CN109725155A - Application of the polypeptide SLE2018-V004 in diagnostic system lupus erythematosus kit - Google Patents

Abstract

The present invention discloses a kind of application of polypeptide SLE2018-V004 in diagnostic system lupus erythematosus kit；The kit is by polypeptide SLE2018-V004 (polypeptide sequence HDGTLLPRSSLH) as antigen, it is connected on BSA by SMCC and is coated with microwell plate, solid phase antigen is made, test serum is sequentially added into the micropore of envelope antigen, the enzyme marking reagent of the anti-Human IgG antibody containing HRP label is added, forms peptide-antibody-ELIAS secondary antibody compound, add 3 after washed, 3', 5,5'- tetramethyl benzidines (TMB) colour developing.TMB converts au bleu under the catalysis of HRP enzyme, and is converted to final yellow under the action of an acid, horizontal with the IgG antibody of the specific recognition polypeptide in the depth test sample of color.Kit provided by the present invention is greatly improved the specificity and sensitivity of systemic loupus erythematosus early diagnosis.

Description

Application of the polypeptide SLE2018-V004 in diagnostic system lupus erythematosus kit

Technical field

The invention belongs to biomedicine fields, and in particular to a kind of polypeptide SLE2018-V004 is in diagnostic system erythema wolf Application in sore kit (composition).

Background technique

Systemic loupus erythematosus (systemic lupus erythematosus, SLE) is that a kind of clinical manifestation is polyphyly The autoimmune disease (autoimmune diseases, ADs) that the autoimmunity of system damage mediates, Chang Yinqi multiple organ system The irreversible lesion of system seriously affects service life and the quality of life of patient.The incidence of systemic loupus erythematosus disease exists In 0.02%~0.07% range, the disease incidence of Urban population is higher, and patient age is mainly distributed on 15~45 years old, and women suffers from Person is in the majority, and men and women's disease rates are about (Lupus 2006.15 (5): 308-318.) 1:9.To the prevalence of systemic loupus erythematosus The research of rate, pathomechanism and its biomarker is three importances of systemic loupus erythematosus research.Systemic erythema The clinical manifestation of lupus has diversity, and serology and amynologic index variability are very big.The autoimmune disease is related to more devices Official, including skin, kidney, brain, in addition to these extraorgans are often with articulate damage, therefore it is systemic for being immunoreacted. Cause autologous tissue's ingredient of autoimmune response to be referred to as autoantigen, including hiding autoantigen (embryonic period, embryonic phase never Contacted with Autologous lymphocyte, body cannot be identified as Self substances) and modification autoantigen (in infection, drug, burning Under the influence of the factors such as wound, ionising radiation, the conformation of autologous tissue changes, and becomes autoantigen).Body generates autoantigen When, due to being identified as dissident part, generates corresponding autoantibody and put up a resistance.These autoantigens or autoantibody exist The early period that disease phenotype shows often has generated, and expression quantity is usually as the evolution of the state of an illness shows certain trend. According to this feature, these autoantigens or autoantibody can be taken as the biomarker to diagnose the illness.

Biomarker is a kind of measuring tool, generation that scientific research personnel is diagnosed the illness using biomarker, recurrence, pre- Afterwards, and be used for therapeutic effect dynamic evaluation.It is still in time a huge challenge to the diagnosis of systemic loupus erythematosus, it can be early The biomarker that phase is accurately diagnosed to be systemic loupus erythematosus is crucial.Excellent systemic loupus erythematosus biomarker The condition that should have is: can accurately diagnose to systemic loupus erythematosus；Diagnosis process has no adverse reaction to patient.Mesh Before, disease biomarkers mainly include gene level marker, cellular level marker and serum levels marker etc..For Systemic loupus erythematosus, it is known that immune targets (or autoantigen) be mainly nuclear components, including ds-DNA chromosome GAP-associated protein GAP, Ro albumen (SSA), La albumen (SSB) and Sm albumen.Diagnosis for systemic loupus erythematosus, from beauty in 1971 (American College of claimed American before Rheumatology, ACR, 1988 years to rheumatism association, state Rheumatism Association, ARA) formulated systemic lupus erythematosus diagnosis standard for the first time since, systemic red yabbi The clinical criteria of sore is just in constantly improve.Increased for the first time in the revision of nineteen eighty-two serological index (antinuclear antibodies, Anti-ds-DNA antibody and anti-Sm antibody), and biometric analysis is used during formulation, clinical diagnosis is used for after verified. In revision in 1997, " the anticardiolipin antibodies positive and lupus anticoagulant are positive " this standard is increased, although without testing Card, but serologic marker object is always research emphasis.(J Nephrol Dialy Transplant[J]Vol.22 No.2 Apr.2013:153-157.)

Serum protein group is one of the effective means of searching system lupus erythematosus.In autoimmunity disease generation, development During, the variation of autoantibodies amount will affect the composition of serum proteins group, this hair in serum biomarkers There is important value in existing.Therefore, more and more scientific research personnel fix sight in the research of serum proteins marker, power Ask and find biomarker in proteomics level, establish diagnosis, prognosis, pharmacodynamic assessment multi-parameters model.

Current existing systemic loupus erythematosus marker includes antinuclear antibodies (ANAs), anti-ds-DNA antibody and anti-Sm anti- Body etc..Antinuclear antibodies as the disease marker generally existing in patient body, in diagnostic criteria, the standard of antinuclear antibodies refers to In the case of unused drug-induced " drug-induced lupus ", immunofluorescence or other experiment antinuclear antibodies titres for being equivalent to the method are different Often；The serum levels of Anti-hCG action are significant related to disease activity and kidney function damage, by detecting Anti-hCG action Level can be with the recurrence of predictive disease, and higher levels of Anti-hCG action is able to detect that before disease progression. (Medical Recapitulate [J], Aug.2015, Vol.21, No.16:2956-2958.) is although antinuclear antibodies height spirit It is quick, but occur in other autoimmunity diseases patients serum, as systemic sclerosis, myositis, Autoimmune hemolytic are poor Blood, rheumatoid and multiple sclerosis (Arthritis&Rheumatism Vol.47, No.4, August 15,2002:434- 444.)；Although Anti-hCG action and the anti-Sm antibody specificity in diagnostic system lupus erythematosus are more significant, it is not (Arthritis&Rheumatism Vol.47, No.5, October 15,2002:546-is generally appeared in patients serum 555).The autoantibodies such as antinuclear antibodies can indicate the generation of disease, but the shortage of specificity hinders them as available pre- The property surveyed biomarker.Autoantibody is still to examine disease early stage because it is in the Central Position of autoimmune disease pathogenesis Disconnected research hotspot.Scientific research personnel wishes to find specificity significantly, and the autoantibody of high sensitivity is as biomarker, in this way The generation of disease can not only be detected, moreover it is possible to distinguish to disease severity.

Biomarker screening technique is promoted to emerge one after another the pursuit of accurate efficient biomarker.In this high pass In the epoch that amount technology continues to develop, scientific research personnel is by way of building library, to be found out in a large amount of candidates in patient and be good for The significant chemical molecular of otherness in health human serum.2013, Jiexia Quan etc. was by synthesis peptidomimetic library as autoantigen Substitute, sifted out for systemic loupus erythematosus specificity be 97.5%, sensitivity be 70% compound marker (Journal of Immunological Methods 402(2014)23–34.).It, can other than chemically synthesized peptidomimetic Also proteins and peptides as candidate.

Protein-chip has had become since appearance probes into biomolecule-protein interaction and screening biomarker Powerful, a large amount of protein molecules are made according to default put in order by certain way and are fixed on solid phase carrier by it Surface forms microarray, is analysed to sample and is incubated for chip, washes away the ingredient failed in conjunction with protein on chip, so It is incubated for afterwards with the antibody of fluorescent marker, the fluorescence signal value of each point is finally read under Fluorescence Scanner.Albumen in serum The content of matter is very inhomogenous, and often abundance is all very low for autoantibody, and common mass spectrum is difficult to solve itself to resist in different samples The huge problem of body differential expression, but protein-chip is then of overall importance with its, unbiasedness, the feature of high throughput can overcome routine Mass spectrographic defect.The difference between patient and Healthy People can be determined in a short time using protein-chip, efficiently found Blood serum designated object.

Polypeptide display library technology is many kinds of, from the point of view of expression vector, there is phage display library, bacteria display library, yeast Show library, cell display library etc.；From the point of view of expressing substance classes, there are the library cDNA, the library mRNA, peptide library etc..Compared to other exhibitions Show that technology, the great advantage of phage display are high-throughput and diversity.Phage display library is the last century 80's development The High Throughput Screening Assay to get up.Since 21 century, with the rise of high throughput sequencing technologies, phage display library technology New development climax is welcome.Phage display library by by gene information together with protein contacts, research protein with The interaction of protein, protein and polypeptide, protein and DNA.Current existing phage display library includes that group learns peptide library And random peptide library, the parallel analysis of thousands of samples is had been achieved with using phage display library.2010, H Benjamin Larman et al. constructs first man proteinoid group phage display technology by the way that human protein's group to be expressed in T7 bacteriophage Show library, paraneoplastic the nervous system disease autoantibody (Nature Biotechnology.Vol is filtered out by the displaying library 29,No.6 JUNE 2011:535-541).After this, phage display library is with its high-throughput advantage cooperation development at present Two generation sequencing technologies, complete the detection operations in terms of many serology.The elutriation of random peptide library adds two generation sequencing technologies It can be used for the determination of epitope, it can also be used to determine the interaction situation of other immune-related proteins such as autoantigen.2015, Christiansen A et al. detects peanut allergy patients serum by the method for being combined phage display library with the sequencing of two generations The effect epitope (Sci Rep.12913 (2015)) of middle IgE and autoantigen.Similar work there are also very much, but by with The biomarker of machine peptide library selection systemic loupus erythematosus has not been reported, and therefore, we pass through high-throughput phage display Library technology screens the polypeptide that discrimination is high in patient and normal person, is desirably to obtain more effectively early to systemic loupus erythematosus Phase diagnosis marker.

Summary of the invention

For the needs that existing technical problem and more accurate SLE Serum marker are found, this hair A kind of bright application for providing polypeptide SLE2018-V004 in diagnostic system lupus erythematosus kit, carrys out qualitative detection human serum In resist the level of the IgG antibody of the polypeptide to be expected to greatly improve and be as a kind of means of auxiliary system lupus erythematosus diagnosis The sensibility and specificity of system property lupus erythematosus early diagnosis.

In order to achieve the above objectives, the technical solution adopted in the present invention is as follows:

In a first aspect, the present invention relates to a kind of polypeptide SLE2018-V004, amino acid sequence HDGTLLPRSSLH.

Second aspect, the present invention relates to a kind of polypeptide SLE2018-V004 to prepare diagnostic system lupus erythematosus composition In purposes.

The third aspect, the present invention relates to a kind of diagnostic kit of diagnostic system Erythematosus Disease, the kits Include polypeptide SLE2018-V004.The kit is using clinical widely used elisa technique, using indirect method qualitative detection people The IgG antibody of anti-polypeptide SLE2018-V004 in serum.

Concrete principle is: passing through hexamethylene -1- carboxylic acid succinimide ester with the polypeptide SLE2018-V004 antigen of synthesis (SMCC) it is connected on bovine serum albumin(BSA) (BSA) and is coated with microwell plate, solid phase antigen is made, successively into the micropore of envelope antigen Test serum is added, adds the enzyme marking reagent of the anti-Human IgG antibody containing horseradish peroxidase (HRP) label, Form polypeptide SLE2018-V004- antibody-ELIAS secondary antibody compound, plus enzyme substrate solution 3,3', 5,5'- after thoroughly washing Tetramethyl benzidine (TMB) colour developing.TMB converts au bleu under the catalysis of HRP enzyme, and is converted under the action of an acid final Yellow judges reaction end with the depth of color, and passes through OD₄₅₀It is worth specific recognition polypeptide SLE2018- in test sample The IgG antibody of V004 is horizontal.

When the kit uses: by polypeptide SLE2018-V004 antigen by being coated on ELISA Plate after coating buffer dilution On micropore in be made solid phase antigen, confining liquid is added.It is each by being added after standard items and test serum sample sample diluting liquid From antigen measuring hole in, the enzyme of the anti-Human IgG antibody containing horseradish peroxidase (HRP) label is added in every hole Reagent is marked, SLE2018-V004- antibody-ELIAS secondary antibody compound is formed, plus enzyme substrate solution is aobvious after washed liquid thoroughly washs Color, the acid terminate liquid of addition after the enzyme substrate solution reaction time arrives.The enzyme substrate solution is converted to indigo plant under the catalysis of HRP enzyme Color, and it is converted to final yellow under the action of an acid, using the depth of color come polypeptide SLE2018- anti-in test sample The level of the IgG antibody of V004.

Preferably, the polypeptide is the elutriation from M13 phage random peptide library.Pass through hexamethylene after synthesizing outside bacteriophage Alkane -1- carboxylic acid succinimide ester (SMCC) is connected on bovine serum albumin(BSA) (BSA), concentration 1mg/mL.

Preferably, the polypeptide SLE2018-V004 is even by hexamethylene -1- carboxylic acid succinimide ester (SMCC) and BSA Connection forms SMCC-BSA- polypeptide coupled product.

Preferably, the kit also includes standard items, coating buffer, confining liquid, sample diluting liquid, contains horseradish mistake Enzyme marking reagent, enzyme substrate solution, cleaning solution and the acid terminate liquid of the anti-Human IgG antibody of oxide enzyme label.

Preferably, it is the standard blood of 0U/mL that the standard items, which include the concentration of the IgG antibody of anti-polypeptide SLE2018-V004, The concentration of the IgG antibody of clear 1 and anti-polypeptide SLE2018-V004 is the standard serum 2 of 100U/mL；The standard serum 1 is positive Ordinary person's serum, standard serum 2 are that SLE2018-V004 antibody is positive serum.

Preferably, the carbonate buffer solution that the coating buffer is 0.05 ± 0.005M pH 9.6 ± 0.05.More preferably Na containing 1.59g in every 1L solution₂CO₃, 2.93g NaHCO₃。

Preferably, the confining liquid is the phosphorus of 0.01 ± 0.005M pH 7.4 ± 0.05 containing 0.5% bovine serum albumin(BSA) Hydrochlorate-NaCl buffer (PBS) solution.Contain 5g bovine serum albumin(BSA) (BSA) in more preferable every 1L, 8g NaCl, 0.2g KH₂PO₄, 2.9g Na₂HPO₄·12H₂O, 0.2g KCl.

Preferably, the sample diluting liquid is 0.01 ± 0.005M pH, 7.4 ± 0.05 phosphate-NaCl buffer.Institute It states standard items and is diluted with test serum sample using sample diluting liquid.

Preferably, the enzyme marking reagent is containing horseradish peroxidase (HRP)-secondary antibody (i.e. horseradish peroxidase-labeled Anti-Human IgG antibody) 0.1-1 μ g/mL.

Preferably, the enzyme substrate solution includes color developing agent A and color developing agent B；Contain acetic acid in every 500mL color developing agent solution A Sodium 13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3mL；350mg containing TMB in every 500mL color developing agent B solution, DMSO 20mL, Citric acid H₂O 5.1g。

Preferably, the cleaning solution is 0.01 ± 0.005M pH, 7.4 ± 0.05 phosphoric acid for including 0.05%Tween-20 Salt-NaCl buffer (PBST).NaCl containing 8g, 0.2g KH in more preferable every 1 liter of solution₂PO₄, 2.9g Na₂HPO₄·12H₂O, 0.2g KCl, 0.5mL Tween-20.

Preferably, the acid terminate liquid is 2 ± 0.1M H₂SO₄Solution.

Preferably, the coating buffer, confining liquid, sample diluting liquid, enzyme marking reagent, cleaning solution, enzyme substrate solution, acid Property one or more of terminate liquid include preservative.Preservative can be added in reagent in kit of the invention, so as to In preservation.

Fourth aspect, it is anti-that the present invention relates to the IgG of anti-polypeptide SLE2018-V004 in qualitative detection human serum sample a kind of The method of body, described method includes following steps:

A, polypeptide SLE2018-V004 is coupled by SMCC and BSA；

B, the polypeptide after coupling is made solid phase and resisted by being coated in the micropore on ELISA Plate after coating buffer dilution Confining liquid is added in original；

C, it will be added in respective antigen measuring hole after standard items and test serum sample sample diluting liquid, every hole is added The enzyme marking reagent of anti-Human IgG antibody containing horseradish peroxidase-labeled forms SLE2018-V004- antibody-enzyme Mark secondary antibody compound；

D, plus enzyme substrate solution develops the color after washed liquid washing, the acid terminate liquid of addition after the enzyme substrate solution reaction time arrives Terminate reaction；Pass through OD₄₅₀It is worth up to the level of the IgG antibody of anti-polypeptide SLE2018-V004 in sample.

Preferably, the above method is anti-polypeptide SLE2018-V004 in the qualitative detection human serum sample of non-diagnoses and treatment IgG antibody method.

Preferably, in step A, the polypeptide SLE2018-V004 is specifically included by the step of SMCC and BSA coupling:

A1, hexamethylene -1- carboxylic acid succinimide ester (SMCC) is added in the buffer PBS containing BSA, is mixed, 25 DEG C 1h is reacted, BSA-SMCC solution is obtained；

A2, BSA-SMCC solution is added into polypeptide SLE2018-V004 solution, it is small that 4 to 6 is statically placed at 25 DEG C after mixing When to get coupled product BSA-SMCC- polypeptide SLE2018-V004.

It is highly preferred that the mass ratio of the SMCC and BSA are 1:5 in step A1；The concentration of the BSA-SMCC solution is 4mg/mL。

Preferably, in step D, pass through OD₄₅₀It is worth up to the level of the IgG antibody of anti-polypeptide SLE2018-V004 in sample It specifically includes:

The optical density OD value for sequentially measuring each hole in 450nm wavelength with enzyme-linked instrument, measures relative unit according to the following formula Value: unit value (U/mL)=(OD value<sample>-OD value<standard serum 1>)/(OD value<2>-OD values of standard serum<standard serum 1 >)×10；

As unit value >=100U/mL, it is judged as that the IgG antibody of anti-polypeptide SLE2018-V004 in human serum sample is horizontal It is high.

Advantage that is high-throughput, quickly analyzing, development is sequenced using phage random polypeptide display library and two generations in the present invention The technology of a set of quick obtaining disease blood serum marker.By to 200 parts of SLE Serums (patient 100, health Patient 100) it analyzes, patient and the reactive difference of Healthy Human Serum IgG are compared in a short time, filter out this hair Bright blood serum designated object-polypeptide SLE2018-V004, the polypeptide can be used for early diagnosing systemic loupus erythematosus.

Compared with prior art, the device have the advantages that are as follows:

1, the specificity of blood serum designated object provided by the invention is 75%, and sensitivity 90% has high specific and height The characteristics of sensitivity.

2, a kind of sensitive, safe and reliable, easy-operating commercial kit, anti-polypeptide in qualitative determination human serum are provided The level of the IgG antibody of SLE2018-V004 facilitates auxiliary early diagnosis systemic loupus erythematosus.

Detailed description of the invention

Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention, Objects and advantages will become more apparent upon:

Fig. 1 is the electrophoresis verifying schematic diagram that polypeptide is coupled situation；

Fig. 2 is ROC curve.

Specific embodiment

The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention Protection scope.

Embodiment 1

The present embodiment is related to a kind of polypeptide SLE2018-V004, the diagnosis of the diagnostic system lupus erythematosus comprising the polypeptide Kit.It is specific as follows:

1, prepare polypeptide and be coupled polypeptide and BSA

1) polypeptide SLE2018-V004 (amino acid sequence are as follows: HDGTLLPRSSLH) is using HPLC method by gill biochemistry The C-terminal of 's synthesis is connected with the polypeptide of cysteine.SMCC (hexamethylene -1- carboxylic acid amber is bought from Thermo Amber imide ester) (article No. 22360).It is dissolved with DMSO (dimethyl sulfoxide), final concentration of 100mg/mL, is saved in -20 DEG C.It protects One month matter phase.

2) 10mg BSA is weighed, 1mL coupling buffer PBS is added, is made into the BSA solution of 10mg/mL.

3) 2mg SMCC (being the relationship of 1:5 with BSA) is taken, is added in above-mentioned carrier protein solution, is mixed, 25 DEG C of reactions 1h。

4) in 10KD bag filter, dialysed overnight.

5) carrier protein solution dialysed is placed in new centrifuge tube, former coupling buffer is added, by activation BSA-SMCC concentration dilution is to 4mg/mL.

6) it takes polypeptide 1mg in Eppendorf pipe, 10 μ L DMSO is added and dissolve polypeptide.200 μ L PBS resuspension is added, And its pH is surveyed in 7.0~7.5.

7) BSA-SMCC (concentration 4mg/mL) that 200 μ L have been activated is added into polypeptide, be statically placed in after mixing 25 DEG C 4 to 6 hours.

8) PBS is added to be settled to 0.8mL after the completion of coupling, solution concentration is 1mg/mL at this time.

9) electrophoresis verifying coupling situation.

Electrophoresis verifies the coupling situation of polypeptide as shown in Figure 1, as shown in Figure 1, protein band is successively from left to right: Marker, BSA, BSA-SMCC, BSA-SMCC- polypeptide SLE2018-V004.It can be seen that BSA molecular weight after being coupled SMCC There is the variation of 20kD or so, lesser the change of molecular weight occurs after BSA-SMCC coupled peptide, since polypeptide is by 12 ammonia Base acid composition, so the change of molecular weight is little, but still it can be seen that polypeptide has been coupled success.

2, the preparation of serum sample:

Whole blood sample in be placed at room temperature for 2 hours or 4 DEG C overnight after in 1000g be centrifuged 20 minutes or so, take supernatant that can stand Detect；Or it is dispensed, and sample is put in -20 DEG C or -80 DEG C preservations, but multigelation should be avoided.Sample after defrosting It should be centrifuged, then detect again.NaN cannot be contained in institute's test sample₃, because of NaN₃Inhibit horseradish peroxidase (HRP) Activity.

3, the preparation method of the various buffers of ELISA method and reagent:

(1) it is coated with buffer: the Na of 0.05M pH 9.6₂CO₃-NaHCO₃

It is coated with buffer	Quality (g)
		Na2CO3	1.59
NaHCO3	2.93
		ddH2O	Add to 1000mL

(2) sample diluting liquid: 7.4 PBS solution of 0.01M pH

Sample diluting liquid	Quality (g)
		NaCl	8.0
KH2PO4	0.2
		Na2HPO4·12H20	2.9
KCl	0.2
		ddH2O	Add to 1000mL

(3) cleaning solution: the PBST solution of 0.01M pH 7.4

Cleaning solution	Quality (g)
		NaCl	8.0
KH2PO4	0.2
		Na2HPO4·12H20	2.9
KCl	0.2
		Tween-20	0.5mL
ddH2O	Add to 1000mL

(4) confining liquid: 7.4 PBS solution of 0.01M pH of 0.5%BSA

Confining liquid	Quality (g)
		BSA	5.0
NaCl	8.0
		KH2PO4	0.2
Na2HPO4·12H20	2.9
		KCl	0.2
ddH2O	Add to 1000mL

(5) enzyme substrate solution: color developing agent A and color developing agent B

Color developing agent A	Quality
		Sodium acetate	13.6g
Citric acid	1.6g
		30% hydrogen peroxide	0.3mL
ddH2O	Add to 500mL

(ready-to-use)

Color developing agent B	Quality
		TMB	350mg
DMSO	20mL
		Citric acid H2O	5.1g
ddH2O	Add to 500mL

(ready-to-use)

(6) terminate liquid: 2mol/L H₂SO₄Solution

Terminate liquid	Quality
		The concentrated sulfuric acid (95%-98%)	22.2mL
ddH2O	Add to 500mL

(concentrated sulfuric acid is slowly dropped into distilled water by timing, and side edged mixes)

4, ELISA method measures the concentration of the IgG antibody of anti-polypeptide SLE2018-V004 in serum, with assistant diagnosis system Lupus erythematosus:

Specific steps are as follows:

(1) it is coated with: the polypeptide solution of purifying being diluted to 1 μ g/mL with coating buffer, is added in 96 hole elisa Plates, Every 100 μ L of hole, 37 DEG C of coatings 2 hours or 4 DEG C are overnight；Cleaning solution board-washing 1 time, drying.

(2) close: be added 200 μ L of confining liquid, incubation at room temperature 2 hours；Cleaning solution board-washing 1 time, drying.

(3) dilution of standard items and sample and sample-adding: standard items and test serum sample 1:100 sample buffer are dilute It releases to 100 μ L, is added in respective antigen measuring orifice plate.It is careful not to bubble, sample is added on plum target bottom hole by sample-adding Portion does not touch hole wall as far as possible, shakes gently mixing, capping or overlay film on ELISA Plate.If test serum sample is more, it is proposed that use Multitube micropipet sample-adding.Standard items and sample to be tested are prepared in 15 minutes before use, are finished discarding, next time, detection used The standard items of Fresh.

(4) incubate: ELISA Plate is placed in 37 DEG C and reacts 120 minutes, gets rid of liquid in clear opening, washs 6 times.

(5) enzyme: 100 μ of enzyme marking reagent of the anti-Human IgG antibody of every Kong Jiahan horseradish peroxidase-labeled L, 37 DEG C, 60 minutes.Liquid in clear opening is got rid of, is ibid patted dry for board-washing 6 times.

(6) it develops the color: patting dry rear each hole and 50 μ L of color developing agent A is first added dropwise, add 50 μ L of color developing agent B, gently concussion mixes, 37 DEG C are protected from light colour developing 15 minutes.

(7) terminate: sequentially every hole adds 50 μ L of terminate liquid, terminates reaction.The addition sequence of terminate liquid should as far as possible with substrate solution Addition sequence it is identical.The substrate reactions time, terminate liquid should be added after as early as possible.

(8) result judgement:

I. the optical density (OD value) in each hole is sequentially measured in 450nm wavelength with enzyme-linked instrument.

Unit value (U/mL)=(A450<sample>-A450<standard serum 1>)/(A450<2>-A450<standard of standard serum Serum 1 >) × 100

* A450 is the abbreviation of absorbance at 450nm.

* the antibody such as current polypeptide there is no the reference standard of the current international practice, therefore use when this test result calibration opposite Unit.

Ii. in serum anti-polypeptide SLE2018-V004 value judgement

Unit value >=100U/mL: can the tentative diagnosis patient be Patients with SLE

Unit value < 100U/mL: cannot diagnose the patient is Patients with SLE

Iii. quality controls

Each testing result has to comply with following standard:

The A450 of standard serum 1 :≤0.100

The A450 of standard serum 2: >=0.700

Above-mentioned standard is not met such as, then result is considered as in vain, it is necessary to detect again.

Iv. the explanation of inspection result

The ROC analysis of 50 Healthy Human Serums, 50 patients serums is established above with reference to value.

5, specificity and sensitivity technique: using serum (the systemic red yabbi of 100 parts of autoimmune disease associated patients 50 parts of sore patient, 50 parts of Healthy People) specificity and sensitivity Detection have been carried out to diagnostic kit of the invention.Detect light absorption value After OD450 using SPSS17.0 obtain ROC curve (result as shown in Fig. 2, abscissa be 1- specificity, ordinate be it is sensitive Degree).The specificity of diagnostic kit assistant diagnosis system lupus erythematosus of the invention is 75%, sensitivity 90%, AUC= 0.83, improve the index of systemic lupus erythematosus diagnosis in the prior art.

Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow Ring substantive content of the invention.

Claims (10)

1. a kind of polypeptide SLE2018-V004, amino acid sequence HDGTLLPRSSLH.

2. a kind of polypeptide SLE2018-V004 is preparing the purposes in diagnostic system lupus erythematosus composition.

3. a kind of diagnostic kit of diagnostic system lupus erythematosus, the kit includes polypeptide SLE2018-V004.

4. diagnostic kit according to claim 3, which is characterized in that the kit also includes standard items, is coated with and delays Fliud flushing, confining liquid, sample diluting liquid, anti-Human IgG antibody containing horseradish peroxidase-labeled enzyme marking reagent, Enzyme substrate solution, cleaning solution and acid terminate liquid.

5. diagnostic kit according to claim 4, which is characterized in that the standard items include anti-polypeptide SLE2018- The concentration of the IgG antibody of standard serum 1 and anti-polypeptide SLE2018-V004 that the concentration of the IgG antibody of V004 is 0U/mL is The standard serum 2 of 100U/mL；The standard serum 1 is normal human serum, and standard serum 2 is that SLE2018-V004 antibody is sun The serum of property.

6. diagnostic kit according to claim 4, which is characterized in that the coating buffer is 0.05 ± 0.005M The carbonate buffer solution of pH 9.6 ± 0.05；The confining liquid is 0.01 ± 0.005M pH containing 0.5% bovine serum albumin(BSA) 7.4 ± 0.05 phosphate-NaCl buffer soln.

7. diagnostic kit according to claim 4, which is characterized in that the enzyme substrate solution includes color developing agent A and shows Toner B；13.6g containing sodium acetate, citric acid 1.6g, 30% hydrogen peroxide 0.3mL in every 500mL color developing agent solution A；Every 500mL is aobvious 350mg containing TMB, DMSO 20mL, citric acid H in toner B solution₂O 5.1g。

8. diagnostic kit according to claim 4, which is characterized in that the sample diluting liquid is 0.01 ± 0.005M 7.4 ± 0.05 phosphate-NaCl buffer of pH；Anti-Human of the enzyme marking reagent containing horseradish peroxidase-labeled IgG antibody 0.1-1 μ g/mL.

9. diagnostic kit according to claim 4, which is characterized in that the cleaning solution is to include 0.05%Tween-20 0.01 ± 0.005M pH, 7.4 ± 0.05 phosphate-NaCl buffer；The acidity terminate liquid is 2 ± 0.1M H₂SO₄It is molten Liquid.

10. a kind of method of the IgG antibody of anti-polypeptide SLE2018-V004 in qualitative detection human serum sample, which is characterized in that Described method includes following steps:

A, polypeptide SLE2018-V004 is coupled by SMCC and BSA；

B, solid phase antigen is made by being coated in the micropore on ELISA Plate after coating buffer dilution in the polypeptide after coupling, added Enter confining liquid；

C, it will be added in respective antigen measuring hole after standard items and test serum sample sample diluting liquid, every hole, which is added, to be contained The enzyme marking reagent of the anti-Human IgG antibody of horseradish peroxidase-labeled forms SLE2018-V004- antibody-enzyme mark two Anti- compound；

D, plus enzyme substrate solution develops the color after washed liquid washing, and the acid terminate liquid of addition terminates after the enzyme substrate solution reaction time arrives Reaction；Pass through OD₄₅₀It is worth up to the level of the IgG antibody of anti-polypeptide SLE2018-V004 in sample.