CN104614535B - TMA detection kit and its preparation method and application - Google Patents

TMA detection kit and its preparation method and application Download PDF

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Publication number
CN104614535B
CN104614535B CN201510068327.7A CN201510068327A CN104614535B CN 104614535 B CN104614535 B CN 104614535B CN 201510068327 A CN201510068327 A CN 201510068327A CN 104614535 B CN104614535 B CN 104614535B
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magnetic ball
kit
anti
tma
thyroid microsomal
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CN201510068327.7A
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CN104614535A (en
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饶微
徐红
陈帆
李武
杨雅丽
李婷华
袁锦云
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深圳市新产业生物医学工程股份有限公司
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Abstract

The invention provides a kind of chemiluminescence immune detection reagent kit for being used to detect TMA and preparation method thereof, the kit includes being marked with the protein A antigens of trace labelling thing or is marked with the anti-human igg of trace labelling thing and anti-human IgM, and the attachment of the thyroid microsomal antigen and protein carrier of the thyroid microsomal antigen of coating magnetic ball or coating magnetic ball.Present invention also offers the detection method of TMA concentration.TMA concentration is detected using kit provided by the invention, is not related to enzymatic reaction, it is not necessary to put up a bridge by enzyme, therefore the reagent holding time is grown, it is stable;It can also be detected by Full-automatic chemiluminescence method, reduce the operating time, reduce manual operation error, while using the specificity of chemical tracing label, improve detection sensitivity and accuracy.

Description

TMA detection kit and its preparation method and application

Technical field

The present invention relates to a kind of detection kit, and in particular to a kind of chemistry hair for being used to detect TMA Light immunity detection reagent.It is micro- the invention further relates to the preparation method of the kit, and using kit detection thyroid gland The method of mitochondrial antibody concentration.

Background technology

TMA (TMA) is a kind of antibody for thyroid microsomal (TM), is by autoimmunity first One of autoantibody caused by shape gland disease.TAM and ATGA (TGA) have been accepted as being that thyroid gland itself is exempted from Important symbol during epidemic disease, it is most representational antibody.TAM and TGA are in the diagnosis side of AITD Face is indispensable index, is one of the limited means of histodiagnosis AITD.

For various thyroid diseases, anti-TM positive rates:Hashimoto thyroiditis is 50%~100%;Thyroid gland work( Disease can be lowered as 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 8.4% anti-TM positive rates.

For autoimmune thyroiditis (i.e. Graves disease) patient, its serum T MA levels be significantly higher than normal person and Other non-self autoimmune thyroid Diseases.Therefore, TMA level has important to antidiastole autoimmune thyroiditis Value, its accuracy rate of diagnosis of the two use in conjunction is up to 98%.

The serum T MA levels of the immunity disease patient such as Hashimoto's thyroiditis, hypothyroidism and hyperthyroidism are also significantly high In normal person, especially Hashimoto's thyroiditis is more prominent, therefore serum T MA levels are that the such disease of diagnosis " specifically refers to Mark ".

Specifically, it is as follows for some specific thyroid diseases, serum T MA horizontal properties.1. hyperthyroidism:The strong sun of TMA performances Property, antibody level is higher than Hashimoto's thyroiditis;TMA can switch to feminine gender, but the first of most clinical cures after some patientss treatment High patient TMA is determined as weakly positive for a long time;Therefore periodic review thyroid function is answered, to prevent recurrence.2. Hashimoto's thyroiditis, Addison's disease:TMA shows strong positive;Methylene inflammation patient TMA is apparently higher than normal person, less than Hashimoto's thyroiditis.It is 3. former The hair property low disease of first:TMA performances are positive, but the low disease of Secondary cases first is negative in TMA, can differentiate that Secondary cases first is low with this.4. thyroid gland Cancer:TMA increases substantially.5. pregnancy period autoimmune disease:TMA can increase.

At present, the method for clinically determining TMA mainly has ELISA, chemiluminescence immunoassay etc..However, enzyme There is many deficiencies, traditional ELISA method detection time length in linked immunosorbent assay, while rely primarily on pure sample-adding etc. by hand Serial troublesome operation, efficiency is low, easily causes experimental results error big;It is dry by outside because enzymatic reaction is not thorough enough, and easily Factor influence is disturbed, such as temperature, time and material concentration influence, therefore specificity is low during detection, poor sensitivity, detection range It is narrow.Chemiluminescence immune assay is will mutually to be tied with highly sensitive chemical luminescent detecting technology and the immune response of high specific Close, be mesh for the detection and analysis technology of various antigens, haptens, antibody, hormone, enzyme, aliphatic acid, vitamin and medicine etc. Before untill most sensitive immunoassay.However, there is Optimality currently used for chemiluminescence immunoassay detection TMA The detection kit of energy is actually rare.Therefore, this area still need badly it is a kind of can be obtained when for detecting TMA high sensitivity and Accuracy, operation are easier, additionally it is possible to the full-automatic detection TMA of detection process detection reagent is realized by analytical instrument Box.

The content of the invention

It is an object of the invention to provide a kind of chemiluminescence immune detection reagent kit for being used to detect TMA, to obtain height Detection sensitivity and accuracy.The kit can also improve the stability of detection reagent, extend the holding time of detection reagent.

The present invention also provides the preparation method of the chemiluminescence immune detection reagent kit for detecting TMA.

In addition, present invention also offers the application according to TMA chemiluminescence immune detection reagent kits provided by the invention.Especially It is to use the kit, the method that TMA detections are carried out by Full-automatic chemiluminescence method, can reduce the operating time, is dropped Low manual operation error, while using the specificity of chemical tracing label, improve detection sensitivity.

According to the present invention, there is provided a kind of chemiluminescence immunoassay detection reagent for being used to detect TMA Box, the kit include being marked with the albumin A of trace labelling thing or are marked with the anti-human igg of trace labelling thing and anti-human IgM, And the thyroid microsomal antigen or the thyroid microsomal antigen of coating magnetic ball and the attachment of protein carrier of coating magnetic ball. Wherein, the direct or indirect labelled protein A of the trace labelling thing, or directly or indirectly mark anti-human igg and anti-human IgM;It is described The attachment of thyroid microsomal antigen or thyroid microsomal antigen and protein carrier is directly or indirectly coated with magnetic ball.

Albumin A is a kind of vegetarian protein A matter, can be specifically (main with people and mammalian antibody If Fc areas IgG) combine.Thus, albumin A is combined in some way with Ago-Gel, can be prepared pure for antibody The affine filler changed.The Fab fragments (antigen-binding fragment) of anti-igg can combine with the Fc fragments (crystallizable fragment) of IgG antibody.It is anti- IgM Fab fragments (antigen-binding fragment) can combine with the Fc fragments (crystallizable fragment) of IgM antibody.

The applicable protein carrier of the present invention can be selected from least one of protein carrier commonly used in the art.For example, institute State protein carrier and be selected from bovine serum albumin(BSA) (BSA), human serum albumins (HSA), albumin rabbit serum (RSA), hemocyanin (KLH), at least one of ox IgG, human IgG, ovalbumin (OVA), myoglobins and thyroglobulin.

According to the present invention, the trace labelling thing can be selected from commonly used in the art for the tracer mark of labelled antigen or antibody Remember thing, be selected from adamantane, luminol and its derivative, different luminol and its derivative, acridinium ester, alkaline phosphatase and peppery At least one of root peroxidase, preferably N- (4- ammonia butyl)-N- ethyls different luminol (ABEI).

According to the present invention, the trace labelling thing preferably passes through 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.

" the directly mark " refers to that trace labelling thing is directly connected with antigen and is marked;" indirect labelling " refers to System trace labelling substance markers albumin A or mark anti-human igg and anti-human IgM, the middle matchmaker are linked by intermediary Jie's link system includes but is not limited to FITC and anti-FITC antibody system or SA and Biotin systems.The inventors discovered that indirectly It is marked with beneficial to three-dimensional effect is weakened, is advantageous to the amplification of signal so that detection is sensitiveer.

According to the present invention, the thyroid microsomal antigen (or its attachment with protein carrier) preferably passes through different sulphur cyanogen Sour fluorescein is coated with magnetic ball indirectly with anti-fluorescein isothiocynate Antibody System or Streptavidin with biotin system.

" direct coated " refers to directly wrap magnetic ball using TM antigens (or its attachment with protein carrier) Quilt;" coating indirectly " refers to link system by intermediary so that TM antigens (or its attachment with protein carrier) Magnetic ball is coated with, intermediary link system include but is not limited to FITC and anti-FITC antibody system or SA with Biotin systems.Equally, the advantages of being coated with indirectly is, is advantageous to weaken three-dimensional effect, is advantageous to the amplification of signal so that inspection Survey sensitiveer.

The present invention some specific embodiments in, the kit include be marked with trace labelling thing albumin A or It is marked with the anti-human igg of trace labelling thing and anti-human IgM, and a kind of component in following components:A) direct coated magnetic The thyroid microsomal antigen (or its attachment with protein carrier) of ball;B) biotinylated thyroid microsomal antigen (or Its attachment with protein carrier) and direct coated magnetic ball Streptavidin;C) it is directly labeled with fluorescein isothiocynate Thyroid microsomal antigen (or its attachment with protein carrier) and the goat-anti fluorescein isothiocynate of direct coated magnetic ball resist Body.

TMA low spot calibration object and high point calibration object can also be included according to kit provided by the invention, and optionally Including buffer solution.Low spot calibration object of the present invention and high point calibration object be both comparatively, wherein " low spot calibration object ", is It is the calibration object that 0-30IU/ml is obtained that TMA is diluted to concentration by finger with 50% cow's serum product;And " high point calibration object " refer to by It is the calibration object that 500-1000IU/ml is obtained that TMA antigens are diluted to concentration with 50% cow's serum product.

Magnetic bead is also referred to as suitable for the magnetic ball of the present invention, can be magnetic microsphere commonly used in the art.Preferably, originally The magnetic ball used is invented, is by nano level Fe2O3Or Fe3O4Magnetic particle and high-molecular organic material carry out compound, formation tool There is the micron-sized solid phase microballoon of superparamagnetism and huge amount protein adsorption capacity, having can quilt rapidly under additional magnetic fields Magnetization, the attribute that remanent magnetism is zero after magnetic field is withdrawn.Wherein, the species of the high-molecular organic material is not particularly limited, can Selected as needed.

Magnetic microsphere used in the present invention should be able to meet a diameter of 0.1-5 μm, and magnetic microsphere can also be changed by surface Property and carry various active functional group, include but is not limited to-OH ,-COOH ,-NH2

In a specific embodiment, the magnetic ball is Fe2O3Or Fe3O4Magnetic nano-particle and high-molecular organic material Complex, and with 0.1-5 μm of particle diameter.

According to the present invention, antibody can be monoclonal antibody or polyclonal antibody.

Present invention also offers a kind of method for preparing kit as described above, the described method comprises the following steps: I) the direct or indirect labelled protein A of trace labelling thing, or directly or indirectly mark anti-human igg and anti-human IgM are utilized;And ii) will Thyroid microsomal antigen (or its attachment with protein carrier) is directly or indirectly coated with magnetic ball;Wherein, the indirect labelling Including trace labelling thing is passed through into fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and life Thing ferritic system labelled protein A or mark anti-human igg and anti-human IgM;It is described indirectly coating include by thyroid microsomal antigen (or Its attachment with protein carrier) pass through fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin It is coated with magnetic ball indirectly with biotin system.

The preparation of low spot calibration object and high point calibration object can also be included according to the reagent box preparation method of the present invention, may be used also To further comprise the assembling of kit.

According to the present invention, a kind of method of detection TMA concentration is additionally provided, methods described carries including the use of according to the present invention The kit as described above supplied, is detected by Chemiluminescence immunoassay to the TMA concentration in testing sample.

Specifically, detection TMA concentration detection methods provided by the invention include by sample to be tested, directly or indirectly mark have The albumin A of trace labelling thing is marked with the anti-human igg of trace labelling thing and anti-human IgM, and directly or indirectly coating magnetic ball TMA or TMA mixed with the attachment of protein carrier, make TMA, albumin A or anti-human igg to be measured and anti-human IgM and coating magnetic ball TMA or the attachment of itself and protein carrier form " sandwich sandwich " shape immune complex, addition excites substrate, and measure is luminous The standard curve of intensity, reference TMA calibration objects concentration and luminous intensity, calculate the TMA concentration of testing sample.

In one embodiment, the detection TMA methods are including the use of according to reagent as described above provided by the invention Box, TMA concentration is detected by chemical illumination immunity analysis instrument.In a preferred embodiment in accordance with this invention, methods described Fully automatically carry out.According to the present invention, the chemical illumination immunity analysis instrument is preferably Maglumi sequence of chemical electrochemiluminescent immunoassay point Analyzer (production of Shenzhen NPD projects biomedical engineering limited company).

The beneficial effects of the present invention are:

1. specific height, sensitivity is good, accuracy is high, detection range is wider.

2. operation is more simple and easy to do, while the kit of the present invention can be with chemical illumination immunity analysis instrument (especially Maglumi sequence of chemical luminescence immunoassays instrument) support the use, full-automation is realized during sample measures so that TMA The detection of concentration can be carried out simply, easily and fast, in bulk, while ensure that the systematic error of detection is smaller.

3. the present invention detects TMA by Chemiluminescence immunoassay, the method detection of conventional ELISA is avoided Time is long, efficiency is low, easily causes that experimental results error is big, easily influenceed by external interference factor, specific low, poor sensitivity etc. Shortcoming;And it need not be put up a bridge by enzyme, therefore the reagent holding time is grown, it is stable.

Brief description of the drawings

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

The present invention is made further explanation and description below in conjunction with non-limiting example.It should be noted, however, that It is that following detailed description of the invention does not make any limitation to the present invention.Those skilled in the art should understand that , the details and form of technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the invention Change, but these modifications and replacement are each fallen within protection scope of the present invention.

In following examples:

It is Bioisystech Co., Ltd that it is refined, which to be purchased from Shenzhen, for albumin A;

The anti-human igg and anti-human IgM for being marked with trace labelling thing are purchased from Medix companies

TM antigens are purchased from Meridian companies of the U.S.;

Goat-anti FITC polyclonal antibodies are purchased from Jackson companies of the U.S.;

Magnetic microsphere, ABEI produce by Shenzhen NPD projects biomedicine limited company;

FITC is purchased from Sigma Co., USA;

Biotin and Streptavidin are purchased from Biosources companies of the U.S.;

TMA calibration objects are purchased from Biodesign companies of the U.S.;

The chemical illumination immunity analysis instruments of Maglumi 2000:Shenzhen NPD projects biomedical engineering limited company gives birth to Production.

Sample to be tested:120 clinical serum samples of Beijing University's Shenzhen hospital are derived from, wherein at least 7 are thyroid function The serum sample of disease patient diagnosed.

Embodiment 1

(1) mark of albumin A

The preparation of dialyzate (F solution):Na is added in 5000ml beakers2CO314.31g NaHCO326.46g add purifying Water is diluted to 4500ml.The F solution prepared is placed in standby on magnetic stirring apparatus.

From the bag filter of suitable interception (conventional 14000), measure suitably sized, one end is tightened after wetting, is purified Water leak test 3 times (need to be without leakage).

100 μ g albumin As are taken to be adjusted to 1ml with 0.1mol/L pH9.5 carbonic acid buffer (F solution).It is put into dialyzate In, dialysis 2 hours is stirred at room temperature, the solution dialysed is added into 300 μ g ABEI Acibenzolars, 37 DEG C are reacted 2 hours.

Pass through G-25 gel column purifying proteins A and ABEI connection product.

D2The preparation of solution:Add 200ml 0.5M phosphate buffers (P001 solution), 20g BSA, 8g NaN3、2g MgCl2·6H2O, 600ml glycerine, purified water constant volume is added to 2000ml, filtering.

By purified connection product D2Solution dilutes, and its concentration is calculated as 0.025 μ g/ml with albumin A.

(2) coating of TM antigens

The preparation of solution A:Weigh after 2.55g sodium acetate trihydrates are dissolved with 4500ml purified waters and add 14ml acetic acid and mix After even, 5000ml (pH 3.6) is settled to.

The solution A of 5 times of coating volumes is added into little Bai bottles, is put into ultrasonic instrument ultrasound on one side while stirring is clear 2-3 minutes are washed, are subsequently placed on magnet, after supernatant is limpid, pour out supernatant.The step is in triplicate.

Magnetic ball is added into the pH3.6 acetate buffer solutions with being coated with volume equivalent, the suspended concentration for making magnetic ball is 20mg/ml, 1- cyclohexyl -2- morpholine ethyl carbodiimide tosilate (CMC) is added, it is 10mg/ml to make its concentration, by certain ratio Rate adds the TM antigens of purifying.

The mixed solution of magnetic ball and TM antigens is put into 37 DEG C of reactions, 24 hours (shaking water bath pots in isothermal vibration water bath Shake speed:260rpm).

PH7.4 phosphate buffers (PBS) are prepared, 2.5g BSA dissolvings is added, mixes, as magnetic ball cleaning fluid.

The mixed solution of the good magnetic ball of warm bath and TM antigens is poured into beaker, is subsequently placed in after being precipitated on magnet, outwells Supernatant, the magnetic ball cleaning fluid stirring and washing of 5 times of volumes is added, is then placed within magnet, supernatant is outwelled after supernatant is limpid Liquid, repeats the cleaning step four times.

C solution is prepared:Weigh methylcellulose (MC) 160g to pour into 5000ml beakers, add purified water to 4000ml, extremely Heat and stir molten 2 hours in 90 DEG C of water bath.It is another to take 4000ml 0.5M phosphate buffers, add 80g NaN3(analysis It is pure), 80ml T-20 (analysis is pure), mix, filtering.200g BSA are added after this two parts of solution are fully mixed, are added water to 40000ml。

Magnetic ball after cleaning is suspended in C solution, suspended concentration 20mg/ml, that is, obtains TM antigen coats Magnetic ball, this suspension vol are that volume is coated with described in the present embodiment.

It is standby that the suspension of the magnetic ball of TM antigen coats is further diluted to 0.5mg/ml.

(3) TMA low spots calibration object, high point calibration object

TMA standard items are diluted into concentration by different proportion with calibration object dilution (50% cow's serum product) is respectively 591.58IU/ml and 25.36IU/ml two high and low calibration object scaling points.

(4) react:Above-mentioned sample to be tested, height calibration object are mixed with mark objects system and magnetic spheroid system respectively, temperature Educate, make the TMA in sample to be tested be combined to obtain reaction product with the albumin A marked through ABEI.

Specifically load procedure is:10 μ l samples to be tested or height calibration object are added, then adds 20 μ l coating TM antigens Magnetic ball solution (above-mentioned steps (2) preparation), reaction 10 minutes after clean, add 100 μ l ABEI mark Protein A solution (on State step (1) preparation), form compound.

(5) detect:Externally-applied magnetic field precipitates above-mentioned reaction product, remove supernatant, and with buffer solution for cleaning after, additionization Learn luminous excimer (NaOH and H2O2), the relative light intensity sent using the detection of the chemical illumination immunity analysis instruments of Maglumi 2000 Spend (concrete operation step may be referred to the chemical illumination immunity analysis instrument specifications of Maglumi 2000), it is to be measured by being calculated The TMA contents of sample.It the results are shown in Table shown in 1.

Embodiment 2

(1) albumin A biotinylation, comprise the following steps that:

The preparation of dialyzate (F solution):Na is added in 5000ml beakers2CO314.31g NaHCO326.46g add purifying Water is diluted to 4500ml.The F solution prepared is placed in standby on magnetic stirring apparatus.

100 μ g biotins and 1mg albumin As is taken to be adjusted to 1ml with 0.1mol/L pH9.5 carbonic acid buffer (F solution). It is put into dialyzate, dialysis 2 hours is stirred at room temperature, 37 DEG C is reacted 2 hours.

Purified by G-25 gel columns.

D2 solution, purified connection product D are prepared according to the method for embodiment 12Solution dilutes, and makes its concentration be 0.025μg/ml。

(2) SA mark

100 μ g SA are taken to be adjusted to 1ml with 0.1mol/L pH9.5 carbonic acid buffer (F solution).It is put into dialyzate, Dialysis 2 hours is stirred at room temperature, the solution dialysed is added into 300 μ g ABEI Acibenzolars, 37 DEG C are reacted 2 hours.

Use G-25 gel columns purifying SA and ABEI connection product.

Purified connection product D2Solution dilutes, and its concentration is calculated as 10 μ g/ml with SA, standby.

(3) TM antigen coats magnetic ball

Method according to embodiment 1 prepares solution A.

The solution A of 5 times of coating volumes is added into little Bai bottles, is put into ultrasonic instrument ultrasound on one side while stirring is clear 2-3 minutes are washed, are subsequently placed on magnet, after supernatant is limpid, pour out supernatant.The step is in triplicate.

Magnetic ball is added to the pH3.6 acetate buffer solutions of coating volume equivalent, the suspended concentration for making magnetic ball is 20mg/ml, then CMC is added, it is 10mg/ml to make its concentration, adds the TM antigens of purifying.

Magnetic ball suspension is put into 37 DEG C of reactions, 24 hours (shaking water bath pot shake speeds in isothermal vibration water bath: 260rpm)。

PH7.4PBS buffer solution 500ml are prepared, 2.5g BSA is added and mixes dissolving, as magnetic ball cleaning fluid.

The good magnetic ball suspension of warm bath is poured into beaker, is subsequently placed in after being precipitated on magnet, outwells supernatant, adds 5 times The magnetic ball cleaning fluid stirring and washing of volume, is then placed within magnet, and supernatant is outwelled after supernatant is limpid, repeats the cleaning Step 4 time.

The magnetic ball of TM antigen coats after cleaning is suspended in the C solution prepared according to embodiment 1, suspended concentration For 20mg/ml, that is, the magnetic ball suspension suspension of TM antigen coats is obtained, this suspension vol is that body is coated with described in this step Product.

Magnetic ball suspension is further diluted to 0.5mg/ml, it is standby.

(4) TMA low spots calibration object, high point calibration object

It is 591.58IU/ml and 15.298IU/ that TMA antigens are diluted into concentration by different proportion with 50% cow's serum product The high and low calibration object scaling points of ml two.

(5) react:Above-mentioned sample to be tested or height calibration object are mixed with mark objects system and magnetic microsphere system respectively Close, incubate, make the TMA in sample to be tested be combined to obtain reaction product with through biotinylated albumin A.Tool Body load procedure is:10 μ l samples to be tested or height calibration object are added, then adds the magnetic ball solution of 20 μ l coating TM antigens (above-mentioned steps (3) prepare gained), reaction add 50 μ l SA solution after being cleaned after 10 minutes (above-mentioned steps (2) prepare gained) With the 50 biotinylated Protein A solutions of μ l (above-mentioned steps (1) prepare gained), compound is formed.

(6) detect:Externally-applied magnetic field precipitates above-mentioned reaction product, remove supernatant, and with buffer solution for cleaning after, additionization Learn luminous excimer (NaOH and H2O2), the relative light intensity sent using the detection of the chemical illumination immunity analysis instruments of Maglumi 2000 Degree, passes through the TMA contents being calculated in sample to be tested.It the results are shown in Table shown in 1.

Embodiment 3

(1) mark of albumin A

The preparation of dialyzate (F solution):Na is added in 5000ml beakers2CO314.31g NaHCO326.46g add purifying Water is diluted to 4500ml.The F solution prepared is placed in standby on magnetic stirring apparatus.

From the bag filter of suitable interception (conventional 14000), measure suitably sized, one end is tightened after wetting, is purified Water leak test 3 times (need to be without leakage).

100 μ g albumin As are taken to be adjusted to 1ml with 0.1mol/L pH9.5 carbonic acid buffer (F solution).It is put into dialyzate In, dialysis 2 hours is stirred at room temperature, the solution dialysed is added into 300 μ g ABEI Acibenzolars, 37 DEG C are reacted 2 hours.

Pass through G-25 gel column purifying proteins A and ABEI connection product.

D2The preparation of solution:Add 200ml 0.5M phosphate buffers (P001 solution), 20g BSA, 8g NaN3、2g MgCl2·6H2O, 600ml glycerine, purified water constant volume is added to 2000ml, filtering.

By purified connection product D2Solution dilutes, and its concentration is calculated as 0.025 μ g/ml with albumin A.

(2) the FITC marks of TM antigens

100 μ g TM antigens are taken to be adjusted to 1ml with 0.1mol/L pH9.5 carbonic acid buffer (F solution).It is put into dialyzate In, dialysis 2 hours is stirred at room temperature, the solution dialysed is added into 300 μ g FITC Acibenzolars, 37 DEG C are reacted 2 hours.

TM antigens and FITC connection product are purified by G-25 gel columns.

By purified connection product D2Solution dilutes, and makes its concentration 0.1 μ g/ml in terms of TM antigens.

(3) goat-anti FITC polyclonal antibodies coating magnetic ball

Weigh after 2.55g sodium acetate trihydrate 4500ml purified waters dissolve again plus after such as 14ml acetic acid mixing, with purifying Water is settled to 5000ml (pH 3.6).

The solution A of 5 times of coating volumes is added into little Bai bottles, is put into ultrasonic instrument ultrasound on one side while stirring is clear 2-3 minutes are washed, are subsequently placed on magnet, after supernatant is limpid, pour out supernatant.The step is in triplicate.

Magnetic ball is added in the pH3.6 acetate buffer solutions of coating volume equivalent, it is 20mg/ml to make magnetic ball suspended concentration, then CMC is added, it is 10mg/ml to make its concentration, and the goat-anti FITC polyclonal antibodies of purifying are added by certain ratio.

It is small that the mixed solution of magnetic ball and goat-anti FITC polyclonal antibodies is put into 37 DEG C of reactions 24 in isothermal vibration water bath When (shaking water bath pot shake speed:260rpm).

PH7.4PBS buffer solutions are prepared, 2.5g BSA dissolvings is added, mixes, as magnetic ball cleaning fluid.

The mixed solution of the good magnetic ball of warm bath and goat-anti FITC polyclonal antibodies is poured into beaker, is subsequently placed on magnet After precipitation, supernatant is outwelled, the magnetic ball cleaning fluid stirring and washing of 5 times of volumes is added, is then placed within magnet, treats that supernatant is limpid After outwell supernatant, repeat the cleaning step four times.

C solution is prepared:Weigh methylcellulose (MC) 160g to pour into 5000ml beakers, add purified water to 4000ml, extremely Heat and stir molten 2 hours in 90 DEG C of water bath.It is another to take 4000ml 0.5M phosphate buffers, add 80g NaN3(analysis It is pure), 80ml T-20 (analysis is pure), mix, filtering.200g BSA are added after this two parts of solution are fully mixed, are added water to 40000ml。

The coated magnetic ball of goat-anti FITC polyclonal antibodies after cleaning is suspended in the C solution of step (3) preparation, Suspended concentration is 20mg/ml, that is, obtains the coated magnetic ball suspension of goat-anti FITC polyclonal antibodies, and this suspension vol is this Volume is coated with described in step.

The suspension of the coated magnetic ball of goat-anti FITC polyclonal antibodies is further diluted to 1mg/ml, it is standby.

(4) TMA low spots calibration object, high point calibration object

TMA standard items are diluted into concentration by different proportion with calibration object dilution and 50% cow's serum product is respectively 591.58IU/ml and 25.36IU/ml two high and low calibration object scaling points.

(5) react:Above-mentioned sample to be tested or height calibration object are mixed with mark objects system and magnetic microsphere system respectively Close, incubate, make the TMA in sample to be tested be combined to obtain reaction product with the albumin A marked.Specific sample-adding Step is:10 μ l samples to be tested or height calibration object are added, adds the FITC solution (above-mentioned steps of 40 μ l mark TM antigens (2) gained is prepared), then adding the 20 coated magnetic ball solution of μ l goat-anti FITC polyclonal antibodies, (above-mentioned steps (3) prepare institute ), the Protein A solution (above-mentioned steps (1) prepare gained) for adding ABEI marks, formation compound are cleaned in reaction after 10 minutes.

(6) detect:Externally-applied magnetic field precipitates above-mentioned reaction product, remove supernatant, and with buffer solution for cleaning after, additionization Learn luminous excimer (NaOH and H2O2), the relative light intensity sent using the detection of the chemical illumination immunity analysis instruments of Maglumi 2000 Degree, passes through the TMA contents being calculated in sample to be tested.It the results are shown in Table shown in 1.

Embodiment 4

(1) anti-human igg and anti-human IgM mark

(1.1) mark of anti-human igg

The preparation of dialyzate (F solution):Na is added in 5000ml beakers2CO314.31g NaHCO326.46g add purifying Water is diluted to 4500ml.The F solution prepared is placed in standby on magnetic stirring apparatus.

From the bag filter of suitable interception (conventional 14000), measure suitably sized, one end is tightened after wetting, is purified Water leak test 3 times (need to be without leakage).

100 μ g anti-human igg are taken to be adjusted to 1ml with 0.1mol/L pH9.5 carbonic acid buffer (F solution).It is put into dialyzate In, dialysis 2 hours is stirred at room temperature, the solution dialysed is added into 300 μ g ABEI Acibenzolars, 37 DEG C are reacted 2 hours.

Anti-human igg and ABEI connection product are purified by G-25 gel columns.

D2The preparation of solution:Add 200ml 0.5M phosphate buffers (P001 solution), 20g BSA, 8g NaN3、2g MgCl2·6H2O, 600ml glycerine, purified water constant volume is added to 2000ml, filtering.By purified connection product D2Solution is dilute Release, its concentration is calculated as 0.025 μ g/ml with anti-human igg.

(1.2) anti-human IgM mark

The preparation of dialyzate (F solution):Na is added in 5000ml beakers2CO314.31g NaHCO326.46g add purifying Water is diluted to 4500ml.The F solution prepared is placed in standby on magnetic stirring apparatus.

From the bag filter of suitable interception (conventional 14000), measure suitably sized, one end is tightened after wetting, is purified Water leak test 3 times (need to be without leakage).

The 100 anti-human IgM of μ g are taken to be adjusted to 1ml with 0.1mol/L pH9.5 carbonic acid buffer (F solution).It is put into dialyzate In, dialysis 2 hours is stirred at room temperature, the solution dialysed is added into 300 μ g ABEI Acibenzolars, 37 DEG C are reacted 2 hours.

Anti-human IgM and ABEI connection product is purified by G-25 gel columns.

D2The preparation of solution:Add 200ml 0.5M phosphate buffers (P001 solution), 20g BSA, 8g NaN3、2g MgCl2·6H2O, 600ml glycerine, purified water constant volume is added to 2000ml, filtering.

By purified connection product D2Solution dilutes, and its concentration is calculated as 0.025 μ g/ml with anti-human IgM.

(2) coating of TM antigens

The preparation of solution A:Weigh after 2.55g sodium acetate trihydrates are dissolved with 4500ml purified waters and add 14ml acetic acid and mix After even, 5000ml (pH 3.6) is settled to.

The solution A of 5 times of coating volumes is added into little Bai bottles, is put into ultrasonic instrument ultrasound on one side while stirring is clear 2-3 minutes are washed, are subsequently placed on magnet, after supernatant is limpid, pour out supernatant.The step is in triplicate.

Magnetic ball is added into the pH3.6 acetate buffer solutions with being coated with volume equivalent, the suspended concentration for making magnetic ball is 20mg/ml, 1- cyclohexyl -2- morpholine ethyl carbodiimide tosilate (CMC) is added, it is 10mg/ml to make its concentration, by certain ratio Rate adds the TM antigens of purifying.

The mixed solution of magnetic ball and TM antigens is put into 37 DEG C of reactions, 24 hours (shaking water bath pots in isothermal vibration water bath Shake speed:260rpm).

PH7.4 phosphate buffers (PBS) are prepared, 2.5g BSA dissolvings is added, mixes, as magnetic ball cleaning fluid.

The mixed solution of the good magnetic ball of warm bath and TM antigens is poured into beaker, is subsequently placed in after being precipitated on magnet, outwells Supernatant, the magnetic ball cleaning fluid stirring and washing of 5 times of volumes is added, is then placed within magnet, supernatant is outwelled after supernatant is limpid Liquid, repeats the cleaning step four times.

C solution is prepared:Weigh methylcellulose (MC) 160g to pour into 5000ml beakers, add purified water to 4000ml, extremely Heat and stir molten 2 hours in 90 DEG C of water bath.It is another to take 4000ml 0.5M phosphate buffers, add 80g NaN3(analysis It is pure), 80ml T-20 (analysis is pure), mix, filtering.200g BSA are added after this two parts of solution are fully mixed, are added water to 40000ml。

Magnetic ball after cleaning is suspended in C solution, suspended concentration 20mg/ml, that is, obtains TM antigen coats Magnetic ball, this suspension vol are that volume is coated with described in the present embodiment.

It is standby that the suspension of the magnetic ball of TM antigen coats is further diluted to 0.5mg/ml.

(3) TMA low spots calibration object, high point calibration object

TMA standard items are diluted into concentration by different proportion with calibration object dilution (50% cow's serum product) is respectively 591.58IU/ml and 25.36IU/ml two high and low calibration object scaling points.

(4) react:Above-mentioned sample to be tested, height calibration object are mixed with mark objects system and magnetic spheroid system respectively, temperature Educate, the TMA in sample to be tested is obtained reaction production with being combined through the anti-human igg that ABEI is marked with anti-human IgM Thing.

Specifically load procedure is:10 μ l samples to be tested or height calibration object are added, then adds 20 μ l coating TM antigens Magnetic ball solution (above-mentioned steps (2) preparation), reaction is cleaned after 10 minutes, each to add 50 μ l mark anti-human igg and anti-human IgM's ABEI solution (above-mentioned steps (1) preparation), form compound.

(5) detect:Externally-applied magnetic field precipitates above-mentioned reaction product, remove supernatant, and with buffer solution for cleaning after, additionization Learn luminous excimer (NaOH and H2O2), the relative light intensity sent using the detection of the chemical illumination immunity analysis instruments of Maglumi 2000 Spend (concrete operation step may be referred to the chemical illumination immunity analysis instrument specifications of Maglumi 2000), it is to be measured by being calculated The TMA contents of sample.It the results are shown in Table shown in 1.

Comparative example 1

Above-mentioned 120 samples to be tested are detected using existing main flow commercialization enzyme linked immunological kit on the market, And contrasted with kit testing result provided by the invention.As a result 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, "-" represents negative (normal).

Understood from what Fig. 1~3 of upper table, 120 clinical samples of this clinical comparison experiment selection, wherein at least 3,14, 28th, 39,50,62, No. 118 samples are thyroid function disease patient diagnosed.For other samples, using the kit of the present invention It is consistent with the testing result for exempting from kit using enzyme.However, for above-mentioned seven Patient Sample As made a definite diagnosis, the inspection-free survey TMA of enzyme shows It is shown as negative, using kit provided by the invention and chemical luminescence detection method test positive, it means that using this hair The technical scheme of bright offer can greatly avoid enzyme from exempting from the detection leakage phenomenon to TMA of platform.In addition, as shown in Figure 1, embodiment 1 and embodiment 4 data dependence it is more consistent, show using mark ABEI albumin A and using mark ABEI mark has The anti-human igg of trace labelling thing is consistent with anti-human IgM effects.From Fig. 1-3, using the measure of embodiment 1-4 kit As a result there is good uniformity.As can be seen here, using the detection knot of TMA detection kits provided by the invention and detection method Fruit is more accurate, it is sensitive, have specificity, can more react clinical truth, thus there is higher application value.

Although the present invention has been described in detail, it will be understood by those skilled in the art that in spirit and scope of the invention Modification will be apparent.However, it should be understood that each side of the invention recorded, different embodiments Each several part and the various features enumerated can be combined or all or part of exchange.In above-mentioned each embodiment, that A little embodiments with reference to another embodiment can be combined suitably with other embodiment, and this is by by this area skill Art personnel are to understand.In addition, it will be understood to those of skill in the art that description above is only the mode of example, not purport In the limitation present invention.

Claims (8)

1. a kind of chemiluminescence immune detection reagent kit for being used to detect TMA, the kit include mark There is the albumin A of trace labelling thing, and the thyroid microsomal of the thyroid microsomal antigen of coating magnetic ball or coating magnetic ball resists The former attachment with protein carrier,
The trace labelling thing in luminol and its derivative, different luminol and its derivative and acridinium ester at least one Kind;
The magnetic ball is Fe2O3Or Fe3O4The complex of magnetic nano-particle and high-molecular organic material.
2. kit according to claim 1, it is characterised in that the protein carrier is selected from bovine serum albumin(BSA), human serum In albumin, albumin rabbit serum, hemocyanin, ox IgG, human IgG, ovalbumin, myoglobins and thyroglobulin It is at least one.
3. kit according to claim 1, it is characterised in that the trace labelling thing is N- (4- ammonia butyl)-N- second Base different luminol.
4. kit according to claim 1, it is characterised in that
The direct or indirect labelled protein A of trace labelling thing, wherein indirect labelling are included by fluorescein isothiocynate with resisting Fluorescein isothiocynate Antibody System or Streptavidin and biotin system indirect labelling;
The attachment of the thyroid microsomal antigen or thyroid microsomal antigen and protein carrier is directly or indirectly coated with magnetic Ball, wherein indirectly coating include by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin with Biotin system is coated with indirectly.
5. kit according to claim 1, it is characterised in that the magnetic ball has 0.1-5 μm of particle diameter.
6. kit according to claim 4, it is characterised in that the kit includes the egg for being marked with trace labelling thing White Staphylococal Protein A, and a kind of component in following components:
A) thyroid microsomal antigen of the thyroid microsomal antigen of direct coated magnetic ball or direct coated magnetic ball carries with albumen The attachment of body;
B) attachment of biotinylated thyroid microsomal antigen or thyroid microsomal antigen and protein carrier, and directly It is coated with the Streptavidin of magnetic ball;
C) thyroid microsomal antigen or thyroid microsomal antigen and protein carrier of fluorescein isothiocynate are directly labeled with Attachment, and the goat-anti fluorescein isothiocynate antibody of direct coated magnetic ball.
7. according to the kit described in any one in claim 1-6, it is characterised in that the kit also includes thyroid gland The low spot calibration object and high point calibration object of microsomal antibody, and optionally include buffer solution.
8. a kind of method for being used to prepare the kit as described in any one in claim 1-7, methods described include following Step:
I) the direct or indirect labelled protein A of trace labelling thing is utilized;With
Ii the attachment of thyroid microsomal antigen or thyroid microsomal antigen and protein carrier directly or indirectly) is coated with magnetic Ball;
Wherein,
The indirect labelling includes trace labelling thing passing through fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System Or Streptavidin and biotin system labelled protein A,
The coating indirectly includes leading to the attachment of thyroid microsomal antigen or thyroid microsomal antigen and protein carrier Cross fluorescein isothiocynate and be coated with magnetic indirectly with biotin system with anti-fluorescein isothiocynate Antibody System or Streptavidin Ball.
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