CN110346576A - A kind of antigen immobilization matrix membrane and the kit and application thereof for being used to detect autoimmunity disease related antinuclear antibodies spectrum comprising it - Google Patents
A kind of antigen immobilization matrix membrane and the kit and application thereof for being used to detect autoimmunity disease related antinuclear antibodies spectrum comprising it Download PDFInfo
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
The invention discloses a kind of antigen immobilization matrix membrane and include its kit and application thereof for being used to detect autoimmunity disease related antinuclear antibodies spectrum.The antigen immobilization matrix membrane includes but not only comprising following 22 antigen detection line independent of each other, and the antigen detection line is by double-stranded DNA, histone, ribosomes P0 albumen, PCNA, SmD1, SmD3, SmB/B ', snRNP-A, snRNP-C, snRNP68/70, SSA/Ro60, SSA/Ro52, SSB/La, Jo-1, PL-7, PL-12, PmScl, Scl-70, CENP-A, CENP-B, RA33 and PR3 form.It is detected using the kit comprising the antigen immobilization matrix membrane, 22 kinds of autoimmune antibodies can be detected simultaneously, to 8 kinds of auxiliary diagnosis common dispersivity connective tissue disease.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of antigen immobilization matrix membrane and comprising its for detecting
Kit of autoimmunity disease related antinuclear antibodies spectrum and application thereof.
Background technique
Autoimmune disease (Autoimmune Diseases) is since body immune system is to antigen existing for itself
A kind of disease for being immunoreacted, and then itself organ-tissue being caused to damage.Its pathogenic process be related to autoantigen exposure,
The microenvironment of the proinflammatory disease of body, the activation of innate immune system, antigen presentation reacting activation acquired immune system, T cell
Activation and B cell release a large amount of anti self antibodies etc. pathomechanism.With the level disorder of immune system and to release
The further attack and damage of the tissue or organ of autoantigen, eventually lead to organic disease, that is, autoimmune disease
Generation.
Special pathomechanism based on autoimmunity class disease it is found that the generation of autoimmune response earlier than the Disease Clinical
The appearance of characterization.Different autoimmune diseases, the distinct of pathogenic autoantigen, therefore resist certainly caused by B cell
The antibody of body antigen also has disease specific.Therefore, the specific autoantibody in blood is detected, is just become to exempting from class disease certainly
Disease carries out the important amynologic index of risk profile, early diagnosis.
Autoimmune disease can break out in any organ and tissue of human body.So far, clinical discovery be more than 100 kinds not
Same exempts from class disease certainly.It is reported according to the authority of the World Health Organization (World Health Organization, WHO), itself
Disease incidence of the immunity disease in crowd accounts for about the 3-5% of total number of persons.In China, the number of patients for suffering from such disease is up to
It is more than 60000000 people.Autoimmune disease is apt to occur in women and the middle-aged and the old, its main feature is that onset is slow, the course of disease is moved repeatedly
It moves, recurrent exerbation is alleviated repeatedly, and some becomes lifelong chronic illness.
The autoantibody immunological detection method that clinical labororatory uses at present includes immunofluorescence technique, Enzyme-linked Immunosorbent Assay
Method and Western blot, distinct methods respectively have superiority and inferiority.
Immunofluorescence technique is using the human laryngeal cancer epithelial cell Hep-2 or mouse liver or mouse kidney composed with complete autoantigen
Cell frozen section is matrix.When serum detection is added, it can specifically bind on native antigen from exempting from antibody in serum, then
The presence that autoantibody is prompted by the method for fluorescent staining, belongs to qualitative detection method.Combined according to antibody itself
The subcellular localization of antigen, dyeing map can be roughly divided into: homogeneous caryogram, kinetochore caryogram, nuclear membrane caryogram, kernel caryogram,
Six major class of spot caryogram and endochylema type.
The advantages of this method is to carry out autoantibody detection using native antigen contained by cell, and spectrotype is complete, and
Antigenic structure is native conformation, the specific highest of detection.The disadvantage is that it is complicated for operation, and qualitative results determine there is subjectivity
Property, probably due to the difference of disease process, the difference of observer and the old and new be sliced the different difference of batch and to coloration result
Paraphrase has differences.Meanwhile this method can not determine the specific type of positive autoantibodies by fluorescence pattern, therefore can not be right
Different types of disease carries out specific antidiastole.
Enzyme linked immunosorbent assay is that a certain particular kind of autoantigen is adsorbed on ELISA Plate.After being incubated for serum, resist
It is former to be specifically bound with autoantibodies in serum, then a kind of quantitative detection means to be developed the color by integrated enzyme reaction.It has sensitive
Degree height, high specificity, operating method simplicity, the features such as having a wide range of application.Meanwhile specificity can be carried out from antibody is exempted from for single class
Quantitative detection eliminates the subjective bias in diagnostic result paraphrase.But the test of single enzyme linked immunological can only detect oneself single
Exempt from antibody, detection flux low cost is high, has great limitation in terms of the auxiliary diagnosis application of autoimmune disease
Property.
Western blot is a kind of detection method based on solid-phase immunoassay technology.Its principle is by dodecyl sulphur
HEP-2 cell lysate is separated by electrophoresis sour sodium-polyacrylamide gel electrophoresis (SDS-PAGE), and then determination is different
Position of the autoantigen on electrophorogram, then comlete antigen is composed from gel by way of electrotransfer and is transferred to cellulose nitrate
Film or nylon membrane.After being incubated for serum, the antigen on autoantibody and immobilon-p in serum is specifically bound, then by not
Same signal detecting mode, such as alkaline phosphatase colour developing or chemiluminescence carry out sxemiquantitative or quantitative detection to antibody is exempted from certainly.
Solid-phase immunoassay technology itself has the advantages that high specific and hypersensitivity.But gel electrophoresis isolation technics is only
Antigen can be separated roughly by molecular size range, not position of the synantigen on film can not be accurately positioned, for detection antibody
It is specific kind of to differentiate that there is subjectivity.Meanwhile the technology also cannot exclude the presence of non-antigen protein, therefore can not differentiate sun
Property detecting signal be specificity or nonspecific result.In addition, dodecyl sodium sulfate etc. becomes during gel electrophoresis
Property agent addition can destroy the native conformation of antigenic determinant to some extent, lead to not identification specificity from exempting from antibody or hair
Raw non-specific binding, causes false positive or false negative result, influences follow-up diagnosis.
With the development of protein engineering techniques, Western blot has obtained further improvement.Pass through different albumen tables
Up to system, can expression and purification obtain specific high-purity recombinant antigen protein, it is then by point sample or the method for scribing line, recombination is anti-
Primordial covering is exempted from antibody certainly to specificity and is detected in a certain determining position of nitrocellulose membrane or nylon membrane.After improvement
Western blot avoid that native antigen is denatured and conformation is destroyed, and can be according to accurate location of the not synantigen on film essence
Antibody type is exempted from quasi- interpretation detection certainly.
Dispersivity connective tissue disease, which refers to go to a doctor, is immunized the set of a major class autoimmune disease of section in rheumatism, is to dredge
Loose connective tissue mucoid oedema and fibrinoid degeneration are one group of disease of pathologic basis.Including systemic red yabbi
Sore, Sjogren syndrome, mixed connective tissue disease, dermatomyositis, polymyositis, chorionitis, rheumatic arthritis, wegener granulomatosis
Deng.This group of disease includes the common ground in terms of various clinical and pathology, such as multisystem is involved simultaneously (skin, muscle, joint,
Heart, kidney, hemopoietic system, central nervous system etc.), can occur together heat, arthralgia, Raynaud's phenomenon, vasculitis, erythrocyte sedimentation rate speed,
γ immunoglobulin such as increases at the presentations, and the long variation of the course of disease is more；But respectively there is characteristic performance.Such disease it is pathogenic
Antigen is mostly cell nuclear protein, therefore generated autoantibody is mostly antinuclear antibodies.Due in such disease incidence mechanism
Often there are a variety of diseases with the overlapping phenomenon occurred in interdependency.Since Characteristic clinical presentation is mostly in course of disease middle and advanced stage
It just gradually appears, just becomes the powerful of such disease early diagnosis for the immunology detection of Specific ANA.
According to the diagnosis and treatment guide of rheumatology branch, Chinese Medical Association, different dispersivity connective tissue disease can be used respective
Special one or more of antinuclear antibodies are as diagnosis index or complementary diagnosis index.For example, the anti-dsDNA that detection is positive,
Anti- SmD1, anti-SmD3, anti-SmB/B ', anti-histone (H2A, H2B, H3 and H4), anti-ribosomal phosphoprotein, antiproliferative nucleus
Antigen etc. exempts from the amynologic index that antibody is diagnostic system lupus erythematosus certainly；Positive anti-SSA/Ro60, anti-SSA/Ro52, resist
The antibody such as SSB/La are the diagnosis index of Sjogren syndrome；The antibody such as anti-snRNPA, anti-snRNPC and anti-snRNP68/70 are mixed
Mould assembly connective tissue disease diagnosis index；The antibody positives such as anti-Jo-1, anti-PL-7, anti-PL-12 and anti-PmScl are dermatomyositis polymyositis
Auxiliary diagnostic index；1 kind of antibody such as anti-Scl-70 is systemic sclerosis index；The antibody such as anti-CENP-A and anti-CENP-B are
Scleroderma circumscriptum index；The antibody such as anti-RA33, anti-CCP, anti-RF are rheumatic arthritis specific index；Anti- PR3, anti-MPO
Equal antibody are the amynologic index of wegener granulomatosis.
But since pathogenesis is interrelated between a variety of dispersivity connective tissue disease, the fortune of detection of plasma diagnosis index
With also comparing, other types disease is increasingly complex, such as: 1) positive rate of the different antinuclear antibodies in same disease is different,
As positive rate is only 10-30% to anti-Jo-1 antibody in myositis patient, it is therefore desirable to increase and detect such as anti-PL-7 and anti-PL-12
Autoantibody is cooperated using many indexes to increase the recall rate of myositis；2) there are overlapped between various disease
Positive antinuclear antibodies, such as the specific marker object that anti-dsDNA and anti-Sm antibody are lupus erythematosus, but patients with SLE serum is also
Often with the positive anti-snRNP-A of appearance, the antibody of-C and -68/70；3) same sufferer may occur in which a variety of rheumatisms with appearance
Overlapping illness, such as 10% myositis patient can detect positive anti-PmScl antibody, and wherein half patient mostly sends out with chorionitis
It is raw, and patients with SLE is often accompanied by secondary Sjogren syndrome, therefore the positive anti-SSA (Ro52/Ro60) of detection and anti-SSB are anti-
Body can effectively prompt the generation of secondary Sjogren syndrome；4) certain Testing index can have suggesting effect to disease, such as sun
Property anti-snRNP class antibody appearance, then prompt the following risk that pulmonary hypertension may occur.
Although there have been a small number of immunoblotting products that can detect a variety of antinuclear antibodies simultaneously in clinical examination field, such as
CN102937468A, CN101329341A and waiting discloses the recombination obtained including the use of recombinant technique in patent documents anti-
Former kit, however its antigen type for including at most is only 17 kinds, the immunological diseases type that can be detected is relatively low, and
The accuracy rate of medical diagnosis on disease and sensitivity are low.
Summary of the invention
The technical problem to be solved by the present invention is to be diagnosed to overcome more antinuclear antibodies in the prior art to detect spectrum
Kinds of Diseases it is not comprehensive enough, specific low and complicated for operation with susceptibility the defects of, a kind of antigen immobilization matrix is provided
Film and comprising its for detecting the kit and application thereof of autoimmunity disease related antinuclear antibodies spectrum, using including the antigen
The kit of immobilization matrix film can detect 22 kinds of autoimmune antibodies simultaneously, to 8 kinds of auxiliary diagnosis common dispersivity knots
Tissue disease is formed, and there is high specificity and sensitivity, of the invention includes that can detect at least 22 kinds of autoimmune antibodies
The kit of reaction film item at home and abroad still belong to the first.
As described above, being used to detect the method for autoimmune disease in the prior art, there are many deficiencies, in consideration of it, this
Inventor obtains a kind of detection film item after making the creative labor, gathered 22 kinds of antibody with adjuvant clinical diagnostic significance
Detection, for the single diagnosis of the great autoimmune disease of 8 major class, cross detection, secondary disease prediction and related disease
Risk profile.
The present invention solution above-mentioned technical problem one of technical solution are as follows: a kind of antigen immobilization matrix membrane, it includes but
It include not only following 22 antigen detection lines independent of each other, the antigen detection line is by double-stranded DNA, histone, ribosomes
P0 albumen, PCNA, SmD1, SmD3, SmB/B ', snRNP-A, snRNP-C, snRNP68/70, SSA/Ro60, SSA/Ro52,
SSB/La, Jo-1, PL-7, PL-12, PmScl, Scl-70, CENP-A, CENP-B, RA33 and PR3 composition.
Wherein, the antigen immobilization matrix membrane can be the antigen immobilization matrix membrane of this field routine, such as nitric acid
Tunica fibrosa or nylon membrane；The antigen detection line is arranged in parallel.
Autoantigen is recombinated used in existing antigen immobilization matrix membrane, mostly from prokaryotic expression system.Due to two
Sulfide linkage forms missing in this expression system of system and posttranslational modification system, generated recombinant antigen conformation mostly with human body
Interior native antigen conformation difference, largely effects on the specific recognition to autoantibody, in turn results in the susceptibility of diagnosis
It is reduced with specificity.The histone, the ribosomes P0 albumen, the snRNP68/70, the PCNA, institute in the present invention
State SmD1, the SmD3, the SmB/B ', the snRNP-A, the snRNP-C, the SSA/Ro52, the SSB/La, institute
State the Jo-1, PL-7, the PL-12, the Scl-70, the CENP-A, the CENP-B, the SSA/Ro60, described
PmScl-75, the RA33 and the PR3 are expressed preferably by mammalian cell system and are obtained, and the mammalian cell is
HEK293 cell.Wherein, histone antigen line of the invention by 4 kinds of different histone H2A, H2B, H3 and H4 antigen group
At.
The present invention solves the two of the technical solution of above-mentioned technical problem are as follows: a kind of anti-core of detection autoimmune disease correlation is anti-
The kit of body spectrum comprising above-mentioned antigen immobilization matrix membrane.The antigen immobilization matrix membrane is preferably immunoblotting
With antigen immobilization matrix membrane；Preferably, the kit further includes the branch for fixing the antigen immobilization matrix membrane
Support carrier, elisa reagent, chromogenic substrate, Sample dilution, concentrated cleaning solution and reaction terminating liquid, the support carrier with it is described
Antigen immobilization matrix membrane anabolic reaction film item.
It is more intuitive accurate in order to make result interpretation, and quantitative analysis can be carried out, the antigen immobilization matrix membrane or
Three quality control bands are preferably also coated on nylon membrane described in person: concentration is the high concentration quality control band of 35-50 μ g/ml, concentration
Middle concentration quality control band and concentration for 15-30 μ g/ml be quality control band described in the low concentration quality control band of 0.5-10 μ g/ml preferably by
Humanized IgG after purification is crossed.
Preferably, the antigen immobilization matrix membrane is also coated with negative control, the negative control has preferably been stripped
The human serum of IgG.
Preferably, the antigen immobilization matrix membrane is also coated with reaction start line, the reaction start line is preferably
Horseradish peroxidase.
Preferably, the elisa reagent is the goat anti-human igg of horseradish peroxidase-labeled；The chromogenic substrate is 5-
The bromo- chloro- 3- indyl-phosphate of 4- and tetrazolyl nitro blue substrate combination (BCIP/NBT) or 3,3', 5,5'- tetramethyl biphenyl
Amine；The Sample dilution is 1 × TBST buffer containing 10%BSA；The concentrated cleaning solution is 20 × TBST buffer；
The reaction terminating liquid is 0.1M/L sulfuric acid.
In the present invention, the autoimmune disease is preferably dispersivity connective tissue disease, the dispersivity connective group
It knits disease and is preferably systemic loupus erythematosus, Sjogren syndrome, mixed connective tissue disease, dermatomyositis/polymyositis, systematicness firmly
Change one of disease, scleroderma circumscriptum, rheumatic arthritis and wegener granulomatosis or a variety of.
Preferably, the kit, which also has reaction, is incubated for disk, it is with independent semiclosed recessed that the reaction, which is incubated for disk,
The plastics of slot are incubated for disk；The quantity of the semiclosed groove of the independence is preferably 10.
The present invention solves the three of the technical solution of above-mentioned technical problem are as follows: a kind of above-mentioned antigen immobilization matrix membrane or on
State purposes of the kit in detection autoimmune disease；The autoimmune disease is preferably dispersivity connective tissue disease,
The dispersivity connective tissue disease is preferably systemic loupus erythematosus, Sjogren syndrome, mixed connective tissue disease, musculus cutaneus
One of inflammation/polymyositis, systemic sclerosis, scleroderma circumscriptum, rheumatic arthritis and wegener granulomatosis are a variety of.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
(1) kit of the invention can detect 22 kinds of autoimmune antibodies simultaneously, to 8 kinds of common disperses of auxiliary diagnosis
Property connective tissue disease, compare market existing procucts have it is higher specificity (form of expression be Healthy People and other without related disorders
It is middle to be detected without any false positive) and high sensitive (disease detection rate is more than market similar-type products)；
(2) single expression, the recombinant antigen that more levels off to human body native antigen protein conformation in the cooperation present invention, it is maximum
Degree avoids the combination of non-specific antibody, significantly improves the accuracy of diagnostic result；
(3) when reaction film item includes quality control band, negative control line and reaction start line, so that result interpretation is more intuitive
Accurately and it is able to carry out quantitative analysis；
(4) kit detection efficiency of the invention is high, and detection can be completed in room temperature 30 minutes；
(5) when kit of the invention includes that reaction is incubated for disk, the semi-enclosed design of independence that the reaction is incubated for disk can
Cross contamination is raw again simultaneously to avoid when detecting multiple samples simultaneously, the design of independent multi-channel reaction slot, so that each incubation
Disk can detect 10 kinds of different samples simultaneously.
Detailed description of the invention
Fig. 1 is quality control band and antigen zone distribution schematic diagram on reaction film item in kit.
Fig. 2 is reaction film schematic cross-section in kit.
Fig. 3 A~G is the class native conformation high-purity recombinant antigen protein of Expression product of the present invention.
Fig. 4 is that Sjogren syndrome patients serum uses existing market product (control) antinuclear antibodies spectrum detection 17S-2-24T
Testing result schematic diagram.
Fig. 5 is the testing result schematic diagram of Healthy People and Sjogren syndrome patients serum using kit of the present invention.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
PcDNA3.1 (+) plasmid used in above-described embodiment is purchased from Invitrogen company；DMEM culture medium, tire ox
Antibiotic P SF used in serum FBS and culture medium is purchased from Gibco company；Transfection reagent JET-PEI is purchased from PolyPlus public affairs
Department；HEK293 cell is purchased from American type culture collection (ATCC)；Ni-NTA nickel ion affinity column is purchased from Qiagen public affairs
Department；Ion exchange column is purchased from GE company.
The preparation of 1 kit of embodiment
1, the preparation of antigen
All autoantigen proteins are all made of mammalian cell expression system and carry out recombinant protein expression.
Biotech firm is entrusted to carry out sequence (the Jo-1:Gene ID 3035 of not synantigen；PL-7:Gene ID6897；PL-
12:Gene ID 16；SnRNP-A:Gene ID 6627；SnRNP-C:Gene ID 6631；SnRNP68/70:Gene ID
6625；SmD1:Gene ID 6632；SmD3:Gene ID 6634；SmB/B':Gene ID 6628；dsDNA:PMID:
6272877；H2A:Gene ID 92815；H2B:Gene ID 8349；H3:Gene ID 8350；H4:Gene ID 8359；
PCNA:Gene ID 5111；P0:Gene ID 6175；SSA/Ro60:Gene ID 6738；SSA/Ro52:Gene ID
6737；SSB/La:Gene ID 6741；CENP-A:Gene ID 1058；CENP-B:Gene ID 1059；Scl-70:Gene
ID 7150；PmScl-75:Gene ID 5393；RA33:Gene ID 3181；PR3:Gene ID 5657) synthesis and by gained
Sequence is cloned into pcDNA3.1 (+) expression vector by ammonia benzyl mycin (final concentration 0.1mg/ml) to resistant positive clone molecule
It is screened, and extracts positive plasmid DNA (being detailed in Molecular Cloning:A Laboratory guide) from positive strain.
Reusing transfection reagent JET-PEI mediates positive colony to transfect to HEK293 cell and using G418 antibiotic to steady
Turn cell strain and carry out resistance screening, concrete operation method are as follows:
1) the positive plasmid DNA of 3 μ g is diluted with the 300mM NaCl solution of 100 μ l；Again with other 100 μ l 300mM
NaCl solution dilutes the JET-PEI reagent of 6 μ l, in being stored at room temperature 5 minutes.
2) above-mentioned two dilutions are sufficiently mixed, and are stored at room temperature 25 minutes.
3) mixed liquor is added with DMEM cell culture medium (10%FBS, 1%PSF) culture to 1 × 106The 6- of a cell
In porocyte culture plates.
4) after transfecting 24 hours, normal cell culture medium is changed to containing final concentration of 1mg/ml G418 antibiotic
DMEM culture medium.
5) replacement in every 2-3 days obtains stable cell strain after about 1 month once containing the fresh culture of G418.
6) it is identified by expression of the detected by Western blot to specific antigen albumen in stable cell strain.
Recombinant protein continues high efficient expression in stable cell strain, most obtains recombination egg in HEK293 cell lysate finally
It is white.The end N- of recombinant protein has 6 histidine tags.
Identification, purifies and separates to gained antigen:
After Optimal Expression condition, successively isolated and purified using the methods of nickel ion affinity purification column-ion exchange column-dialysis
Recombinant protein identifies that single recombinant antigen purity is up to 90% or more (see Fig. 3 A~G) through SDS-PAGE.Recombination after purification
Antigen protein carries out clinical verification by using negative and positive standard serum.
2, it is coated with
It is 100- that autoantigen protein, which is diluted in confining liquid (1%BSA is dissolved in 1 × PBS solution) to final concentration range,
500ng/ μ l, then using full-automatic point sample instrument to 22 antigen zones on film item, 3 quality control bands, 1 negative control band and 1
It reacts initial tape and carries out point sample coating and sequence, it is 1 μ l/cm that point sample instrument, which sprays volume,2。
Specifically as shown in Figure 1, being wherein not limited to represented form for the form of scribing line, the arrangement of various antigens is suitable
Sequence can be adjusted arbitrarily.
3, after the completion of being coated with, film item is placed in confining liquid, is closed 1 hour in room temperature.Confining liquid component are as follows: 1% ox blood
Pure albumen (BSA) is dissolved in 1 × PBS solution.
4, fixed nitrocellulose membrane
The prepared nitrocellulose membrane with 22 kinds of antigen is fixed on the support carrier, reaction film item is obtained, sees Fig. 2.
5, the assembling of kit
Reaction film item 20, enzyme connect 1 × 30ml of reagent, 1 × 30ml of chromogenic substrate, 3 × 50ml of Sample dilution, are concentrated and wash
Wash 1 × 40ml of 1 × 50ml of liquid and reaction terminating liquid.
The application method of 2 kit of embodiment
Preparation of samples: human serum or the blood plasma for having added anti-coagulants.Sample preservation is in 2-4 DEG C, if cannot be tested in 24 hours,
Sample need to be saved to -20 or -80 DEG C.Significant hemolysis has floccule or the sample of mustiness object to will affect testing result, should not make
With.
Cleaning solution is prepared: diluting 1 part of 20 × concentrated cleaning solution with 19 parts of distilled water, mixing, which is formulated as standard wash liquid, (to be needed
It is ready-to-use).
(1) reaction film item is put into reaction and is incubated in disk semiclosed independent reaction groove together, 1ml standard wash is added
Liquid shakes gently to thorough wet film item, moves and abandon dilution.
(2) with Sample dilution according to 1 (sample serum): the dilution proportion sample to be detected of 250 (dilutions), by 1ml
Film reactive tank is added in diluted sample.
(3) disk will be incubated for be put on the shaking table gently shaken with 60rpm/min speed, is incubated for 5 minutes in (25 degree) of room temperature
Afterwards, sample in reactive tank to the greatest extent is abandoned.
(4) 2ml standard wash liquid is added in film reactive tank, is placed in shaking table room temperature jog, reject cleaning solution after 2 minutes,
It is repeated 3 times.
(5) 1ml elisa reagent is added, after five minutes in incubation at room temperature, abandons sample in reactive tank to the greatest extent.
(6) step 4 is repeated.
(7) 1ml chromogenic substrate is added, is placed on shaking table, room temperature is protected from light incubation after five minutes, abandons substrate solution to the greatest extent.
(8) 2ml is added and distills water washing film item, reject distilled water after shaking table jog 1 minute.
(9) 1ml reaction terminating liquid is added, is stored at room temperature 1 minute, reject terminate liquid.
(10) step 8 is repeated.
(11) film item is taken out, blots residual liquid with blotting paper.
After the reaction was completed, it by related quantitative interpretation software such as ImageJ or can be used entirely certainly visually or after scanning
Dynamic interpretation analysis instrument such as full automatic gel imaging analysis instrument analyzes testing result.By with negative control line and 3
The result of nature controlling line is compared, can interpretation from exempt from antibody titer be feminine gender, weakly positive, moderate positive, strong positive or export be
The detection numerical value of quantization.
Effect example 1
Use kit of the present invention and existing sub- brightness dragon antinuclear antibodies spectrum detection 17S-2-24T (control) difference in domestic market
Healthy Human Serum and Patients with Sjogren Syndrome serum are detected, it is possible to find there is no any signal to detect in healthy serum
(non-false positive signal, i.e. specificity are high)；In Sjogren syndrome patients serum, above-mentioned existing products in markets is only capable of detecting faint
Anti- SSA/Ro52 antibody and anti-snRNP antibody signal；And 3 classics that kit of the present invention has detected this disease completely are immune
Diagnosis index is learned, i.e., anti-SSA/Ro52, anti-SSA/Ro60 and anti-SSB/La exempt from antibody certainly, and signal is strong, and sensitivity is high.Together
When, due to the high specific and high sensitivity of kit of the present invention, also while detecting positive anti-Scl-70, anti-PmScl, having resisted
The autoantibodies such as PL-7 and anti-snRNP68/70, prompting patient, there may be secondary myositis and the possibility of systemic sclerosis
Property, it need to develop keeping continuous observation, to disease to guarantee good prognosis (Fig. 4, Fig. 5).
The different detection reagents of effect example 2 compare the recall rate of dispersivity connective tissue autoimmune disease
The different detection reagents of table 1 compare the recall rate of dispersivity connective tissue autoimmune disease (faces more than 200
Bed sample statistics result)
Table 1 is shown: kit of the present invention and Europe traditional Mongolian medicine diagnose Co., Ltd (hereinafter referred to as Ou Meng) antinuclear antibodies spectrum
Specific good, (such as jaundice patient, nephropathy patient, as a result do not show) does not produce in healthy population and other diseases crowd
Raw non-specific result；And antinuclear antibodies detection spectrum specificity is slightly for Ya Huilong Biotechnology Co., Ltd (hereinafter referred to as sub- brightness dragon)
Difference, there are nearly 30% false positive recall rates in healthy population blood sample, and the diagnostic marker for false positive occur is mostly erythema
Lupus specific marker object.
Further: compared to Ou Meng, kit of the present invention to lupus erythematosus, scleroderma circumscriptum/systemic sclerosis,
The sensibility of the diseases such as dermatomyositis/polymyositis is stronger, shows higher recall rate；And in mixed connective tissue disease and drying
In two class disease of syndrome, detection result is consistent with Ou Meng product.
In addition, compared to sub- brightness dragon, recall rate of the kit of the present invention to diseases such as Sjogren syndrome, dermatomyositis polymyositis
More preferably, the recall rate in the diseases such as mixed connective tissue disease and scleroderma circumscriptum/systemic sclerosis is consistent with it.Although
Sub- brightness dragon product is up to 98% to the recall rate of lupus erythematosus, but since its lupus erythematosus specific index is deposited in healthy population
False positive 30% detects, therefore the data reliability is not high, can not temporarily compare.
To sum up, kit of the present invention, which compares market existing procucts, has preferably specificity and sensitivity.
Claims (10)
1. a kind of antigen immobilization matrix membrane, which is characterized in that include but not only comprising following 22 antigen inspections independent of each other
Survey line, the antigen detection line is by double-stranded DNA, histone, ribosomes P0 albumen, PCNA, SmD1, SmD3, SmB/B ',
snRNP-A、snRNP-C、snRNP68/70、SSA/Ro60、SSA/Ro52、SSB/La、Jo-1、PL-7、PL-12、PmScl、
Scl-70, CENP-A, CENP-B, RA33 and PR3 composition.
2. antigen immobilization matrix membrane as described in claim 1, which is characterized in that the antigen immobilization matrix membrane is nitric acid
Tunica fibrosa or nylon membrane；The antigen detection line is arranged in parallel；
And/or it is the histone, the ribosomes P0 albumen, the snRNP68/70, the PCNA, the SmD1, described
It is SmD3, the SmB/B ', the snRNP-A, the snRNP-C, the SSA/Ro52, the SSB/La, the Jo-1, described
PL-7, the PL-12, the Scl-70, the CENP-A, the CENP-B, the SSA/Ro60, the PmScl-75, institute
It states RA33 and the PR3 is obtained using mammalian cell system expression, the mammalian cell is HEK293 cell.
3. a kind of kit for detecting autoimmunity disease related antinuclear antibodies spectrum, which is characterized in that it includes such as claim 1
Or antigen immobilization matrix membrane described in 2.
4. kit as claimed in claim 3, which is characterized in that the antigen immobilization matrix membrane is Western blot use
Antigen immobilization matrix membrane；Preferably, the kit further includes the support for fixing the antigen immobilization matrix membrane
Carrier, elisa reagent, chromogenic substrate, Sample dilution, concentrated cleaning solution and reaction terminating liquid, the support carrier resist with described
Former immobilization matrix film anabolic reaction film item.
5. kit as claimed in claim 3, which is characterized in that be also coated with three Quality Controls on antigen immobilization matrix membrane
Band: the middle concentration quality control band and concentration that concentration is the high concentration quality control band of 35-50 μ g/ml, concentration is 15-30 μ g/ml be
The low concentration quality control band of 0.5-10 μ g/ml, preferably, the quality control band is crossed by humanized IgG after purification；
And/or negative control is also coated on the antigen immobilization matrix membrane.
6. kit as claimed in claim 5, which is characterized in that the negative control is the human serum for having stripped IgG.
7. kit as claimed in claim 3, which is characterized in that be also coated with and react on the antigen immobilization matrix membrane
Initial line, the reaction start line are preferably horseradish peroxidase.
8. kit as claimed in claim 3, which is characterized in that the elisa reagent is the sheep of horseradish peroxidase-labeled
Anti-human igg；
The chromogenic substrate is the chloro- 3- indyl-phosphate of the bromo- 4- of 5- and tetrazolyl nitro blue substrate combination (BCIP/NBT) or 3,
3', 5,5'- tetramethyl benzidine；
The Sample dilution is 1 × TBST buffer containing 10%BSA；
The concentrated cleaning solution is 20 × TBST buffer；
And/or the reaction terminating liquid is 0.1M/L sulfuric acid.
9. kit as claimed in claim 3, which is characterized in that the autoimmune disease is dispersivity connective tissue
Disease, the dispersivity connective tissue disease be preferably systemic loupus erythematosus, Sjogren syndrome, mixed connective tissue disease,
One of dermatomyositis/polymyositis, systemic sclerosis, scleroderma circumscriptum, rheumatic arthritis and wegener granulomatosis or
It is a variety of；
And/or the kit also has reaction and is incubated for disk, it is the modeling with independent semiclosed groove that the reaction, which is incubated for disk,
Material is incubated for disk；The quantity of the semiclosed groove of the independence is preferably 10.
10. a kind of antigen immobilization matrix membrane as claimed in claim 1 or 2, or as described in any one of claim 3~9
Kit detection autoimmune disease in purposes；The autoimmune disease is preferably dispersivity connective tissue disease,
The dispersivity connective tissue disease is preferably systemic loupus erythematosus, Sjogren syndrome, mixed connective tissue disease, musculus cutaneus
One of inflammation/polymyositis, systemic sclerosis, scleroderma circumscriptum, rheumatic arthritis and wegener granulomatosis are a variety of.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113267629A (en) * | 2021-05-13 | 2021-08-17 | 广东优尼德生物科技有限公司 | Antinuclear antibody spectrum detection kit and preparation method thereof |
| CN115656154A (en) * | 2022-10-26 | 2023-01-31 | 广东优尼德生物科技有限公司 | anti-RNP antibody chemiluminescence detection kit based on recombinant RNP multiple antigens |
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| WO1992014148A1 (en) * | 1991-02-08 | 1992-08-20 | Bio-Line S.A. | Kit for detection of antinuclear antibodies and method for detecting such antibodies |
| US20030013134A1 (en) * | 1998-02-23 | 2003-01-16 | Mark Roth | Method of diagnosing autoimmune disease |
| CN101329341A (en) * | 2008-07-09 | 2008-12-24 | 黑龙江美康汇融生物技术股份有限公司 | Reagent kit for detecting autoimmunity disease related antinuclear antibodies spectrum and preparation method thereof |
| CN102937648A (en) * | 2012-11-14 | 2013-02-20 | 四川省新成生物科技有限责任公司 | Kit for detecting antinuclear antibody spectrum related to autoimmune diseases (AIDs) |
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| WO1992014148A1 (en) * | 1991-02-08 | 1992-08-20 | Bio-Line S.A. | Kit for detection of antinuclear antibodies and method for detecting such antibodies |
| US20030013134A1 (en) * | 1998-02-23 | 2003-01-16 | Mark Roth | Method of diagnosing autoimmune disease |
| CN101329341A (en) * | 2008-07-09 | 2008-12-24 | 黑龙江美康汇融生物技术股份有限公司 | Reagent kit for detecting autoimmunity disease related antinuclear antibodies spectrum and preparation method thereof |
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| CN113267629A (en) * | 2021-05-13 | 2021-08-17 | 广东优尼德生物科技有限公司 | Antinuclear antibody spectrum detection kit and preparation method thereof |
| CN113267629B (en) * | 2021-05-13 | 2022-06-24 | 广东优尼德生物科技有限公司 | Antinuclear antibody spectrum detection kit and preparation method thereof |
| CN115656154A (en) * | 2022-10-26 | 2023-01-31 | 广东优尼德生物科技有限公司 | anti-RNP antibody chemiluminescence detection kit based on recombinant RNP multiple antigens |
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