CN101324579A - Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method - Google Patents
Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method Download PDFInfo
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Abstract
The invention relates to a magnetic corpuscule chemiluminescent enzyme immunoassay kit for detecting carbohydrate antigen and the application method thereof. The kit comprises FITC antibody-coated magnetic corpuscules; a marker solution prepared by mixing the FITC-marked carbohydrate antigen monoclonal antibody and the enzyme-marked carbohydrate antigen monoclonal antibody; a carbohydrate antigen standard sample solution; a concentrated washing solution; and a luminescent substrate solution, wherein carbohydrate antigen optionally adopts one of CA72-4, CA50, CA19-9, CA242, CA15-3, CA27-29 and CA125. The enzyme-marked antibody and the FITC-marked antibody are the monoclonal antibodies corresponding to the antigens. The FITC antibody coating the magnetic corpuscules adopts a polyclonal antibody or a monoclonal antibody. The marker solution is prepared by mixing an FITC-marked capture antibody working solution and an enzyme-marked antibody pair working solution by the volume ratio of 1:(1-3). Compared with the known kit for mensurating the carbohydrate antigen, the kit has the advantages of high flux, high sensitivity, wide linear range, rapidness, etc., and has a wide application prospect for the clinical inspection, etc.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of magnetic microparticle chemiluminescence enzyme immune analytic reagent kit and using method thereof that detects sugar antigen.
Background technology
Cancer has constituted serious threat to human existence health.The key of oncotherapy at present is early detection, early treatment, and this has been people's common recognition.But the hospital diagnosis infantile tumour mainly is by iconography and cell pathology diagnostic techniques at present, just made a definite diagnosis after having tangible occupying lesion and clinical symptoms, but this has not been the very early time of canceration.Along with early diagnosis of tumor enters the protein epoch, be that the body fluid protein of representative becomes the focus in the tumor-marker research field with blood.The discovery of more and more tumor markerses is had higher requirement to setting up development novel detection technique quick, highly sensitive, high special tumor markers.
Sugar antigen is the wide class tumour capacity marking thing of the fast application of development, and it comprises glycoprotein and glycolipid, often shows as the mucin form.Its antigenic determinant can be positioned on the sugar chain or on pyrenoids.According to the difference of glycosyl epi-position and the difference of epitope size, generally can be divided into sugar antigen 72-4 (carbonhydrate antigen 72-4, CA72-4), sugar antigen 50 (carbonhydrate antigen 50, CA50), sugar antigen 19-9 (carbonhydrate antigen 19-9, CA19-9), carbohydrate antigen 242 (carbonhydrate antigen 242, CA242), sugar antigen 15-3 (carbonhydrate antigen 15-3, CA15-3), sugar antigen 27-29 (carbonhydrate antigen 27-29, CA27-29) and sugar antigen 125 (carbonhydrate antigen 125, CA125).At present to these marks carry out examination in early stage that single inspection or joint inspection can be disease, clinical diagnosis, curative effect monitoring, cancer metastasis and more back recurrence situation reference frame is provided.
Be used to detect the immunization method of sugar antigen at present except enzyme immunoassay (EIA) (enzymeimmunoassay is arranged, EIA), radiommunoassay (Radio immunoassay, RIA), immunofluorescence assay (fluoroimmunoassay, FIA), radio-immunity development (radioimmunoimaging, RII) and outside antibody chip (antibody microarrays) technology, chemiluminescence immune assay (Chemiluminescence immunoassay, CLIA) technology is highly sensitive because of it, high special, fast, characteristics such as high flux obtain application more and more widely.
In the immune detection of reality, because impurity component contained in the testing sample is more, having influenced detection sensitivity and accuracy to a certain extent, so separate, be purified into the purpose determinand fast from complicated background, is one of difficult problem of facing of clinical examination worker.Immune magnetic particulate technology is to utilize magnetic solid phase particle of the synthetic certain particle size size of macromolecular material to make carrier, with method bags such as physisorption, chemical coupling by on have specificity affinity various immunologic active materials (antigen or antibody), making its sensitization is the immune magnetic particle, has that velocity of separation is fast, efficient is high, favorable repeatability; Instrument and equipment simple to operate, as not need costliness; Do not influence characteristics such as the biological character of separated cell or other biomaterial and function, adding directed movement under the action of a magnetic field, make some special composition be separated, concentrate or purifying.As a kind of general solid phase isolation methods that is used for the CLIA detection technique, thereby magnetic particle can improve effective package amount conservation greatly, thereby the range of linearity that can widen detection is simultaneously effectively avoided the generation of crotch effect, will promote application and the development of CLIA technology in clinical examination/detection.
Summary of the invention
The purpose of this invention is to provide a kind of magnetic microparticle chemiluminescence enzyme immune analytic method and dedicated kit thereof that detects sugar antigen.Promptly adopt highly specific monoclonal antibody and bag by anti-fluorescein isothiocynate (fluorescein-iso-thiocyanate, FITC) the magnetic particle solid phase isolation technics of antibody, utilize FITC and enzyme mark two strain monoclonal antibodies respectively, in liquid phase, form immune sandwich complex, add bag then by the magnetic particle separating agent of anti-FITC antibody, wash separation, and in the tubular type chemiluminescent analyzer, measure.
The magnetic particle chemical luminescence immune analysis reagent box of detection sugar antigen provided by the present invention comprises the magnetic particle that is coated with anti-FITC antibody; The sugar antigen monoclonal antibody of flag F ITC and the sugar antigen monoclonal antibody of marker enzyme are mixed the thing liquid that serves as a mark; The sugar antigen standard solution; Concentrated cleaning solution; Luminous substrate liquid.
Described bag is that particle diameter is that 2-3 μ m, working concentration are that 5-10mg/mL, kernel are that tri-iron tetroxide (Fe3O4), surface parcel have reactive group as amino (NH2-), carboxyl (polymkeric substance COOH) by the magnetic particle of anti-FITC antibody; The bag of described anti-FITC antibody is by the glutaraldehyde two-step approach magnetic particle and anti-FITC antibody to be carried out coupling, and to contain 0.5% bovine serum albumin(BSA) (bovine serum albumin, BSA), 1% caseic pH is 7.2, the phosphate buffer of 0.02mol/L seals to be finished.
The conjugate of described FITC molecule and sugar antigen monoclonal antibody is to carry out mark with common alkalies labelling method antagonist.Described labelled antibody is capture antibody, comprising: anti-CA72-4 monoclonal antibody, anti-CA50 monoclonal antibody, anti-CA19-9 monoclonal antibody, anti-CA242 monoclonal antibody, anti-CA15-3 monoclonal antibody, anti-CA27-29 monoclonal antibody and anti-CA 125 monoclonal antibody.FITC label form can be freeze-dried powder, concentrate and working fluid; Described FITC labelled antibody liquid is deposited with the dilution of 20% calf serum.
The marker enzyme of described enzymic-labelled antibody is horseradish peroxidase or alkaline phosphatase; The mark of enzyme can adopt several different methods of the prior art such as glutaraldehyde method or sodium periodate method, if marker enzyme is an alkaline phosphatase, then is preferably glutaraldehyde method, if marker enzyme is a horseradish peroxidase, then is preferably the sodium periodate method.Enzyme labeling thing form can be freeze-dried powder, concentrate and working fluid; Described labelled antibody is pairing antibody, comprising: anti-CA72-4 monoclonal antibody, anti-CA50 monoclonal antibody, anti-CA19-9 monoclonal antibody, anti-CA242 monoclonal antibody, anti-CA15-3 monoclonal antibody, anti-CA27-29 monoclonal antibody and anti-CA 125 monoclonal antibody.The used dilution of described enzyme labeling thing working fluid is the thimerosal antiseptic solution that contains 0.05% glycerine, 0.2g/L (the enzyme labeling thing that can prevent to put into-20 ℃ freezes, and keeps the biologically active of enzyme labeling thing).
Described mixed mark thing liquid is that volume ratio is 1: 1-1: 3 FITC mark capturing antibody working fluid and enzyme labeling pairing antibody working fluid mix.
Described standard solution is the solution that six concentration gradients contain sugar antigen; The matrix of described preparation standard items is 50% horse serum; The sugar antigen of described formation standard items comprises: CA72-4, CA50, CA19-9, CA242, CA15-3, CA27-29 and CA125.
Described concentrated cleaning solution is that the pH value is 7.4, contains the 0.1-0.5% Tween-20, the phosphate buffer of 0.1% sodium azide antiseptic.
Described when marker enzyme is horseradish peroxidase luminous substrate liquid constitute by A liquid and B liquid, described luminous substrate liquid A liquid is superoxol, described luminous substrate liquid B liquid is luminol solution; When marker enzyme is alkaline phosphatase, described luminous substrate liquid is 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 "-hydroxyl) benzene-1; 2-dioxetane sodium phosphate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1; 2-dioxetane-3; 2 '-adamantane, AMPPD) damping fluid.
Used reaction tube material can be transparent polystyrene, tygon, polypropylene, glass etc.
The method of detection sugar antigen provided by the invention is to utilize described magnetic particle chemiluminescence Enzymoimmune reagent kit to detect sugar antigen.
The method of described detection sugar antigen also can comprise the pre-treatment of sample.
The method of detection sugar antigen provided by the present invention may further comprise the steps:
1) sample pre-treatments
To clinical serum blood plasma, centrifugal 5 minutes of 3000rpm gets upper strata liquid and can carry out assay determination.
2) utilize the magnetic microparticle chemiluminescence enzyme immune analytic reagent kit test sample of above-mentioned detection sugar antigen.
But sugar antigen content among the kit qualitative and quantitative analysis human serum sample of the present invention.
The magnetic microparticle chemiluminescence enzyme immune analytic reagent kit of detection sugar antigen of the present invention mainly adopts the magnetic particle solid phase to separate and the reaction pattern of double antibodies sandwich is qualitative or sample such as detection by quantitative human serum blood plasma in sugar antigen content; The pre-treatment of sample is required low, sample pretreatment process is simple and reliable, can be fast, the high throughput testing gross sample.
Sugar antigen is the big molecule of an albuminoid, generally adopts the method for double-antibody sandwich to measure to this quasi-molecule.The seventies in last century, scientists has obtained the monoclonal antibody of sugar antigen successively by hybridoma technology, thereby provides assurance for the immune detection pattern of setting up double-antibody sandwich.
Detection principle of the present invention is when the pairing antibody of capture antibody that the FITC mark is arranged in the reactant liquor and enzyme labeling, after adding series standard product or sample solution, by the antigenic determinant on " identification " antigen molecule, in solution, form FITC labelled antibody-antigen-enzymic-labelled antibody compound.Add the magnetic particle that is coated with anti-FITC antibody then, it is well-dispersed in the solution, the immune complex coupling can be enriched to the magnetic particle sub-surface by immune response, after applying magnetic field, the test tube bottom coupling can be had the magnetic particle of immune complex to be enriched in the test tube bottom, add luminous substrate, measure luminous intensity with the tubular type light-emitting appearance, antigenic content becomes positive correlation in the luminous intensity of sample and the sample, relatively can draw corresponding antigen content with typical curve.
The magnetic microparticle chemiluminescence enzyme immune analytic reagent kit of detection sugar antigen of the present invention mainly adopts the content of sugar antigen in the qualitative or samples such as detection by quantitative human serum, blood plasma of direct sandwich method; Pre-treatment requirement to sample is low, and the pre-treatment process is simple, and energy is quick, the high throughput testing gross sample; High special sugar antigen monoclonal antibody and super paramagnetic, high dispersive, magnetic particle that specific surface area is big have been adopted, main agents provides with the form of working fluid, the method of inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit of the present invention, simple in structure, easy to use, low price, carrying convenience, compare with the enzyme linked immunological kit on the market, board-like chemical luminescence reagent kit, the range of linearity is wide, effectively avoid the crotch effect, do not need diluted sample, be applicable to the qualitative, quantitative of batch samples screening.
Description of drawings
Fig. 1 is the typical curve (double logarithmic curve) that detects CA50 with magnetic microparticle chemiluminescence enzyme immune analytic reagent kit
Fig. 2 is the typical curve (double logarithmic curve) that detects CA125 with magnetic microparticle chemiluminescence enzyme immune analytic reagent kit
Embodiment
The method of following embodiment is conventional method if no special instructions.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The preparation and the detection method thereof of the magnetic microparticle chemiluminescence enzyme immune analytic reagent kit of embodiment 1, detection CA50.
The magnetic microparticle chemiluminescence enzyme immune analytic reagent kit that detects CA50 comprises:
(1) be coated with the magnetic particle of anti-FITC antibody: particle diameter is that 2-3 μ m, working concentration are 5-10mg/mL, 20mL/ bottle, 1 bottle;
(2) the CA50 monoclonal antibody (pairing antibody) of the CA50 monoclonal antibody (capture antibody) of flag F ITC and marker enzyme is mixed the thing liquid that serves as a mark: for volume ratio is that 1: 1 FITC mark capturing antibody working fluid and enzyme labeling pairing antibody working fluid mixes, the 15mL/ bottle, 1 bottle.Wherein FITC labelled antibody liquid is 1.25 μ g/mL with the dilution of 20% calf serum, the used dilution of the enzyme working fluid of mark alkaline phosphatase is that the thimerosal antiseptic solution that contains 0.05% glycerine, 0.2g/L (the enzyme labeling thing that can prevent to put into-20 ℃ freezes, and keeps the biologically active of enzyme labeling thing) and dilution are 1/2000;
(3) CA50 standard solution: the dense standard items of CA50 are diluted to 6 bottles of standard solution, 0U/mL, 20U/mL, 40U/mL, 80U/mL, 140U/mL, 300U/mL, 0.5mL/ bottle with 50% horse serum;
(4) luminous substrate liquid: AMPPD damping fluid, 6mL/ bottle, 1 bottle;
(5) concentrated cleaning solution: the pH value is 7.4, contains the 0.1-0.5% Tween-20, the phosphate buffer of 0.1% sodium azide antiseptic.The 30mL/ bottle, 1 bottle.Use 20 times of distilled water dilutings during use;
(6) reaction tube: used reaction tube material is transparent polystyrene, tygon, polypropylene, glass etc., aluminium film Vacuum Package, 100/bag, 1 bag.
Wherein, the CA50 MONOCLONAL ANTIBODIES SPECIFIC FOR method of the CA50 monoclonal antibody of anti-FITC antibody and magnetic particle conjugate, FITC mark, alkali phosphatase enzyme mark is as follows:
One, the preparation of anti-FITC antibody and magnetic particle conjugate
1, the preparation principle of anti-FITC antibody and magnetic particle conjugate: magnetic particle is that kernel is that the polymkeric substance that has reactive group amino has been wrapped up on Fe3O4, surface, can adopt the glutaraldehyde two-step approach that magnetic particle and anti-FITC antibody are carried out coupling.The so synthetic particle that is coated with anti-FITC antibody, not only effectively package amount increases, and has " long-armed " at micro-space, thereby reduces sterically hindered, make IgG antibody fully launch the differential threshold of antigen, the space coupling that helps antibody-antigen combines with non-covalent.
2, the preparation method of anti-FITC antibody and magnetic particle conjugate: with particle diameter is that the magnetic particle of 2-3 μ m activates with glutaraldehyde, stirring at room, behind the mixing 3 hours, add magnetic field, leaving standstill 10-15min, pour out supernatant, is that 7.4 0.01mol/L phosphate buffer cleans three times with the pH value, and suspend with this solution, concentration is 50-100mg/mL; Add anti-FITC antibody 20-80 μ g in every milliliter of suspending liquid, after 4 ℃ stirring is spent the night down, add magnetic field, leave standstill 10-15min, pour out supernatant, with contain 0.5% bovine serum albumin(BSA), 1% caseic pH is 7.2, the phosphate buffer of 0.02mol/L was in room temperature sealing 3-4 hour; Be 7.4, contain the 0.02mol/LTris-HCl buffer solution for cleaning three times of 0.5%BSA with the pH value at last, and be mixed with the working fluid of 5-10mg/mL with this solution.The magnetic particle separating agent should be not frozen 4 ℃ of preservations, and the time spent shakes up gently.
Two, the CA50 MONOCLONAL ANTIBODIES SPECIFIC FOR of FITC mark
1, the CA50 MONOCLONAL ANTIBODIES SPECIFIC FOR principle of FITC mark: the conjugate of FITC molecule and sugar antigen monoclonal antibody is to carry out mark with common alkalies labelling method antagonist.
2, the CA50 MONOCLONAL ANTIBODIES SPECIFIC FOR method of FITC mark:
(1) A liquid: getting CA50 monoclonal antibody 0.5mg, to place 50mmol/L pH value be 9.3 Na2CO3-NaHCO3 damping fluid;
(2) B liquid: getting FITC2 mg, to be dissolved in 1mL 50mmol/L pH value be in 9.3 the Na2CO3-NaHCO3 damping fluid;
(3) A liquid is placed magnetic stirring apparatus, get B liquid and slowly drop in the A liquid, between 5-8 minute, add, prevent bubble, put 4 ℃ then and stirred 12～16 hours;
(4) reactant liquor is adorned bag filter, put 10mmol/L pH value and be in 7.4 the phosphate buffer dialysis 2 days, change dislysate every day 2 times;
(5) be 1.25 μ g/mL with label with the dilution of 20% calf serum, add an amount of proclin300, packing is preserved down for-20 ℃.
Three, the CA50 MONOCLONAL ANTIBODIES SPECIFIC FOR of alkali phosphatase enzyme mark
1, the CA50 MONOCLONAL ANTIBODIES SPECIFIC FOR principle of alkali phosphatase enzyme mark: can adopt several different methods of the prior art such as glutaraldehyde method or sodium periodate method that enzyme is crosslinked on antibody, preferred glutaraldehyde method;
2, the CA50 MONOCLONAL ANTIBODIES SPECIFIC FOR method of alkali phosphatase enzyme mark:
(1) gets alkaline phosphatase 20mg and be dissolved in 1.5% glutaraldehyde solution room temperature standing over night;
(2) reacted enzyme solutions is used the physiological saline wash-out through Sephadex G-2 5 chromatographic columns, flow speed control 1.2mL/min, and collect brown effluent.Be positioned in the 25mL beaker electromagnetic agitation;
(3) getting CA50 monoclonal antibody 2.5mg, is that 7.4 phosphate buffer is diluted to 5mL with 0.1mol/L pH value, stirs dropwise to add in the enzyme solutions down;
(4) add 0.2mol/L lysine 0.2mL, mixing was put room temperature 2-3 hour;
(5) the reactant liquor bag filter of packing into is that 7.4 phosphate buffer dialysis is after 24 hours, with the thimerosal antiseptic solution dilution in 1: 2000 that contains 0.05% glycerine, 0.2g/L, packing, stored frozen with 0.1mol/L pH value.
Utilize the method for CA50 content in this kit test sample as follows:
One, sample pre-treatments
To human serum blood plasma: get on an empty stomach morning blood sample this, the centrifugal 5min of 3000rpm gets upper strata liquid 25 μ L and analyzes.Must not deposit above 48 hours for testing sample 2-8 ℃,, preserve below Ying Yu-20 ℃ if do not detect in 48 hours, but should be above 30 days.
Two, detection method
In test tube, add 25 μ L blood serum samples or, add label mixed liquor 50 μ L again, 37 ℃ of constant temperature oscillating reactions 30min with the series standard product solution of volume.Add magnetic particle solution 50 μ L, behind 37 ℃ of constant temperature oscillating reactions 5min, test tube rack placed separate 5min on the magnetic separator, the separation vessel that reverses is then poured out supernatant, and the test tube of reversing is placed on the filter paper, bounces separation vessel to remove wall built-up liquid.Every pipe adds cleansing solution 500 μ L, makes its abundant mixing with circular motion, places and separates 5min on the magnetic separator, and the separation vessel that reverses is then poured out supernatant, and the test tube that reverses is placed on the filter paper, bounces separation vessel to remove wall built-up liquid, repeats 3 times.Add luminous substrate 300 μ L, circular motion makes its abundant mixing, and the dark place is placed 2min and is placed on magnetic separator, treat that the magnetic particle is enriched in the bottom after, place the tubular type light-emitting appearance to measure in test tube.
Three, interpretation of result
Luminous counting-dose-effect curve represents that with double logarithmic curve each the concentration standard solution that is promptly obtained or the mean value (B) of sample luminous value deduct first zero standard luminous value (B0) on schedule, take the logarithm, promptly again
The longitudinal axis (Y-axis)=log (B-B0)
Natural logarithm value with CA50 concentration is transverse axis (X-axis), the drawing standard curve, as shown in Figure 1.CA50 concentration can be read from typical curve in corresponding each sample.Also can carry out match, calculate the concentration of CA50 in the sample solution with the multiparameter regression equation method.Because the range of linearity of the board-like kit of most of chemiluminescences is between 0-150U/mL on the market, and the range of linearity that this kit provides increases (0-300U/mL) greatly, so avoided the dilution of enriched sample, the crotch effect also can be avoided.Use the whole testing process of this kit only to need just can finish in 1.5 hours, lowest detection is limited to 1.0U/mL.
The preparation and the detection method thereof of the magnetic microparticle chemiluminescence enzyme immune analytic reagent kit of embodiment 2, detection CA125.
The magnetic microparticle chemiluminescence enzyme immune analytic reagent kit that detects CA125 comprises:
(1) be coated with the magnetic particle of anti-FITC antibody: particle diameter is that 2-3 μ m, working concentration are 5-10mg/mL, 20mL/ bottle, 1 bottle;
(2) the CA125 monoclonal antibody (pairing antibody) of the CA125 monoclonal antibody (capture antibody) of flag F ITC and marker enzyme is mixed the thing liquid that serves as a mark: for volume ratio is that 1: 2 FITC mark capturing antibody working fluid and enzyme labeling pairing antibody working fluid mixes, the 15mL/ bottle, 1 bottle.Wherein FITC labelled antibody liquid is 1.25 μ g/mL with the dilution of 20% calf serum, the used dilution of the enzyme working fluid of mark horseradish peroxidase is that the thimerosal antiseptic solution that contains 0.05% glycerine, 0.2g/L (the enzyme labeling thing that can prevent to put into-20 ℃ freezes, and keeps the biologically active of enzyme labeling thing) and dilution are 1/1500;
(3) CA125 standard solution: the dense standard items of CA125 are diluted to 6 bottles of standard solution, 0U/mL, 50U/mL, 100U/mL, 200U/mL, 500U/mL, 1000U/mL, 0.5mL/ bottle with 50% horse serum;
(4) luminous substrate liquid: A liquid: superoxol, 7mL/ bottle, 1 bottle; B liquid: luminol solution, 7mL/ bottle, 1 bottle.Volume ratio is mixing in 1: 1 during use.
(5) concentrated cleaning solution: the pH value is 7.4, contains the 0.1-0.5% Tween-20, the phosphate buffer of 0.1% sodium azide antiseptic.The 30mL/ bottle, 1 bottle.Use 20 times of distilled water dilutings during use;
(6) reaction tube: used reaction tube material is transparent polystyrene, tygon, polypropylene, glass etc., aluminium film Vacuum Package, 100/bag, 1 bag.
Wherein, the CA125 MONOCLONAL ANTIBODIES SPECIFIC FOR method of the CA125 monoclonal antibody of anti-FITC antibody and magnetic particle conjugate, FITC mark, alkali phosphatase enzyme mark is as follows:
One, the preparation of anti-FITC antibody and magnetic particle conjugate
1, the preparation principle of anti-FITC antibody and magnetic particle conjugate: magnetic particle is that kernel is that the polymkeric substance that has reactive group amino has been wrapped up on Fe3O4, surface, can adopt the glutaraldehyde two-step approach that magnetic particle and anti-FITC antibody are carried out coupling.
2, the preparation method of anti-FITC antibody and magnetic particle conjugate: with particle diameter is that the magnetic particle of 2-3 μ m activates with glutaraldehyde, stirring at room, behind the mixing 3 hours, add magnetic field, leaving standstill 10-15min, pour out supernatant, is that 7.4 0.01mol/L phosphate buffer cleans three times with the pH value, and suspend with this solution, concentration is 50-100mg/mL; Add anti-FITC antibody 20-80 μ g in every milliliter of suspending liquid, after 4 ℃ stirring is spent the night down, add magnetic field, leave standstill 10-15min, pour out supernatant, with contain 0.5% bovine serum albumin(BSA), 1% caseic pH is 7.2, the phosphate buffer of 0.02mol/L was in room temperature sealing 3-4 hour; Be 7.4, contain the 0.02mol/LTris-HCl buffer solution for cleaning three times of 0.5%BSA with the pH value at last, and be mixed with the working fluid of 5-10mg/mL with this solution.The magnetic particle separating agent should be not frozen 4 ℃ of preservations, and the time spent shakes up gently.
Two, the CA125 MONOCLONAL ANTIBODIES SPECIFIC FOR of FITC mark
1, the CA125 MONOCLONAL ANTIBODIES SPECIFIC FOR principle of FITC mark: the conjugate of FITC molecule and sugar antigen monoclonal antibody is to carry out mark with common alkalies labelling method antagonist.
2, the CA125 MONOCLONAL ANTIBODIES SPECIFIC FOR method of FITC mark:
(1) A liquid: getting CA50 monoclonal antibody 0.5mg, to place 50mmol/L pH value be 9.3 Na2CO3-NaHCO3 damping fluid;
(2) B liquid: getting FITC2 mg, to be dissolved in 1mL 50mmol/L pH value be in 9.3 the Na2CO3-NaHCO3 damping fluid;
(3) A liquid is placed magnetic stirring apparatus, get B liquid and slowly drop in the A liquid, between 5-8 minute, add, prevent bubble, put 4 ℃ then and stirred 12～16 hours;
(4) reactant liquor is adorned bag filter, put 10mmol/L pH value and be in 7.4 the phosphate buffer dialysis 2 days, change dislysate every day 2 times;
(5) be 1.25 μ g/mL with label with the dilution of 20% calf serum, add an amount of proclin300, packing is preserved down for-20 ℃.
Three, the CA125 MONOCLONAL ANTIBODIES SPECIFIC FOR of the enzyme labeling of horseradish peroxidase
1, horseradish peroxidase-labeled CA125 MONOCLONAL ANTIBODIES SPECIFIC FOR principle: use the periodates oxidizing process.Sodium periodate is oxidized to aldehyde radical with the polysaccharide of horseradish peroxidase molecular surface, and the amino on aldehyde radical and the antibody molecule forms Schiff alkali and combines.The latter can further use NaBH4 (or monoethanolamine) reduction to generate stable enzymic-labelled antibody.
2, the enzyme labeling CA125 MONOCLONAL ANTIBODIES SPECIFIC FOR method of horseradish peroxidase:
(1) the 8-10mg horseradish peroxidase is put into the 1mL vial, adds the dissolving of 1mlL redistilled water, and it is brown that liquid is;
(2) dropwise slowly add the new NaIO4 0.2mL for preparing under the electromagnetic agitation, continue to stir 30-40min under the room temperature, it is green that liquid is;
(3) with reactant liquor to 1mmol/L, pH value be 4.4 sodium-acetate buffer dialysed overnight under 4 ℃, liquid is changed 3～5 times in the centre, each 300mL, solution is light brown;
(4) 1mg CA125 monoclonal antibody is dissolved in 2mLDMF, and is diluted to 4mL with 0.2mol/L Na2CO3 damping fluid;
(5) the good horseradish peroxidase solution vial of packing into of will dialysing adds 0.2mol/LNa2CO3 solution the pH value is adjusted to 9.0-10.0, adds antibody-solutions behind the mixing immediately, limit edged electromagnetic agitation; Continue to stir 3-5 hour;
(6) in reactant liquor, add new NaBH4 solution 100～200 μ L that prepare, placed 2 hours for 4 ℃.
(7) the reactant liquor bag filter of packing into is 7.4 phosphate buffer dialysis after 24 hours with 0.1mol/L pH value, changes liquid 4 times.With the thimerosal antiseptic solution dilution in 1: 2000 that contains 0.05% glycerine, 0.2g/L, packing, stored frozen.
Utilize the method for CA125 content in this kit test sample as follows:
One, sample pre-treatments
To human serum blood plasma: get on an empty stomach morning blood sample this, the centrifugal 5min of 3000rpm gets upper strata liquid 25 μ L and analyzes.Must not deposit above 48 hours for testing sample 2-8 ℃,, preserve below Ying Yu-20 ℃ if do not detect in 48 hours, but should be above 30 days.
Two, detection method
In test tube, add 25 μ L blood serum samples or, add label mixed liquor 50 μ L again, 37 ℃ of constant temperature oscillating reactions 30min with the series standard product solution of volume.Add magnetic particle solution 50 μ L, behind 37 ℃ of constant temperature oscillating reactions 5min, test tube rack placed separate 5min on the magnetic separator, the separation vessel that reverses is then poured out supernatant, and the test tube of reversing is placed on the filter paper, bounces separation vessel to remove wall built-up liquid.Every pipe adds cleansing solution 500 μ L, makes its abundant mixing with circular motion, places and separates 5min on the magnetic separator, and the separation vessel that reverses is then poured out supernatant, and the test tube that reverses is placed on the filter paper, bounces separation vessel to remove wall built-up liquid, repeats 3 times.Add luminous substrate 300 μ L, circular motion makes its abundant mixing, and the dark place is placed 2min and is placed on magnetic separator, treat that the magnetic particle is enriched in the bottom after, place the tubular type light-emitting appearance to measure in test tube.
Three, interpretation of result
Luminous counting-dose-effect curve represents that with double logarithmic curve each the concentration standard solution that is promptly obtained or the mean value (B) of sample luminous value deduct first zero standard luminous value (B0) on schedule, take the logarithm, promptly again
The longitudinal axis (Y-axis)=log (B-B0)
Natural logarithm value with CA125 concentration is transverse axis (X-axis), the drawing standard curve, as shown in Figure 2.CA125 concentration can be read from typical curve in corresponding each sample.Also can carry out match, calculate the concentration of CA125 in the sample solution with the multiparameter regression equation method.Because the range of linearity of the board-like kit of most of chemiluminescences is between 0-500U/mL on the market, and the range of linearity that this kit provides increases (0-1000U/mL) greatly, so avoided the dilution of enriched sample, the crotch effect also can be avoided.Use the whole testing process of this kit only to need just can finish in 1.5 hours, lowest detection is limited to 1.0U/mL.
Embodiment 3, kit precision, accuracy and stability experiment
1, kit precision is measured
(1) standard items precision experiment
The kit of preparation among embodiment 1 and the embodiment 2 is got three batches respectively carry out the precision experiment, every batch is extracted 10 kits.With the CA50 standard items of the kit measurement 40U/mL that extracted among the embodiment 15 times, with the CA125 standard items of the kit measurement 100U/mL that extracted among the embodiment 25 times.Calculate the coefficient of variation of measuring concentration.The measurement result of three batches of kits among the embodiment 1 is as shown in table 1, the result show the coefficient of variation 4.0%-8.2% between.
The repeatable experiment of table 1 CA50 standard
The measurement result of three batches of kits among the embodiment 2 is as shown in table 2, and the result shows that the coefficient of variation is between 5.8%-8.8%.
The repeatable experiment of table 2 CA125 standard
(2) the repeatable experiment of sample
Get two portions of normal human serums, add CA50 and CA125 standard items respectively to 40U/mL and 100U/mL.Get each 3 of the kits of three different batches among embodiment 1 and the embodiment 2,, calculate the coefficient of variation respectively sample replication 5 times.Measurement result is shown in table 3, table 4, the coefficient of variation that shows the CA50 magnetic microparticle chemiluminescence enzyme immune analytic reagent kit mensuration serum sample that the present invention is prepared is less than 8.6%, and the CA125 magnetic microparticle chemiluminescence enzyme immune analytic reagent kit is measured the coefficient of variation of serum sample less than 5.0%.
The repeatable experiment of table 3 CA50 blood serum sample
The repeatable experiment of table 4 CA125 blood serum sample
2, kit accuracy determination
Get two clinical definite value serum of CA50, concentration is respectively 42.56U/mL and 83.54U/mL, respectively to standard solution 20U/mL, the 40U/mL and the 80U/mL that wherein add CA50, according to the method for embodiment 1 to each concentration do 3 parallel, calculate recovery rate.The result is as shown in table 5, and the interpolation recovery that shows CA50 is between 82.4%-112%.
Get two clinical definite value serum of CA125, concentration is respectively 27.78U/mL and 93.58U/mL, respectively to standard solution 50U/mL, the 100U/mL and the 200U/mL that wherein add CA125, according to the method for embodiment 2 to each concentration do 3 parallel, calculate recovery rate.The result is as shown in table 6, and the interpolation recovery that shows CA125 is between 98.1%-106%.
The recovery of the kit of table 5 embodiment 1
The recovery of the kit of table 6 embodiment 2
3, kit stability experiment
After the kit of embodiment 1 and embodiment 2 carried out 37 ℃ of 5 days and 7 days accelerated tests respectively, measure the recovery of standard addition of the maximum of kit, minimum luminous intensity, determinand, the kit index that shows embodiment 1 and embodiment 2 is all within normal range.Kit key component to embodiment 1 and embodiment 2: label working fluid, standard items, luminous substrate liquid, magnetic particle working fluid carry out 2-8 ℃ of 8 months tracking test, and the result shows that every index meets the requirements fully.Consider the generation of the freezing situation of kit, with the kit of embodiment 1 and embodiment 2 put into-20 ℃ freezing 7 days, measurement result shows that also every index of kit is normal fully.Kit can be preserved more than 6 months at least at 2-8 ℃ as can be seen from the above results.
Claims (19)
1, a kind of magnetic microparticle chemiluminescence enzyme immune analytic reagent kit that is used to detect sugar antigen is characterized in that: comprise the magnetic particle that is coated with anti-FITC antibody; The sugar antigen monoclonal antibody of flag F ITC and the sugar antigen monoclonal antibody of marker enzyme are mixed the thing liquid that serves as a mark; The sugar antigen standard solution; Concentrated cleaning solution; Luminous substrate liquid.
2, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 1 is characterized in that: described sugar antigen is selected from CA72-4, CA50, CA19-9, CA242, CA15-3, CA27-29 or CA125.
3, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 1 is characterized in that: described FITC labeled monoclonal antibody and enzyme labeling monoclonal antibody all are the monoclonal antibody specifics of sugar antigen to be checked.
4, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 1 is characterized in that: sugar antigen to be checked is CA50, and used FITC labelled antibody and enzymic-labelled antibody are the monoclonal antibody specific of CA50.
5, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 1 is characterized in that: sugar antigen to be checked is CA125, and used FITC labelled antibody and enzymic-labelled antibody are the monoclonal antibody specific of CA125.
6, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 1 is characterized in that: described bag be can be polyclonal antibody or monoclonal antibody by the anti-FITC antibody of magnetic particle.
7, according to each described magnetic microparticle chemiluminescence enzyme immune analytic reagent kit among the claim 1-6, it is characterized in that: described bag is that particle diameter is that 2-3 μ m, kernel are that tri-iron tetroxide (Fe3O4), surface parcel have reactive group (polymkeric substance COOH), its working concentration are 5-10mg/mL as amino (NH2-), carboxyl by the magnetic particle of anti-FITC antibody.
8, according to each described magnetic microparticle chemiluminescence enzyme immune analytic reagent kit among the claim 1-6, it is characterized in that: the bag of described anti-FITC antibody is by the glutaraldehyde two-step approach magnetic particle and anti-FITC antibody to be carried out coupling, and the confining liquid of use is that to contain 0.5% bovine serum albumin(BSA), 1% casein, pH value be 7.2 0.02mol/L phosphate buffer.
9, according to each described magnetic microparticle chemiluminescence enzyme immune analytic reagent kit among the claim 1-6, it is characterized in that: the conjugate of described FITC molecule and sugar antigen monoclonal antibody is to carry out mark with alkalies labelling method antagonist, and FITC label form can be freeze-dried powder, concentrate and working fluid; The solution of the described FITC of depositing labelled antibody liquid is 20% calf serum.
10, according to each described magnetic microparticle chemiluminescence enzyme immune analytic reagent kit among the claim 1-6, it is characterized in that: the marker enzyme of described enzymic-labelled antibody is horseradish peroxidase or alkaline phosphatase; Enzyme labeling thing form can be freeze-dried powder, concentrate and working fluid; The used dilution of described enzyme labeling thing working fluid is the thimerosal antiseptic solution that contains 0.05% glycerine, 0.2g/L.
11, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 10 is characterized in that: marker enzyme is an alkaline phosphatase, and labeling method is preferably glutaraldehyde method.
12, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 10 is characterized in that: marker enzyme is a horseradish peroxidase, and labeling method is preferably the sodium periodate method.
13, according to each described magnetic microparticle chemiluminescence enzyme immune analytic reagent kit among the claim 1-6, it is characterized in that: described mixed mark thing liquid is that volume ratio is 1: 1-1: 3 FITC mark capturing antibody working fluid and enzyme labeling pairing antibody working fluid mix.
14, according to each described magnetic microparticle chemiluminescence enzyme immune analytic reagent kit among the claim 1-6, it is characterized in that: described standard solution is the solution that 6 concentration gradients contain sugar antigen; The matrix of described preparation standard items is 50% horse serum; The sugar antigen of described formation standard items comprises: CA72-4, CA50, CA19-9, CA242, CA15-3, CA27-29 and CA125.
15, according to each described magnetic microparticle chemiluminescence enzyme immune analytic reagent kit among the claim 1-6, it is characterized in that: described concentrated cleaning solution is that the pH value is 7.4, contain the 0.1-0.5% Tween-20, the phosphate buffer of 0.1% sodium azide antiseptic.
16, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 1, it is characterized in that: described marker enzyme is a horseradish peroxidase, luminous substrate liquid is made of A liquid and B liquid, and luminous substrate liquid A liquid is superoxol, and luminous substrate liquid B liquid is luminol solution.
17, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 1, it is characterized in that: described when marker enzyme be alkaline phosphatase, described luminous substrate liquid is 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 "-hydroxyl) benzene-1; 2-dioxetane sodium phosphate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1; 2-dioxetane-3; 2 '-adamantane, AMPPD) damping fluid.
18, magnetic microparticle chemiluminescence enzyme immune analytic reagent kit according to claim 1 is characterized in that: also comprise the reaction tube of use, the reaction tube material can be transparent polystyrene, tygon, polypropylene, glass.
19, a kind of magnetic microparticle chemiluminescence enzyme immune analytic method that detects sugar antigen is characterized in that: may further comprise the steps:
(1) sample pre-treatments:
To clinical serum blood plasma, centrifugal 5 minutes of 3000rpm gets upper strata liquid and can carry out assay determination;
(2) utilize the magnetic microparticle chemiluminescence enzyme immune analytic reagent kit test sample of arbitrary described detection sugar antigen among the claim 1-18.
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| CNA2007101188855A CN101324579A (en) | 2007-06-13 | 2007-06-13 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
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| CNA2007101188855A CN101324579A (en) | 2007-06-13 | 2007-06-13 | Magnetic microparticle chemiluminescence enzyme immune analytic reagent kit for detecting saccharide antigen and its use method |
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