CN104614535A - Thyroid microsomal antibody detection reagent kit as well as preparation method and application thereof - Google Patents
Thyroid microsomal antibody detection reagent kit as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a chemical luminescent immunodetection thyroid microsomal antibody detection reagent kit and a preparation method of the reagent kit. The reagent kit comprises a protein A antigen labeled with a tracer marker, or an anti-human IgG and anti-human IgM labeled with the tracer marker, a thyroid microsomal antigen coated with magnetic spheres, or a junction complex of a protein carrier and the thyroid microsomal antigen coated with the magnetic spheres. The invention also provides a method for detecting the concentration of the thyroid microsomal antibody. When the reagent kit provided by the invention is used for detecting the concentration of the thyroid microsomal antibody, the enzymatic reaction is not involved, and the bridging of enzyme is not needed, so that the reagent is long in preservation time and stable; the detection also can be carried out by virtue of a full-automatic chemical luminescent method, so that the operation time is shortened, the manmade operation error is reduced, and the sensitivity and the accuracy in detection can be improved by utilizing the specificity of the chemical tracer marker.
Description
Technical field
The present invention relates to a kind of detection kit, being specifically related to a kind of chemiluminescence immune detection reagent kit for detecting thyroid microsomal antibody.The invention still further relates to the preparation method of this kit, and use this kit to detect the method for thyroid microsomal antibody concentration.
Background technology
Thyroid microsomal antibody (TMA) is a kind of antibody for thyroid microsomal (TM), is one of autoantibody caused by autoimmune thyroid disease.It is important symbol in thyroid autoimmune process that TAM and antithyroglobulin antibodies (TGA) are generally acknowledged, is the representational antibody of most.TAM and TGA is indispensable index in the diagnosis of autoimmune thyroid disease, is one of limited means of histodiagnosis autoimmune thyroid disease.
For various thyroid disease, anti-TM positive rate: Hashimoto thyroiditis is 50% ~ 100%; Hypothyroidism is 88.9%; Thyroid tumors are 13.1%; Simplex goiter is 8.6%; Subacute thyroiditis is 17.2% ~ 25%; Systemic loupus erythematosus (SLE) is 15.4% ~ 44.7%; Other rheumatism are 30%.Normal person also has the anti-TM positive rate of 8.4%.
For autoimmune thyroiditis (namely Graves is sick) patient, its serum T MA level is significantly higher than normal person and other non-self autoimmune thyroid Diseases.Therefore, TMA level has important value to antidiastole autoimmune thyroiditis, and its accuracy rate of diagnosis of the two use in conjunction can reach 98%.
The serum T MA level of the immunity disease patients such as Hashimoto's thyroiditis, hypothyroidism and hyperthyroidism is also significantly higher than normal person, and especially Hashimoto's thyroiditis is more outstanding, and therefore serum T MA level is this type of disease of diagnosis " specific parameters ".
Particularly, for some concrete thyroid diseases, serum T MA horizontal properties is as follows.1. hyperthyroidism: TMA shows strong positive, and antibody horizontal is higher than Hashimoto's thyroiditis; After some patients treatment, TMA can transfer feminine gender to, but the hyperthyroid patient TMA of most clinical cure is determined as the weak positive for a long time; Therefore periodic review thyroid function is answered, in case recurrence.2. Hashimoto's thyroiditis, Addison's disease: TMA shows strong positive; The scorching patient TMA of methylene apparently higher than normal person, lower than Hashimoto's thyroiditis.3. the low disease of primary hypothyroidism: TMA shows the positive, but the low disease of Secondary cases first is TMA feminine gender, this can differentiate that Secondary cases first is low.4. thyroid cancer: TMA increases obviously.5. pregnancy period autoimmune disease: TMA can increase.
At present, the method measuring TMA clinically mainly contains euzymelinked immunosorbent assay (ELISA), chemiluminescence immunoassay etc.But euzymelinked immunosorbent assay (ELISA) exists many deficiencies, traditional euzymelinked immunosorbent assay (ELISA) method is long for detection time, and mainly rely on the serial troublesome operation such as pure manual application of sample, efficiency is low, easily causes experimental result error large simultaneously; Because enzymatic reaction is thorough not, and be subject to external interference factor impact, as impacts such as temperature, time and material concentrations, when therefore detecting, specificity is low, and poor sensitivity, sensing range is narrow.Chemiluminescence immune assay combines having highly sensitive chemical luminescent detecting technology with the immune response of high specific, for the detection analytical technology of various antigen, haptens, antibody, hormone, enzyme, fatty acid, vitamin and medicine etc., it is immunoassay the sensitiveest so far.But the detection kit with premium properties at present for chemiluminescence immunoassay detection TMA is actually rare.Therefore, this area still needs one badly can obtain high sensitivity and accuracy when for detecting TMA, operates easier, can also realize the detection kit of the detection TMA of testing process full-automation by analytical instrument.
Summary of the invention
The object of the present invention is to provide a kind of chemiluminescence immune detection reagent kit for detecting TMA, to obtain high detection sensitivity and accuracy.This kit can also improve the stability detecting reagent, extends the holding time detecting reagent.
The present invention is also provided for the preparation method of the chemiluminescence immune detection reagent kit detecting TMA.
In addition, present invention also offers the application according to TMA chemiluminescence immune detection reagent kit provided by the invention.Especially adopt described kit, carried out the method for TMA detection by Full-automatic chemiluminescence method, can the running time be reduced, reduce manual operation error, utilize the specificity of chemical tracing label simultaneously, improve detection sensitivity.
According to the present invention, provide a kind of chemiluminescence immune detection reagent kit for detecting thyroid microsomal antibody, described kit comprises the albumin A being marked with trace labelling thing or the anti-human igg being marked with trace labelling thing and anti-human IgM, and bag by the thyroid microsomal antigen of magnetic ball or bag by the connector of the thyroid microsomal antigen of magnetic ball and protein carrier.Wherein, the direct or indirect labelled protein A of described trace labelling thing, or directly or indirectly mark anti-human igg and anti-human IgM; The connector of described thyroid microsomal antigen or thyroid microsomal antigen and protein carrier directly or indirectly wraps by magnetic ball.
Albumin A is a kind of vegetarian protein A matter, can be combined with the Fc district of people and mammalian antibody (mainly IgG) specifically.Thus, albumin A is combined in some way with Ago-Gel, can for the preparation of the affine filler of antibody purification.The Fab fragment (Fab) of anti-igg can combine with the Fc fragment (crystallizable fragment) of IgG antibody.The Fab fragment (Fab) of anti-IgM can combine with the Fc fragment (crystallizable fragment) of IgM antibody.
The protein carrier that the present invention is suitable for can be selected from least one in the protein carrier commonly used this area.Such as, described protein carrier is selected from least one in bovine serum albumin(BSA) (BSA), human serum albumins (HSA), albumin rabbit serum (RSA), hemocyanin (KLH), ox IgG, human IgG, ovalbumin (OVA), myoglobins and thyroglobulin.
According to the present invention, described trace labelling thing can be selected from this area the trace labelling thing being usually used in labelled antigen or antibody, such as be selected from least one in diamantane, luminol and derivant thereof, different luminol and derivant, acridinium ester, alkaline phosphatase and horseradish peroxidase, be preferably N-(4-ammonia butyl) the different luminol of-N-ethyl (ABEI).
According to the present invention, described trace labelling thing is preferably by fluorescein isothiocynate (FITC) and anti-FITC antibody system or Streptavidin (SA) and biotin (Biotin) system indirect labelling albumin A or indirect labelling anti-human igg and anti-human IgM.
Described " directly marking " refers to that trace labelling thing is directly connected with antigen and marks; Described " indirect labelling " refers to and makes trace labelling substance markers albumin A or mark anti-human igg and anti-human IgM by intermediary link system, and described intermediary link system includes but not limited to FITC and anti-FITC antibody system or SA and Biotin system.The present inventor finds, indirect labelling is conducive to weakening steric effect, is conducive to the amplification of signal, makes detection sensitiveer.
According to the present invention, described thyroid microsomal antigen (or connector of itself and protein carrier) wraps by magnetic ball indirectly preferably by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system.
Described " direct coated " refers to and utilizes TM antigen (or connector of itself and protein carrier) directly to carry out bag quilt to magnetic ball; Described " indirectly wrapping quilt " refers to and links system by intermediary, make TM antigen (or connector of itself and protein carrier) carry out bag quilt to magnetic ball, described intermediary link system includes but not limited to FITC and anti-FITC antibody system or SA and Biotin system.Equally, the advantage of indirect bag quilt is, is conducive to weakening steric effect, is conducive to the amplification of signal, makes detection sensitiveer.
In specific embodiments more of the present invention, described kit comprises the albumin A being marked with trace labelling thing or the anti-human igg being marked with trace labelling thing and anti-human IgM, and is selected from a kind of component in following component: a) thyroid microsomal antigen (or connector of itself and protein carrier) of direct coated magnetic ball; B) Streptavidin of biotinylated thyroid microsomal antigen (or connector of itself and protein carrier) and direct coated magnetic ball; C) thyroid microsomal antigen (or connector of itself and protein carrier) of fluorescein isothiocynate and the goat-anti fluorescein isothiocynate antibody of direct coated magnetic ball is directly marked with.
The low spot calibration object of TMA and high some calibration object can also be comprised according to kit provided by the invention, and optionally comprise damping fluid.Low spot calibration object of the present invention and high some calibration object be both comparatively speaking, wherein " low spot calibration object ", refer to that it is the calibration object that 0-30IU/ml obtains that TMA 50% cow's serum goods are diluted to concentration; And " high some calibration object " refers to that it is the calibration object that 500-1000IU/ml obtains that TMA antigen 50% cow's serum goods are diluted to concentration.
Being applicable to magnetic ball of the present invention also referred to as magnetic bead, can be magnetic microsphere conventional in this area.Preferably, the magnetic ball that the present invention uses is by nano level Fe
2o
3or Fe
3o
4magnetic particle and high-molecular organic material carry out compound, and form the micron-sized solid phase microballoon with superparamagnetism and huge amount protein adsorption capacity, have and can be magnetized rapidly under additional magnetic fields, after withdrawing magnetic field, remanent magnetism is the attribute of zero.Wherein, the kind of described high-molecular organic material is not particularly limited, and can select as required.
It is 0.1-5 μm that magnetic microsphere used in the present invention should be able to meet diameter, and magnetic microsphere with various active functional group, can also include but not limited to-OH ,-COOH ,-NH by surface modification
2.
In a specific embodiment, described magnetic ball is Fe
2o
3or Fe
3o
4the complex of magnetic nano-particle and high-molecular organic material, and the particle diameter with 0.1-5 μm.
According to the present invention, antibody can be monoclonal antibody or polyclonal antibody.
Present invention also offers a kind of method for the preparation of kit described above, said method comprising the steps of: i) utilize the direct or indirect labelled protein A of trace labelling thing, or directly or indirectly mark anti-human igg and anti-human IgM; And ii) thyroid microsomal antigen (or connector of itself and protein carrier) is directly or indirectly wrapped by magnetic ball; Wherein, described indirect labelling comprises trace labelling thing by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system labelled protein A or mark anti-human igg and anti-human IgM; Described indirect bag is included and is indirectly wrapped by magnetic ball by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system by thyroid microsomal antigen (or connector of itself and protein carrier).
Kit preparation method according to the present invention can also comprise the preparation of low spot calibration object and high some calibration object, can further include the assembling of kit.
According to the present invention, additionally provide a kind of method detecting TMA concentration, described method comprises use according to kit as above provided by the invention, is detected the TMA concentration in testing sample by Chemiluminescence immunoassay.
Particularly, detection TMA concentration detection method provided by the invention comprises sample to be tested, the albumin A being directly or indirectly marked with trace labelling thing or the anti-human igg being marked with trace labelling thing and anti-human IgM, and directly or indirectly bag is mixed with the connector of protein carrier by TMA or TMA of magnetic ball, make TMA to be measured, albumin A or anti-human igg and anti-human IgM and bag are formed " sandwich sandwich " shape immune complex by the connector of the TMA of magnetic ball or itself and protein carrier, add and excite substrate, measure luminous intensity, the typical curve of reference TMA calibration object concentration and luminous intensity, calculate the TMA concentration of testing sample.
In one embodiment, described detection TMA method comprises use according to kit described above provided by the invention, detects TMA concentration by chemical illumination immunity analysis instrument.In a preferred embodiment in accordance with this invention, described method is fully automatically carried out.According to the present invention, described chemical illumination immunity analysis instrument is preferably Maglumi sequence of chemical luminescence immunoassay instrument (production of Shenzhen NPD projects biomedical engineering incorporated company).
Beneficial effect of the present invention is:
1. specificity is high, sensitivity good, accuracy is high, sensing range is wider.
2. operation is more simple and easy to do, kit of the present invention can support the use with chemical illumination immunity analysis instrument (especially Maglumi sequence of chemical luminescence immunoassay instrument) simultaneously, full-automation is achieved in sample mensuration process, the detection of TMA concentration simply, easily and fast, in bulk can be carried out, ensure that the systematic error detected is less simultaneously.
3. the present invention detects TMA by Chemiluminescence immunoassay, the method detection time avoiding conventional euzymelinked immunosorbent assay (ELISA) is long, efficiency is low, easily cause experimental result error large, be subject to the shortcomings such as external interference factor impact, specificity are low, poor sensitivity; And do not need to be put up a bridge by enzyme, therefore the reagent holding time is long, stable.
Accompanying drawing explanation
Fig. 1 is the experimental result data comparison diagram of embodiment 1 and embodiment 2;
Fig. 2 is the experimental result data comparison diagram of embodiment 1 and embodiment 3;
Fig. 3 is the experimental result data comparison diagram of embodiment 1 and embodiment 4.
Embodiment
Below in conjunction with non-limiting example, the present invention is made further explanation and description.It is to be noted, however, that following detailed description of the present invention does not make any restriction to the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
In following examples:
It is Bioisystech Co., Ltd that albumin A is purchased from Shenzhen refined;
The anti-human igg and the anti-human IgM that are marked with trace labelling thing are purchased from Medix company
TM antigen is purchased from Meridian company of the U.S.;
Goat-anti FITC polyclonal antibody is purchased from Jackson company of the U.S.;
Magnetic microsphere, ABEI produce by the biomedical incorporated company of Shenzhen NPD projects;
FITC purchased from American Sigma company;
Biotin and Streptavidin equal purchased from American Biosources company;
TMA calibration object purchased from American Biodesign company;
Maglumi 2000 chemical illumination immunity analysis instrument: Shenzhen NPD projects biomedical engineering incorporated company produces.
Sample to be tested: the 120 routine clinical serum samples taking from Beijing University's Shenzhen hospital, wherein at least 7 examples are the serum sample of thyroid function disease patient diagnosed.
Embodiment 1
(1) mark of albumin A
The preparation of dislysate (F solution): add Na in 5000ml beaker
2cO
314.31g, NaHCO
326.46g, adds purified water and is diluted to 4500ml.The F solution prepared is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional 14000), measure suitable size, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g albumin A 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying protein A and ABEI.
D
2the preparation of solution: add the 0.5M phosphate buffer (P001 solution) of 200ml, 20g BSA, 8g NaN
3, 2g MgCl
26H
2o, 600ml glycerine, adds purified water constant volume to 2000ml, filters.
By connection product D good for purifying
2solution dilution, makes its concentration count 0.025 μ g/ml with albumin A.
(2) the bag quilt of TM antigen
The preparation of solution A: take after adding the mixing of 14ml acetic acid again after 2.55g sodium acetate trihydrate 4500ml purified water is dissolved, be settled to 5000ml (pH is 3.6).
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic ball is added with bag by the pH3.6 acetate buffer solution of volume equivalent, the suspended concentration of magnetic ball is made to be 20mg/ml, add 1-cyclohexyl-2-morpholine ethyl carbodiimide tosilate (CMC) again, make its concentration be 10mg/ml, add the TM antigen of purifying by certain ratio.
The mixed solution of magnetic ball and TM antigen is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
Preparation pH7.4 phosphate buffer (PBS), adds 2.5g BSA and dissolves, and mixing, is magnetic ball cleaning fluid.
The magnetic ball of temperature being bathed and the mixed solution of TM antigen are poured in beaker, are then placed in after magnet precipitates, outwell supernatant, add the magnetic ball cleaning fluid stirring and washing of 5 times of volumes, then be placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared: take methylcellulose (MC) 160g and pour in 5000ml beaker, add purified water to 4000ml, stirs molten 2 hours as heating in the water baths of 90 DEG C.Separately get 4000ml 0.5M phosphate buffer, add 80g NaN
3(analyzing pure), 80ml T-20 (analyzing pure), mixing, filters.Add 200g BSA after fully being mixed by these two parts of solution, add water to 40000ml.
Be suspended in C solution by the magnetic ball after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball that TM is antigen coated, this suspension vol is described in the present embodiment and wraps by volume.
The suspending liquid of magnetic ball antigen coated for TM is diluted to 0.5mg/ml further for subsequent use.
(3) TMA low spot calibration object, high some calibration object
TMA standard items calibration object dilution (50% cow's serum goods) is diluted to by different proportion two high and low calibration object scaling points that concentration is respectively 591.58IU/ml and 25.36IU/ml.
(4) react: above-mentioned sample to be tested, height calibration object are mixed with label system and magnetic spheroid system respectively, incubation, the thyroid microsomal antibody in sample to be tested is combined with the albumin A marked through ABEI and obtains reaction product.
Concrete load procedure is: add 10 μ l samples to be tested or height calibration object, then add 20 μ l to wrap by the magnetic ball solution of TM antigen (above-mentioned steps (2) preparation), react after 10 minutes and clean, add the Protein A solution (above-mentioned steps (1) preparation) that 100 μ l ABEI mark, form compound.
(5) detect: above-mentioned reaction product precipitates by externally-applied magnetic field, remove supernatant, and with after buffer solution for cleaning, add chemiluminescence excimer (NaOH and H
2o
2), the relative light intensity that uses Maglumi 2000 chemical illumination immunity analysis instrument to detect to send (concrete operation step can with reference to Maglumi 2000 chemical illumination immunity analysis instrument instructions), by calculating the TMA content of sample to be tested.The results are shown in Table shown in 1.
Embodiment 2
(1) albumin A biotinylation, concrete steps are as follows:
The preparation of dislysate (F solution): add Na in 5000ml beaker
2cO
314.31g, NaHCO
326.46g, adds purified water and is diluted to 4500ml.The F solution prepared is placed on magnetic stirring apparatus for subsequent use.
The carbonic acid buffer (F solution) getting 100 μ g biotins and 1mg albumin A 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, and 37 DEG C are reacted 2 hours.
By G-25 gel column purifying.
According to the method preparation D2 solution of embodiment 1, the connection product D that purifying is good
2solution dilution, makes its concentration be 0.025 μ g/ml.
(2) mark of SA
The carbonic acid buffer (F solution) getting 100 μ g SA 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
Use the connection product of G-25 gel column purifying SA and ABEI.
The connection product D that purifying is good
2solution dilution, makes its concentration count 10 μ g/ml with SA, for subsequent use.
(3) the antigen coated magnetic ball of TM
Solution A is prepared according to the method for embodiment 1.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic ball is added bag by the pH3.6 acetate buffer solution of volume equivalent, the suspended concentration making magnetic ball is 20mg/ml, then adds CMC, makes its concentration be 10mg/ml, adds the TM antigen of purifying.
Magnetic ball suspending liquid is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
Preparation pH7.4PBS damping fluid 500ml, adds 2.5g BSA and mixes dissolving, be magnetic ball cleaning fluid.
The magnetic ball suspending liquid of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic ball cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
Be suspended in the C solution prepared according to embodiment 1 by magnetic ball antigen coated for the TM after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball suspension suspending liquid that TM is antigen coated, this suspension vol is described in this step and wraps by volume.
Magnetic ball suspending liquid is diluted to 0.5mg/ml further, for subsequent use.
(4) TMA low spot calibration object, high some calibration object
It is the high and low calibration object scaling point of 591.58IU/ml and 15.298IU/ml two that TMA antigen is diluted to concentration with 50% cow's serum goods by different proportion.
(5) react: above-mentioned sample to be tested or height calibration object are mixed with label system and magnetic microsphere system respectively, incubation, the thyroid microsomal antibody in sample to be tested is combined with through biotinylated albumin A and obtains reaction product.Concrete load procedure is: add 10 μ l samples to be tested or height calibration object, then add 20 μ l to wrap by the magnetic ball solution of TM antigen (above-mentioned steps (3) prepares gained), react after 10 minutes and add 50 μ l SA solution (above-mentioned steps (2) prepares gained) and the biotinylated Protein A solution of 50 μ l (above-mentioned steps (1) prepares gained) after cleaning, form compound.
(6) detect: above-mentioned reaction product precipitates by externally-applied magnetic field, remove supernatant, and with after buffer solution for cleaning, add chemiluminescence excimer (NaOH and H
2o
2), use Maglumi 2000 chemical illumination immunity analysis instrument to detect the relative light intensity sent, by calculating the TMA content in sample to be tested.The results are shown in Table shown in 1.
Embodiment 3
(1) mark of albumin A
The preparation of dislysate (F solution): add Na in 5000ml beaker
2cO
314.31g, NaHCO
326.46g, adds purified water and is diluted to 4500ml.The F solution prepared is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional 14000), measure suitable size, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g albumin A 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying protein A and ABEI.
D
2the preparation of solution: add the 0.5M phosphate buffer (P001 solution) of 200ml, 20g BSA, 8g NaN
3, 2g MgCl
26H
2o, 600ml glycerine, adds purified water constant volume to 2000ml, filters.
By connection product D good for purifying
2solution dilution, makes its concentration count 0.025 μ g/ml with albumin A.
(2) the FITC mark of TM antigen
The carbonic acid buffer (F solution) getting 100 μ g TM antigen 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g FITC Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying TM antigen and FITC.
By connection product D good for purifying
2solution dilution, makes its concentration in TM antigen 0.1 μ g/ml.
(3) goat-anti FITC polyclonal antibody bag is by magnetic ball
Take after 2.55g sodium acetate trihydrate 4500ml purified water is dissolved and add again as after the mixing of 14ml acetic acid, be settled to 5000ml (pH is 3.6) by purified water.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic ball is added bag by the pH3.6 acetate buffer solution of volume equivalent, make magnetic ball suspended concentration be 20mg/ml, then add CMC, make its concentration be 10mg/ml, add the goat-anti FITC polyclonal antibody of purifying by certain ratio.
The mixed solution of magnetic ball and goat-anti FITC polyclonal antibody is put into isothermal vibration water bath 37 DEG C react 24 hours (shaking water bath pot jolting speed: 260rpm).
Preparation pH7.4PBS damping fluid, add 2.5g BSA and dissolve, mixing, is magnetic ball cleaning fluid.
The mixed solution of the magnetic ball of temperature being bathed and goat-anti FITC polyclonal antibody is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic ball cleaning fluid stirring and washing of 5 times of volumes, then be placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared: take methylcellulose (MC) 160g and pour in 5000ml beaker, add purified water to 4000ml, stirs molten 2 hours as heating in the water baths of 90 DEG C.Separately get 4000ml 0.5M phosphate buffer, add 80g NaN
3(analyzing pure), 80ml T-20 (analyzing pure), mixing, filters.Add 200g BSA after fully being mixed by these two parts of solution, add water to 40000ml.
The magnetic ball of goat-anti FITC polyclonal antibody bag quilt after cleaning is suspended in C solution prepared by step (3), suspended concentration is 20mg/ml, namely obtain the magnetic ball suspending liquid of goat-anti FITC polyclonal antibody bag quilt, this suspension vol is described in this step and wraps by volume.
The suspending liquid of the magnetic ball of goat-anti FITC polyclonal antibody bag quilt is diluted to 1mg/ml further, for subsequent use.
(4) TMA low spot calibration object, high some calibration object
TMA standard items calibration object dilution and 50% cow's serum goods are diluted to by different proportion two high and low calibration object scaling points that concentration is respectively 591.58IU/ml and 25.36IU/ml.
(5) react: above-mentioned sample to be tested or height calibration object are mixed with label system and magnetic microsphere system respectively, incubation, the thyroid microsomal antibody in sample to be tested is combined with the albumin A of mark and obtains reaction product.Concrete load procedure is: add 10 μ l samples to be tested or height calibration object, add the FITC solution (above-mentioned steps (2) prepares gained) that 40 μ l mark TM antigen again, then the magnetic ball solution (above-mentioned steps (3) prepares gained) of 20 μ l goat-anti FITC polyclonal antibody bag quilts is added, react cleaning after 10 minutes and add the Protein A solution (above-mentioned steps (1) prepares gained) of ABEI mark, form compound.
(6) detect: above-mentioned reaction product precipitates by externally-applied magnetic field, remove supernatant, and with after buffer solution for cleaning, add chemiluminescence excimer (NaOH and H
2o
2), use Maglumi 2000 chemical illumination immunity analysis instrument to detect the relative light intensity sent, by calculating the TMA content in sample to be tested.The results are shown in Table shown in 1.
Embodiment 4
(1) mark of anti-human igg and anti-human IgM
(1.1) mark of anti-human igg
The preparation of dislysate (F solution): add Na in 5000ml beaker
2cO
314.31g, NaHCO
326.46g, adds purified water and is diluted to 4500ml.The F solution prepared is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional 14000), measure suitable size, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g anti-human igg 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying anti-human igg and ABEI.
D
2the preparation of solution: add the 0.5M phosphate buffer (P001 solution) of 200ml, 20g BSA, 8g NaN
3, 2g MgCl
26H
2o, 600ml glycerine, adds purified water constant volume to 2000ml, filters.By connection product D good for purifying
2solution dilution, makes its concentration count 0.025 μ g/ml with anti-human igg.
(1.2) mark of anti-human IgM
The preparation of dislysate (F solution): add Na in 5000ml beaker
2cO
314.31g, NaHCO
326.46g, adds purified water and is diluted to 4500ml.The F solution prepared is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional 14000), measure suitable size, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g anti-human IgM 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of anti-human IgM and the ABEI of G-25 gel column purifying.
D
2the preparation of solution: add the 0.5M phosphate buffer (P001 solution) of 200ml, 20g BSA, 8g NaN
3, 2g MgCl
26H
2o, 600ml glycerine, adds purified water constant volume to 2000ml, filters.
By connection product D good for purifying
2solution dilution, makes its concentration count 0.025 μ g/ml with anti-human IgM.
(2) the bag quilt of TM antigen
The preparation of solution A: take after adding the mixing of 14ml acetic acid again after 2.55g sodium acetate trihydrate 4500ml purified water is dissolved, be settled to 5000ml (pH is 3.6).
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic ball is added with bag by the pH3.6 acetate buffer solution of volume equivalent, the suspended concentration of magnetic ball is made to be 20mg/ml, add 1-cyclohexyl-2-morpholine ethyl carbodiimide tosilate (CMC) again, make its concentration be 10mg/ml, add the TM antigen of purifying by certain ratio.
The mixed solution of magnetic ball and TM antigen is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
Preparation pH7.4 phosphate buffer (PBS), adds 2.5g BSA and dissolves, and mixing, is magnetic ball cleaning fluid.
The magnetic ball of temperature being bathed and the mixed solution of TM antigen are poured in beaker, are then placed in after magnet precipitates, outwell supernatant, add the magnetic ball cleaning fluid stirring and washing of 5 times of volumes, then be placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared: take methylcellulose (MC) 160g and pour in 5000ml beaker, add purified water to 4000ml, stirs molten 2 hours as heating in the water baths of 90 DEG C.Separately get 4000ml 0.5M phosphate buffer, add 80g NaN
3(analyzing pure), 80ml T-20 (analyzing pure), mixing, filters.Add 200g BSA after fully being mixed by these two parts of solution, add water to 40000ml.
Be suspended in C solution by the magnetic ball after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball that TM is antigen coated, this suspension vol is described in the present embodiment and wraps by volume.
The suspending liquid of magnetic ball antigen coated for TM is diluted to 0.5mg/ml further for subsequent use.
(3) TMA low spot calibration object, high some calibration object
TMA standard items calibration object dilution (50% cow's serum goods) is diluted to by different proportion two high and low calibration object scaling points that concentration is respectively 591.58IU/ml and 25.36IU/ml.
(4) react: above-mentioned sample to be tested, height calibration object are mixed with label system and magnetic spheroid system respectively, incubation, the thyroid microsomal antibody in sample to be tested and the anti-human igg marked through ABEI are combined with anti-human IgM and obtain reaction product.
Concrete load procedure is: add 10 μ l samples to be tested or height calibration object, then add 20 μ l to wrap by the magnetic ball solution of TM antigen (above-mentioned steps (2) preparation), react after 10 minutes and clean, respectively add the ABEI solution (prepared by above-mentioned steps (1)) that 50 μ l mark anti-human igg and anti-human IgM, form compound.
(5) detect: above-mentioned reaction product precipitates by externally-applied magnetic field, remove supernatant, and with after buffer solution for cleaning, add chemiluminescence excimer (NaOH and H
2o
2), the relative light intensity that uses Maglumi 2000 chemical illumination immunity analysis instrument to detect to send (concrete operation step can with reference to Maglumi 2000 chemical illumination immunity analysis instrument instructions), by calculating the TMA content of sample to be tested.The results are shown in Table shown in 1.
Comparative example 1
Adopt existing main flow commercialization enzyme linked immunological kit on the market to detect above-mentioned 120 routine samples to be tested, and contrast with kit testing result provided by the invention.Result is as shown in table 1 and Fig. 1 ~ 3.
Table 1
*: through statistical analysis, decision threshold is: normal person <10IU/ml, the positive >=10IU/ml; "+" represents positive, and "-" represents negative (normally).
From what Fig. 1 ~ 3 of upper table, this clinical comparison tests the 120 routine clinical samples chosen, and wherein at least 3,14,28,39,50,62, No. 118 samples are thyroid function disease patient diagnosed.For other samples, adopt kit of the present invention consistent with the testing result that employing enzyme exempts from kit.But, for above-mentioned seven Patient Sample A made a definite diagnosis, enzyme inspection-free survey TMA is shown as feminine gender, adopts kit provided by the invention and chemical luminescence detection method test positive, this means to adopt technical scheme provided by the invention can greatly avoid enzyme to exempt from the detection leakage phenomenon to TMA of platform.In addition, as shown in Figure 1, the data dependence of embodiment 1 and embodiment 4 is more consistent, shows to adopt the albumin A of mark ABEI consistent with anti-human IgM effect with the anti-human igg being marked with trace labelling thing of employing mark ABEI.From Fig. 1-3, the measurement result of the kit of embodiment 1-4 is adopted to have good consistance.As can be seen here, adopt the testing result of TMA detection kit provided by the invention and detection method more accurate, sensitive, have specificity, more can react clinical truth, thus there is higher using value.
Although the present invention is described in detail, for a person skilled in the art, the amendment in spirit and scope of the invention will be apparent.In addition, should be understood that, each side that the present invention records, each several part of different embodiment and the various features enumerated can be combined or all or part of exchange.In each above-mentioned embodiment, those embodiments with reference to another embodiment can suitably combine with other embodiment, and this is by understand by those skilled in the art.In addition, the description that it will be understood to those of skill in the art that above is only the mode of example, is not intended to limit the present invention.
Claims (11)
1. one kind for detecting the chemiluminescence immune detection reagent kit of thyroid microsomal antibody, described kit comprises the albumin A being marked with trace labelling thing or the anti-human igg being marked with trace labelling thing and anti-human IgM, and bag by the thyroid microsomal antigen of magnetic ball or bag by the connector of the thyroid microsomal antigen of magnetic ball and protein carrier.
2. kit according to claim 1, it is characterized in that, described protein carrier is selected from least one in bovine serum albumin(BSA), human serum albumins, albumin rabbit serum, hemocyanin, ox IgG, human IgG, ovalbumin, myoglobins and thyroglobulin.
3. kit according to claim 1, it is characterized in that, described trace labelling thing is selected from least one in diamantane, luminol and derivant thereof, different luminol and derivant, acridinium ester, alkaline phosphatase and horseradish peroxidase.
4. kit according to claim 3, is characterized in that, described trace labelling thing is N-(4-ammonia butyl) the different luminol of-N-ethyl.
5. kit according to claim 1, is characterized in that,
The direct or indirect labelled protein A of described trace labelling thing, or directly or indirectly mark anti-human igg and anti-human IgM, wherein indirect labelling comprises by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system indirect labelling;
The connector of described thyroid microsomal antigen or thyroid microsomal antigen and protein carrier directly or indirectly wraps by magnetic ball, and wherein indirectly bag is included and indirectly wraps quilt by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system.
6. kit according to claim 1, is characterized in that, described magnetic ball is Fe
2o
3or Fe
3o
4the complex of magnetic nano-particle and high-molecular organic material, and the particle diameter with 0.1-5 μm.
7. kit according to claim 5, is characterized in that, described kit comprises the protein A antigens being marked with trace labelling thing or the anti-human igg being marked with trace labelling thing and anti-human IgM, and is selected from a kind of component in following component:
A) thyroid microsomal antigen of direct coated magnetic ball or the thyroid microsomal antigen of direct coated magnetic ball and the connector of protein carrier;
The Streptavidin of the b) connector of biotinylated thyroid microsomal antigen or thyroid microsomal antigen and protein carrier, and direct coated magnetic ball;
C) thyroid microsomal antigen of fluorescein isothiocynate or the connector of thyroid microsomal antigen and protein carrier is directly marked with, and the goat-anti fluorescein isothiocynate antibody of direct coated magnetic ball.
8. according to the kit in claim 1-7 described in any one, it is characterized in that, described kit also comprises the low spot calibration object of thyroid microsomal antibody and high some calibration object, and optionally comprises damping fluid.
9., for the preparation of the method as the kit in claim 1-8 as described in any one, said method comprising the steps of:
I) utilize the direct or indirect labelled protein A of trace labelling thing, or directly or indirectly mark anti-human igg and anti-human IgM; With
Ii) connector of thyroid microsomal antigen or thyroid microsomal antigen and protein carrier is directly or indirectly wrapped by magnetic ball;
Wherein,
Described indirect labelling comprises by trace labelling thing by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system labelled protein A or mark anti-human igg and anti-human IgM,
Described indirect bag is included and is indirectly wrapped by magnetic ball by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system by the connector of thyroid microsomal antigen or thyroid microsomal antigen and protein carrier.
10. one kind is detected the method for thyroid microsomal antibody concentration, it is characterized in that, described method comprises use as the kit in claim 1-8 as described in any one, is detected the thyroid microsomal antibody concentration in testing sample by Chemiluminescence immunoassay.
11. methods according to claim 10, is characterized in that, described method comprises use as the kit in claim 1-8 as described in any one, detects thyroid microsomal antibody concentration by chemical illumination immunity analysis instrument.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1595161A (en) * | 2004-06-22 | 2005-03-16 | 北京倍爱康生物技术股份有限公司 | Magnetic separation enzyme-linked immune detecting method for thyroid gland peroxidase antibody |
| CN1928560A (en) * | 2006-05-26 | 2007-03-14 | 深圳市新产业生物医学工程有限公司 | Immune analysis reagent for magnetic separation alkali phosphatase enzyme mark and analytical detection method for same |
| CN102072957A (en) * | 2011-01-18 | 2011-05-25 | 威海威高生物科技有限公司 | Hepatitis C virus antibody diagnostic kit and preparation method thereof |
| CN102680456A (en) * | 2011-03-16 | 2012-09-19 | 北京联众泰克科技有限公司 | ECLI (Electro ChemiLuminescence Immunoassay) determining method |
| CN103109190A (en) * | 2010-08-19 | 2013-05-15 | 霍夫曼-拉罗奇有限公司 | An assay for measurement of antibodies binding to a therapeutic monoclonal antibody |
| CN103336115A (en) * | 2013-07-17 | 2013-10-02 | 江阴泽成生物技术有限公司 | Magnetic particle chemiluminescence immune assay kit for TPOAb and detecting method thereof |
| CN104237513A (en) * | 2014-09-30 | 2014-12-24 | 博奥赛斯（天津）生物科技有限公司 | Thyroid peroxidase antibody magnetic-particle chemiluminescence immune quantitative testing kit |
-
2015
- 2015-02-10 CN CN201510068327.7A patent/CN104614535B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1595161A (en) * | 2004-06-22 | 2005-03-16 | 北京倍爱康生物技术股份有限公司 | Magnetic separation enzyme-linked immune detecting method for thyroid gland peroxidase antibody |
| CN1928560A (en) * | 2006-05-26 | 2007-03-14 | 深圳市新产业生物医学工程有限公司 | Immune analysis reagent for magnetic separation alkali phosphatase enzyme mark and analytical detection method for same |
| CN103109190A (en) * | 2010-08-19 | 2013-05-15 | 霍夫曼-拉罗奇有限公司 | An assay for measurement of antibodies binding to a therapeutic monoclonal antibody |
| CN102072957A (en) * | 2011-01-18 | 2011-05-25 | 威海威高生物科技有限公司 | Hepatitis C virus antibody diagnostic kit and preparation method thereof |
| CN102680456A (en) * | 2011-03-16 | 2012-09-19 | 北京联众泰克科技有限公司 | ECLI (Electro ChemiLuminescence Immunoassay) determining method |
| CN103336115A (en) * | 2013-07-17 | 2013-10-02 | 江阴泽成生物技术有限公司 | Magnetic particle chemiluminescence immune assay kit for TPOAb and detecting method thereof |
| CN104237513A (en) * | 2014-09-30 | 2014-12-24 | 博奥赛斯（天津）生物科技有限公司 | Thyroid peroxidase antibody magnetic-particle chemiluminescence immune quantitative testing kit |
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