with protein bound such as antibody and enzymes.The carboxyl that utilizes biotin can be made into the derivant (activation biotin) of various reactive groups after the chemical modification in addition, with the needs that are fit to combine with various biomacromolecules.
(isoelectric point is that pH value 10-10.5. Avidin is rich in tryptophane to Avidin for avidin, A) molecular weight 68kDa, closes by the imidazolone loops of trp residue and biotin.Affinity between Avidin and the biotin is extremely strong, and is higher at least 10,000 times than the affinity of antigen and antibody, and have high degree of specificity and stability.
Setting up amplification system by the biotin-avidin reaction is the basis, be to use the biotin labeling coated antibody, combine back label mixing with solid phase antigen after, add Avidin-bond polystyrene solid phase again, because an antibody can connect a plurality of biotin molecules, therefore can combine with a plurality of Avidin molecules when reacting at a biotinylated antibody, Avidin character is very stable, per molecule can be in conjunction with 4 biotin derivatives, it plays amplification as the bridge material between the biotinylated molecule.
Simultaneously, it is again a glycoprotein, can be directly be coupled with various biomolecule such as IgG and theophylline, phenytoinum naticum, protein etc., thereby the reaction of mensuration is amplified.We have been used for the method bag by plate, and have obtained good effect: biotin combine the affinity with height with Avidin, and the coupled antibody of equal energy, antigen and horseradish peroxidase and do not influence biologically active.
In the process of technical optimization next, we will be applied to the biotin-avidin system technology in the bag quilt of magnetic particle, combine with the magnetic particle by formed Avidin-biotin-multienzyme complex antibody, follow the trail of the antigen or the antibody of biotin enzyme or acridinium ester mark, by the substrate for enzymatic activity colour developing, can detect corresponding antibody or antigen.
The benefit of the method is can shared reagent in different projects, makes it have more special, sensitive, easy, characteristics fast.
Process innovation of the present invention is:
Adopt novel reaction amplification system in the coating technique---adopt Streptavidin one biotin coating technique.This is a kind of novel reaction amplification system.With magnetic microsphere or polystyrene solid phase as carrier, the bag of maximum is by last Streptavidin from the teeth outwards, with mark the antigen of biotin or antibody response form Streptavidin one biotin coated antibody, form the strong bonded and the multistage enlarge-effect of high affinity again with label, feasible detection is sensitive more, can reach the purpose of detectable concentration less than the ultramicron material of 1Pmol/L.
Streptavidin is a kind of slightly slant acidity albumen, and its molecular weight is 65000, is made of 4 identical subunits of sequence, and each subunit can be in conjunction with 1 biotin molecule, and therefore 1 Streptavidin molecule has 4 sites that can combine with biotin molecule.Strepto-affinity element does not contain glycosyl, with histocyte, when DNA combines, being difficult for producing non-specific binding. its activity can reach 18u. in chemiluminescence immune assay, magnetic microsphere or polystyrene solid phase are as carrier, can wrap by the Streptavidin of maximum, and effectively combine with biotin reagent, accelerate immune response speed greatly.
Biotin claims biotin again, structure ringwise, be divided into I ring and II ring, the I ring is the main position of Streptavidin combination, the II ring is unique structure of labelled antibody and enzyme, can be effectively in conjunction with different antigen and various enzyme, be used to measure multiple protein, hormone, antigen and multiple medicine, antibody labeling behind the biotin, its conjugated antigen active unaffected, plurality of enzymes in conjunction with biotin after its catalytic force remain unchanged basically, play amplification effectively so adopt biotin to combine with multiple label; The multivalence reagent of many " haptic elements " that its biotinylation forms makes the entire reaction system multistage amplification reaction occur, the detection of determinand is had high sensitivity, and reaction velocity satisfies and the scientific research demand than very fast clinical.
Technology of the present invention has been accelerated reaction velocity greatly.In test process, the incubation time is 9～18min, and 20min can go out the result, has shortened the time greatly than radio immunoassay, has improved work efficiency.The antibody reagent of high specific and high-affinity can adsorb more antigen or antibody.The wide linearity that luminous signal detects adds and the signal amplification of Streptavidin-biotin coating technique, makes the range of linearity of chemiluminescence detection wideer.
Bag of the present invention is by technology, and it rationally is used in the actual production.This has guaranteed that not only the product consumption of producing according to whole process flow reduces to minimumly, has also guaranteed the safe and reliable of product quality.Various and become one when serial when product item, product all will carry out the bag quilt of different antibodies (antigen), obscuring and pollute when avoiding production run.The coating technique of novel process can be unified the solid phase carrier component, avoids unnecessary error, makes detection more succinct, convenient.
The present invention utilizes technology of the present invention to carry out the biological medicine in-vitro diagnosis, and development product is mainly used in the clinical treatment diagnosis kits, preferred as: the external immunodiagnosis kit of preparation thyroxine (T4).Main audient is hospitals at different levels, and other audients also comprise the food safety supervision system, and public security system is engaged in and is checked relevant scientific research system.Chemiluminescence has vast market prospect as the most promising technique direction of immunodiagnosis detection means.
Compare with other immune diagnostic method such as radiommunoassay, EIA enzyme immunoassay and time resolved fluoro-immunoassay, advantages such as that chemiluminescence immune assay of the present invention has is highly sensitive, high specificity, sensing range is wide, keeping life is long, favorable reproducibility, cost are cheap relatively can be used for sxemiquantitative and quantitative test.It is quick in use for the method, all only needs 1-3 minute detection times.Easy and simple to handle can carrying out whenever and wherever possible.Becoming in the immunodiagnosis field global most popular analytical approach at present at present, also is one of important developing direction of immunoreagent.Country " 863 " supports the exploitation and the application of this technology in the works for this field emphasis.Table 1 is project technical advantage figure, and table 2 is the contrast of each analytical approach.
Table 1, project technical advantage figure
| The advantage of immune diagnostic technique | The immune diagnostic technique shortcoming | |
| Biochemical diagnosis | Cheap, be more suitable for extensive examination, can before the clinical symptoms performance, detect, need not oral drugs. | Price is more expensive than biochemical reagents, and product is complete not as biogenetic products |
| Instrument diagnoses | Wide application, running programization, less demanding to operator quality. | Not can be used as the standard of making a definite diagnosis |
| Other diagnosis | Wide application, running programization, less demanding to operator quality. | Specific aim is not strong, so major part can't be as making a definite diagnosis standard |
The contrast of table 2, each analytical approach
| Advantage | Shortcoming | |
| Radiommunoassay | Technology maturation with a long history, favorable reproducibility as a result. | Radioactive waste aftertreatment difficulty, complicated operation, the running time is long, and the product shelf life is short, is difficult for robotization. |
| EIA enzyme immunoassay | Easy and simple to handle, the running time is short, visual result, and the product shelf life is long. | Sensitivity and sensing range are exempted from not as putting, and testing result is affected by environment big |
| Enzymatic chemical luminous immunoassay | Easy and simple to handle, the running time is short, and the product shelf life is long, and is highly sensitive, and sensing range is wide. | The result is affected by environment big, poor reproducibility |
| Time resolved fluoro-immunoassay | Easy and simple to handle, the product shelf life is long, and is highly sensitive, and sensing range is wide, and favorable reproducibility as a result is not with environmental change | Detection time is long |
| The flash type chemiluminescence immune assay | Easy and simple to handle, shelf life is long, and is highly sensitive, and sensing range is wide, and favorable reproducibility does not become with environment as a result | Need self-reacting device to cooperate |
| Change, detection time is short |
Flash type chemiluminescence of the present invention mainly is to form with the technology of the direct labelled antigen of acridinium ester (antibody), highly sensitive is the key of flash type chemiluminescence analytical technique superiority, it can detect the material that methods such as radiommunoassay and enzyme-linked immuno assay can't detect, the meaning important again to the early diagnosis tool of disease.The acridinium ester labelled reagent term of validity is long, and stable in properties can reach 1 year.
The acridinium ester thing that serves as a mark in the flash type chemiluminescence, we accomplish the user without the replication typical curve, can two-point calibrations, and fast, accurately obtain data.Purpose is exactly to make it systematizedly make the patient can obtain to detect data easier, fast, accurately simultaneously.
Embodiment:
Further specify the present invention by the following examples, but not as restriction of the present invention.
Embodiment 1
In the labelling technique of acridinium ester, the present invention adopts a kind of special process, avoids on the antibody and antigen reactive variable region, acridinium ester is marked on the fixed area of antibody, makes it both keep due characteristic, is difficult for inactivation again.In further development, horseradish peroxidase, alkaline phosphatase, acridinium ester are marked on the antigen (antibody) simultaneously, accomplishing does not influence enzymatic activity not influencing antigen (antibody) activity, does not interfere with each other combined mark under the situation of influence.
The present invention adopts the coating technique based on the biotin-avidin reaction, and this technology can be so that participating in immunoreactive antibody concentration increases greatly, and then raising detection sensitivity and reaction rate.The bag that the biotin-avidin system technology is applied to the magnetic particle by in be the technical optimization of company in this project, combine with the magnetic particle by formed Avidin-biotin-antibody, follow the trail of the antigen or the antibody of enzyme or acridinium ester mark, can detect corresponding antibody or antigen.
Specific embodiment of the present invention is to be used to prepare thyroxine (T4) diagnosis kits, this kit
Leading indicator is:
(1) to the detection error of national quality-control product in positive and negative 20% scope, sensitivity≤5ng/ml, accuracy is analyzed within variance coefficient CV%＜15%;
(2) coefficient of variation CV%＜20% between the analysis.The linearly dependent coefficient absolute value | r| 〉=0.9900 variation within batch coefficient and batch between cheap coefficient less than 15%.To the above normal person's testing result false drop rates of 300 examples less than 15%, with the related coefficient of the correlation analysis of import like product testing result greater than 0.9.
(3) the product shelf life was greater than 12 months.
Embodiment 2
Thyroxine (T4) immunodiagnosis kit
Adopt Streptavidin-biotin reaction amplification system, in conjunction with the polystyrene coating technique, horseradish peroxidase-labeled antigen (antibody) utilizes Streptavidin-biotin reaction amplification system, make that participating in immunoreactive antibody concentration increases greatly, and then improve detection sensitivity and reaction rate.
Utilizing polystyrene bag quilt, the technology of acridinium ester labelled antigen (antibody) is marked at acridinium ester on the fixed area of antibody in forming, and makes it in the coating technique of polystyrene solid phase, and its labelled antibody had both kept due characteristic, was difficult for inactivation again
Adopt the acridinium ester marking process that T4 antigen is carried out mark and purifying.In the coating technique of magnetic particle, use horseradish peroxidase-labeled, by combining of antibody and magnetic particle, follow the trail of the antigen or the antibody of horseradish peroxidase-labeled, can detect corresponding antibody or antigen.
With being coated with the magnetic particle that anti-fluorescein antibody or anti-sheep two resist, with behind the how anti-mark fluorescent element of goat-anti T4 in advance with magnetic particle reaction that is coated with anti-fluorescein antibody or directly that the T4 goat-anti is anti-with being coated with anti-sheep two magnetic particle reaction, final realization resists goat-anti T4 indirectly to be coated on the magnetic particle, and by washing separation back preservation.
Utilize biotin-avidin system, with the how anti-mark biotin of goat-anti T4, combine with the magnetic particle by formed Avidin-biotin-antibody, antigen or antibody response with horseradish peroxidase-labeled detect corresponding antibody or antigen.
Antibody sandwich on the magnetic particle, is adopted the flash type chemiluminescent substance acridinium ester thing that serves as a mark, labelled antibody (antigen), with antigen (antibody) triplicity in the sample, the direct luminous qualitative and quantitative analysis that carries out.
The method that adopts magnetic sheet absorption back washing to separate realizes separating.
Embodiment 3
1. the preparation of acridinium ester label:
The acridinium ester label has the luminous specific groups of generation on chemical constitution, participate in luminescence-producing reaction directly in the luminescence immunoassay process.Usually this class material does not have background luminescence, can be used to detect the sample of low concentration or micro-concentrations in reaction, is the very high luminous agent of a class luminescence efficiency.Acridinium ester I, II molecule and acridinium carboxamide III all can combine with antibody (or antigen), generate to have the labelled antibody that chemiluminescence is active by force, the immune response specificity is high.Acridinium ester is marked on the amino of antigen (antibody), with antigen reactive variable region several amino is arranged on the antibody, and these several amino have determined immunoreactive specificity and compatibility.So want can more high efficiency mark on acridinium ester can not damage antibody activity again, a good approach is exactly that acridinium ester is marked on the fixed area of antibody, makes it both keep due characteristic, is difficult for inactivation again.
With antibody sandwich on the magnetic particle:
Particulate is microballoon or the particle that is aggregated into by high polymer monomer, and diameter mostly is micron or millimeter level.Particulate have can with the functional group of protein bound (as-NH
2,-COOH ,-OH ,-CHO etc.), be easy to antibody (antigen) and form chemical coupling, and binding capacity is big.In addition, when reaction, particulate can be distributed in the entire reaction solution uniformly, and reaction area is increased, and reaction velocity is accelerated.
Magnetic-particle can make reactant (antibody) effectively be attached to its surface rapidly in liquid phase, also can use Avidin-biotin amplification system here.
3. Avidin-biotin application of sending out system big:
Biotin can be used for mark range protein (comprising antigen, antibody, enzyme etc.), formation biotinylated protein matter derivant by after the carboxyl on the thiphene ring valeric acid side chain and the coupled activation of multiple big molecule.A protein molecule can combine with a plurality of biotin molecules, thereby has higher specific activity.The macromolecular polyvalency of biotinylation is the material base of the multistage amplification of biotin-avidin system.
The application of Avidin-biotin system in chemiluminescence has various ways, also can be used for wrapping indirectly quilt.Can on solid phase, wrap in advance, formerly be combined with biotin by the antibody of solid phase or antigen, biotinylated antibody or antigen be combined with Avidin by Avidin one biotin reaction with the absorption method bag by Avidin.This bag not only can be increased the antibody or the antigen amount of absorption by method, and its binding site is fully exposed.
In addition, the also available biotinylated antibody surrogate of the enzyme labelled antibody in the conventional chemical luminescence technology connects Avidin-enzyme conjugates then, to amplify reaction signal.
1) at first utilizes methodology, prescription and the raw material of the ready-made T4CLEIA technology maturation of company, can guarantee that so at least immunoreaction process is rationally believable, the variable that minimizing that like this can maximum possible is studied later.
2) the acridinium ester marking process that adopts me to do to grope and achieve success carries out mark and purifying to T4 antigen.
3) certain company of buying Italy is coated with the anti-magnetic particle of anti-fluorescein antibody or anti-sheep two, with behind the how anti-mark fluorescent element of goat-anti T4 in advance with magnetic particle reaction that is coated with anti-fluorescein antibody or directly that the T4 goat-anti is anti-with being coated with anti-sheep two magnetic particle reaction, final realization resists goat-anti T4 indirectly to be coated on the magnetic particle, and by washing separation back preservation.
4) magnetic particle after instrument can not be realized automatically reaction being finished in early days separates under the prerequisite with reactant liquor, and company takes to realize separating with the method that magnetic sheet absorption back washing separates.
5) the CLIA methodology for the treatment of stably to set up T4 evolutionary operation step progressively more later on realizes robotization.
Claims (10)
1, a kind of employing acridinium ester labelled antigen or antibody analysis method is characterized in that, acridinium ester is combined with antibody or antigen, generate labelled antibody.
2, side's analysis method according to claim 1, it is characterized in that acridinium ester is marked on the amino of antigen or antibody, on the antibody with antigen reactive variable region, mainly by being made up of several amino, these several amino have determined immunoreactive specificity and compatibility.
3, side's analysis method according to claim 1, it is characterized in that, wherein also comprise a kind of coating technique, utilize polystyrene bag quilt, the technology of acridinium ester labelled antigen or antibody is marked at acridinium ester on the fixed area of antibody in forming, and makes it in the coating technique of polystyrene solid phase, its labelled antibody had both kept due characteristic, was difficult for inactivation again.
4, side's analysis method according to claim 1 is characterized in that, also comprises a kind of reaction amplification system, it is characterized in that, adopts the reaction of biotin-avidin to set up amplification system.
5, side's analysis method according to claim 4 is characterized in that, described reaction amplification system is characterized in that, uses the biotin labeling coated antibody, after label mixes after combining with solid phase antigen, adds Avidin-bond polystyrene solid phase again.
6, side's analysis method according to claim 4 is characterized in that, described reaction amplification system is characterized in that, adopts the reaction method system of Streptavidin-biotin.
7, side's analysis method according to claim 4 is characterized in that, described reaction amplification system, and as carrier, the bag of maximum is by last Streptavidin from the teeth outwards with magnetic microsphere or polystyrene solid phase,
(1) with mark the antigen of biotin or antibody response form Streptavidin one biotin coated antibody,
(2) form the strong bonded and the multistage enlarge-effect of high affinity again with label, can reach the ultramicron material of detectable concentration less than 1Pmol/L.
8, side's analysis method according to claim 4, it is characterized in that, described reaction amplification system, biotin-avidin reaction amplification system is applied in the bag quilt of magnetic particle, combine with the magnetic particle with formed Avidin-biotin-multienzyme complex antibody, follow the trail of the antigen or the antibody of biotin enzyme or acridinium ester mark,, can detect corresponding antibody or antigen by the substrate for enzymatic activity colour developing.
9, a kind of thyroxine (T4) immunodiagnosis kit that utilizes the method preparation of claim 1, it is characterized in that, this kit adopts the acridinium ester marking process that T4 antigen is carried out mark and purifying, in the coating technique of magnetic particle, use horseradish peroxidase-labeled,, follow the trail of the antigen or the antibody of horseradish peroxidase-labeled by combining of antibody and magnetic particle, can detect corresponding antibody or antigen
With being coated with the magnetic particle that anti-fluorescein antibody or anti-sheep two resist, with behind the how anti-mark fluorescent element of goat-anti T4 in advance with magnetic particle reaction that is coated with anti-fluorescein antibody or directly that the T4 goat-anti is anti-with being coated with anti-sheep two magnetic particle reaction, the final realization indirectly with many anti-being coated on the magnetic particle of goat-anti T4, and separate the back by washing and preserve
Utilize biotin-avidin system, with the how anti-mark biotin of goat-anti T4, combine with the magnetic particle by formed Avidin-biotin-antibody, antigen or antibody response with horseradish peroxidase-labeled detect corresponding antibody or antigen,
Antibody sandwich on the magnetic particle, is adopted the flash type chemiluminescent substance acridinium ester thing that serves as a mark, labelled antibody (antigen), with antigen (antibody) triplicity in the sample, the direct luminous qualitative and quantitative analysis that carries out.
10, according to thyroxine (T4) immunodiagnosis kit of claim 9, it is characterized in that, comprise following content
The preparation of acridinium ester label:
The acridinium ester label has the luminous specific groups of generation on chemical constitution, in the luminescence immunoassay process, participate in luminescence-producing reaction directly, usually this class material does not have background luminescence, in reaction, can be used to detect the sample of low concentration or micro-concentrations, it is the very high luminous agent of a class luminescence efficiency, acridinium ester I, II molecule and acridinium carboxamide III all can combine with antibody (or antigen), it is active strong that generation has chemiluminescence, the labelled antibody that the immune response specificity is high, acridinium ester is marked on the amino of antigen (antibody), with antigen reactive variable region several amino are arranged on the antibody, these several amino have determined immunoreactive specificity and compatibility, so want can more high efficiency mark on acridinium ester can not damage antibody activity again, a good approach is exactly that acridinium ester is marked on the fixed area of antibody, make it both keep due characteristic, be difficult for inactivation again
With antibody sandwich on the magnetic particle:
Particulate is microballoon or the particle that is aggregated into by high polymer monomer, and diameter mostly is micron or millimeter level, particulate have can with the functional group of protein bound (as-NH
2,-COOH ,-OH ,-CHO etc.), be easy to antibody (antigen) and form chemical coupling, and binding capacity is big, in addition, when reaction, particulate can be distributed in the entire reaction solution uniformly, and reaction area is increased, and reaction velocity is accelerated,
Magnetic-particle can make reactant (antibody) effectively be attached to its surface rapidly in liquid phase, also can use Avidin-biotin amplification system here,
The application that Avidin-biotin sends out system big:
Biotin by carboxyl and the coupled activation of multiple big molecule on the thiphene ring valeric acid side chain after, can be used for mark range protein (comprising antigen, antibody, enzyme etc.), form biotinylated protein matter derivant, a protein molecule can combine with a plurality of biotin molecules, thereby have higher specific activity, the macromolecular polyvalency of biotinylation is the material base of the multistage amplification of biotin-avidin system.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2008101270464A CN101609090A (en) | 2008-06-19 | 2008-06-19 | A kind of employing acridinium ester labelled antigen or antibody analysis method |
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| CNA2008101270464A CN101609090A (en) | 2008-06-19 | 2008-06-19 | A kind of employing acridinium ester labelled antigen or antibody analysis method |
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| CN102408416A (en) * | 2011-09-15 | 2012-04-11 | 常州市思杰生物化学有限公司 | Thiophene derivative for DNA detection and preparation method thereof |
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| CN103185796A (en) * | 2013-03-17 | 2013-07-03 | 南昌大学 | Food-borne pathogenic bacteria quick detection method based on Gamma-Fe2O3@Au nano particle indirect enrichment and immunomagnetic separation |
| CN103217529A (en) * | 2013-03-17 | 2013-07-24 | 南昌大学 | Gamma-Fe2O3 nanoparticle indirect enrichment immunomagnetic separation based method for rapid detection of food-borne pathogenic bacteria |
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| CN109852669A (en) * | 2019-01-04 | 2019-06-07 | 南京航思生物科技有限公司 | A kind of method of acridinium ester label nucleic acid |
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