CN105353138A - Methods for improving accuracy and expanding linear range of immunodetection as well as reagent - Google Patents
Methods for improving accuracy and expanding linear range of immunodetection as well as reagent Download PDFInfo
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- CN105353138A CN105353138A CN201510952436.5A CN201510952436A CN105353138A CN 105353138 A CN105353138 A CN 105353138A CN 201510952436 A CN201510952436 A CN 201510952436A CN 105353138 A CN105353138 A CN 105353138A
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- Prior art keywords
- antibody
- ferritin
- sample
- biotin
- acridinium ester
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention provides a method for improving detection accuracy of an immunity analyzer, a method for expanding a linear range of chemiluminesent immunoassay of an analyte in a sample as well as a reagent. The improvement of the methods and the reagent is as follows: a biotin labeled antibody and an avidin substance fixed solid phase are added to the sample before an acridinium ester or acridine sulfonamide labelled antibody for incubation, and then the acridinium ester or acridine sulfonamide labelled antibody is added for incubation. According to the improvement, the luminous quantity of an analyte calibration material with higher concentration can be remarkably or better distinguished, so that the linear range of the chemiluminesent immunoassay of the analyte is expanded, meanwhile, the chemiluminescence background value is remarkably reduced, and further, the concentration of the analyte in the sample such as clinical serum or blood plasma can be measured more accurately. The methods and the reagent are matched with a chemiluminesent immunoassay analyzer for use and can be applied to detection of tumor markers, infectious diseases, hormones, liver diseases, acute anemia and the like.
Description
Technical field
The present invention relates to analyte chemical electrochemiluminescent immunoassay in the method and raising sample improving immunity analysis instrument detection accuracy and detect method and the agents useful for same thereof of the range of linearity, described method and described reagent all relate to biotin labelled antibodies, acridinium ester or acridine sulfonamide labelled antibody, the solid phase that Avidin class material is fixing, cleaning fluid and inspire liquid, belongs to immunological technique field.
Background technology
Ferritin (ferritin) is a kind of water miscible iron-binding protein, is present in various tissue and body fluid.The globular preteins complex that it is made up of 24 protein protomers is also iron storage protein main in cell.When in conjunction with iron atom, it is referred to as apoferritin (apoferritin).Each ferritin generally can store 2500 ~ 3000 iron atoms, can reach at most 45000 iron atoms.Containing a small amount of ferritin in normal human serum, but when the gestational period and acute anemia, the ferritin levels in serum usually can reduce, and when diseases such as acute and chronic liver damage and liver cancer, the ferritin levels in serum usually can raise.Ferritin Levels change in serum can be used as and judges whether iron deficiency or the excessive index of iron load.The ferritin levels measured in serum is particularly suitable for distinguishing because iron in body utilizes the inadequate hypoferric anemia causing iron to store initiation on the low side.
In human body, ferritin exists with different molecular forms, often kind of molecular forms is called as isoferritin (isoferritin), this is because the subunit of ferritin exists with H and L two kinds of forms, H subunit in different ferritin and L subunit by the existence of different ratios, but two subunits add up to 24.The strongest acid isoferritin contains 24 H subunits, and the strongest isoferritin of alkalescence contains 24 L subunits.Different reactions may be there is in different isoferritins in different immune detection.
Sugar antigen CA19-9 is the Research of predicting markers of cancer of pancreas, cancer of the stomach, colorectal cancer and carcinoma of gallbladder, and large quantity research proves that CA19-9 concentration is relevant with these tumor sizes, is the mark the highest to cancer of pancreas susceptibility reported so far.Pancreas cancer patients 85% ~ 95% is positive, and CA19-9 measures the antidiastole and the state of illness monitoring that contribute to cancer of pancreas.When CA19-9 is less than 1000U/ml, have certain Surgical Significance, after tumor resection, CA19-9 concentration can decline, and as risen, then can represent recurrence again.Also there is higher positive rate to the diagnosis of cancer of pancreas transfer, when serum CA 19-9 levels is higher than 10000U/ml, almost all there is periphery transfer.The positive rate of cancer of the stomach, colorectal cancer, carcinoma of gallbladder, cholangiocarcinoma, liver cancer also can be very high, if detect CEA and AFP to can further improve positive detection rate (for cancer of the stomach, CA72-4 and CEA joint-detection is done in suggestion) simultaneously.The multiple optimum and inflammatory disorders of intestines and stomach and liver, as pancreatitis, slight cholestasia and jaundice, CA19-9 concentration also can increase, and its concentration is many lower than 120U/ml, must be differentiated.
Because people's ferritin and CA19-9 are high molecular weight protein, when adopting the immunoassay of such as radio immunoassay, enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetry etc. and so on to detect the ferritin levels in the sample such as serum or blood plasma, the double-antibody sandwich immunoassay method that adopts detects more.
Since chemiluminescence immunoassay (chemiluminesentimmunoassay, CLIA) since eighties of last century the seventies and eighties comes out, because it adopts nonradioactive labeling, and antigen-antibody immune response and chemiluminescence signal are combined, thus the sensitivity having excellent specificity and significantly improve, is at home and abroad used widely.At present, in market product, the main CLIA technology utilized can be divided into three major types: (1) enzymatic chemical luminous immunoassay method, as Chinese patent application CN101545910A just adopts in this way, chemical luminous substrate is luminous through the degradation of enzyme, different according to enzyme type, mainly be divided into: a) horseradish peroxidase (HRP) chemiluminescence immunoassay, its conventional chemical luminous substrate can be selected from luminol, different luminol and derivant thereof; B) alkaline phosphatase (ALP) chemiluminescence immunoassay, its conventional chemical luminous substrate is 1,2-dichloroethane analog derivative, common are PPD, Lumi-Phos and Lumi-Plus etc. of AMPPD, CSPD, CDP and CDP-Star and Lumigen company; (2) Electrochemiluminescence assay, as Roche company Elecsys series and Cobas Series Electrochemical luminescence immunoassay system adopt exactly in this way, in the specific chemical luminescence-producing reaction that electrode surface is caused by galvanochemistry, in fact galvanochemistry and chemiluminescence two processes are included, its conventional electron accepter is tripropyl amine (TPA) (TPA), and its conventional electrochemiluminescence reagent is tris (bipyridine) ruthenium; (3) based on the chemiluminescence immunoassay of acridinium ester (acridiniumester) or acridine sulfonamide (acridiniumsulfonylamide), its chemical illuminating reagent is acridinium ester or acridine sulfonamide, do not need catalyzer, only need change the conditions such as the pH of solution just can be luminous.
In above-mentioned three kinds of CLIA technology, the major defect of enzymatic chemical luminous immunoassay method is that working curve may drift about in time, and low end slope easily moves down in non-linear, thus sensitivity is lower, the less stable of enzyme addition, active easily forfeiture, often need strengthen the consumption of enzyme, cost is relatively high.And Electrochemiluminescence assay accuracy of measurement is higher, but it is complete in the flow cell of Electrogenerated chemiluminescent immunoassay instrument supporting specially that the electrochemiluminescence reaction involved by it and luminous signal detect, its instrument cost and after-sales service are costly, metering system is complicated, be not suitable for large-scale promotion to use, and in detection infectious disease intermediate item as five indexes of hepatitis b, hepatitis C antigen and/or antibody, HIV antibody and/or antigen, Treponema pallidum antigen and/or antibody, human herpes simplex vicus I type/II type antigen and/or antibody, Human Cytomegalovirus Antigen and/or antibody, toxoplasma antigen and/or antibody, when rubella virus antigen and/or antibody etc., because flow cell is recycling, even if cleaning fluid is utilized repeatedly to clean flow cell when detecting each, the strong positive sample formerly detected still likely marginally remains in flow cell, this will affect the testing result of follow-up negative sample to be measured, thus likely cause false positive results to occur.And in the third method, acridinium ester or acridine sulfonamide are relative simple with the coupling of antigen or antibody, conjugate stable luminescence, luminous quantity and acridinium ester or acridine sulfonamide concentration linear, and luminescence is flash type, luminescence is concentrated fast and intensity is large, is convenient to quick detection, in addition, luminescence-producing reaction disturbing factor is few, background value is low, and signal to noise ratio (S/N ratio) is high, is thus more and more subject to the attention of immune diagnostic reagent developer.
Such as, Siemens ADVIACentaur chemical illumination immunity analysis instrument detect ferritin time adopt the third method above-mentioned to detect exactly: add in sample acridinium ester label goat-anti ferritin polyclonal antibody and on the surface coupling the magnetic bead of mouse Anti-ferritin monoclonal antibody, after cleaning, add acid solution and alkali lye successively, inspire acridinium ester chemiluminescent, according to the ferritin levels in luminous quantity determination serum or plasma sample.In addition, the ARCHITECT ferritin detection kit of Abbott is also utilize the magnetic bead of ferritin antibody bag quilt and acridinium ester label ferritin antibody to detect ferritin levels in serum or plasma sample.But they all do not integrate biotin-avidin system, its detection sensitivity still treats further raising.When the Elecsys1010/2010 Electrogenerated chemiluminescent immunoassay instrument of Roche Holding Ag detects ferritin (or CA19-9), in serum or plasma sample, first add biotin labeling iron-resistant albumen (or CA19-9) monoclonal antibody and tris (bipyridine) ruthenium mark iron-resistant albumen (or CA19-9) monoclonal antibody, then the magnetic particle of streptomysin Avidin bag quilt is added, to electrode application voltage, detect electrochemical luminescence signals, although improve chemiluminescence signal further by integrating biotin-avidin system, but still there is defect mentioned in Electrochemiluminescence assay.
In addition, US Patent No. 6986913B2 embodiment 35 discloses first blood plasma and biotin labeling goat-anti PTH polyclonal antibody and acridinium ester label goat-anti PTH polyclonal antibody to be hatched and produces a kind of immune complex, then the magnetic bead of Streptavidin bag quilt is added, by the interaction of biotin and Streptavidin, this immune complex is allowed to be attached on magnetic bead, then utilize cleaning fluid to clean, after inspiring, utilize Liaison immunity analysis instrument to detect chemiluminescence signal.But, utilize and thisly first add method that then biotin labelled antibodies and acridinium ester label antibody add the magnetic bead of Streptavidin bag quilt and detect ferritin or CA19-9 also has its weak point: first, when profit detects the luminous quantity of variable concentrations ferritin or CA19-9 calibration object in this way, the luminous quantity that the ferritin of higher concentration or CA19-9 calibration object produce is difficult to effectively or more effectively make a distinction; Secondly, when measuring the sample such as clinical serum or blood plasma, the measuring error of this method is larger, poor accuracy; Moreover the chemiluminescence background value of this method is higher, can affect measurement accuracy.
Summary of the invention
For the deficiencies in the prior art part, the present invention find utilize detect sample based on the direct chemiluminescence immunoassay of acridinium ester or acridine sulfonamide time, first sample and biotin labelled antibodies (Biotin-Ab) and the fixing solid phase of Avidin class material are hatched, then acridinium ester is added or acridine sulfonamide labelled antibody (AE-Ab) is hatched again, then cleaning fluid is utilized repeatedly to clean to remove unconjugated Biotin-Ab and AE-Ab, add and inspire liquid and detect luminous value, the luminous quantity difference amplitude that higher concentration calibration object when detecting variable concentrations calibration object produces can be increased significantly, and reduce chemiluminescence background value significantly, improve the detection accuracy of the sample such as clinical serum or blood plasma simultaneously.Described sample is selected from the samples such as calibration object, clinical serum or blood plasma.
Antibody in " biotin labelled antibodies " in the present invention comprises the antibody analyzing thing (being designated as A) at least one specific binding sample, antibody in " acridinium ester or acridine sulfonamide labelled antibody " in the present invention comprises the antibody analyzing thing A at least one specific binding sample, and described analysis thing A can be selected from ferritin; Tumor associated antigen, as AFP, CEA, PSA, CA19-9, CA125, CA153, CA724 etc.; Steroids antigen is as FSH, TSH, LH etc.; Infectious disease class antigen, as HBsAg, HBcAg, HBeAg, HBxAg, HCV antigen etc.For double-antibody sandwich immunoassay method, the antibody of at least one specific binding assay thing A in " biotin labelled antibodies " and the different antigenic determinants of at least one specific binding assay thing A specific binding assay thing A in " acridinium ester or acridine sulfonamide labelled antibody ".
The present invention's mentioned " luminous quantity of sample " refers to after the solid phase of sample through fixing with biotin labelled antibodies, Avidin class material and acridinium ester or acridine sulfonamide labelled antibody are hatched, form " the analysis thing-acridinium ester in solid phase-biotin labelled antibodies-sample or acridine sulfonamide labelled antibody " compound, remove unconjugated biotin labelled antibodies and unconjugated acridinium ester or acridine sulfonamide labelled antibody, then add after inspiring liquid, chemiluminescence signal produces, luminous quantity measured when utilizing immunity analysis instrument to detect described chemiluminescence signal.Sample can be analyze thing calibration object, may also be the sample such as clinical serum or blood plasma, and therefore, the present invention's mentioned " luminous quantity of sample " is identical with the implication of " analyte immunoassay luminous quantity "." luminous quantity of calibration object " mentioned in the present invention refers to " luminous quantity of sample " when sample is analysis thing calibration object.
The invention provides a kind of method improving immunity analysis instrument detection accuracy, its step comprises: (1) provides immunity analysis instrument; (2) by sample and biotin labelled antibodies and the fixing solid-phase incubation of Avidin class material, forming reactions mixed liquor; (3) in described reaction mixture, add acridinium ester or acridine sulfonamide labelled antibody, then hatch; (4) unconjugated biotin labelled antibodies and unconjugated acridinium ester or acridine sulfonamide labelled antibody is removed; (5), after adding and inspiring liquid, chemiluminescence signal produces, and utilizes described immunity analysis instrument to detect the luminous quantity of described chemiluminescence signal.
Further, the antibody in the antibody in described biotin labelled antibodies and described acridinium ester or acridine sulfonamide labelled antibody is selected from ferritin antibody or anti-CA19-9 antibody simultaneously.
Further, described sample is selected from calibration object, serum or blood plasma.
Further, described solid phase is selected from microwell plate, plastic cement particulate, magnetic bead, elastoplast pipe or plastic bead.
Further, the concentration of described biotin labelled antibodies is 0.1 ~ 5.0 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 0.1 ~ 5.0 μ g/ml.
Further, the concentration of described biotin labelled antibodies is 0.4 ~ 0.8 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 2.5 ~ 5.0 μ g/ml.
The present invention also provides a kind of chemiluminescence immune assay reagent detecting analyte concentration in sample, described reagent comprises biotin labelled antibodies, Avidin class material fixing solid phase, acridinium ester or acridine sulfonamide labelled antibody, cleaning fluid and inspires liquid, and described biotin labelled antibodies and the fixing solid phase of described Avidin class material join in described sample prior to described acridinium ester or acridine sulfonamide labelled antibody.
Further, hatch after the solid phase that described biotin labelled antibodies and described Avidin class material are fixed joins described sample, forming reactions mixed liquor, then adds described acridinium ester in described reaction mixture or acridine sulfonamide labelled antibody is hatched again.
Further, the antibody in the antibody in described biotin labelled antibodies and described acridinium ester or acridine sulfonamide labelled antibody is selected from ferritin antibody or anti-CA19-9 antibody simultaneously.
Further, described sample is selected from calibration object, serum or blood plasma.
Further, described solid phase is selected from microwell plate, plastic cement particulate, magnetic bead, elastoplast pipe or plastic bead.
Further, the concentration of described biotin labelled antibodies is 0.1 ~ 5.0 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 0.1 ~ 5.0 μ g/ml.
Further, the concentration of described biotin labelled antibodies is 0.4 ~ 0.8 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 2.5 ~ 5.0 μ g/ml.
The present invention also provides a kind of kit for detecting analyte concentration in sample, described kit comprises biotin labelled antibodies, Avidin class material fixing solid phase, acridinium ester or acridine sulfonamide labelled antibody, cleaning fluid and inspires liquid, and described biotin labelled antibodies and the fixing solid phase of described Avidin class material join in described sample prior to described acridinium ester or acridine sulfonamide labelled antibody.
Further, hatch after the solid phase that described biotin labelled antibodies and described Avidin class material are fixed joins described sample, forming reactions mixed liquor, then adds described acridinium ester in described reaction mixture or acridine sulfonamide labelled antibody is hatched again.
Further, the antibody in the antibody in described biotin labelled antibodies and described acridinium ester or acridine sulfonamide labelled antibody is selected from ferritin antibody or anti-CA19-9 antibody simultaneously.
Further, described sample is selected from calibration object, serum or blood plasma.
Further, described solid phase is selected from microwell plate, plastic cement particulate, magnetic bead, elastoplast pipe or plastic bead.
Further, the concentration of described biotin labelled antibodies is 0.1 ~ 5.0 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 0.1 ~ 5.0 μ g/ml.
Further, the concentration of described biotin labelled antibodies is 0.4 ~ 0.8 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 2.5 ~ 5.0 μ g/ml.
The present invention also provides a kind of method improving the analyte chemical electrochemiluminescent immunoassay detection range of linearity in sample, its step comprises: (1) adds the fixing solid phase of biotin labelled antibodies and Avidin class material in described sample, hatch, forming reactions mixed liquor; (2) in described reaction mixture, add acridinium ester or acridine sulfonamide labelled antibody, then hatch; (3) add cleaning fluid and remove unconjugated biotin labelled antibodies and unconjugated acridinium ester or acridine sulfonamide labelled antibody; (4), after adding and inspiring liquid, chemiluminescence signal produces, and measures its luminous quantity.
Further, the antibody in the antibody in described biotin labelled antibodies and described acridinium ester or acridine sulfonamide labelled antibody is selected from ferritin antibody or anti-CA19-9 antibody simultaneously.
Further, described sample is selected from calibration object, serum or blood plasma.
Further, described solid phase is selected from microwell plate, plastic cement particulate, magnetic bead, elastoplast pipe or plastic bead.
Further, the concentration of described biotin labelled antibodies is 0.1 ~ 5.0 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 0.1 ~ 5.0 μ g/ml.
Further, the concentration of described biotin labelled antibodies is 0.4 ~ 0.8 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 2.5 ~ 5.0 μ g/ml.
Accompanying drawing explanation
Fig. 1. Application way 1 measures the luminous quantity of variable concentrations ferritin calibration object, according to the typical curve that the funtcional relationship of luminous quantity and ferritin calibration object concentration is made.
Fig. 2. Application way 2 measures the luminous quantity of variable concentrations ferritin calibration object, according to the typical curve that the funtcional relationship of luminous quantity and ferritin calibration object concentration is made.
Fig. 3. Application way 1 measures the ferritin levels value of 50 routine clinical serum samples, and by comparing the measured value of its measured value and Roche, draws clinical accordance analysis graph.
Fig. 4. Application way 2 measures the ferritin levels value of 50 routine clinical serum samples, and by comparing the measured value of its measured value and Roche, draws clinical accordance analysis graph.
Embodiment
Direct chemiluminescence immunoassay based on acridinium ester or acridine sulfonamide detects the analyte concentration in sample, in the mensuration sample adopted, the kit of analyte concentration comprises biotin labelled antibodies (Biotin-Ab), acridinium ester or acridine sulfonamide labelled antibody (AE-Ab), the solid phase that Avidin class material is fixing, calibration object, cleaning fluid and inspire liquid; In the mensuration sample adopted, the reagent of analyte concentration comprises biotin labelled antibodies (Biotin-Ab), acridinium ester or acridine sulfonamide labelled antibody (AE-Ab), the solid phase that Avidin class material is fixing, calibration object, cleaning fluid and inspire liquid.Described sample is selected from calibration object or the sample such as clinical serum or blood plasma.When described analysis thing is ferritin, antibody in Biotin-Ab and the antibody in AE-Ab are ferritin antibody, are biotin labeling ferritin antibody (Biotin-ferritin-Ab) and acridinium ester or acridine sulfonamide and mark ferritin antibody (AE-ferritin-Ab); When described analysis thing is CA19-9, the antibody in Biotin-Ab and the antibody in AE-Ab are anti-CA19-9 antibody.Below respectively to fixing solid phase, the calibration object of Biotin-Ab, AE-Ab, Avidin class material, inspire liquid and cleaning fluid launches to illustrate, for convenience of explanation, be described for Biotin-ferritin-Ab and AE-ferritin-Ab simultaneously.
The ferritin antigenic determinant identified Biotin-ferritin-Ab and AE-ferritin-Ab, Biotin-ferritin-Ab and AE-ferritin-Ab is not identical.Ferritin antibody in ferritin antibody in described Biotin-ferritin-Ab and described AE-ferritin-Ab can be selected from Anti-ferritin monoclonal antibody or polyclonal antibody, utilize the animal of ferritin or its fragment immunizing non-human be separated to produce, in addition, ferritin antibody in ferritin antibody in described Biotin-ferritin-Ab and described AE-ferritin-Ab also can be the fragment that Anti-ferritin monoclonal antibody or polyclonal antibody still have ferritin binding activities after chemical reagent or ferment treatment, as after papain digestion the Fab fragment that produces, or the F produced after pepsin digestion (ab')
2, or F (ab')
2the Fab' fragment produced after enzyme IdeS digests.Described non-human animal is optional from animals such as mouse, rat, cavy, rabbit, chicken, pig, sheep, goat, horse, mule and camels.Preferably, the ferritin antibody in described Biotin-ferritin-Ab and the ferritin antibody in described AE-ferritin-Ab are selected from mouse Anti-ferritin monoclonal antibody or rabbit Anti-ferritin monoclonal antibody.
The ferritin be separated or its fragment can be utilize conventional protein separation technology isolated from the sample such as human serum or blood plasma, also can be utilize conventional technique for gene engineering to express in prokaryotes (as Escherichia coli and bacillus subtilis etc.) or eukaryotic cells (as yeast cells, insect cell, vegetable cell and mammalian cell etc.) by the DNA fragmentation of encoding human ferritin or its fragment, then produce through purifying, can also be utilize in in-vitro transcription/translation system allow this DNA fragmentation express after, more purified and produce.
In biotin labeling ferritin antibody (Biotin-ferritin-Ab), the antibody biotin method that its labeling method can refer to US Patent No. 5089423A announcement is carried out.Before use, utilize biotin labelled antibodies dilution to dilute Biotin-ferritin-Ab, wherein said biotin labelled antibodies dilution can be selected from ddH
2o, physiological saline, buffering range comprise the damping fluid of pH7.0, preferably from the 0.01MPBS damping fluid of pH7.4 or the 10mMTris-HCl damping fluid of pH7.4.In described biotin labelled antibodies dilution, the stabilizing agents such as appropriate BSA, glutin or casein can be added as required, also appropriate antiseptic can be added as required, as Sodium azide, thimerosal, Proclin-150, Proclin-200, Proclin-300 or Proclin-5000 etc.
In addition, in biotin labeling ferritin antibody (Biotin-ferritin-Ab), the implication of described biotin (Biotin) is biotin and derivant thereof, and its common derivant can be selected from 2-iminobiotin, long-armed activated biotin, biotin hydrazides, biotin-N-mercaptoethylmaine, biotin-O-amino, biotin methyl ether, Sulfo-NHS-LC-Biotin, Sulfo-NHS-Biotin, NHS-PEO
4-Biotin, PEO
3-Amine-Biotin, NHS-LC-LC-Biotin, NHS-LC-Biotin, Sulfo-NHS-Biotin and NHS-Biotin etc.
In acridinium ester or acridine sulfonamide mark ferritin antibody (AE-ferritin-Ab), the acridinium ester class chemical illuminating reagent that acridinium ester can select US Patent No. 4745181A, US4946958A, US5521103A and US5656500A etc. to disclose, the acridine sulfonamides chemical illuminating reagent that acridine sulfonamide can select US Patent No. 5783699A, US5669819A, US5565570A, US5545739A and US5468646A etc. to disclose.Its labeling method can adopt together with acridinium ester or acridine sulfonamide be coupled at ferritin antibody by cross-linking reagent, the crosslinking chemical adopted can be selected from N-succinimido-3-(4-hydroxyphenyl) propionic ester [N-succinimidyl-3-(4-hydroxyphenyl) propionate, SHPP], succinimide pyridine dimercapto propyl ester [N-Succinimidyl3-(2-pyridyldithio) propionate, SPDP], 4-maleimidobutyric acid-N-succinimide ester (N-γ-maleimidobutyryloxysuccinimideester, GMBS), S-acetyl mercapto succinic anhydride (S-acetylmercaptosuccinicanhydride, AMSA), dicyclohexyl carbon (dicyclohexylcarbodiimide, DCC), SMCC, Sulfo-SMCC, glutaraldehyde, sodium metaperiodate and other conventional cross-linking reagents etc.Before using, acridinium ester label antibody diluent can be utilized to be diluted by a certain percentage by AE-ferritin-Ab solution, wherein said acridinium ester label antibody diluent can be selected from ddH
2o, physiological saline, buffering range comprise the damping fluid of pH7.0, preferably from the 0.01MPBS damping fluid of pH7.4 or the 10mMTris-HCl damping fluid of pH7.4.In described acridinium ester label antibody diluent, the stabilizing agents such as appropriate BSA, glutin or casein can be added as required, also appropriate antiseptic can be added as required, as Sodium azide, thimerosal, Proclin-150, Proclin-200, Proclin-300 or Proclin-5000 etc.
In the solid phase that Avidin class material is fixing, Avidin class material be selected from Avidin (avidin), Streptavidin (streptavidin), neutral Avidin (neutravidin) or other can in conjunction with the Avidin class material of biotin, preferably, described Avidin class material is Streptavidin, and solid phase is selected from microwell plate, plastic cement particulate, magnetic bead, elastoplast pipe, plastic bead etc.Microwell plate can be selected from 16 common hole ELISA Plate, 48 hole ELISA Plate and 96 hole ELISA Plate etc., and plastic bead can be selected from the PS02N/PS03N/PS04N/PS05N/PS06N/PS07N/PS08N model polystyrene microsphere etc. of Niu Bang bio tech ltd, Shenzhen.Magnetic bead be using paramagnets such as tri-iron tetroxides as kernel, surface is through amino, carboxyl, hydroxyl, the magnetic particle of sulfydryl isoreactivity base group modification or magnetic microsphere.Bead diameter is generally 0.01 ~ 100 μm, is preferably 0.1 ~ 10 μm.
The fixing means of Avidin class material and solid phase can be divided into physisorption and chemical crosslinking two kinds of modes, wherein physical adsorption process is fairly simple, Avidin class material and solid phase is allowed to hatch at 4 ~ 37 DEG C, Avidin class material just can be allowed to be coated in solid phase, Chemical Crosslinking Methods then relates to use cross-linking reagent, and the cross-linking reagent used can be selected from SHPP, SPDP, GMBS, AMSA, DCC, SMCC, Sulfo-SMCC, glutaraldehyde, sodium metaperiodate and other conventional cross-linking reagents etc.In addition, the solid phase that affine class material is fixed also can be bought from existing supplier, as bought from Streptavidin MagneSpheres such as DynabeadsM-270, M-280, C1 and T1 of Thermo Fischer Scient Inc., wherein, the biotin binding capacity of 1mgDynabeadsM-270 is >=950pmol, can in conjunction with about 10 μ g biotin labelled antibodies, and the biotin binding capacity of 1mgDynabeadsT1 is 1100 ~ 1700pmol, can in conjunction with about 20 μ g biotin labelled antibodies.
When detecting the ferritin levels in the sample such as serum or blood plasma, ferritin calibration object production standard curve first should be utilized.For this reason, need to utilize calibration object dilution that the high concentration ferritin calibration object prepared in advance is carried out a series of dilution, thus obtain the ferritin calibration object of variable concentrations, or directly utilize calibration object dilution and ferritin directly to prepare the ferritin calibration object of variable concentrations, in addition using the ferritin calibration object of the calibration object dilution of not ferritin as zero-dose.When detecting CA19-9, then need to utilize CA19-9 and calibration object diluent preparing CA19-9 calibration object.Described calibration object dilution is optional from calf serum, NBCS or hyclone, also can be selected from ddH
2o, PBS damping fluid, physiological saline, HEPES damping fluid, PIPES damping fluid, MOPS damping fluid, Tricine damping fluid, triethanolamine-hydrochloride buffer, Tris-HCl damping fluid or barbital sodium-hydrochloride buffer etc.In described calibration object dilution, the stabilizing agents such as appropriate BSA, glutin or casein can be added as required, also appropriate antiseptic can be added as required, as Sodium azide, thimerosal, Proclin-150, Proclin-200, Proclin-300 or Proclin-5000 etc.
Inspire liquid and contain superoxide, comprise and inspire liquid A and inspire liquid B.Inspire liquid A and can be selected from 0.01 ~ 0.5M salpeter solution, sulfuric acid solution, hydrochloric acid solution or citrate-phosphate salt buffer; Inspire liquid B and can be selected from 0.05 ~ 1.5M sodium hydroxide solution; Superoxide is included in and inspires liquid A or inspire in liquid B, and its mass concentration is 0.1% ~ 3%, and common superoxide has hydrogen peroxide, urea peroxide and perborate etc.In addition, in order to enhanced chemiluminescence efficiency, also can add reinforcing agent toward inspiring liquid A or inspiring in liquid B, the reinforcing agent added is inspiring liquid A or the volumetric concentration inspired in liquid B is 0.1 ~ 2%, described reinforcing agent mostly is surfactant, can be selected from other reinforcing agents disclosed in SDS, Tween-20, Tween-21, Tween-40, Tween-60, Tween-61, Tween-80, Tween-81, Tween-85, TritonX-100, Brij, CTAC or US Patent No. 4927769A.
When reality detects sample, no matter sample analyzes thing calibration object or the sample such as clinical serum or blood plasma, after the present invention finds the solid-phase incubation first allowing described sample and Biotin-Ab and Avidin class material fixing, add AE-Ab again to carry out hatching (method 1), still after first allowing described sample and Biotin-Ab and AE-Ab hatch, the solid phase adding Avidin class material more fixing carries out hatching (method 2, i.e. contrast method) there is marked difference, instead of two kinds can the method for phase trans-substitution: compared to method 2 (contrast method), the luminous quantity of higher concentration calibration object can make a distinction by method 1 significantly or better, and chemiluminescence background value is significantly on the low side.What is more important, when detecting analyte immunoassay luminous quantity in clinical sample, compared to method 2 (contrast method), the measurement accuracy of method 1 significantly improves.By system of selection 1, clinical sample ferritin levels linear detection range of the present invention is 0 ~ 2000ng/ml, and clinical sample CA19-9 concentration linear detection range of the present invention is 5 ~ 1200U/ml.For method 2 (contrast method) and method 1, follow-up step is all the same, namely after twice hatches, adds cleaning fluid and removes unconjugated Biotin-Ab and unconjugated AE-Ab, and then adds and inspire liquid, detects luminous quantity.Common cleaning fluid can be selected from ddH
2o, PBS damping fluid, physiological saline, HEPES damping fluid, PIPES damping fluid, MOPS damping fluid, Tricine damping fluid, triethanolamine-hydrochloride buffer, Tris-HCl damping fluid or barbital sodium-hydrochloride buffer etc.In described cleaning fluid, can add Tween-20, Tween-21, Tween-40, Tween-60, Tween-61, Tween-80, Tween-81, Tween-85 or TritonX-100 as required makes its volumetric concentration be 0.01 ~ 1.0%, also appropriate antiseptic can be added as required, as Sodium azide, thimerosal, Proclin-150, Proclin-200, Proclin-300 or Proclin-5000 etc.The PBS damping fluid of described cleaning fluid preferably certainly containing 0.05 ~ 0.1%Tween-20, i.e. PBST damping fluid, or the Tris-HCl damping fluid containing 0.05 ~ 0.1%Tween-20, i.e. TBST damping fluid.
In mensuration sample provided by the present invention, in the kit of analyte concentration and the mensuration sample that provides, the reagent of analyte concentration is all applicable to supporting the use with immunity analysis instrument.Described immunity analysis instrument can be automanual.For semi-automatic immunity analysis instrument, except utilizing semi-automatic immunity analysis instrument to detect except luminous quantity, other steps can be all artificial, also can be partial automations, as Chinese patent CN202854137U and CN201440139U etc.Described immunity analysis instrument also can be full automatic.Utilizing automatic lmunoassays analyzer (automatedimmunoassayanalyzer, AIA) Aulomatizeted Detect is carried out, the manual operation error produced when can reduce craft or semi-hand operation, also conveniently utilizes automatic lmunoassays analyzer high flux to detect the analyte concentration of multiple different sample simultaneously
When measuring the analyte immunoassay luminous quantity in sample, if the solid phase in the solid phase that Avidin class material is fixed is magnetic bead, the concrete structure example of so matching used automatic lmunoassays analyzer can see US Patent No. 5637275A, US5358691A, US5863506A, US5849247A; Chinese patent CN103443629A, CN103399161B, CN103217541A, CN102147406B, CN202075285U, CN104345158A and CN201765226U etc.Automatic lmunoassays analyzer mainly comprises sample disc, reagent disc, reaction tray, reaction cup, sample/agent transfer unit, cleaning unit and detecting unit etc.Preferably, reaction cup is placed in reaction tray.Described sample/agent transfer unit can be single buanch unit, but can by the agent transfer of placing in the sample placed in sample disc and reagent disc in reaction cup, also can be made up of sample buanch unit and agent transfer unit, wherein sample buanch unit is used for the sample placed in sample disc to transfer in reaction cup, agent transfer unit be used for will the agent transfer of placing in reagent disc in reaction cup, in addition agent transfer unit can be single agent transfer unit, also can be multiple agent transfer unit, if multiple agent transfer unit, then each agent transfer unit can shift the reagent placed in one or more reagent discs in reaction cup.Analyze thing calibration object can be placed on as required in sample disc or reagent disc.Described reagent comprises the fixing solid phase of Biotin-Ab, AE-Ab, Avidin class material, inspires liquid and cleaning fluid etc., and wherein cleaning fluid can be placed in reagent disc, also can be placed on separately in soda liquor container; Mention above, inspire liquid to comprise and inspire liquid A and inspire liquid B, inspire liquid A and inspire liquid B and can be placed in reagent disc simultaneously, also can be placed on separately and respective inspire in liquid container, in addition, inspire liquid A or B also can be placed in kit dish and inspire liquid B or A and be placed on and inspire in liquid container.When analysis thing calibration object or the sample such as clinical serum or blood plasma are transferred to after in reaction cup by sample/agent transfer unit, the solid phase that Biotin-Ab and Avidin class material fix first is transferred in reaction cup and is hatched by recycling sample/agent transfer unit, and then utilizes sample/agent transfer unit to be joined in reaction cup by AE-Ab and hatch.After hatching end, under the magnetic fields of magnet, cleaning unit is utilized to remove unconjugated Biotin-Ab and AE-Ab in reaction cup, then add in reaction cup and inspire liquid A, then reaction cup is placed in detecting unit, to be added inspire liquid B after, detect its luminous quantity, or after reaction cup is placed on detecting unit, successively adds and inspire liquid A and inspire liquid B, detect its luminous quantity.
When measuring the analyte immunoassay luminous quantity in sample, if the solid phase in the solid phase that Avidin class material is fixed is microwell plate, the concrete structure example of so matching used automatic lmunoassays analyzer can see Chinese patent CN201945557U, CN104142407A and CN103267867A etc.
Following examples further illustrate the present invention.These embodiments are not used to limit the scope of the invention, and are to provide a further understanding of the present invention.
Embodiment 1: biotin labelled antibodies method
The present embodiment is described for biotin derivative NHS-LC-Biotin (buying from PierceChemicalCo.) and mouse Anti-ferritin monoclonal antibody (ferritin-mAb), and its labeling method is as follows:
1. get 1mgferritin-mAb, add appropriate 0.01MPBS damping fluid (pH7.2 ~ 7.4), obtain the ferritin-mAb solution that final concentration is 1mg/mL, and add in bag filter;
2. in bag filter, add the 0.01MPBS damping fluid (pH7.2 ~ 7.4) being not less than 500ml dialyse, each dialysis time is not less than 4h, repeats to change dislysate 3 ~ 4 times, is then transferred in Eppendorf pipe by the Ab solution after dialysis;
3. take 1mgNHS-LC-Biotin, add suitable quantity of water, obtain the NHS-LC-Biotin solution that final concentration is 2mg/mL;
4. mark by the molar ratio of 5 ~ 50 times of NHS-LC-Biotin and ferritin-mAb, the NHS-LC-Biotin solution getting proper volume joins in step 2 in the Eppendorf pipe that ferritin-mAb is housed and mixes, and be placed on rotatable reactor, room temperature reaction 30min, forming reactions mixed liquor;
5. the reaction mixture in step 4 is transferred in bag filter, and bag filter is inserted in the 0.01MPBS damping fluid (pH7.2 ~ 7.4) being not less than 500ml and dialyse, repeat to change dislysate 3 ~ 4 times, each dialysis time is for being not less than 4h, then collection of biological element mark ferritin-mAb (Bio-ferritin-mAb) stoste, its volume is V1;
6. in the stoste of collecting, add the 0.01MPBS damping fluid (pH7.2 ~ 7.4) containing 5%BSA of (0.1 × V1) volume, obtain Bio-ferritin-mAb mother liquor, preserve at 2 ~ 8 DEG C;
Before use, utilize biotin labelled antibodies dilution (select the 0.01MPBS damping fluid of pH7.4 be described for example) to dilute the Bio-ferritin-mAb mother liquor obtained in step 6, obtain Bio-ferritin-mAb working fluid.
Wherein, the NHS-LC-Biotin solution in step 3 is preferably now with the current; In steps of 5, the concentration of collected Bio-ferritin-mAb stoste can also be measured: the OD first detecting this solution
280value, calculates its concentration.
Embodiment 2: acridinium ester or acridine sulfonamide labelled antibody method
The present embodiment illustrates acridinium ester or acridine sulfonamide labeling method for acridinium ester and mouse Anti-ferritin monoclonal antibody (ferritin-mAb), as follows:
1. get 1mgferritin-mAb, add appropriate 0.1MNaHCO
3solution (pH8.0 ~ 9.6), obtains the ferritin-mAb solution that final concentration is 1mg/mL, and adds in bag filter;
2. in bag filter, add the 0.1MNaHCO being not less than 500ml
3solution (pH8.0 ~ 9.6) is dialysed, and each dialysis time is not less than 4h, repeats to change dislysate 3 ~ 4 times, is then transferred in Eppendorf pipe by the ferritin-mAb solution after dialysis;
3. taking 1mg acridinium ester is dissolved in appropriate dimethyl formamide (DMF) reagent, obtains the acridinium ester solution that final concentration is 1mg/mL;
4. mark by the molar ratio of 5 ~ 50 times of acridinium esters and ferritin-mAb, the acridinium ester solution getting proper volume joins in step 2 in the Eppendorf pipe that ferritin-mAb is housed and slightly mixes, and be placed on rotatable reactor, room temperature reaction 30min, forming reactions mixed liquor;
5. the reaction mixture in step 4 is transferred in bag filter, and bag filter is inserted in the 0.01MPBS damping fluid (pH6.0 ~ 6.5) being not less than 500ml and dialyse, repeat to change dislysate 3 ~ 4 times, each dialysis time is for being not less than 4h, then collect acridinium ester label ferritin-mAb (AE-ferritin-mAb) stoste, its volume is V2;
6. in the stoste of collecting, add the 0.01MPBS damping fluid (pH6.0 ~ 6.5) containing 5%BSA of (0.1 × V2) volume, obtain AE-ferritin-mAb mother liquor, preserve at 2 ~ 8 DEG C;
Before use, utilize acridinium ester label antibody diluent (select the 0.01MPBS damping fluid of pH7.4 be described for example) to dilute the AE-ferritin-mAb mother liquor obtained in step 6, obtain AE-ferritin-mAb working fluid.
Wherein, the acridinium ester solution in step 3 is preferably now with the current; In step 5, also can measure the concentration of collected AE-ferritin-mAb stoste: the OD first detecting this solution
280value, calculates its concentration.
Embodiment 3: the luminous quantity detecting sample
In the solid phase that Avidin class material is fixing, solid phase can be selected from microwell plate, plastic cement particulate, magnetic bead, elastoplast pipe or plastic bead.The present embodiment is described for the magnetic bead that Streptavidin is fixing.When the magnetic bead using Streptavidin fixing, now relevant immune response and chemiluminescence reaction can be carried out in the reaction vessels such as cuvette, reaction cup, test tube, ELISA Plate hole or cuvette, and the present embodiment is described for reaction cup.
In the present embodiment, sample can be ferritin calibration object, also can be the sample such as clinical serum or blood plasma.
For convenience of explanation, the liquid A that inspires selected in the present embodiment is the 0.1N salpeter solution containing mass concentration 0.25% hydrogen peroxide, and triggering liquid B is the 0.25N sodium hydroxide solution containing volumetric concentration 0.5%TritonX-100; Selected cleaning fluid is 0.01MPBST damping fluid.
The step of method 1 is as follows:
1. according to the explanation in embodiment 1, biotin labelled antibodies dilution is utilized to dilute Bio-ferritin-mAb mother liquor, obtain the Bio-ferritin-mAb working fluid that final concentration is 0.1 ~ 5.0 μ g/ml, utilize acridinium ester label antibody diluent to dilute AE-ferritin-mAb mother liquor simultaneously, obtain the AE-ferritin-mAb working fluid that final concentration is 0.1 ~ 5.0 μ g/ml;
2. in reaction cup, add the Biotin-ferritin-mAb working fluid and 50 μ l1.0mg/mLDynabeadsM-270 Streptavidin MagneSphere suspending liquid (0.01MPBS damping fluids that obtain in 10 μ l samples, 50 μ l steps 1, pH7.4, containing 0.1%BSA and 0.02% Sodium azide), at 37 DEG C, hatch 20min, obtain reaction mixture;
3. add the AE-ferritin-mAb working fluid obtained in 50 μ l steps 1 in the reaction mixture in step 2, at 37 DEG C, hatch 10min, subsequently under the influence of a magnetic field, magnetic bead is adsorbed onto on the inwall of reaction cup, the solution in removing reaction cup;
4. in reaction cup, add the resuspended magnetic bead of cleaning fluid, and then under the influence of a magnetic field, the solution in removing reaction cup, utilizes cleaning fluid repeated washing magnetic bead 3 times, thus removes unconjugated Biotin-ferritin-mAb and unconjugated AE-ferritin-mAb;
5. successively add 250 μ l and inspire liquid A and 250 μ l inspire liquid B, then detect luminous quantity (RLUs).
What the difference of the step of method 2 (contrast method) and the step of method 1 was to add in step 2 is sample, Biotin-ferritin-mAb working fluid and AE-ferritin-mAb working fluid instead of sample, Biotin-ferritin-mAb working fluid and DynabeadsM-270 Streptavidin MagneSphere suspending liquid, what step 3 added is DynabeadsM-270 Streptavidin MagneSphere suspending liquid instead of AE-ferritin-mAb working fluid, and other conditions remain unchanged.
Embodiment 4: utilize ferritin calibration object production standard curve
Sample in the present embodiment is ferritin calibration object.For this reason, first utilize calf serum and ferritin to make the ferritin calibration object of variable concentrations, its concentration is respectively 0ng/ml, 10ng/ml, 25ng/ml, 100ng/ml, 500ng/ml, 1000ng/ml and 2000ng/ml.
Bio-ferritin-mAb working fluid concentration in embodiment 3 step 2 is chosen as 0.8 μ g/ml, AE-ferritin-mAb working fluid concentration in embodiment 3 step 2 is chosen as 5.0 μ g/ml, then in reaction cup, the luminous quantity (RLUs) of the ferritin calibration object of variable concentrations is detected according to the method 2 (contrast method) in embodiment 3 and method 1, wherein the ferritin calibration object Parallel testing twice of each concentration, asks its average luminescence amount
testing result respectively as shown in Table 1 and Table 2, then utilizes
with the funtcional relationship of ferritin calibration object concentration, production standard curve, respectively as depicted in figs. 1 and 2.
Convenient in order to describe, represent the luminous quantity of 1000ng/ml ferritin calibration object solution with A1, A2 represents the luminous quantity of 2000ng/ml ferritin calibration object solution, and Bio represents Bio-ferritin-mAb working fluid, and AE represents AE-ferritin-mAb working fluid.
The testing result of the variable concentrations ferritin calibration object in table 1 method 1
The testing result of the variable concentrations ferritin calibration object in table 2 method 2
Known by the data in comparison sheet 1 and table 2, in method 1, Parallel testing 1 (RLUs), Parallel testing 2 (RLUs) and average luminescence amount
a2/A1 ratio be respectively 1.704,1.615 and 1.658, and the relative A1 of A2 adds 70.4%, 61.5% and 65.8% respectively; And in method 2 (contrast method), Parallel testing 1 (RLUs), Parallel testing 2 (RLUs) and
a2/A1 ratio be respectively 1.352,1.243 and 1.298, and the relative A1 of A2 only increases 35.2%, 24.3% and 29.8% respectively.In view of 2 times that 2000ng/ml ferritin calibration object concentration is 1000ng/ml ferritin calibration object concentration, and the luminous quantity of ferritin calibration object and its concentration proportional, therefore in theory, the luminous quantity of 2000ng/ml ferritin calibration object should be 2 times of 1000ng/ml ferritin calibration object, but be in fact limited to the restriction of the factors such as detecting instrument sensitivity, always there are some deviations, but this ratio should close to 2.Based on this, can the A2/A1 ratio of perception method 1 all close to 2, significantly the luminous quantity of 1000ng/ml and 2000ng/ml ferritin calibration object can be made a distinction, and the A2/A1 ratio of method 2 (contrast method) is all much smaller than 2, can not effectively the luminous quantity of 1000ng/ml and 2000ng/ml ferritin calibration object be made a distinction.
Meanwhile, also known according to the data of table 1 and table 2, chemiluminescence background value (i.e. the luminous quantity of 0ng/ml ferritin calibration object) in chemiluminescence background value (i.e. the luminous quantity of 0ng/ml ferritin calibration object) ratio method 1 in method 2 (contrast method) significantly increases, with regard to average luminescence amount
, increasing degree is up to 60.98%.
In addition, when the luminous value of the ferritin calibration object of twice Parallel testing variable concentrations, the %CV of method 1 is all far smaller than 10%, therefore detects precision good.And the %CV of method 2 (contrast method) is more higher comparatively speaking, wherein the %CV of 100.00ng/ml ferritin calibration object is even also greater than 10%, and therefore, the detection precision of method 2 (contrast method) is poor.
Embodiment 5:Bio-ferritin-mAb and the combination of AE-ferritin-mAb variable concentrations
In the present embodiment, A1, A2, Bio are identical with embodiment 4 with the implication of AE.
In example 4, the luminous quantity that the ferritin calibration object of high concentration produces can significantly distinguish by discover method 1 of the present invention.On this basis, by changing the concentration of Bio and AE, measure A1 and A2 according to method 1, its result is as shown in table 3 below.
The average luminescence amount of table 3 variable concentrations combination
In known table 3, the luminous quantity of 1000ng/ml and 2000ng/ml ferritin calibration object all can make a distinction by Bio and the AE combination of variable concentrations effectively.
Embodiment 6: clinical sample ferritin levels detects
Sample in the present embodiment is the sample such as clinical serum or blood plasma.
Bio-ferritin-mAb working fluid concentration in embodiment 3 step 2 is chosen as 0.8 μ g/ml, AE-ferritin-mAb working fluid concentration in embodiment 3 step 2 is chosen as 5.0 μ g/ml, in reaction cup, then detect the ferritin levels of 50 routine different clinical serum samples according to the method 2 (contrast method) in embodiment 3 and method 1.In addition, in view of the ferritin Electrogenerated chemiluminescent immunoassay detection accuracy of Roche (Roche) is higher, therefore the ferritin Electrogenerated chemiluminescent immunoassay kit of Roche and Roche Electrogenerated chemiluminescent immunoassay instrument E170 matching used with it is also utilized to detect this 50 routine clinical sample, and in order to the measurement accuracy of appraisal procedure 2 (contrast method) and method 1, its result is as shown in table 4:
Table 4 clinical sample ferritin levels testing result
In his-and-hers watches 4, the measured value of method 1 and the measured value of Roche carry out clinical accordance analysis, and its result as shown in Figure 3.
In his-and-hers watches 4, the measured value of method 2 (contrast method) and the measured value of Roche carry out clinical accordance analysis, and its result as shown in Figure 4.
From the data in table 4, when measuring the clinical serum sample of Low Concentration Iron albumen (sample 1 ~ 6), the measured value of method 1 and the measured value ratio of Roche float 0.82 ~ 0.95, with the measured value deviation of Roche-18% ~-5%, reach the normal range specified in the industry, and the measured value of method 2 (contrast method) and the measured value ratio of Roche float 0.58 ~ 0.77, with the measured value deviation-42% ~-23% of Roche, can the measured value of perception method 2 (contrast method) significantly on the low side, do not reach the normal range specified in the industry, to in measuring during clinical serum sample (sample 16 ~ 37) of concentration ferritin, the measured value of method 1 and the measured value ratio of Roche float 0.92 ~ 1.16, with the measured value deviation of Roche-8% ~+16%, reach the normal range specified in the industry, and the measured value of method 2 (contrast method) and the measured value ratio of Roche float 0.93 ~ 1.50, except the measured value of only a few sample is in normal range, the measured value of most sample and the measured value deviation of Roche at least+21%, be up to+50%, can the measured value of perception method 2 (contrast method) significantly higher, do not meet the normal range specified in the industry, when measuring the clinical serum sample of high concentration ferritin (sample 47 ~ 50), the measured value of method 1 and the measured value ratio of Roche float 0.88 ~ 0.98, with the measured value deviation of Roche-12% ~-2%, reach the normal range specified in the industry, and the measured value of method 2 (contrast method) and the measured value ratio of Roche float 0.76 ~ 0.89, with the measured value deviation-11% ~-24% of Roche, except the known measured value except sample 48 and sample 50 can accept reluctantly, the measured value of method 2 (contrast method) to sample 47 and sample 49 is significantly on the low side, do not reach the normal range specified in the industry.
Again according in Fig. 3 and Fig. 4, the slope of Fig. 3 is 0.995, closely 1.0, R values are greater than 0.99, because of the measured value of the method 1 and the measured value anastomose property of Roche better, accuracy is better, and the slope of Fig. 4 is 0.919, be significantly less than 1.0, and R value is less than 0.99, because of the measured value of the method 2 (contrast method) and the measured value anastomose property of Roche poor, measuring error is significantly bigger than normal.
In sum, known based on the result in embodiment 4, embodiment 5 and embodiment 6, if system of selection 2 (contrast method) detects the ferritin calibration object of variable concentrations, difference amplitude so between the luminous value that produces of the ferritin calibration object of higher concentration is too small, and they can not be made a distinction effectively; And, if system of selection 2 (contrast method) detects clinical serum or plasma sample, so it can not detect the clinical sample of low concentration and high concentration ferritin exactly, and during the clinical sample of concentration ferritin in the detection, the testing result of method 2 (contrast method) is significantly higher.This just means that the detection accuracy of method 2 (contrast method) is poor.Otherwise, if system of selection 1 detects the ferritin calibration object of variable concentrations, then the luminous value that the ferritin calibration object of higher concentration produces can be distinguished effectively; And during ferritin levels in Application way 1 clinical serum or plasma sample, the clinical sample of Low Concentration Iron albumen and the clinical sample of middle concentration ferritin can not only be detected exactly, and the clinical sample of high concentration ferritin can be detected exactly, this just shows that the detection accuracy of method 1 significantly improves compared to method 2 (contrast method), and the linear detection range of method 1 significantly increases.
Moreover when employing method 1 detects the ferritin calibration object of variable concentrations, its chemiluminescence background value can significantly reduce, thus greatly can improve detection accuracy and signal to noise ratio (S/N ratio), therefore helps the detection accuracy improving clinical sample further.On the contrary, the chemiluminescence background value of method 2 (contrast method) is significantly higher, can reduce the detection accuracy of clinical sample further.
Embodiment 7: clinical sample CA19-9 Concentration Testing
The present embodiment selects sugar antigen CA19-9 to be described.Prepare biotin labeling little mouse-anti CA19-9 monoclonal antibody (Bio-CA199-mAb) according to the method in embodiment 1, prepare acridinium ester label little mouse-anti CA19-9 monoclonal antibody (AE-CA199-mAb) according to the method in embodiment 2.
The 0.01MPBS damping fluid of pH7.4 and CA19-9 compound concentration is utilized to be respectively the CA19-9 calibration object of 5U/ml, 50U/ml, 200U/ml, 600U/ml and 1200U/ml, Bio-CA199-mAb working fluid concentration is chosen as 0.65 μ g/ml, AE-CA199-mAb working fluid concentration is chosen as 3.0 μ g/ml, then in reaction cup, the luminous quantity (RLUs) of the CA19-9 calibration object of variable concentrations is detected according to the method 2 (contrast method) in embodiment 3 and method 1, wherein the CA19-9 calibration object Parallel testing twice of each concentration, asks its average luminescence amount
testing result is as shown in table 5.The average luminescence amount of note 600U/mlCA19-9 calibration object is A11, and the average luminescence amount of note 1200U/mlCA19-9 calibration object is A12.
Table 5
As shown in Table 5, the A12/A11 ratio of method 1 is 1.722, be greater than 1.600 of method 2 (contrast method), this means that, relative to method 2 (contrast method), the luminous value of high concentration CA19-9 calibration object can make a distinction by method 1 better.
Bio-CA199-mAb working fluid concentration is chosen as 0.65 μ g/ml, AE-CA199-mAb working fluid concentration is chosen as 3.0 μ g/ml, then detects the CA19-9 concentration in 7 routine clinical serum samples according to the method 2 (contrast method) in embodiment 3 and method 1.In addition, in view of the CA19-9 Electrogenerated chemiluminescent immunoassay detection accuracy of Roche (Roche) is higher, therefore the CA19-9 Electrogenerated chemiluminescent immunoassay kit of Roche and Roche Electrogenerated chemiluminescent immunoassay instrument E170 matching used with it is also utilized to detect this 7 routine clinical sample, and in order to the measurement accuracy of appraisal procedure 2 (contrast method) and method 1, its result is as shown in table 6, and wherein the clinical sample CA19-9 concentration linearity test upper limit of Roche is 1000U/ml.
Table 6 clinical sample CA19-9 Concentration Testing result
From table 6 data, method 2 (contrast method) and method 1 in the detection low concentration CA19-9 blood serum sample (sample 1 ~ 2) phase difference are little, but when detecting high concentration CA19-9 blood serum sample (sample 3 ~ 7), the testing result of method 1 is more accurate, and the testing result of method 2 (contrast method) is significantly on the low side.It can thus be appreciated that the accuracy that Application way 1 detects CA19-9 concentration in high concentration CA19-9 blood serum sample is better, and can significantly improve its linear detection range.
Claims (10)
1. improve a method for immunity analysis instrument detection accuracy, its step comprises: (1) provides immunity analysis instrument; (2) by sample and biotin labelled antibodies and the fixing solid-phase incubation of Avidin class material, forming reactions mixed liquor; (3) in described reaction mixture, add acridinium ester or acridine sulfonamide labelled antibody, then hatch; (4) unconjugated biotin labelled antibodies and unconjugated acridinium ester or acridine sulfonamide labelled antibody is removed; (5), after adding and inspiring liquid, chemiluminescence signal produces, and utilizes described immunity analysis instrument to detect the luminous quantity of described chemiluminescence signal.
2. the method for claim 1, the antibody in the antibody in described biotin labelled antibodies and described acridinium ester or acridine sulfonamide labelled antibody is selected from ferritin antibody or anti-CA19-9 antibody simultaneously.
3. the method for claim 1, described sample is selected from calibration object, serum or blood plasma.
4. the method as described in one of claim 1-3, the concentration of described biotin labelled antibodies is 0.1 ~ 5.0 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 0.1 ~ 5.0 μ g/ml.
5. one kind for detecting the chemiluminescence immune assay reagent of analyte concentration in sample, described reagent comprises biotin labelled antibodies, Avidin class material fixing solid phase, acridinium ester or acridine sulfonamide labelled antibody, cleaning fluid and inspires liquid, it is characterized in that, the solid phase that described biotin labelled antibodies and described Avidin class material are fixed joins in described sample prior to described acridinium ester or acridine sulfonamide labelled antibody.
6. reagent as claimed in claim 5, the antibody in the antibody in described biotin labelled antibodies and described acridinium ester or acridine sulfonamide labelled antibody is selected from ferritin antibody or anti-CA19-9 antibody simultaneously.
7. reagent as claimed in claim 6, described sample is selected from calibration object, serum or blood plasma.
8. the reagent as described in one of claim 5-7, the concentration of described biotin labelled antibodies is 0.1 ~ 5.0 μ g/ml, and the concentration of described acridinium ester or acridine sulfonamide labelled antibody is 0.1 ~ 5.0 μ g/ml.
9. improve the method that analyte chemical electrochemiluminescent immunoassay in sample detects the range of linearity, its step comprises: (1) adds the solid phase that biotin labelled antibodies and Avidin class material are fixed toward described sample in, hatches, forming reactions mixed liquor; (2) in described reaction mixture, add acridinium ester or acridine sulfonamide labelled antibody, then hatch; (3) add cleaning fluid and remove unconjugated biotin labelled antibodies and unconjugated acridinium ester or acridine sulfonamide labelled antibody; (4), after adding and inspiring liquid, chemiluminescence signal produces, and measures its luminous quantity.
10. method as claimed in claim 9, the antibody in the antibody in described biotin labelled antibodies and described acridinium ester or acridine sulfonamide labelled antibody is selected from ferritin antibody or anti-CA19-9 antibody simultaneously.
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