CN109725158A - Application of the polypeptide SLE2018-V001 in diagnostic system lupus erythematosus kit - Google Patents
Application of the polypeptide SLE2018-V001 in diagnostic system lupus erythematosus kit Download PDFInfo
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Abstract
The present invention discloses a kind of application of polypeptide SLE2018-V001 in diagnostic system lupus erythematosus kit.The amino acid sequence of the polypeptide SLE2018-V001 are as follows: YEHAMYRSAVLL.The present invention is using clinical widely used MBP enzyme linked immuno-adsorbent assay (ELISA) technology, using the level of the IgG antibody of anti-YEHAMYRSAVLL polypeptide in indirect method qualitative detection human serum.Provided kit through the invention, a kind of means that will be early diagnosed as auxiliary system lupus erythematosus are greatly improved the specificity and sensitivity of systemic loupus erythematosus early diagnosis, and specificity is 74%, sensitivity 88%.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of polypeptide SLE2018-V001 is in diagnostic system erythema wolf
Application in sore kit.
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.22No.2Apr.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.
(MedicalRecapitulate [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.6JUNE 2011:535-541).After this, phage display library is developed at present with its high-throughput advantage cooperation
Two generation sequencing technologies, complete the detection operations in terms of many serology.The elutriation of random peptide library can plus two generation sequencing technologies
Determination for 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
It is bright to provide a kind of polypeptide SLE2018-V001 (polypeptide sequence YEHAMYRSAVLL) in the reagent of diagnostic system lupus erythematosus
Application in box carrys out in qualitative detection human serum to resist the level of the IgG antibody of the polypeptide, examines as auxiliary system lupus erythematosus
A kind of disconnected means are expected to greatly improve the sensibility and specificity of systemic loupus erythematosus early diagnosis.
The purpose of the present invention is what is be achieved through the following technical solutions:
In a first aspect, the present invention provides a kind of polypeptide SLE2018-V001, the amino acid sequence of the polypeptide are as follows:
YEHAMYRSAVLL。
Second aspect, the present invention provides a kind of polypeptide SLE2018-V001 in the group for preparing diagnostic system lupus erythematosus
Close the application in object.
Specifically: passing through hexamethylene -1- carboxylic acid succinimide ester with the polypeptide SLE2018-V001 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-V001- 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
Then yellow terminates reaction, passes through OD450It is worth the IgG antibody water of the anti-polypeptide SLE2018-V001 of specific recognition in test sample
It is flat.
Preferably, the polypeptide SLE2018-V001 be from M13 phage random peptide library elutriation and obtain.
The third aspect, the present invention provides a kind of diagnostic kits of diagnostic system lupus erythematosus, including polypeptide
SLE2018-V001。
Preferably, the polypeptide SLE2018-V001 is even by hexamethylene -1- carboxylic acid succinimide ester (SMCC) and BSA
Connection forms SMCC-BSA- polypeptide coupled product.
Preferably, the kit further includes standard items, coating buffer, confining liquid, sample diluting liquid, terminate liquid, enzyme
Mark reagent, enzyme substrate solution and cleaning solution.
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-V001,
The concentration of the IgG antibody of clear 1 and anti-polypeptide SLE2018-V001 is the standard serum 2 of 100U/mL；The standard serum 1 is positive
Ordinary person's serum, standard serum 2 are that SLE2018-V001 antibody is positive serum；
Preferably, the polypeptide SLE2018-V001 antigen is using coating buffer dilution, the coating buffer
The carbonate buffer solution of 0.05 ± 0.005M, pH 9.6 ± 0.05, i.e., Na containing 1.59g in every 1L solution2CO3, 2.93g
NaHCO3；
Preferably, the confining liquid is the phosphorus of 0.01 ± 0.005M containing 0.5% bovine serum albumin(BSA), pH 7.4 ± 0.05
Hydrochlorate-NaCl buffer (PBS) solution contains 5g bovine serum albumin(BSA) (BSA) in that is, every 1L, 8g NaCl, 0.2g KH2PO4,
2.9g Na2HPO4·12H2O, 0.2g KCl.
Preferably, in step C, the enzyme substrate solution includes: 13.6g containing sodium acetate in color developing agent A:500mL solution, lemon
Lemon acid 1.6g, 30% hydrogen peroxide 0.3mL；350mg containing TMB, DMSO 20mL, citric acid H in color developing agent B:500mL solution2O
5.1g。
Preferably, in step B, the standard items are diluted with test serum sample using sample diluting liquid, the sample
Product dilution is 7.4 phosphate-NaCl buffer (PBS) of 0.01M pH；
The cleaning solution used that washs is containing 0.05%Tween-20,0.01 ± 0.005M, 7.4 ± 0.05 phosphorus of pH
Hydrochlorate-NaCl buffer (PBST), i.e., NaCl containing 8g, 0.2g KH in every 1 liter of solution2PO4, 2.9g Na2HPO4·12H2O,
0.2g KCl, 0.5mL Tween-20；
The terminate liquid is 2 ± 0.1M H2SO4Solution；
The enzyme marking reagent is the enzyme marking reagent of the anti-Human IgG antibody containing horseradish peroxidase-labeled.
Preferably, each reagent used in the kit also includes preservative, in order to save.
Fourth aspect, the present invention provides the IgG antibodies of anti-polypeptide SLE2018-V001 in qualitative detection human serum a kind of
Method, comprising the following steps:
A, polypeptide SLE2018-V001 is coupled by SMCC and BSA；
B, solid phase antigen is made by being coated in the micropore on ELISA Plate after dilution in the polypeptide after coupling, closing is added
Liquid；
C, it will be added in respective antigen measuring hole after standard items and the dilution of test serum sample, after incubation, every hole is added
The enzyme marking reagent of anti-Human IgG antibody containing horseradish peroxidase-labeled forms SLE2018-V001- antibody-enzyme
Mark secondary antibody compound；
D, it after step C processing, thoroughly washs, the colour developing of plus enzyme substrate solution, terminate liquid is then added and terminates reaction, passes through
OD450It is worth up to the level of the IgG antibody of anti-polypeptide SLE2018-V001 in sample.
Preferably, in step A, the polypeptide SLE2018-V001 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-V001 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-V001.
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.
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-V001, the polypeptide are expected to be used for early diagnosis systemic loupus erythematosus.
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, the specificity of blood serum designated object provided by the invention is 74%, and sensitivity 88% has high specific and height
The characteristics of sensitivity.
2, the present invention provide it is a kind of it is sensitive, safe and reliable, easy-operating can commercial kit, qualitative determination human serum
In anti-polypeptide SLE2018-V001 IgG antibody level, facilitate 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 verification result of the polypeptide of the preparation coupling of the embodiment of the present invention 1 PBS；
Fig. 2 is the ROC curve that the embodiment of the present invention 1 obtains.
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, several changes and improvements can also be made.These belong to the present invention
Protection scope.
Embodiment 1
1, prepare polypeptide and be coupled polypeptide and BSA
1) polypeptide SLE2018-V001 (amino acid sequence are as follows: YEHAMYRSAVLL) 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 SLE2018-V001 1mg in Eppendorf pipe, 10 μ L DMSO is added and dissolve polypeptide.It is added
Polypeptide SLE2018-V001 solution is resuspended to obtain in 200 μ L PBS, and surveys its pH in 7.0~7.5.
1, the 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.
2, PBS is added to be settled to 0.8mL after the completion of coupling, solution concentration is 1mg/mL at this time.
3, electrophoresis verifying coupling situation.
As a result as shown in Figure 1, protein band is successively from left to right as shown in the figure: Marker, BSA, BSA-SMCC, BSA-
SMCC- polypeptide SLE2018-V001.It can be seen that there is the variation of 20kD or so, BSA- in BSA molecular weight after being coupled SMCC
Occurs lesser the change of molecular weight after SMCC coupled peptide, since polypeptide is made of 12 amino acid, so the change of molecular weight
Less, 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, composition is as shown in table 1 below.
Table 1
(2) sample diluting liquid: pH 7.4PBS solution, composition are as shown in table 2 below.
Table 2
(3) cleaning solution: the PBST solution of pH 7.4, composition are as shown in table 3 below.
Table 3
(4) confining liquid: the pH 7.4PBS solution of 0.5%BSA, composition are as shown in table 4 below.
Table 4
(5) enzyme substrate solution: ((ready-to-use) is formed as shown in the following table 5 and 6 by color developing agent A and color developing agent B.
5 color developing agent A of table
6 color developing agent A of table
(6) terminate liquid: 2mol/L H2SO4Solution (concentrated sulfuric acid is slowly dropped into distilled water by timing, and side edged mixes), group
At as shown in table 7 below.
Table 7
4, ELISA method measures the concentration of the IgG antibody of anti-polypeptide SLE2018-V001 in serum, with assistant diagnosis system
Lupus erythematosus, specific steps are as follows:
(1) it is coated with: the coupled product BSA-SMCC- polypeptide SLE2018-V001 of purifying is diluted to 1 μ with coating buffer
G/mL is added in 96 hole elisa Plates, and 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: by standard items (IgG antibody of anti-polypeptide SLE2018-V001 it is dense
Degree is that the concentration of the IgG antibody of 0U/mL standard serum 1 and anti-polypeptide SLE2018-V001 is 100U/mL standard serum 2；It is described
Standard serum 1 is normal human serum, and standard serum 2 is that SLE2018-V001 antibody is positive serum) and test serum sample
1:100 is diluted to 100 μ L with sample buffer, is added in respective antigen measuring orifice plate.It is careful not to bubble, sample-adding will
Sample is added on plum target hole bottom, does not touch hole wall as far as possible, shakes gently mixing, capping or overlay film on ELISA Plate.If blood to be measured
Final proof product are more, it is proposed that are loaded using multitube micropipet.Standard items and sample to be tested are prepared in 15 minutes before use, are used
Complete 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, 60 minutes, forms SLE2018-V001- antibody-ELIAS secondary antibody compound by 37 DEG C.Liquid in clear opening is got rid of, ibid board-washing 6 times bats
It is dry.
(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-V001 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.
Specificity and sensitivity technique: using the serum (systemic loupus erythematosus of 100 parts of autoimmune disease associated patients
50 parts of patient, 50 parts of Healthy People) specificity and sensitivity Detection have been carried out to diagnostic kit of the invention.Detect light absorption value
OD450Obtaining ROC curve using SPSS 17.0 afterwards, (for result as shown in Fig. 2, abscissa is 1- specificity, ordinate is sensitive
Degree).The specificity of diagnostic kit assistant diagnosis system lupus erythematosus of the invention is 74%, sensitivity 88%, AUC=
0.81, 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 a variety of changes or modify within the scope of the claims, this not shadow
Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase
Mutually combination.
Claims (10)
1. a kind of polypeptide SLE2018-V001, which is characterized in that the amino acid sequence of the polypeptide are as follows: YEHAMYRSAVLL.
2. it is a kind of according to claim 1 shown in polypeptide SLE2018-V001 in the composition for preparing diagnostic system lupus erythematosus
In application.
3. polypeptide SLE2018-V001 according to claim 1 or 2 be from M13 phage random peptide library elutriation and obtain
It arrives.
4. a kind of diagnostic kit of diagnostic system lupus erythematosus, which is characterized in that including polypeptide described in claim 1
SLE2018-V001。
5. the diagnostic kit of diagnostic system lupus erythematosus according to claim 4, which is characterized in that the kit
It further include standard items, coating buffer, confining liquid, sample diluting liquid, terminate liquid, enzyme marking reagent, enzyme substrate solution and cleaning solution.
6. the diagnostic kit of diagnostic system lupus erythematosus according to claim 5, which is characterized in that the standard items
The standard serum 1 and anti-polypeptide SLE2018-V001 that the concentration of IgG antibody including anti-polypeptide SLE2018-V001 is 0U/mL
The concentration of IgG antibody is the standard serum 2 of 100U/mL；The standard serum 1 is normal human serum, and standard serum 2 is
SLE2018-V001 antibody is positive serum；
The polypeptide SLE2018-V001 antigen using coating buffer dilution, the coating buffer be 0.05 ± 0.005M,
The carbonate buffer solution of pH 9.6 ± 0.05；
The confining liquid is that 0.01 ± 0.005M, the phosphate-NaCl of pH 7.4 ± 0.05 containing 0.5% bovine serum albumin(BSA) are slow
Fliud flushing.
7. the diagnostic kit of diagnostic system lupus erythematosus according to claim 5, which is characterized in that the zymolyte
Solution includes: 13.6g containing sodium acetate in color developing agent A:500mL solution, citric acid 1.6g, 30% hydrogen peroxide 0.3mL；Color developing agent B:
350mg containing TMB in 500mL solution, DMSO 20mL, citric acid H2O 5.1g。
8. the diagnostic kit of diagnostic system lupus erythematosus according to claim 5, which is characterized in that the standard items
It is diluted using sample diluting liquid, the sample diluting liquid is 0.01 ± 0.005M pH, 7.4 phosphate-NaCl buffer；
The cleaning solution is containing 0.05%Tween-20,0.01 ± 0.005M, 7.4 ± 0.05 phosphate-NaCl of pH buffering
Liquid；
The terminate liquid is 2 ± 0.1M H2SO4Solution；
The enzyme marking reagent is the enzyme marking reagent of the anti-Human IgG antibody containing horseradish peroxidase-labeled.
9. the diagnostic kit of diagnostic system lupus erythematosus according to claim 5, which is characterized in that the kit
Each reagent of middle use also includes preservative.
10. a kind of method of the IgG antibody of anti-polypeptide SLE2018-V001 in qualitative detection human serum, which is characterized in that including
Following steps:
A, polypeptide SLE2018-V001 is coupled by SMCC and BSA；
B, the polypeptide after coupling is diluted and then is coated in the micropore on ELISA Plate by coating buffer and solid phase antigen is made,
Confining liquid is added；
C, it is added in respective antigen measuring hole after diluting standard items and test serum sample with sample diluting liquid, after incubation,
The enzyme marking reagent of the anti-Human IgG antibody containing horseradish peroxidase-labeled is added in every hole, forms SLE2018-
V001- antibody-ELIAS secondary antibody compound；
D, it after step C processing, thoroughly washs, the colour developing of plus enzyme substrate solution, terminate liquid is then added and terminates reaction, passes through OD450
It is worth up to the level of the IgG antibody of anti-polypeptide SLE2018-V001 in sample.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811594481.8A CN109725158B (en) | 2018-12-25 | 2018-12-25 | Application of polypeptide SLE2018-V001 in kit for diagnosing systemic lupus erythematosus |
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| CN201811594481.8A CN109725158B (en) | 2018-12-25 | 2018-12-25 | Application of polypeptide SLE2018-V001 in kit for diagnosing systemic lupus erythematosus |
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| CN110687285A (en) * | 2019-10-29 | 2020-01-14 | 安徽医科大学 | Diagnostic kit and application of MAK16 in preparation of early diagnosis reagent for systemic lupus erythematosus |
| CN112986587A (en) * | 2021-02-22 | 2021-06-18 | 江苏省中医院 | Application of serum lipid in preparation of reagent and kit for diagnosing systemic lupus erythematosus |
| WO2021227364A1 (en) * | 2020-05-15 | 2021-11-18 | 上海交通大学 | Diagnostic marker and application thereof in diagnosis of covid-19 and detection of previous coronavirus infections |
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