CN108414766A

CN108414766A - Kit for quantitatively detecting diabetes autoantibody and its application

Info

Publication number: CN108414766A
Application number: CN201810085130.8A
Authority: CN
Inventors: 孙玉龙; 杨亚云; 何林富; 王�义
Original assignee: SHANGHAI LIANGRUN BIOLOGICAL PHARMACEUTICAL TECHNOLOGY Co Ltd
Current assignee: SHANGHAI LIANGRUN BIOLOGICAL PHARMACEUTICAL TECHNOLOGY Co Ltd
Priority date: 2018-01-29
Filing date: 2018-01-29
Publication date: 2018-08-17

Abstract

The present invention relates to a kind of for the quantitatively kit of detection diabetes autoantibody and its application, kit, which includes coupling, the magnetic particle of Streptavidin, biotinylation capture antigen, the detection antigen and substrate exciting liquid of acridinium ester label, wherein antigen are selected from one or more of 8 antigen protein of zinc transporter, glutamate decarboxylase antigen protein, tyrosine phosphatase antigen protein, pancreatic island cell antigen albumen and insulin antigen protein；The kit can be used for accurately measuring the diabetes autoantibody content in biological sample, to provide foundation for the auxiliary of type 1 diabetes and antidiastole, and applied to the early screening of youngsters and children diabetes autoantibody.Quantitative combined detection kit of the present invention have many advantages, such as it is easy, sensitive, stable, reproducible, with practical value.

Description

Kit for quantitatively detecting diabetes autoantibody and its application

Technical field

The invention belongs to diabetes diagnosis field, it is related to, for quantitatively detecting the kit of diabetes autoantibody, also relating to And application of the kit in diagnosing diabetes.

Background technology

Diabetes are as the third-largest non-communicable diseases at present after angiocardiopathy and tumour, it has also become serious prestige Coerce the worldwide public health problem of human health.There are about 10% in diabetic population to belong to type 1 diabetes (TIDM), China Large population base, therefore the absolute number of cases of such patient is more.TIDM is mediated by autoreactive T cell, with B cell Selective destruction is characterized, and the usual onset age is lighter, and faster, blood glucose is more difficult to control, is easy out for the speed of islet function failure Existing diabetic complication, therefore the key treated is to early diagnose or screening goes out this kind of patient and give appropriate intervention to arrange It applies, delays or even prevent islet function failure, to reduce complication generation and quality of making the life better.

T1DM is a kind of autoimmune disease, mainly due to the beta Cell of islet in pancreas by autoimmune destruction, Caused by leading to pancreas element hyposecretion, therefore it can show occur itself for being directed to beta Cell of islet relevant molecule in peripheral blood Antibody.Immune system may need several months or several years to make a response the β cell proteins of body generation insulin, fasting blood syrup Putting down can just increase in the β cell damages of the moon 80%.Therefore, extensive Risk Screening is to the identification of early stage β cell damage and pre- Prediction is essential afterwards.In New-Onset Diabetes Mellitus people, anti-testing result is to differentiate 1 type and non-self immunity diabetes such as The most important standard of diabetes B.Islet cell autoantibodies are identification initial stage or already present autoimmune process, detection The best marker of disease process.Childhood, adolescence and the first time type 1 diabetes in adolescence (25 years old or less) itself it is anti- The screening of body should include that CLIA, ELISA, RIA or IFT are detected, in order to assess autoantibody reaction.It should regularly (1-3 Year, depend on age and diabetes risk) these parameters, especially Children and teenager are monitored, because of their autoantibody Reactions change is more preferably frequently quick.90% type 1 diabetes just can detect a kind or several before symptom occurs in clinic in serum Diabetes related auto-antibodies.Autoantibody reaction more more early more serious (positive islet cell autoantibodies quantity, autoantibody Affinity and autoantibody titer), diabetes risk is higher.Since the 1970s, scientific researchers have been found that A variety of islet autoantibodies.The main antibody for having been used for detection T1DM at present has GADA (glutamic acid decarboxylase antibody), IA2A (Protein tyrosine phosphatase antibodies), IAA (insulin autoantibodies) and ICA (islet cell antibodies), these antibody are 1 type sugar The important immunological marker object of urine disease diagnosis.In addition ZnT8A has height as a kind of new islet autoantibody to beta Cell of islet The specificity of degree, is also increasingly being applied in the research of type 1 diabetes.

In current existing detection architecture, the diagnostic significance of single autoantibody detection all has certain limitation. Such as the positive rate of IAA detections increases with the age and can be decreased obviously, the positive rate in 30 years old or more patient is significantly lower than 10 years old Child patient below.Positive rates of the GADA in newly hair TIDM is higher and antibody existence time is longer, but it is not pancreas islet Cell high special also can detect in other relevant autoimmune diseases of thyroid gland and rheumatism.Type 1 diabetes patient Positive rate is high at the initial stage of a disease for the detection of ICA, and general population is only 0.15%, but it is short to hold time, clinical onset several weeks extremely In several months, proceeding by property of ICA concentration declines.IA2A is later in the time that patient's body occurs, and positive rate also can be with the course of disease Extend and declines.Studies have found that ZnT8 antibody newly sends out type 1 diabetes people, diabetes B people and normal person in Caucasian Recall rate is respectively 60-80% in group,<3% and<2%, specificity is up to 95-99%.Some researches show that ZnT8 in for the country Positive rate is 24.5% respectively in compatriots T1DM patient, with the 27.8% of Japan Report without significant difference, but less than in report Caucasian, it may be possible to since the ethnic difference opposite sex causes.And joint-detection ZnT8, GADA, IA-2A in initial patient T1DM 98% is can reach with the positive rate of IAA autoantibodies, illustrates that the diagnostic of different autoantibodies can be complementary, joint-detection can Improve the clinical efficacies indexs such as the sensitivity and specificity of detection.

At present for the product of type 1 diabetes autoantibody joint-detection, it is only limitted to the qualitative detection to each autoantibody, Including protein chip and Strip Immunoblot method, it is only capable of reflecting autoantibody and whether there is and can not accomplish quantitative detection, This makes the accuracy of detection be restricted, while sensitivity is also limited to.For in detection method, we have abandoned tradition Indirect method, because the indirect method person of presence itself is unconquerable：First, sensitivity is low, because enzyme marker is that enzyme mark is anti-human IgG, in order to avoid non-specific adsorption, serum specimen necessary 1:10 or 1:20 or more are diluted, reaction sensitivity be not required to The dual-antigen sandwich method of dilute serum sample be easy to cause missing inspection than low 10-20 times；Secondly, poor specificity, even if serum passes through 1:10-1:20 dilutions, IgG in individual samples still has a non-specific adsorption to coating micro reaction plate, and enzyme mark anti-human igg pair The human IgG of diabetes autoantibody and the other manner absorption of specific adsorption necessarily causes a part of false sun without resolution capability Property.Therefore the present invention is based on Magnetism particulate immuno chemistry luminescence methods to realize that diabetes autoantibody quantifies joint-detection, high-efficient simple it is real Quantitatively detected while existing GADA, IAA, IA2A, ICA and ZnT8A, be the auxiliary of type 1 diabetes and antidiastole improve according to According to, and applied to the early screening of youngsters and children diabetes autoantibody.

Invention content

In view of this, one of the objects of the present invention is to provide a kind of reagents for quantitatively detecting diabetes autoantibody Application in preparing the kit of type 1 diabetes auxiliary diagnosis or early screening, the second object of the present invention are to provide one Kit of the kind for quantitatively detecting diabetes autoantibody can carry out the diabetes autoantibody in biological sample accurate It is quantitative, overcome indirect method and the drawbacks of single marker sensitivity is low, poor specificity.Glycosuria is prepared by gene engineering method The gene engineering antigen of sick autoantibody；Antigen is coupled to form biotinylated antigen with biotin, and antigen is coupled shape with acridinium ester At acridinium ester label antigen；Biotinylated antigen can have the magnetic bead of Streptavidin to react with coupling, also can be anti-with acridinium ester mark It is former to carry out dual-antigen sandwich method detection to the diabetes autoantibody in serum together.

In order to achieve the above objectives, the present invention provides the following technical solutions：

1, type 1 diabetes auxiliary diagnosis or early screening are being prepared for quantitatively detecting the reagent of diabetes autoantibody Application in kit, the reagent of the quantitatively detection diabetes autoantibody, which includes coupling, has the magnetism of Streptavidin micro- Grain, biotinylation capture antigen, the detection antigen and substrate exciting liquid of acridinium ester label, and the antigen resists selected from zinc transporter 8 Former albumen, glutamate decarboxylase antigen protein, tyrosine phosphatase antigen protein, pancreatic island cell antigen albumen and insulin antigen One or more of albumen.

Preferably, the amino acid sequence of 8 antigen protein of zinc transporter is as shown in SEQ ID NO.1；The tyrosine The amino acid sequence of phosphatase antigen albumen is as shown in SEQ ID NO.2；The amino acid of the glutamate decarboxylase antigen protein Sequence is as shown in SEQ ID NO.5, and the amino acid sequence of the pancreatic island cell antigen albumen is as shown in SEQ ID NO.6, pancreas islet The amino acid sequence of plain antigen protein is as shown in SEQ ID NO.7.

2, a kind of kit for quantitatively detecting diabetes autoantibody, the kit, which includes coupling, has strepto- affine Magnetic particle, the biotinylation of element capture antigen, the detection antigen and substrate exciting liquid of acridinium ester label, and the antigen is selected from zinc 8 antigen protein of transporter, glutamate decarboxylase antigen protein, tyrosine phosphatase antigen protein, pancreatic island cell antigen albumen and One or more of insulin antigen protein.

Preferably, the substrate exciting liquid includes preexciting liquid and exciting liquid, described as follows with exciting liquid each component concentration： The hydrogen peroxide of mass fraction 0.1%, the Triton X-100 of mass fraction 0.5%, the DMF and concentration of mass fraction 0.25% The HCl of 1mM；The exciting liquid each component concentration is as follows：The sodium hydroxide of concentration 0.5M, the 0.5%Triton X- of mass fraction 100 and mass fraction 0.25% n,N-Dimethylformamide.

Preferably, the kit further includes calibration object, and the calibration object is 8 antigen protein monoclonal antibody of anti-zinc transporter, resists Glutamate decarboxylase antigen protein monoclonal antibody, anti-pancreatic island cell antigen albumen monoclonal antibody, resists anti-tyrosine phosphatase antigen protein monoclonal antibody Insulin antigen protein monoclonal antibody.

Preferably, the grain size of the magnetic particle is 0.3-2 μm.

Preferably, biotinylation capture antigen dosage is 100 μ L, 8 antigen protein of biotinylation zinc transporter A concentration of 0.8-1.2 μ g/ml, a concentration of 0.3-0.9 μ g/ml of biotinylation glutamate decarboxylase antigen protein, biotinylation junket Propylhomoserin phosphatase antigen albumen concentration 0.3-0.7 μ g/ml, a concentration of 0.4-1.2 μ g/ml of biotinylation pancreatic island cell antigen albumen With a concentration of 0.5-1.5 μ g/ml of biotinylation insulin antigen protein.

It is furthermore preferred that the detection antigen dosage of the acridinium ester label is 100 μ L, wherein acridinium ester label zinc transporter 8 A concentration of 0.5-1.5 μ g/ml of antigen protein, a concentration of 2-4 μ g/ml of acridinium ester label glutamate decarboxylase antigen protein, acridine Ester marks a concentration of 1-3 μ g/ml of tyrosine phosphatase antigen protein, a concentration of 2-4 μ of acridinium ester label pancreatic island cell antigen albumen A concentration of 2-6 μ g/ml of g/ml, acridinium ester label insulin antigen protein.

More preferably, it is characterised in that the detection antigen of biotinylated antigen and acridinium ester label protection liquid is The phosphate buffer of 0.01mol/L, pH=7.4, BSA and matter containing mass fraction 2% in the phosphate buffer Measure the thimerosal of score 0.1%.

The beneficial effects of the present invention are：

One, kit high sensitivity：Not only serum sample does not dilute or only needs 1:1、1:2 dilutions, it is sensitiveer than indirect method Degree improves 10 times or so；And multiple markers quantify the limitation that joint-detection overcomes single marker poor sensitivity；

Two, kit high specificity：Due to capture antigen and detection antigen be it is specific, indirect method some can not keep away The non-specific sample overwhelming majority exempted from can be excluded, and improve the accuracy of clinical diagnosis.

Three, kit accuracy is good：Quantitative detection overcomes restriction of the conventional method for accuracy.Four, kit is practical Property is strong：Magnetic particle semi-liquid phase system, Avidin-Biotin amplification system, acridinium ester flash light emission form and multiple diabetes Autoantibody markers joint-detection so that kit is easy, sensitive, efficient.

Description of the drawings

In order to keep the purpose of the present invention, technical solution and advantageous effect clearer, the present invention provides following attached drawing and carries out Explanation：

Fig. 1 is the SDS-PAGE figures of 1 antigen protein ZnT8CR of embodiment.

Fig. 2 is the SDS-PAGE figures of 2 antigen protein IA-2 of embodiment.

Fig. 3 is the SDS-PAGE figures of 3 antigen protein GAD65 of embodiment.

Fig. 4 is the SDS-PAGE figures of 4 antigen protein ICA69 of embodiment.

Fig. 5 is the SDS-PAGE figures of 5 antigen protein insulin of embodiment.

Fig. 6 is the standard curve of the ZnT8A calibration objects of 8 dual-antigen sandwich method of embodiment.Wherein, Y-axis represents absorbance hair Light logarithm, X-axis represent the log concentration value of ZnT8A calibration objects.

Fig. 7 is the standard curve of the IAA calibration objects of 8 dual-antigen sandwich method of embodiment.Wherein, Y-axis represents absorbance and shines Logarithm, X-axis represent the log concentration value of IAA calibration objects.

Fig. 8 is the standard curve of the IA-2A calibration objects of 8 dual-antigen sandwich method of embodiment.Wherein, Y-axis represents absorbance hair Light logarithm, X-axis represent the log concentration value of IA-2A calibration objects.

Fig. 9 is the standard curve of the ICA calibration objects of 8 dual-antigen sandwich method of embodiment.Wherein, Y-axis represents absorbance and shines Logarithm, X-axis represent the log concentration value of ICA calibration objects.

Figure 10 is the standard curve of the GADA calibration objects of 8 dual-antigen sandwich method of embodiment.Wherein, Y-axis represents absorbance hair Light logarithm, X-axis represent the log concentration value of GADA calibration objects.

Figure 11 is the ROC curve that five kinds of autoantibody logistic regressions generate in embodiment 9.

Specific implementation mode

Additive amount, content and the concentration of many kinds of substance is referred to herein, wherein the percentage composition, except special instruction Outside, all refer to mass percentage.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are only used for illustrating mesh , rather than limiting the invention.In addition, it should also be understood that, after the design for having read the present invention, those skilled in the art couple Its various changes made or adjustment, should all fall within the scope of protection of the present invention, such equivalent forms also belong to the application The range that the appended claims limit.

The instrument and material used in the embodiment of the present invention is as follows：

Streptavidin：Purchased from Beijing Suo Laibao Science and Technology Ltd；Antigen protein (ZnT8-RW, insulin, IA-2, ICA-69、GAD65)：Upper current chart profit biological medicine Science and Technology Ltd. provides；Biotin：Purchased from Thermo Scientific； Concentrate washing lotion：0.15%PBST makees 25 times of dilutions with preceding with purified water；Acridinium ester：Purchased from sigma；Other chemical reagent are It analyzes pure, is purchased from China Medicine (Group) Shanghai Chemical Reagent Co.,.

The preparation of 1 antigen ZnT8-RW of embodiment

ZnT8 is Zinc transporter 8, the study found that C-terminal (c-terminus) section (ZnT8 of ZnT8：Aa268-369) have There are higher detection sensitivity and specificity, becomes preferred antigen section.The antigen section has good antigenicity, can make For the preferred antigens of type 1 diabetes patient's auxiliary diagnostic.It is SEQ ID including amino acid sequence to study more at present NO：3

(LKDFSILLMEGVPKSLNYSGVKELILAVDGVLSVHSLHIWSLTMNQVILSAHVATAASRDSQVVRREIAKALSKSF TMHSLTIQMESPVDQDPDCLFCEDPCD ZnT8CR (aa268-369,325R) and amino acid sequence) is SEQ ID NO：4

(LKDFSILLMEGVPKSLNYSGVKELILAVDGVLSVHSLHIWSLTMNQVILSAHVATAASWDSQVVRREIAKALSKSF TMHSLTIQMESPVDQDPDCLFCEDPCD ZnT8CW (aa268-369,325W)), the two the difference is that ZnT8 amino acid The 325th amino acids of c-terminus are different in sequence, the former is R, the latter W.The amino acid sequence information foundation of ZnT8 albumen UniportKB databases, number Q8IWU4, GeneBank database mRNA coded sequence accession number are NM_001172815. ZnT8CR and ZnT8CW are cascaded to form synthetic gene ZnT8RW, amino acid sequence such as SEQ ID NO：Shown in 1.

Using the method based on PAS (PCR-based Accurate Synthesis), design overall length splices primer, is drawing The both ends of object respectively devise protectiveness base synthetic gene ZNT8-RW, are connected into carrier pYE-GAP；The recombinant plasmid pYE- of acquisition GAP-ZNT8-RW is transferred to TOP10 clone strains.After digestion and sequence verification are errorless, and extract 10 μ g of plasmid or more.It utilizes BspHI linearizes recombinant plasmid PYE-GAPY-ZNT8-RW, and electrotransformation Pichia pastoris GS115 selects positive strain and passes through PCR Protein expression and purifying are carried out after verification, Westernblot testing results are as shown in Figure 1.

Then IA-2, GAD65, ICA69 and insulin antigen sequence, wherein IA-2 amino acid sequences such as SEQ ID are selected Shown in NO.2, GAD65 amino acid sequences are as shown in SEQ ID NO.5；ICA69 amino acid sequences are as shown in SEQ ID NO.6； For insulin amino acid sequences as shown in SEQ ID NO.7, expression protein SDS-PAGE result is as shown in Figure 2-5.

Each antigen protein amino acid sequence is as follows

Following (the SEQ ID NO of ZnT8RW amino acid sequences：1)：

EFMEGVPKSLNYSGVKELILAVDGVLSVHSLHIWSLTMNQVILSAHVATAASRDSQVVRREIAKALSKSFTMHSLTI QMESPVDQDPDCLFCEDPCDPSTPPGSSGGGLKDFSILLMEGVPKSLNYSGVKELILAVDGVLSVHSLHIWSLTMNQ VILSAHVATAASWDSQVVRREIAKALSKSFTMHSLTIQMESPVDQDPDCLFCEDPCDHHHHHH.

IA-2 amino acid sequences are as follows：(SEQ ID NO.2)

ARQQDKERLAALGPEGAHGDTTFEYQDLCRQHMATKSLFNRAEGPPEPSRVSSVSSQFSDAAQASPSSH SSTPSWCEEPAQANMDISTGHMILAYMEDHLRNRDRLAKEWQALCAYQAEPNTCATAQGEGNIKKNRHPDFLPYDHA RIKLKVESSPSRSDYINASPIIEHDPRMPAYIATQGPLSHTIADFWQMVWESGCTVIVMLTPLVEDGVKQCDRYWPD EGASLYHVYEVNLVSEHIWCEDFLVRSFYLKNVQTQETRTLTQFHFLSWPAEGTPASTRPLLDFRRKVNKCYRGRSC PIIVHCSDGAGRTGTYILIDMVLNRMAKGVKEIDIAATLEHVRDQRPGLVRSKDQFEFALTAVAEEVNAILKALPQ

GAD65 amino acid sequences are as follows：(SEQ ID NO.5)

MASPGSGFWSFGSEDGSGDSENPGTARAWCQVAQKFTGGIGNKLCALLYGDAEKPAESGGSQPPRAAAR KAACACDQKPCSCSKVDVNYAFLHATDLLPACDGERPTLAFLQDVMNILLQYVVKSFDRSTKVIDFHYPNELLQEYN WELADQPQNLEEILMHCQTTLKYAIKTGHPRYFNQLSTGLDMVGLAADWLTSTANTNMFTYEIAPVFVLLEYVTLKK MREIIGWPGGSGDGIFSPGGAISNMYAMMIARFKMFPEVKEKGMAALPRLIAFTSEHSHFSLKKGAAALGIGTDSVI LIKCDERGKMIPSDLERRILEAKQKGFVPFLVSATAGTTVYGAFDPLLAVADICKKYKIWMHVDAAWGGGLLMSRKH KWKLSGVERANSVTWNPHKMMGVPLQCSALLVREEGLMQNCNQMHASYLFQQDKHYDLSYDTGDKALQCGRHVDVFK LWLMWRAKGTTGFEAHVDKCLELAEYLYNIIKNREGYEMVFDGKPQHTNVCFWYIPPSLRTLEDNEERMSRLSKVAP VIKARMMEYGTTMVSYQPLGDKVNFFRMVISNPAATHQDIDFLIEEIERLGQDL

ICA69 amino acid sequences are as follows：(SEQ ID NO.6)

MSGHKCSYPWDLQDRYAQDKSVVNKMQQKYWETKQAFIKATGKKEDEHVVASDADLDAKLELFHSIQRTCLDLSKAI VLYQKRICFLSQEENELGKFLRSQGFQDKTRAGKMMQATGKALCFSSQQRLALRNPLCRFHQEVETFRHRAISDTWL TVNRMEQCRTEYRGALLWMKDVSQELDPDLYKQMEKFRKVQTQVRLAKKNFDKLKMDVCQKVDLLGASRCNLLSHML ATYQTTLLHFWEKTSHTMAAIHESFKGYQPYEFTTLKSLQDPMKKLVEKEEKKKINQQESTDAAVQEPSQLISLEEE NQRKESSSFKTEDGKSILSALDKGSTHTACSGPIDELLDMKSEEGACLGPVAGTPEPEGADKDDLLLLSEIFNASSL EEGEFSKEWAAVFGDGQVKEPVPTMALGEPDPKAQTGSGFLPSQLLDQNMKDLQASLQEPAKAASDLTAWFSLFADL DPLSNPDAVGKTDKEHELLNAinsulin amino acid sequences are as follows：(SEQ ID NO.7)

MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELG GGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

The preparation of embodiment 2, biotinylation insulin antigen

The biotin labeling of insulin antigen：The 50mM carbonic acid buffer dialysed overnights of insulin pH9.5.By antigen： Biotin=1:10 molar ratios mix insulin antigen and biotin, and the DMSO of final volume 10% is added, and 25 DEG C are protected from light and shake Reaction 3 hours is swung, reaction mixture is then moved into room temperature, the ethanol amine that final volume is 10% is added, shaken at room temperature reaction 2 is small When terminate reaction.Bag filter is filled to dialyse to the Tris buffer solutions of pH8.5 12 hours.It is mixed that isometric glycerine is added after the completion of dialysis Even, -80 DEG C of packing storages obtain biotinylation insulin antigen (bio-insulin).

Biotinylation insulin, IA-2, ICA-69 and GAD65 in the same manner.

The preparation of embodiment 3, acridinium ester label glutamate decarboxylase GAD65

The acridinium ester label of glutamate decarboxylase antigen：Pure GAD65 antigens dialysed to the 100mM phosphate buffers of pH8 At night, a concentration of 1mg/mL is adjusted, acridinium ester (5mmol/L DMF solutions) is separately taken, with 10-50:1 molar ratio is to antigen protein The desired amount of acridine ester solution is added in solution.Lysine placement 15min is added to terminate instead after being protected from light 30min in room temperature It answers.The antigen marked bag filter is filled later to dialyse to the PBS buffer solution of pH7 12 hours.It is added after the completion of dialysis isometric Glycerine mixing, -80 DEG C of packing storages, that is, obtain the antigen of acridinium ester label.

Embodiment 4 establishes autoantibody magnetic microparticle chemiluminescence double antigens sandwich detection method

First, the standard curve of ZnT8A is drawn out.Concrete operations are as follows：100 μ L biotinylations ZnT8,100 μ L acridinium esters Label ZnT8 and 100 μ L calibration objects (concentration is respectively 0,10,20,75,500U/mL) have chain with coupling after 37 DEG C of incubation 20min The magnetic bead of mould Avidin is incubated 15min altogether, washs 3 times；Then 100 μ L substrates exciting liquids are added, and (wherein substrate exciting liquid includes Preexciting liquid and exciting liquid, it is described as follows with exciting liquid each component concentration：The hydrogen peroxide of mass fraction 0.1%, mass fraction 0.5% Triton X-100, the HCl of the DMF and concentration 1mM of mass fraction 0.25%；The exciting liquid each component concentration is such as Under：The sodium hydroxide of concentration 0.5M, the N of the 0.5%Triton X-100 and mass fraction 0.25% of mass fraction, N- dimethyl Formamide) 37 DEG C be incubated 2min after detect luminous signal value, and record.Using four parameter fitting modes, calibration curve is established, is counted Calculate measurement result.Wherein antigen protection liquid is the phosphate buffer of 0.01mol/L, pH=7.4, is contained in phosphate buffer The thimerosal of the BSA and mass fraction 0.1% of mass fraction 2%.

By having determined the chief component of detection architecture above, and it is micro- to establish the magnetic detected for ZnT8A Grain dual-antigen sandwich method detection architecture, the range of linearity 10-500U/mL, linear R²>0.99。

Secondly, using above-mentioned identical step, need to only calibration object be changed into detected sample, you can detect measuring samples Luminous value, and corresponding standard curve calculates the ZnT8A contents of the measuring samples.

Fig. 6 shows that the standard curve of ZnT8A calibration objects, wherein Y-axis represent luminous value logarithm, and X-axis represents the schools ZnT8 The logarithm of quasi- product concentration.

According to just as method establish IAA, IA-2A, ICA and GADA standard curve.

Fig. 7 shows that the standard curve of IAA calibration objects, wherein Y-axis represent luminous value logarithm, and X-axis represents IAA calibration objects The logarithm of concentration.

Fig. 8 shows that the standard curve of IA-2A calibration objects, wherein Y-axis represent luminous value logarithm, and X-axis represents the schools IA-2A The logarithm of quasi- product concentration.

Fig. 9 shows that the standard curve of ICA calibration objects, wherein Y-axis represent luminous value logarithm, and X-axis represents ICA calibration objects The logarithm of concentration.

Figure 10 shows that the standard curve of GADA calibration objects, wherein Y-axis represent luminous value logarithm, and X-axis represents the schools GADA The logarithm of quasi- product concentration.

Embodiment 5, diabetes autoantibody detection kit clinical application

Inventor collects T1DM serum samples 100, T2DM serum samples 100, normal human serum sample 100 from hospital Example, lupus erythematosus patients (autoantibodies) serum sample 50,20, Hashimoto thyroiditis patients serum sample, Rheumatoid arthritis human serum sample (RF is positive) 30, sample, is detected according to the process in embodiment 8, detects institute The results are shown in Table 1, statistics index is as shown in table 2, and the AUC of 5 kinds of autoantibodies is as shown in table 3.Figure 11 shows five kinds The ROC curve that autoantibody logistic regression generates.

The statistical result of 1. kit measurement diabetes autoantibody of table

The statistics index of 2. kit measurement diabetes autoantibody of table

Special recall rate：Other four kinds of markers are the independent recall rate of some marker under negative condition

The AUC of 3 five kinds of autoantibodies of table

Marker	AUC	P values	95%CI
				IAA	0.7046	＜ 0.05	0.598-0.811
IA-2A	0.8672	＜ 0.05	0.800-0.934
				ICA	0.5732	＜ 0.05	0.450-0.696
ZnT8A	0.7612	＜ 0.05	0.669-0.853
				GADA	0.8678	＜ 0.05	0.800-0.936
Joint	0.9744	＜ 0.05	0.951-0.998

If data are shown in table 1, table 2 and table 3, the check sample (T2D and NC) with 99% is that negative concentration value is Cutoff values, the cutoff values of IAA, IA-2A, ICA, ZnT8A and GADA be respectively 0.4U/mL, 1U/mL,>10U/mL、15U/ ML and 5U/mL.Under conditions of specificity 99%, the sensitivity of IAA, IA-2A, ICA, ZnT8A and GADA is respectively 52%, 42%, 44%, 38%, 54%.The sensitivity of single marker is respectively less than five kinds of marker joint sensitivity-and 74%, and combine Area is all higher than single autoantibody markers under the ROC curve of detection.It is single in the case where other four kinds of markers are negative A marker all has specific sensitivity, the specific sensitivity of IAA, IA-2A, ICA, ZnT8A and GADA is respectively 2%, 6%, 2%, 4% and 6%.

Embodiment 6, diabetes autoantibody quantify combined detection kit performance evaluation

Sensitivity technique：With reference to CLSI EP17-A file recommendation experimental programs, calculates diabetes autoantibody and quantitatively combine The sensitivity of detection kit, it is as a result as follows：The sensitivity of insulin autoantibodies is 0.2U/mL, glutamate decarboxylase itself The sensitivity of antibody is 2.5U/mL, and the sensitivity of tyrosine phosphatase autoantibody is 0.4U/mL, the spirit of islet cell antibodies Sensitivity is 5U/mL, and the sensitivity of 8 autoantibody of zinc transporter is 5U/mL.

Linearity test：Insulin autoantibodies are to a concentration of school 0U/mL, 0.4U/mL, 1.0U/mL, 10U/mL, 50U/mL Quasi- product do linear analysis, calculate linearly dependent coefficient, r>0.99, range of linearity 0.4-500U/mL；Glutamic acid decarboxylase antibody Linear analysis is done to a concentration of 5U/mL, 18U/mL, 35U/mL, 120U/mL, 250U/mL calibration object, calculates linearly dependent coefficient, r>0.99, range of linearity 5-250U/mL；Tyrosine phosphatase autoantibody to a concentration of 0U/mL, 0.75U/mL, 2U/mL, 10U/mL, 50U/mL calibration object do linear analysis, calculate linearly dependent coefficient, r>0.99, range of linearity 0.75-50U/mL； Islet cell antibodies do linear analysis to a concentration of 0U/mL, 10U/mL, 50U/mL, 150U/mL, 500U/mL calibration object, calculate Linearly dependent coefficient, r>0.99, range of linearity 10-500U/mL；8 autoantibody of zinc transporter is to a concentration of 0U/mL, 10U/ ML, 20U/mL, 75U/mL, 500U/mL calibration object do linear analysis, calculate linearly dependent coefficient, r>0.99, the range of linearity is 10-500U/mL；

Precision measures：Take two insulin antibody samples of a concentration of 1U/mL and 10U/mL, 35U/mL and 120U/mL two A glutamic acid decarboxylase antibody sample, two tyrosine phosphatase antibody samples of 2U/mL and 10U/mL, 20U/mL and 50U/mL two Two a islet cell antibodies sample, 20U/mL and 75U/mL 8 antibody samples of zinc transporter, each each concentration of sample respectively do 10 It is a parallel, it is detected with three batches of kits, calculating kit criticizes interior and difference between batch, the results showed that in the kit batch and between criticizing Difference is respectively less than 5%.

Interference is tested：Pooled serum is taken to add chaff interferent, including bilirubin, hemoglobin, ascorbic acid, glycerine respectively Ester, adding proportion 1:20, it measures pooled serum respectively and is added to the detected value of pooled serum after various chaff interferents.According to 《WST416-2013 interference experiment guides》It is evaluated.The result shows that interference has reached file standard, can with clinic The accurate evaluation of laboratory diabetes autoantibody.

In conclusion the kit of the present invention can be used for the diabetes autoantibody content of quantitative detection clinical sample, from And the auxiliary and antidiastole for type 1 diabetes provide foundation, and applied to the early stage of youngsters and children diabetes autoantibody Screening.

Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.

Sequence table

<110>Upper current chart profit biological medicine Science and Technology Ltd.

<120>Kit for quantitatively detecting diabetes autoantibody and its application

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 217

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 1

Glu Phe Met Glu Gly Val Pro Lys Ser Leu Asn Tyr Ser Gly Val Lys

1 5 10 15

Glu Leu Ile Leu Ala Val Asp Gly Val Leu Ser Val His Ser Leu His

20 25 30

Ile Trp Ser Leu Thr Met Asn Gln Val Ile Leu Ser Ala His Val Ala

35 40 45

Thr Ala Ala Ser Arg Asp Ser Gln Val Val Arg Arg Glu Ile Ala Lys

50 55 60

Ala Leu Ser Lys Ser Phe Thr Met His Ser Leu Thr Ile Gln Met Glu

65 70 75 80

Ser Pro Val Asp Gln Asp Pro Asp Cys Leu Phe Cys Glu Asp Pro Cys

85 90 95

Asp Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Gly Leu Lys Asp Phe

100 105 110

Ser Ile Leu Leu Met Glu Gly Val Pro Lys Ser Leu Asn Tyr Ser Gly

115 120 125

Val Lys Glu Leu Ile Leu Ala Val Asp Gly Val Leu Ser Val His Ser

130 135 140

Leu His Ile Trp Ser Leu Thr Met Asn Gln Val Ile Leu Ser Ala His

145 150 155 160

Val Ala Thr Ala Ala Ser Trp Asp Ser Gln Val Val Arg Arg Glu Ile

165 170 175

Ala Lys Ala Leu Ser Lys Ser Phe Thr Met His Ser Leu Thr Ile Gln

180 185 190

Met Glu Ser Pro Val Asp Gln Asp Pro Asp Cys Leu Phe Cys Glu Asp

195 200 205

Pro Cys Asp His His His His His His

210 215

<210> 2

<211> 376

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 2

Ala Arg Gln Gln Asp Lys Glu Arg Leu Ala Ala Leu Gly Pro Glu Gly

1 5 10 15

Ala His Gly Asp Thr Thr Phe Glu Tyr Gln Asp Leu Cys Arg Gln His

20 25 30

Met Ala Thr Lys Ser Leu Phe Asn Arg Ala Glu Gly Pro Pro Glu Pro

35 40 45

Ser Arg Val Ser Ser Val Ser Ser Gln Phe Ser Asp Ala Ala Gln Ala

50 55 60

Ser Pro Ser Ser His Ser Ser Thr Pro Ser Trp Cys Glu Glu Pro Ala

65 70 75 80

Gln Ala Asn Met Asp Ile Ser Thr Gly His Met Ile Leu Ala Tyr Met

85 90 95

Glu Asp His Leu Arg Asn Arg Asp Arg Leu Ala Lys Glu Trp Gln Ala

100 105 110

Leu Cys Ala Tyr Gln Ala Glu Pro Asn Thr Cys Ala Thr Ala Gln Gly

115 120 125

Glu Gly Asn Ile Lys Lys Asn Arg His Pro Asp Phe Leu Pro Tyr Asp

130 135 140

His Ala Arg Ile Lys Leu Lys Val Glu Ser Ser Pro Ser Arg Ser Asp

145 150 155 160

Tyr Ile Asn Ala Ser Pro Ile Ile Glu His Asp Pro Arg Met Pro Ala

165 170 175

Tyr Ile Ala Thr Gln Gly Pro Leu Ser His Thr Ile Ala Asp Phe Trp

180 185 190

Gln Met Val Trp Glu Ser Gly Cys Thr Val Ile Val Met Leu Thr Pro

195 200 205

Leu Val Glu Asp Gly Val Lys Gln Cys Asp Arg Tyr Trp Pro Asp Glu

210 215 220

Gly Ala Ser Leu Tyr His Val Tyr Glu Val Asn Leu Val Ser Glu His

225 230 235 240

Ile Trp Cys Glu Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Val

245 250 255

Gln Thr Gln Glu Thr Arg Thr Leu Thr Gln Phe His Phe Leu Ser Trp

260 265 270

Pro Ala Glu Gly Thr Pro Ala Ser Thr Arg Pro Leu Leu Asp Phe Arg

275 280 285

Arg Lys Val Asn Lys Cys Tyr Arg Gly Arg Ser Cys Pro Ile Ile Val

290 295 300

His Cys Ser Asp Gly Ala Gly Arg Thr Gly Thr Tyr Ile Leu Ile Asp

305 310 315 320

Met Val Leu Asn Arg Met Ala Lys Gly Val Lys Glu Ile Asp Ile Ala

325 330 335

Ala Thr Leu Glu His Val Arg Asp Gln Arg Pro Gly Leu Val Arg Ser

340 345 350

Lys Asp Gln Phe Glu Phe Ala Leu Thr Ala Val Ala Glu Glu Val Asn

355 360 365

Ala Ile Leu Lys Ala Leu Pro Gln

370 375

<210> 3

<211> 103

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 3

Leu Lys Asp Phe Ser Ile Leu Leu Met Glu Gly Val Pro Lys Ser Leu

1 5 10 15

Asn Tyr Ser Gly Val Lys Glu Leu Ile Leu Ala Val Asp Gly Val Leu

20 25 30

Ser Val His Ser Leu His Ile Trp Ser Leu Thr Met Asn Gln Val Ile

35 40 45

Leu Ser Ala His Val Ala Thr Ala Ala Ser Arg Asp Ser Gln Val Val

50 55 60

Arg Arg Glu Ile Ala Lys Ala Leu Ser Lys Ser Phe Thr Met His Ser

65 70 75 80

Leu Thr Ile Gln Met Glu Ser Pro Val Asp Gln Asp Pro Asp Cys Leu

85 90 95

Phe Cys Glu Asp Pro Cys Asp

100

<210> 4

<211> 103

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 4

Leu Lys Asp Phe Ser Ile Leu Leu Met Glu Gly Val Pro Lys Ser Leu

1 5 10 15

Asn Tyr Ser Gly Val Lys Glu Leu Ile Leu Ala Val Asp Gly Val Leu

20 25 30

Ser Val His Ser Leu His Ile Trp Ser Leu Thr Met Asn Gln Val Ile

35 40 45

Leu Ser Ala His Val Ala Thr Ala Ala Ser Trp Asp Ser Gln Val Val

50 55 60

Arg Arg Glu Ile Ala Lys Ala Leu Ser Lys Ser Phe Thr Met His Ser

65 70 75 80

Leu Thr Ile Gln Met Glu Ser Pro Val Asp Gln Asp Pro Asp Cys Leu

85 90 95

Phe Cys Glu Asp Pro Cys Asp

100

<210> 5

<211> 585

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 5

Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Phe Gly Ser Glu Asp Gly

1 5 10 15

Ser Gly Asp Ser Glu Asn Pro Gly Thr Ala Arg Ala Trp Cys Gln Val

20 25 30

Ala Gln Lys Phe Thr Gly Gly Ile Gly Asn Lys Leu Cys Ala Leu Leu

35 40 45

Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gly Gly Ser Gln Pro Pro

50 55 60

Arg Ala Ala Ala Arg Lys Ala Ala Cys Ala Cys Asp Gln Lys Pro Cys

65 70 75 80

Ser Cys Ser Lys Val Asp Val Asn Tyr Ala Phe Leu His Ala Thr Asp

85 90 95

Leu Leu Pro Ala Cys Asp Gly Glu Arg Pro Thr Leu Ala Phe Leu Gln

100 105 110

Asp Val Met Asn Ile Leu Leu Gln Tyr Val Val Lys Ser Phe Asp Arg

115 120 125

Ser Thr Lys Val Ile Asp Phe His Tyr Pro Asn Glu Leu Leu Gln Glu

130 135 140

Tyr Asn Trp Glu Leu Ala Asp Gln Pro Gln Asn Leu Glu Glu Ile Leu

145 150 155 160

Met His Cys Gln Thr Thr Leu Lys Tyr Ala Ile Lys Thr Gly His Pro

165 170 175

Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu Asp Met Val Gly Leu Ala

180 185 190

Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr Asn Met Phe Thr Tyr Glu

195 200 205

Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Val Thr Leu Lys Lys Met

210 215 220

Arg Glu Ile Ile Gly Trp Pro Gly Gly Ser Gly Asp Gly Ile Phe Ser

225 230 235 240

Pro Gly Gly Ala Ile Ser Asn Met Tyr Ala Met Met Ile Ala Arg Phe

245 250 255

Lys Met Phe Pro Glu Val Lys Glu Lys Gly Met Ala Ala Leu Pro Arg

260 265 270

Leu Ile Ala Phe Thr Ser Glu His Ser His Phe Ser Leu Lys Lys Gly

275 280 285

Ala Ala Ala Leu Gly Ile Gly Thr Asp Ser Val Ile Leu Ile Lys Cys

290 295 300

Asp Glu Arg Gly Lys Met Ile Pro Ser Asp Leu Glu Arg Arg Ile Leu

305 310 315 320

Glu Ala Lys Gln Lys Gly Phe Val Pro Phe Leu Val Ser Ala Thr Ala

325 330 335

Gly Thr Thr Val Tyr Gly Ala Phe Asp Pro Leu Leu Ala Val Ala Asp

340 345 350

Ile Cys Lys Lys Tyr Lys Ile Trp Met His Val Asp Ala Ala Trp Gly

355 360 365

Gly Gly Leu Leu Met Ser Arg Lys His Lys Trp Lys Leu Ser Gly Val

370 375 380

Glu Arg Ala Asn Ser Val Thr Trp Asn Pro His Lys Met Met Gly Val

385 390 395 400

Pro Leu Gln Cys Ser Ala Leu Leu Val Arg Glu Glu Gly Leu Met Gln

405 410 415

Asn Cys Asn Gln Met His Ala Ser Tyr Leu Phe Gln Gln Asp Lys His

420 425 430

Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Ala Leu Gln Cys Gly Arg

435 440 445

His Val Asp Val Phe Lys Leu Trp Leu Met Trp Arg Ala Lys Gly Thr

450 455 460

Thr Gly Phe Glu Ala His Val Asp Lys Cys Leu Glu Leu Ala Glu Tyr

465 470 475 480

Leu Tyr Asn Ile Ile Lys Asn Arg Glu Gly Tyr Glu Met Val Phe Asp

485 490 495

Gly Lys Pro Gln His Thr Asn Val Cys Phe Trp Tyr Ile Pro Pro Ser

500 505 510

Leu Arg Thr Leu Glu Asp Asn Glu Glu Arg Met Ser Arg Leu Ser Lys

515 520 525

Val Ala Pro Val Ile Lys Ala Arg Met Met Glu Tyr Gly Thr Thr Met

530 535 540

Val Ser Tyr Gln Pro Leu Gly Asp Lys Val Asn Phe Phe Arg Met Val

545 550 555 560

Ile Ser Asn Pro Ala Ala Thr His Gln Asp Ile Asp Phe Leu Ile Glu

565 570 575

Glu Ile Glu Arg Leu Gly Gln Asp Leu

580 585

<210> 6

<211> 483

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 6

Met Ser Gly His Lys Cys Ser Tyr Pro Trp Asp Leu Gln Asp Arg Tyr

1 5 10 15

Ala Gln Asp Lys Ser Val Val Asn Lys Met Gln Gln Lys Tyr Trp Glu

20 25 30

Thr Lys Gln Ala Phe Ile Lys Ala Thr Gly Lys Lys Glu Asp Glu His

35 40 45

Val Val Ala Ser Asp Ala Asp Leu Asp Ala Lys Leu Glu Leu Phe His

50 55 60

Ser Ile Gln Arg Thr Cys Leu Asp Leu Ser Lys Ala Ile Val Leu Tyr

65 70 75 80

Gln Lys Arg Ile Cys Phe Leu Ser Gln Glu Glu Asn Glu Leu Gly Lys

85 90 95

Phe Leu Arg Ser Gln Gly Phe Gln Asp Lys Thr Arg Ala Gly Lys Met

100 105 110

Met Gln Ala Thr Gly Lys Ala Leu Cys Phe Ser Ser Gln Gln Arg Leu

115 120 125

Ala Leu Arg Asn Pro Leu Cys Arg Phe His Gln Glu Val Glu Thr Phe

130 135 140

Arg His Arg Ala Ile Ser Asp Thr Trp Leu Thr Val Asn Arg Met Glu

145 150 155 160

Gln Cys Arg Thr Glu Tyr Arg Gly Ala Leu Leu Trp Met Lys Asp Val

165 170 175

Ser Gln Glu Leu Asp Pro Asp Leu Tyr Lys Gln Met Glu Lys Phe Arg

180 185 190

Lys Val Gln Thr Gln Val Arg Leu Ala Lys Lys Asn Phe Asp Lys Leu

195 200 205

Lys Met Asp Val Cys Gln Lys Val Asp Leu Leu Gly Ala Ser Arg Cys

210 215 220

Asn Leu Leu Ser His Met Leu Ala Thr Tyr Gln Thr Thr Leu Leu His

225 230 235 240

Phe Trp Glu Lys Thr Ser His Thr Met Ala Ala Ile His Glu Ser Phe

245 250 255

Lys Gly Tyr Gln Pro Tyr Glu Phe Thr Thr Leu Lys Ser Leu Gln Asp

260 265 270

Pro Met Lys Lys Leu Val Glu Lys Glu Glu Lys Lys Lys Ile Asn Gln

275 280 285

Gln Glu Ser Thr Asp Ala Ala Val Gln Glu Pro Ser Gln Leu Ile Ser

290 295 300

Leu Glu Glu Glu Asn Gln Arg Lys Glu Ser Ser Ser Phe Lys Thr Glu

305 310 315 320

Asp Gly Lys Ser Ile Leu Ser Ala Leu Asp Lys Gly Ser Thr His Thr

325 330 335

Ala Cys Ser Gly Pro Ile Asp Glu Leu Leu Asp Met Lys Ser Glu Glu

340 345 350

Gly Ala Cys Leu Gly Pro Val Ala Gly Thr Pro Glu Pro Glu Gly Ala

355 360 365

Asp Lys Asp Asp Leu Leu Leu Leu Ser Glu Ile Phe Asn Ala Ser Ser

370 375 380

Leu Glu Glu Gly Glu Phe Ser Lys Glu Trp Ala Ala Val Phe Gly Asp

385 390 395 400

Gly Gln Val Lys Glu Pro Val Pro Thr Met Ala Leu Gly Glu Pro Asp

405 410 415

Pro Lys Ala Gln Thr Gly Ser Gly Phe Leu Pro Ser Gln Leu Leu Asp

420 425 430

Gln Asn Met Lys Asp Leu Gln Ala Ser Leu Gln Glu Pro Ala Lys Ala

435 440 445

Ala Ser Asp Leu Thr Ala Trp Phe Ser Leu Phe Ala Asp Leu Asp Pro

450 455 460

Leu Ser Asn Pro Asp Ala Val Gly Lys Thr Asp Lys Glu His Glu Leu

465 470 475 480

Leu Asn Ala

<210> 7

<211> 110

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 7

Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu

1 5 10 15

Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly

20 25 30

Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe

35 40 45

Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly

50 55 60

Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu

65 70 75 80

Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys

85 90 95

Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

100 105 110

Claims

1. the reagent for quantitatively detection diabetes autoantibody is in the reagent for preparing type 1 diabetes auxiliary diagnosis or early screening Application in box, it is characterised in that：The reagent of the quantitatively detection diabetes autoantibody, which includes coupling, Streptavidin Magnetic particle, biotinylation capture antigen, the detection antigen and substrate exciting liquid of acridinium ester label, and the antigen is transported selected from zinc 8 antigen protein of body, glutamate decarboxylase antigen protein, tyrosine phosphatase antigen protein, pancreatic island cell antigen albumen and pancreas islet One or more of plain antigen protein.

2. application according to claim 1, it is characterised in that：The amino acid sequence of 8 antigen protein of zinc transporter is such as Shown in SEQ ID NO.1；The amino acid sequence of the tyrosine phosphatase antigen protein is as shown in SEQ ID NO.2；The paddy The amino acid sequence of propylhomoserin decarboxylase antigen albumen is as shown in SEQ ID NO.5, the amino acid of the pancreatic island cell antigen albumen Sequence is as shown in SEQ ID NO.6, and the amino acid sequence of insulin antigen protein is as shown in SEQ ID NO.7.

3. a kind of kit for quantitatively detecting diabetes autoantibody, it is characterised in that：The kit, which includes coupling, to be had Magnetic particle, the biotinylation of Streptavidin capture antigen, and the detection antigen and substrate exciting liquid of acridinium ester label are described anti- Original is anti-selected from 8 antigen protein of zinc transporter, glutamate decarboxylase antigen protein, tyrosine phosphatase antigen protein, islet cells One or more of former albumen and insulin antigen protein.

4. a kind of kit for quantitatively detecting diabetes autoantibody according to claim 3, it is characterised in that：It is described Substrate exciting liquid includes preexciting liquid and exciting liquid, described as follows with exciting liquid each component concentration：The peroxide of mass fraction 0.1% Change hydrogen, the Triton X-100 of mass fraction 0.5%, the HCl of the DMF and concentration 1mM of mass fraction 0.25%；The exciting liquid Each component concentration is as follows：The sodium hydroxide of concentration 0.5M, the 0.5%Triton X-100 and mass fraction 0.25% of mass fraction N,N-Dimethylformamide.

5. a kind of kit for quantitatively detecting diabetes autoantibody according to claim 3, it is characterised in that：It is described Kit further includes calibration object, and the calibration object is 8 antigen protein monoclonal antibody of anti-zinc transporter, anti-glutamic acid decarboxylase antigen protein Monoclonal antibody, anti-tyrosine phosphatase antigen protein monoclonal antibody, anti-pancreatic island cell antigen albumen monoclonal antibody, anti-insulin antigen protein monoclonal antibody.

6. a kind of kit for quantitatively detecting diabetes autoantibody according to claim 3, it is characterised in that：It is described The grain size of magnetic particle is 0.3-2 μm.

7. a kind of kit for quantitatively detecting diabetes autoantibody according to claim 3, it is characterised in that：It is described Biotinylation capture antigen dosage be 100 μ L, a concentration of 0.8-1.2 μ g/ml of 8 antigen protein of biotinylation zinc transporter, A concentration of 0.3-0.9 μ g/ml of biotinylation glutamate decarboxylase antigen protein, biotinylation tyrosine phosphatase antigen protein are dense Spend 0.3-0.7 μ g/ml, a concentration of 0.4-1.2 μ g/ml of biotinylation pancreatic island cell antigen albumen and biotinylation insulin antigen A concentration of 0.5-1.5 μ g/ml of albumen.

8. a kind of kit for quantitatively detecting diabetes autoantibody according to claim 3, it is characterised in that：It is described The detection antigen dosage of acridinium ester label is 100 μ L, wherein a concentration of 0.5-1.5 μ of 8 antigen protein of acridinium ester label zinc transporter A concentration of 2-4 μ g/ml of g/ml, acridinium ester label glutamate decarboxylase antigen protein, acridinium ester label tyrosine phosphatase antigen A concentration of 1-3 μ g/ml of albumen, a concentration of 2-4 μ g/ml of acridinium ester label pancreatic island cell antigen albumen, acridinium ester label insulin A concentration of 2-6 μ g/ml of antigen protein.

9. a kind of kit for quantitatively detecting diabetes autoantibody according to claim 3, it is characterised in that：It is special Sign is：The detection antigen of biotinylated antigen and acridinium ester label protection liquid is the phosphate of 0.01mol/L, pH=7.4 Buffer solution, the thimerosal of the BSA containing mass fraction 2% and mass fraction 0.1% in the phosphate buffer.