CN108828234A - A kind of protein chip and preparation method thereof for autoimmunity disease marker detection - Google Patents
A kind of protein chip and preparation method thereof for autoimmunity disease marker detection Download PDFInfo
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Abstract
The invention discloses a kind of protein chip for autoimmunity disease marker detection, the preparation method of the protein chip includes the following steps：（1）Black slide pretreatment；（2）Antigenic solution point sample；（3）Closing process；The protein chip is made.Protein chip of the present invention realizes that joint-detection can effectively improve detection efficiency for detecting autoimmunity disease marker, using the protein chip technology of our company's original creation, reduces testing cost；Inventor developed simultaneously include anti-SSA52 antibody, anti-SSA60 antibody, anti-SSB antibody, Anti-hCG action, anti-U1-snRNP antibody, anti-CENP-B antibody, anti-Histone antibody, anti-P0 antibody, anti-Sm antibody, anti-Nucleosome antibody, 12 indexs of Anti-Scl-70 and anti-Jo-1 antibody protein chip diagnostic kit, with quick, efficiently, the advantages that inexpensive.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to a kind of for detecting the albumen core of autoimmunity disease marker
Piece and preparation method thereof.
Background technique
Autoimmune disease is that immune system is immunoreacted the composition of body, damages and causes disease
Disease.Under normal circumstances, immune system only generates the exotic of intrusion body, such as bacterium, virus, helminth and graft
Reaction, eliminates or repels these foreign matters.Under the influence of certain factors, there is certain in the morphological element of body or immune system itself
It is a little abnormal, cause immune system accidentally to attack itself composition as exotic.At this time immune system can be generated for body
The antibody and activated lymphocytes of itself some composition, damage destroy autologous tissue's internal organs, lead to disease.If not in time effectively
Be controlled, consequence is extremely serious：The attack of immune system will affect each human organ, and would generally all the life to its into
Row attack；Sometimes it is also possible to simultaneously the numerous positions of body be caused to damage.Early diagnosis is extremely important from for exempting to management,
Early stage discovery can avoid or postpone target organ or the damage that can not be remedied occurs for tissue.The common phase of autoimmune disease
It is as follows to close index：
(1) anti-SSA/Ro antibody
Anti- SSA/Ro antibody is that itself found in Sjogren syndrome (SS) patient (Ro) serum the 1950s resists
Body.SSA/Ro antigen is a kind of micro ribonucleic acid albumen, is located at nucleus, participates in mRNA to the process for translating bioactive molecule.?
1984, the protein of 60kDa was considered as one of component of ribonucleoprotein；By 1988, detected by Western blot
The antibody of an anti-52kDa (Ro52) is detected in SSA/Ro positive serum.But SSA/Ro 52 is not natural ribose core
The stabilization component of protein body.Anti- SSA/Ro antibody is related to all kinds of autoimmune diseases, is most commonly in Sjogren syndrome
(40-80%), it also sees in systemic loupus erythematosus (30-40%) and primary biliary cirrhosis (20%), occasionally in slow
Sexuality hepatitis.In addition, Anti SS-A antibody is positive in 100% neonatal lupus erythematosus.The antibody can be transmitted to through placenta
Fetus causes inflammatory reaction and Neonatal Congenital heart block.
(2) anti-SSB/La antibody
Anti- SSB/La antibody is found in Sjogren syndrome (SS) patient (La) serum the 1970s.SSB/La
Antigen belongs to micronuclear ribonucleoprotein (snRNPs), polymerize by the phosphorylating protein of 48kDa (or 51kDa) and through RNA
The RNAs that enzyme III is transcribed forms compound.SSB/La antigen rich content in caryoplasm, also sees kernel, about 10% it is anti-
Original may also appear in cytoplasm.The function of SSB/La antigen in vivo is not clear, may translate bioactive molecule in mRNA
It works in the process.Anti SS-B antibody is almost detected in Sjogren syndrome (40-80%) and systemic loupus erythematosus (10-20%)
Female patient in.Male to female ratio is 1:29.Often occur Anti SS-A antibody and Anti SS-B antibody simultaneously in Sjogren syndrome.
(3) Anti-hCG action (anti-dsDNA antibody)
The flexibility of double-stranded DNA main chain is higher than single stranded DNA, and corresponding affinity of antibody is also higher.Thus Anti-hCG action
Affinity is higher, is easier to form immune complex deposit in conjunction with double-stranded DNA and cause a disease in glomerular basement membrane.Or it is anti-
DsDNA antibody directly acts on glomerulus antigen and causes the renal damage of SLE patient.In the serum of lupus vegetans nephritis patient
In the Anti-hCG action of high titre can be detected, the time of occurrence of clinical manifestation and antibody is not consistent, more before the onset of lupus
Year, Anti-hCG action may be present in serum, and Anti-hCG action may play certain work to the state of an illness of SLE is judged
With.It has defined at present, the activity of Anti-hCG action and disease has apparent correlation.
(4) anti-U1-snRNP antibody (anti-nuclear ribonucleoprotein antibody)
Anti-RNP antibody, is the abbreviation of anti-u1-RNP or anti-u1-SnRNP, and full Chinese name is that anti-U1 micronuclear ribonucleoprotein is anti-
Body, antigen are U1 small molecule cell nucleus ribonucleoprotein particles, belong to one of anti-ENA (Extractable nuclear antigen antibody).
The anti-RNP antibody of high titre is mostly related to mixed connective tissue disease, and the positive rate in systemic loupus erythematosus is 40%-
55%, related to Raynaud's phenomenon, often prognosis is preferable for positive.Other connective tissue disease such as Sjogren syndrome etc. also may occur in which sun
Property.
(5) anti-CENP-B antibody (anti-centromere antibody)
Kinetochore is the major site that sister chromatid closely connects together in eukaryotic chromosome, is correctly divided in chromosome
It plays an important role from.Anti-centromere antibody is the autoantibody for being directed to Kinetochore antigen and generating, and can be divided into several
The different protein of kind：CENP-A, CENP-B, CENP-C, CENP-D, CENP-E, CENP-F, CENP-G etc., wherein main anti-
It originally was tri- kinds of CENP-A, CENP-B, CENP-C, kinetochore GAP-associated protein GAP B (CENP-B) can always resist with containing various kinetochores
The serum of body reacts.Anti-centromere antibody (ACA) is related with topical type progressive systemic sclerosis, positive rate 70-
90%.The antibody also can be detected in patients with primary biliary cirrhosis (positive is 10-30%).
(6) anti-Histone antibody (histonic antibody)
Histone is a kind of basonuclin, is made of 5 subunits (Hl, HZA, HZB, H3, H4).Histonic antibody
Related to various autoimmune diseases, in systemic lupus erythematosus (SLE) patient, histonic antibody recall rate is 30% 1
70%, in uncomplicated patient with rheumatoid arthritis positive rate 15% one 50%, young type patient with rheumatoid arthritis
Positive rate is 60%, the recall rate of drug-induced lupus patient's anti-histone it is very high (>95%).On these sLE patients clinicals with
Ephritis person is common.
(7) anti-P0 antibody (anti-ribosomes P0 protein antibodies)
Ribosomal protein (ribosome proteins) is a kind of acidoglycoprotein, passes through core after synthesizing in cytoplasm
Positioning signal (NLS) is transported into nucleus, and kernel is then gathered in.It is a small number of antibody forming systems of anti cytoplasmic antigen in SLE
One of, antigen is mainly P0, P1 and P2 albumen.Anti- P0 antibody is the more popular significant antibody of SLE of recent research, is to make
For the autoantibody of anti-cytoplasmic antigen, mainly damaged with SLE patients' neural's psychiatric system related.In the SLE of about 10%-20%
It is detected in patient, and discovery is related to mental disease and renal damage, is one of SLE specific antibody.
(8) anti-Sm antibody
Anti-Sm antibody is that Tan and Kunkel in 1966 et al. is had found in the serum of patient for the first time with ID method, and with head
Patient name (Smith) name of example discovery.The target antigen of anti-Sm antibody, which is located at, to be made of nucleoprotein and RNA in nucleus
On one group of molecule particles.This histone be referred to as small nuclear ribonucleoprotein (small nuclear ribonucleo-protein,
snRNP).Due to uracil (uridine) content very abundant in the snRNP of this group of small molecule particle, therefore snRNP is otherwise known as
UsnRNP。
(9) anti-Nucleosome antibody (Anti-nucleosome antibodies)
Nucleosome is the fundamental structural unit of chromosome, is the autoantigen that pathogenic T auxiliary cell identifies in SLE, draws
It plays cognate B cells and generates nucleosome specific autoantibody.Exclusive source is apoptotic cell in vivo for it.Experiments have shown that SLE suffers from
The Apoptosis of person accelerates, and since dysimmunity not can effectively clear nucleosome again, accumulates nucleosome in vivo then, pierce
Swash body and generates AnuA.Clinical research prompt, AnuA and the generation of SLE Disease Activity and lupus nephritis are obviously related.AnuA
Sensibility and specificity in SLE patient is respectively 69.3% and 98%, the positive of the AnuA in lupus nephritis (LN) patient
Rate 85.4%, the positive rate in non-LN patient are 40.7%, and the two difference is extremely significant.AnuA and Anti-ds-DNA antibodies in SLE patient
Antibody and anti-Sm antibody compare, AnuA positive rate highest, respectively 72.4%, 48.3%, 31.0%.
(10) Anti-Scl-70 (anti-topoisomerase I antibody)
Douvas is equal to 1979 annual reports, there is a kind of antinuclear antibodies in Patients with scleroderma's serum, and target antigen is nucleus
A kind of middle nonhistone proteins, molecular weight 70kD, therefore referred to as Scl-70.1986, Shero etc. was confirmed, the sheet of Scl-70
Matter is Topoisomerase -1 DNA, and natural molecule amount is 100kD, and 70kD antigen is its degradation fragment.Anti-Scl-70 is regarded
For the serum marker antibody of systemic sclerosis, the positive often involves with diffusivity cutaneous lesions, proximal end skin, Cardiac Involvemant, simultaneously
Tumor of swelling and pulmonary interstitial fibrosis are closely related, it is considered to be the index of prognosis mala.
(11) anti-JO-1 antibody (anti-Histidyl-tRNA-synthetase antibody)
Anti- JO-1 antibody is a kind of anti-aminoacyl-GEKC synthesis enzyme antibody, is had to the diagnosis of polymyositis stronger
Specificity is the serum marker antibody of polymyositis generally acknowledged at present (PM).Positive rate reaches in polymyositis (PM)
25% or so.Other connective tissue disease are feminine gender；Positive, about 80% polymyositis and skin in anti-JO-1 and anti-SSA binomial
Myositis (DM) patient is associated with Sjogren syndrome；And anti-JO-1 and two positives of anti-RNP have clinically had been found that Lei Nuoshi is existing
As；
China exempts from about 20,000,000 people of Disease certainly, wherein about 3,000,000 people of patient with severe symptoms；Women suffers from from the probability for exempting from disease
It is higher than male.It should be noted that major part is the chronic disease for causing injuries of tissues and organs from related disease is exempted from all, due to me
Base of state doctor level it is lower, facility is not complete, encounter performance come from the patient for exempting from symptom after, base doctor is not usually to this
Direction considers and suspects, causes many from Disease is exempted from by mistaken diagnosis, illness rate is underestimated by a degree of.
The detection of autoimmunity disease index of correlation mostly uses immunological method, and the most common detection method of existing market has immune
Blotting (spot immune, band are immune), enzyme linked immunological, chemoluminescence method etc..Western blot is suitable for from the inspection for exempting from primary dcreening operation
It tests, method is simple, and sensitivity and specificity are general, does not need complicated instrument, and it is at low cost, but can sometimes obtain determining
As a result, it is desirable to do further inspection, and this method detection is only able to achieve qualitative observation；Enzyme linked immunological (ELISA), single index
Luminescence method is the most common method of immunodiagnosis, and sensitivity and specificity are relatively high, and result reliability is compared with Western blot
It improves, but combination project need to individually detect each project, detection efficiency is lower；
Protein chip technology realizes that joint-detection can be with more than ten of index of single-time measurement.Detection efficiency is effectively improved,
It realizes quantitative detection, reduce testing cost.And this method has high sensitivity, specific good, full-automatic degree height, using model
Enclose the advantages that wide.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, the applicant provides one kind to be used for autoimmune disease mark
Protein chip of analyte detection and preparation method thereof.Protein chip of the present invention is for detecting autoimmune disease marker, using this
The protein chip technology of company's original creation realizes that joint-detection can effectively improve detection efficiency, reduces testing cost；The present invention
People develops while including anti-SSA52 antibody, anti-SSA60 antibody, anti-SSB antibody, Anti-hCG action, anti-U1-snRNP anti-
Body, anti-CENP-B antibody, anti-Histone antibody, anti-P0 antibody, anti-Sm antibody, anti-Nucleosome antibody, Anti-Scl-70
With the protein chip diagnostic kit of ten indexs of anti-Jo-1 antibody, have quickly, efficiently, it is inexpensive the advantages that.
Technical scheme is as follows：
A kind of protein chip for autoimmune disease marker detection, the preparation method of the protein chip include such as
Lower step：
(1) black slide pretreatment；
(2) antigenic solution point sample；
(3) protein chip is made in closing process.
The black pretreated method of slide described in step (1) is：
1. by black slide be placed in the slide pretreatment fluid containing NaOH impregnate 16~for 24 hours, later using purified water cleaning 2
~8 times；
2. black slide is placed in solution of silane (medium is 25% ethyl alcohol) that mass concentration is 0.05~1% impregnate 20~
60min；
3. soaked black slide nitrogen purging is put into baking oven, 0.2~0.6h is toasted under the conditions of 100~180 DEG C.
Antibody-solutions described in step (2) include SSA52 antigen, SSA60 antigen, SSB antigen, dsDNA antigen, U1-
SnRNP antigen, CENP-B antigen, Histone antigen, P0 antigen, Sm antigen, Nucleosome antigen, Scl-70 antigen and Jo-
1 antigenic solution.
The method of point sample described in step (2) is Machine automated point sample.
Closed process described in step (3) is：The good black slide of point sample is submerged 1 in confining liquid~for 24 hours, it takes out later black
Slide, and it is centrifuged the remaining confining liquid of removal, the protein chip is made.
The confining liquid is the buffer solution containing closed protein；The closed protein is bovine serum albumin(BSA) or egg white egg
It is white；The buffer is one of PBS buffer solution, Tris buffer, HEPS buffer, MOPS buffer or a variety of.
A kind of application of the protein chip, is made kit for the protein chip.
The kit further includes the secondary antibody solution for being marked with HRP enzyme or alkali phosphorus enzyme, the chemistry sensitive to marker
Luminous substrate.
Described mark has the secondary antibody solution concentration of enzyme for 1ug/ml, and wherein solvent is the Sai Mofei company of outsourcing
ELIAS secondary antibody dilution, pH=6.0；The chemiluminescent substrate be respectively containing hydrogen peroxide, luminol detection liquid A and
Detect liquid B.
The present invention is beneficial to be had technical effect that：
Protein chip of the present invention is realized using protein chip technology and is joined for detecting 12 important autoimmunity markers
Detection efficiency can be effectively improved by closing detection, reduce testing cost；Simultaneously include anti-SSA52 antibody, anti-SSA60 antibody,
Anti-SSB antibody, Anti-hCG action, anti-U1-snRNP antibody, anti-CENP-B antibody, anti-Histone antibody, anti-P0 antibody, anti-Sm
Antibody, anti-Nucleosome antibody, 12 indexs of Anti-Scl-70 and anti-Jo-1 antibody protein chip diagnostic kit,
With quick, efficiently, it is inexpensive the advantages that.Automation inspection may be implemented in the automation protein chip reading apparatus for cooperating our company
It surveys.Since effectively 12 index integrations being detected in a chip, it is only necessary to which a patient's blood sample can be real
It is quickly detected while existing 12 indexs.This product and technology do not have also in the world at present as a kind of novel detection method
There is similar product to appear on the market.
The present invention is using classical immunology indirect method.The fixed trapped antigen in the chip matrix that glass is carrier, this
A little antigens can capture antibody specific in tested sample, captured antibody and be marked with the second of HRP enzyme or alkali phosphorus enzyme
Antibody combines, and forms compound.The chemiluminescent substrate sensitive to marker is added and carries out chemiluminescence, optical signal passes through
CCD camera acquisition, may determine that the concentration of special marker antigen in tested sample by the power of optical signal.
This kit has used autoimmunity chip technology platform, and what the individual event detection method of other domestic producers used
It is the luminous means of general chemistry.In contrast, the advantage of this product is：1. special due to antibody and this product antigen binding
Property it is high, affinity is strong, and is influenced by other impurities lower, therefore, the requirement to biological sample is very low, can simplify sample
Pretreatment process；2. being capable of fast high-flux, a large amount of protein example of parallelization quantitative analysis；3. it is easy to operate, as a result
Accuracy is high；4. patient need to only adopt a blood sample, and can provide monitoring result rapidly.5. required reagent and sample are few, price
It is cheap.
Detailed description of the invention
Fig. 1 is antibody spot sample schematic diagram of the present invention；
In figure, column 1：Positive quality control column；Column 2：Blank；Column 3 are to column 14：Respectively anti-SSA52 antibody, anti-SSA60 antibody,
Anti-SSB antibody, Anti-hCG action, anti-U1-snRNP antibody, anti-CENP-B antibody, anti-Histone antibody, anti-P0 antibody, anti-Sm
Antibody, anti-Nucleosome antibody, Anti-Scl-70 and anti-Jo-1 antibody measurement column；Column 15：Negative Quality Control column.
Specific embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
Embodiment 1
A kind of protein chip for autoimmunity disease marker detection, the preparation method of the protein chip include as follows
Step：
(1) black slide pretreatment；
1. black slide is placed in the slide pretreatment fluid containing 2%NaOH and impregnates 16h, later using purified water cleaning 2
~8 times；
2. black slide is placed in the solution of silane (25% ethyl alcohol) that mass concentration is 0.05% and impregnates 60min；
3. toasting 0.2h under the conditions of 180 DEG C for being put into baking oven after soaked black slide nitrogen purging.
(2) antibody-solutions point sample；
With reference to Fig. 1, using Machine automated point sample SSA52 antigen, SSA60 antigen, SSB antigen, dsDNA antigen, U1-
SnRNP antigen, CENP-B antigen, Histone antigen, P0 antigen, Sm antigen, Nucleosome antigen, Scl-70 antigen and Jo-
1 antigenic solution, antigen solution concentration 0.1mg/mL, every point sample 20nL, each antibody spot sample distribution is as shown in Figure 1.
(3) closing process；
The good black slide of point sample is submerged into 10h in confining liquid (PBS buffer solution for including 1% bovine serum albumin(BSA)), later
Black slide is taken out, and is centrifuged the remaining confining liquid of removal, the protein chip is made.
(4) kit
By the protein chip be marked with HRP enzyme secondary antibody solution (concentration 1ug/ml, wherein medium be outsourcing
Sai Mofei company ELIAS secondary antibody dilution, pH=6.0), detection liquid A (containing 1% luminol and 2%Tris) and detect liquid B
(1% hydrogen peroxide) is packaged into kit jointly.
Embodiment 2
A kind of protein chip for autoimmunity disease marker detection, the preparation method of the protein chip include as follows
Step：
(1) black slide pretreatment；
It is impregnated for 24 hours 1. being placed in black slide in the slide pretreatment fluid containing 2%NaOH, later using purified water cleaning 2
~8 times；
2. black slide is placed in the solution of silane (medium is 25% ethyl alcohol) that mass concentration is 0.5% and impregnates 30min；
3. toasting 0.5h under the conditions of 140 DEG C for being put into baking oven after soaked black slide nitrogen purging.
(2) antibody-solutions point sample；
With reference to Fig. 1, using Machine automated point sample SSA52 antigen, SSA60 antigen, SSB antigen, dsDNA antigen, U1-
SnRNP antigen, CENP-B antigen, Histone antigen, P0 antigen, Sm antigen, Nucleosome antigen, Scl-70 antigen and Jo-
1 antigenic solution, antibody-solutions concentration are 0.1mg/mL, and every point sample 20nL, each antibody spot sample distribution is as shown in Figure 1.
(3) closing process；
The good black slide of point sample is submerged in confining liquid (PBS buffer solution for including 2% ovalbumin) for 24 hours, is taken out later
Black slide, and it is centrifuged the remaining confining liquid of removal, the protein chip is made.
(4) kit
By the protein chip be marked with HRP enzyme secondary antibody solution (concentration 1ug/ml, wherein medium be outsourcing
Sai Mofei company ELIAS secondary antibody dilution, pH=6.0), detection liquid A (containing 1% luminol and 2%Tris) and detect liquid B
(containing 1% hydrogen peroxide) is packaged into kit jointly.
Embodiment 3
A kind of protein chip for autoimmunity disease marker detection, the preparation method of the protein chip include as follows
Step：
(1) black slide pretreatment；
1. black slide is placed in the slide pretreatment fluid containing 2%NaOH and impregnates 20h, later using purified water cleaning 2
~8 times；
2. black slide is placed in the solution of silane (medium is 25% ethyl alcohol) that mass concentration is 1% and impregnates 20min；
3. toasting 0.6h under the conditions of 100 DEG C for being put into baking oven after soaked black slide nitrogen purging.
(2) antibody-solutions point sample；
With reference to Fig. 1, using Machine automated point sample SSA52 antigen, SSA60 antigen, SSB antigen, dsDNA antigen, U1-
SnRNP antigen, CENP-B antigen, Histone antigen, P0 antigen, Sm antigen, Nucleosome antigen, Scl-70 antigen and Jo-
1 antigenic solution, antibody-solutions concentration are 0.1mg/mL, and every point sample 20nL, each antibody spot sample distribution is as shown in Figure 1.
(3) closing process；
The good black slide of point sample is submerged into 8h in confining liquid (the Tris buffer for including 3% bovine serum albumin(BSA)), later
Black slide is taken out, and is centrifuged the remaining confining liquid of removal, the protein chip is made.
(4) kit
By the protein chip be marked with HRP enzyme secondary antibody solution (concentration 1ug/ml, wherein medium be outsourcing
Sai Mofei company ELIAS secondary antibody dilution, pH=6.0), detection liquid A is (containing containing 1% luminol and 2%Tris) and detecting liquid
B (1% hydrogen peroxide) is packaged into kit jointly.
Test case：
Using our company produce SLXP-001 type biological chip reading apparatus clinical serum is detected, testing result with
The number of autoimmune kit (reference reagent) of Ou Meng company production compares.
The course of work of SLXP-001 type biological chip reading apparatus is as follows：
For sample to be tested (serum dilutes 100 times) after the dilution of instrument automatic sucking 200ul into reaction cup, instrument incite somebody to action this
Protein chip made from inventive embodiments is automatically put into test serum, and 37 DEG C are incubated for 40 minutes, and subsequent instrument clamping jaw is by core
Piece takes out, and puts into after instrument auto-flushing and is marked with the secondary antibody solution of HRP enzyme (200ul, instrument are inhaled in advance automatically
It is good), it is incubated for again after forty minutes, instrument clamping jaw takes out chip again, and it is molten to put into luminous substrate after instrument auto-flushing
It (is mixed by the detection liquid B of detection the liquid A and 100ul of 100ul, by instrument automatic sucking and mixing) in liquid, finally to egg
White chip carries out imaging of taking pictures, and software automatically analyzes picture, provides analysis result.Testing result such as table 1, table 2, table 3,4 institute of table
Show.
Table 1
Table 2
Table 3
Table 4
As seen from the above table, kit provided by the present invention, while detecting anti-SSA52 antibody, anti-SSA60 antibody, anti-SSB
Antibody, Anti-hCG action, anti-U1-snRNP antibody, anti-CENP-B antibody, anti-Histone antibody, anti-P0 antibody, anti-Sm antibody,
12 anti-Nucleosome antibody, Anti-Scl-70 and anti-Jo-1 antibody indexs, can obtain similar to single index kit
Result (relative error majority is within 5%), in sensitivity, the range of linearity etc. is also not significantly different.This kit
The measurement that single index kit carries out various autoimmune disease antibody can effectively be substituted.It may be implemented using this kit
High efficiency, letter operation, low cost, multiple advantages such as the used time is short are remarkably contributing in time, accurately find autoimmunity disease and prison
Control the development degree of autoimmunity disease.
Claims (8)
1. a kind of protein chip for autoimmunity disease marker detection, it is characterised in that the preparation method of the protein chip
Include the following steps：
（1）Black slide pretreatment；
（2）Antigenic solution point sample；
（3）The protein chip is made in closing process.
2. protein chip according to claim 1, it is characterised in that step（1）Described in the black pretreated method of slide
For：
1. by black slide be placed in the slide pretreatment fluid containing NaOH impregnate 16 ~ for 24 hours, later using purified water clean 2 ~ 8 times；
2. black slide is placed in the solution of silane that mass concentration is 0.05 ~ 1% and impregnates 20 ~ 60min；
3. toasting 0.2 ~ 0.6h under the conditions of 100 ~ 180 DEG C for being put into baking oven after soaked black slide nitrogen purging.
3. protein chip according to claim 1, it is characterised in that step（2）Described in antigenic solution include SSA52 anti-
Original, SSA60 antigen, SSB antigen, dsDNA antigen, U1-snRNP antigen, CENP-B antigen, Histone antigen, P0 antigen, Sm
Antigen, Nucleosome antigen, Scl-70 antigen and Jo-1 antigenic solution.
4. protein chip according to claim 1, it is characterised in that step（2）Described in point sample method be machine it is automatic
Change point sample.
5. protein chip according to claim 1, it is characterised in that step（3）Described in closed process be：Point sample is good
Black slide submerge 1 in confining liquid ~ for 24 hours, take out black slide later, and be centrifuged the remaining confining liquid of removal, the albumen core be made
Piece.
6. protein chip according to claim 5, it is characterised in that the confining liquid is that the buffering containing closed protein is molten
Liquid；The closed protein is bovine serum albumin(BSA) or ovalbumin；The buffer is PBS buffer solution, Tris buffer, HEPS
One of buffer, MOPS buffer are a variety of.
7. a kind of application of any one of claim 1 ~ 6 protein chip, it is characterised in that examination is made in the protein chip
Agent box.
8. application according to claim 7, it is characterised in that the kit further includes being marked with HRP enzyme or alkali phosphorus enzyme
Secondary antibody solution, the chemiluminescent substrate sensitive to marker.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109596840A (en) * | 2019-01-10 | 2019-04-09 | 江苏三联生物工程有限公司 | A kind of protein chip and preparation method thereof for primary biliary cirrhosis marker detection |
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| CN111423505A (en) * | 2020-04-20 | 2020-07-17 | 四川携光生物技术有限公司 | Ribosome P protein modified protein and preparation method and application thereof |
| CN111423505B (en) * | 2020-04-20 | 2022-04-08 | 四川携光生物技术有限公司 | Ribosome P protein modified protein and preparation method and application thereof |
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