CN103185780B - Reagent device for detecting anti-U1-snRNP antibody and method thereof - Google Patents
Reagent device for detecting anti-U1-snRNP antibody and method thereof Download PDFInfo
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Abstract
The invention discloses a reagent device for detecting an anti-U1-snRNP antibody and a method thereof; the reagent device is long strip-shaped, comprises a substrate with eight hole sites and a handle disposed at one end of the substrate; the eight hole sites comprise a sample hole, an adjuvant hole, an enzyme conjugate hole, a substrate hole, a stopping solution hole, a diluent hole, a reaction hole, and a dilution hole, which are arranged in sequence from the end near the handle. The reagent device of the invention realizes anti-U1-snRNP antibody detection with the device based on a principle of enzyme-linked immunoassay, is an independent, single-sample analytic detection method, and can be used cooperatively with corresponding specific analytical instruments; during detection, a detection reagent or a sample is injected by using a full automatic precision liquid filling device, which provides advantages of operation automation, accurate filling amount, high accuracy and precision of detection results; and the reagent device has wide application prospects.
Description
Technical field
The present patent application relates to a kind of reagent device and the method thereof that detect anti-U1-snRNP antibody, belongs to clinical immunology detection technique field.
Background technology
Mixed connective tissue disease (Mixed connective tissue disease, MCTD) be a kind of with systemic loupus erythematosus (SLE), systemic sclerosis (SSc), the symptom of the diseases such as polymyositis/dermatomyositis (PM/DM) and rheumatoid arthritis (RA) overlaps as the rheumatic syndrome of feature, and its outstanding feature is in its serum, have the spotted type antinuclear antibodies (ANA) of very high titre and anti-U1-snRNP antibody.
At eukaryotic, heterogeneous karyon ribonucleoprotein (hnRNP) and micronuclear ribonucleoprotein (snRNP) participate in the process of mRNA maturation jointly.SnRNA is one group of microRNA in eukaryotic core, about containing 50 ~ 200 nucleotide, therefore is called small nuclear rna.It is combined the snRNP formed with associated proteins, mainly at processing RNA precursor, play a significant role when excising unnecessary fragment (as introne).Because this organizes uracil rich content in micromolecular snRNP, the U-snRNP therefore snRNP is otherwise known as.Have now found that, the U-snRNP in mammalian cell has at least 13 kinds (U1 ~ U13), is mostly distributed in caryoplasm.6 kinds of snRNA (U1 ~ U6) being rich in U are present in zooblast core in a large number.Often kind of snRNP is formed by a kind of snRNA (respectively corresponding title U1, U2, U4, U5 and U6) albumen corresponding to 6 ~ 10.
The antibody reacted with U1-snRNP comprises Sm antibody and RNP antibody.RNP antibody contains anti-U1-snRNP antibody, and anti-U1-snRNP antibody positive and the anti-Sm antibody positive are the features of MCTD.But find that in patients serum the situation of anti-U1-snRNP antibody and anti-Sm antibody is much shown in, this may expand relevant with autoimmunity simultaneously.
The common methods of the anti-U1-snRNP antibody of clinical detection comprises Western blot, countercurrent immunoelectrophoresis and ELISA method, but these methods all also exist weak point.
One, Western blot
Western blotting moves on on film by protein transduction, then utilizes antibody to detect.To known expressing protein, available corresponding antibodies detects as primary antibodie, to the expression product of new gene, by merging the antibody test of part.Its weak point is:
(1) can only quantitative and semi-quantitative analysis be carried out, the amount that analyte is concrete cannot be drawn.
(2) complex operation step, the test used time is longer.
(3) sensitivity detected need to improve.
Two, countercurrent immunoelectrophoresis
Counter immunoelectrophoresis is the easy and method fast of the one set up in conjunction with electrophoretic techniques on AGP test basis.Can there is result in the method, therefore can be used for quick diagnosis at short notice.But there is also weak point:
(1) when antigen-antibody ratio is not suitable for, obvious visible precipitation line can not all be there is;
(2) different electrophoretic buffers is selected to have a certain impact to its sensitivity;
(3) selection of voltage and current can interference experiment result.
Three, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used in detecting anti-U1-snRNP antibody, but the method also also exists following weak point:
(1) use 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate as antigen coated apparatus and reaction vessel, 12 batches, 6 batches, 8 batches can only be divided in use or whole plate once uses, cannot carry out independently, the detection of single part;
(2) reagent type that quantitative measurement is used is more, each detects reagent and all carrys out splendid attire with reagent bottle, and all need to change imbibition nozzle when often using a kind of reagent to be filled into respectively in the micropore of microwell plate, not only reagent bottle kind is many, and the operation of filling reagent is also very loaded down with trivial details;
(3) the corresponding mark to Detection Information is lacked, can only by checking that the product batch number and term of validity information detecting reagent could be understood or know to the mark of kit external packing box, and the information known is not controlled in testing process, there is very large randomness;
(4) detect reagent in testing process, be in open space, easily cause the cross pollution between various reagent and affect the accuracy of testing result;
(5) the many employing manual operationss of testing process, the dosage of reagent or sample is not bery accurate, and operating process is very loaded down with trivial details and complicated, easily bust occurs, and accuracy and the precision of testing result are poor;
(6) in the quantity configuration and use of test item reagent set, item number × 48/96 person-portion is, if need detection 10 projects, then reagent configuration and use number must be 10 × 48/96 person-portions, if only have the project that a sample needs detection 10 different, also need the reagent of configuration 10 × 48/96 person-portion, there is the shortcoming of inadequate economical rationality.
Summary of the invention
Patented claim of the present invention be namely for current for above shortcomings part in the detection of anti-U1-snRNP antibody, a kind of reagent device and the detection method thereof that can carry out the detection of separate single person-portion are provided.
Patented claim of the present invention realizes the detection of anti-U1-snRNP antibody based on the principle of enzyme linked immunosorbent detection, be a kind of independently, single part, the disposable method of inspection; Further, the present patent application additionally provides the corresponding reagent device supporting to the method, plurality of reagents required for anti-U1-snRNP antibody ELISA immune detection is contained in one independently, in the reagent device of porous structure, moved abandoned detecting step by substep filling by this reagent device; Further, described reagent device can also carry out corresponding fully-automated synthesis by special immunity analysis instrument.
One of object of the present patent application is to provide a kind of reagent device detecting anti-U1-snRNP antibody, and this object is realized by following technical scheme:
Specifically, the reagent device of the anti-U1-snRNP antibody of the detection described in the present patent application is strip, comprise the matrix with position, eight holes and the handle being positioned at matrix one end, putting in order of position, eight holes is followed successively by sample aperture near handle end, assistant agent hole, enzyme conjugates hole, substrate hole, stop fluid apertures, dilution fluid apertures, reacting hole and dilution holes, sample to be tested is filled in sample aperture, assistant agent hole adds auxiliary reagent whenever necessary for detection, enzyme conjugates solution is filled in enzyme conjugates hole, substrate fills substrate solution in hole, stop filling stop buffer in fluid apertures, dilution is filled in dilution fluid apertures, reacting hole is flat and has high light penetrability, hole endoperidium has U1-snRNP antigen, and to annotate liquid sample to be measured and detect reagent and cleansing solution as reaction vessel, absorbance measurement is carried out after reaction terminating, dilution holes is as Sample Dilution container, during for diluted sample.
Further, in described reagent device, assistant agent hole, enzyme conjugates hole, substrate hole, termination fluid apertures and dilution fluid apertures are coated with sealing film, sealing film mainly plays and prevent the effect of overflow in the process of transport and movement, in addition, the effect that preventing dust pollutes and strengthens stability is also played.
Further, described handle is pasted with bar code, this bar code identification has the relevant information of anti-U1-snRNP antibody test reagent and analytical equipment.
Further, described information comprises test item code, detects the sequence number of reagent product batch number, the reagent term of validity, quantitative measurement standard parameter of curve, enzyme linked immunoassay type, reagent and analytical equipment.
Further, described reagent device also comprises several support columns, described support column is positioned at below described reagent device matrix, is separated with position, more than one hole between adjacent supports post, and the effect of support column is physical strength in order to strengthen matrix and balance.
Further, in described reagent device, reacting hole is made up of exit orifice and endoporus, the bottom of exit orifice has bottom outlet, endoporus through bottom outlet and with bottom outlet wringing fit, leave anti-overflow chamber between exit orifice and endoporus, the effect in anti-overflow chamber is in course of reaction, if overflow, can stay in chamber, preventing pollution instrument and other reagent devices.
Further, in described reagent device, the U1-snRNP antigen of the endoporus bag quilt of reacting hole comprises natural U1-snRNP or the U1-snRNP of restructuring.
It is clearly understood that, in the present patent application, in described reagent device, the size of position, each hole, shape are not limited, such as the open peristoma of position, each hole can be square or circular, the shape of its section also can be square, " U " type or " V " type, but for reacting hole, in order to obtain good light penetrability, bottom should be flat.
Further, the sample added in described sample aperture is test serum or blood plasma, and the amount added is determined according to the multiple of the size of sample aperture and the dilution of detection needs.
Described assistant agent hole is the use adding auxiliary reagent when being necessary, such as adsorbent, neutralizing agent, inhibitor, damping fluid etc., to remove the interference to detection reaction and result of other materials in testing process.
Enzyme conjugates solution is added in described enzyme conjugates hole, Main Ingredients and Appearance is anti-human igg or IgA or IgM or Ig (GAM) antibody of horseradish peroxidase-labeled, the anti-human igg of alkali phosphatase enzyme mark or IgA or IgM or Ig (GAM) antibody, the anti-human igg of glucose oxidase thing enzyme labeling or IgA or IgM or Ig (GAM) antibody, or beta galactosidase mark anti-human igg or IgA or IgM or Ig (GAM) antibody.
The substrate solution Main Ingredients and Appearance added in described substrate hole is TMB, OPD or ABTS.
TMB is as a kind of chromogen reagent of new type of safe, progressively replace the benzidine derivative of strong carcinogen biphenylamine and other carcinogenicity, be applied to the fields such as clinical assay, forensic medical examination, criminal detection and environmental monitoring, especially in biochemistry test, TMB, as the new substrate of peroxidase, obtains a wide range of applications in enzyme immunoassay (EIA) (EIA) and enzyme linked immunosorbent assay method (ELISA).
Sulfuric acid or the hydrochloric acid solution of the stop buffer Main Ingredients and Appearance added in described termination fluid apertures to be concentration be 0.1 ~ 0.5M.
The dilution Main Ingredients and Appearance added in described dilution fluid apertures is phosphate buffer, Tris damping fluid, borate buffer solution or carbonate buffer solution, and containing components such as certain density bovine serum albumin(BSA), sodium azide, for getting rid of the interference of the nonspecific reaction of sample small molecular material and increasing stability.
Another object of the present patent application is to provide the detection method utilizing mentioned reagent device to carry out anti-U1-snRNP antibody, and described method comprises following step:
1) in described sample aperture, undiluted sample is added;
2) from dilution fluid apertures, drawing dilution adds in dilution holes;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw partially liq return dilution fluid apertures;
4) from sample aperture, draw sample add in dilution fluid apertures, sample is diluted;
5) from dilution fluid apertures, imbitition adds in dilution holes, is further diluted by sample;
6) from dilution holes, imbitition adds in reacting hole, under the condition of 25 ~ 37 DEG C, hatches 30 ~ 60min;
7) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
8) from enzyme conjugates hole, drawing enzyme conjugates solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, removes liquid after reaction 30 ~ 60min;
9) step 7 is repeated);
10) from substrate hole, drawing substrate solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, and reaction 10 ~ 20min;
11) from termination fluid apertures, draw stop buffer filling in reacting hole, read OD value in 450nm in 1 ~ 10min, or Double wavelength method measures, wavelength coverage is at 620nm ~ 690nm.
Further, described method comprises following step:
1) in described sample aperture, add undiluted sample, volume is at least 1/4 of sample aperture capacity;
2) from dilution fluid apertures, drawing dilution adds in dilution holes, and the amount of absorption is more than 7/10 of total amount of liquid in hole;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw partially liq according to expection extension rate and return dilution fluid apertures;
4) from sample aperture, draw sample add in dilution fluid apertures, diluted by sample, extension rate is 1 ~ 10 times;
5) from dilution fluid apertures, imbitition adds in dilution holes, and further diluted by sample, extension rate is 1 ~ 100 times;
6) from dilution holes, imbitition adds in reacting hole, under the condition of 25 ~ 37 DEG C, hatches 30 ~ 60min;
7) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
8) from enzyme conjugates hole, drawing enzyme conjugates solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, removes liquid after reaction 30 ~ 60min;
9) step 7 is repeated);
10) from substrate hole, drawing substrate solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, and reaction 10 ~ 20min;
11) from termination fluid apertures, drawing stop buffer adds in reacting hole, reads OD value in 1 ~ 10min in 450nm, or Double wavelength method measures, and wavelength coverage is at 620nm ~ 690nm.
Further, described method comprises following step:
1) in described sample aperture, add sample 80 ~ 150 μ L;
2) from dilution fluid apertures, 360 μ L liquid are drawn in dilution holes with accurate charger;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw 180 μ L liquid return dilution fluid apertures;
4) from sample aperture, 20 μ L liquid are drawn in dilution fluid apertures, the dilution of 10 times, sample with accurate charger;
5) with accurate charger from dilution fluid apertures imbitition 20 μ L in dilution holes, the dilution of 100 times, sample;
6) in reacting hole, at 25 ~ 37 DEG C, after hatching 60min, liquid is removed with accurate charger imbitition 100 μ L from dilution fluid apertures;
7) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
8) react to reacting hole with the abzyme bond solution that 100 μ L horseradish peroxidase-labeled drawn by accurate charger from enzyme conjugates hole, at 25 ~ 37 DEG C, after reaction 30min, remove liquid;
9) step 7 is repeated);
10) to reacting hole, 10 ~ 15min is reacted with the tmb substrate solution that 100 μ L drawn by accurate charger from substrate hole;
11) annotate in reacting hole from the stop buffer stopping drawing fluid apertures 100 μ L with accurate charger, detects under 450nm and 630nm dual wavelength condition in 1 ~ 10min, read OD value.
Described cleansing solution comprises PBST solution, PBST solution refers to that PBS solution adds Tween-20, PBS solution refers to phosphate buffer (Phosphate Buffer Solution), osmotic pressure is similar to physiological condition, has again very strong pH surge capability, is commonly used to wash cell and the basal liquid as cell chulture, and Tween-20 is a kind of non-ionic surfactant, cell growth can have an impact, and Tween-20 has the effect of renaturation antigen, can improve specific recognition capability.
Reagent device and the method thereof of anti-U1-snRNP antibody is measured described in the present patent application, not only there is high specificity that other detection methods have, highly sensitive, accuracy is good, cost is lower, request for utilization is not high, operation is comparatively easy, obtain the advantage that the testing result time is short, be widely used etc., and solve many deficiencies of other detection methods, be embodied in the following aspects:
1. the detection method described in uses enzyme-linked immuno assay principle, utilize specific analytical instrument, adopt supporting special detection kit and analytical reagent device, automatically realizing the quantitative measurement of anti-U1-snRNP antibody, is a kind of completely newly, be suitable for, scheme that is practical, that detect anti-U1-snRNP antibody efficiently, fast;
2. it be a kind of independently, the detection reagent of single part and analytical equipment, without the need to using 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole special enzyme-linked immune microwell plate as antigen coated articles for use and reaction vessel as general ELISA method, as long as have a sample can carry out the detection of respective items object in use and without the waste of reagent, if the quantity of sample exceedes portion, use this reagent and analytical equipment by actual sample number;
3. each is detected required reagent and is contained in the reagent wells position of an analytical reagent device by it, and detection reagent need not be carried out splendid attire with different reagent bottles respectively, not only operate very easy, and be not easy to cause bust, thus ensure the correctness of testing result;
4. it has a Special bar code to each analytical reagent device, the numerical value of bar code comprises test item code corresponding to detection, detects reagent product batch number, the reagent term of validity, quantitative measurement typical curve parameter, concrete enzyme linked immunoassay type, reagent and analytical equipment the information such as sequence number, can not arbitrarily be changed, strictly controlled during use, especially when use exceedes term of validity detection reagent, to be identified and stop and send examining report, thus the accuracy of detection can be guaranteed;
5. each detection reagent is effectively separated and seals by it, can not cause the cross pollution between various reagent and affect testing result;
6. it is a kind of analytical reagent device being exclusively used in particular analysis instrument, and annotate with full automatic accurate charger in testing process and detect reagent, cleansing solution or sample, operation automation, dosage is accurate, and accuracy and the precision of testing result are high;
7., in the quantity configuration and use of test item reagent set, all use needs to carry out being equipped with by reality, especially detecting entry, are equipped with more appropriate, there will not be super configuration and service condition.
Accompanying drawing explanation
Fig. 1 is the cross-sectional view of an embodiment of reagent device described in the present patent application;
Fig. 2 is the top plan view of embodiment described in Fig. 1;
Fig. 3 is the cross-sectional view of another embodiment of reagent device described in the present patent application;
Fig. 4 is the top plan view of reacting hole in another embodiment of reagent device described in the present patent application;
Fig. 5 and Fig. 6 is the assembling schematic diagram of endoporus and exit orifice in reacting hole in reagent device described in Fig. 4;
Wherein, 10 be matrix, 20 be handle, 30 be bar code, 40 be sealing film, 50 be support column, 11 be sample aperture, 12 be assistant agent hole, 13 be enzyme conjugates hole, 14 be substrate hole, 15 for stop fluid apertures, 16 for dilution fluid apertures, 17 be reacting hole, 18 be dilution holes, 171 be exit orifice, 172 be endoporus, 173 be anti-overflow chamber, 174 for bottom outlet.
Embodiment
Below in conjunction with concrete pick-up unit and implementation step, the reagent device described in the present patent application and detection method are conducted further description, object is in order to the public better understands technical scheme described in the present patent application instead of the restriction to described technical scheme.In fact, with identical or approximate principle, to the improvement that described reagent device carries out, comprise the change of its shape, size, material used, and the increase and decrease of corresponding construction or replacement, all within the present patent application technical scheme required for protection.
Embodiment one detects the reagent device one of anti-U1-snRNP antibody
As shown in Figure 1-2, the reagent device of the anti-U1-snRNP antibody of the detection described in the present patent application is strip, comprise the matrix 10 with position, eight holes and the handle 20 being positioned at matrix 10 one end, putting in order of position, eight holes is followed successively by sample aperture 11 from nearly handle 10 end, assistant agent hole 12, enzyme conjugates hole 13, substrate hole 14, stop fluid apertures 15, dilution fluid apertures 16, reacting hole 17 and dilution holes 18, sample to be tested is filled in described sample aperture 11, assistant agent hole 12 adds auxiliary reagent when needing for detection, any reagent is not added in assistant agent hole 12 in the detection method described in the present patent application, enzyme conjugates solution is added in enzyme conjugates hole 13, substrate solution is added in substrate hole 14, stop fluid apertures 15 and add stop buffer, dilution is added in dilution fluid apertures 16, reacting hole 17 is flat and has very high light penetrability, when splendid attire is colourless or Reagent blank solutions time, to the absorbance of visible ray or ultraviolet light or fluorescence close to zero, highly purified U1-snRNP antigen has been coated with in hole, and to annotate liquid sample to be measured and detect reagent and cleansing solution as reaction vessel, absorbance measurement is carried out after reaction terminating, dilution holes 18 is as Sample Dilution container, during for diluted sample.
Further, in described reagent device, assistant agent hole 12, enzyme conjugates hole 13, substrate hole 14, termination fluid apertures 15 and dilution fluid apertures 16 are coated with sealing film 40, sealing film 40 mainly play transport and movement process in, prevent the effect of overflow, in addition, the effect that preventing dust pollutes and increases stability is also played.
Further, described handle 20 is pasted with bar code 30, this bar code 30 mark has the information of anti-U1-snRNP antibody test reagent and analytical equipment, and described information comprises test item code, detects the sequence number of reagent product batch number, the reagent term of validity, quantitative measurement standard parameter of curve, enzyme linked immunoassay type, reagent and analytical equipment.
Embodiment two detects the reagent device two of anti-U1-snRNP antibody
As shown in Figure 3, the reagent device of the anti-U1-snRNP antibody of the detection in the present embodiment, its basic structure is identical with the reagent device in embodiment one, several support columns 50 are also comprised at described reagent device, described support column 50 is arranged in below described reagent device matrix 10, be separated with position, more than one hole between adjacent supports post 50, the effect of support column 50 is physical strength in order to strengthen matrix and balance.
Embodiment three detects the reagent device three of anti-U1-snRNP antibody
As Figure 4-Figure 6, for detecting the preferred embodiment of the reagent device of anti-U1-snRNP antibody described in the present patent application, its basic structure is identical with embodiment one or embodiment two, difference is that reacting hole 17 is detachable structure, reacting hole 17 is made up of exit orifice 171 and endoporus 172, the bottom of exit orifice 171 has bottom outlet 174, endoporus 172 through bottom outlet 174 and with bottom outlet 174 wringing fit, anti-overflow chamber 173 is left between exit orifice 171 and endoporus 172, the effect in anti-overflow chamber 173 is in course of reaction, if overflow, can stay in chamber, preventing pollution instrument and other reagent devices.
Further, the cooperation fixed form of described endoporus and exit orifice can also be that the outer wall of endoporus is designed to two sections, the thickness of epimere outer wall is greater than the thickness of hypomere outer wall, the diameter of the bottom outlet of exit orifice is equal with the external diameter of endoporus hypomere, like this endoporus hypomere is inserted in bottom outlet, the epimere of endoporus is stuck on bottom outlet just, can play fastening effect equally.
Making one indirect method of embodiment four reagent device or kit detects anti-U1-snRNP antibody
The present invention is simpler based on the technical one of enzyme linked immunosorbent detection, accurately, effective methodology, its ultimate principle adopted is Dot-ELISA: will by highly purified U1-snRNP Antigen adsorption in solid phase, by hatch make dilution human serum or blood plasma in specific antibody be combined with antigen, the antibody be not combined with solid phase is removed in washing, add the human immunoglobulins's enzyme connection thing by horseradish peroxidase-labeled, hatch, remove unconjugated enzyme connection thing, add enzyme chromogen substrate, the color produced is directly proportional to the specific antibody concentrations detected in sample.This method is mainly by realizing the immune detection of anti-U1-snRNP antibody for the analytical reagent device of enzyme linked immunosorbent detection and matched reagent.By this enzyme-linked immunoassay method, use analytical reagent device and the reagent of particular analysis instrument simultaneously, can be quick, judge accurately and diagnose for adjuvant clinical.
Using position, hole 11 as sample aperture, add liquid sample in use, take for during detection;
Using position, hole 12 as assistant agent hole, with sealing film sealing, for subsequent use for detection;
Using position, hole 13 as enzyme conjugates hole, add enzyme conjugates solution sealing film and seal, take for during detection;
Using position, hole 14 as substrate hole, seal with sealing film after adding tmb substrate solution, during for detection;
Using position, hole 15 as termination fluid apertures, seal with sealing film after adding stop buffer, during for detection;
Using position, hole 16 as dilution fluid apertures, seal with sealing film after adding Sample dilution, during for detection;
Hole/reacting hole/colorimetric hole using position, hole 17 as encrusting substance, has been coated with highly purified U1-snRNP antigen, and to annotate liquid sample to be measured and detect reagent and cleansing solution as reaction vessel, carries out absorbance measurement after reaction terminating;
Using position, hole 18 as dilution holes, during for diluted sample;
Be pasted with the bar code 30 of anti-U1-snRNP antibody test reagent and analytical equipment information at handle 20, this bar code comprises test item code, detects the sequence number of reagent product batch number, the reagent term of validity, quantitative measurement typical curve parameter, enzyme linked immunoassay type, reagent and analytical equipment.
Prepare some analytical equipments according to the method described above, prepare corresponding calibration object and Quality Control thing in addition.So namely, constitute complete anti-U1-snRNP TPPA kit component.By detecting the reagent device of anti-U1-snRNP antibody and supporting the use the external packing box of component loading kit, namely make and detect anti-U1-snRNP antibody kit.
The detection method of the anti-U1-snRNP IgG antibody of embodiment five
The present patent application provides a kind of method utilizing mentioned reagent device to carry out the detection of anti-U1-snRNP IgG antibody, and described method comprises following step:
1. in described sample aperture, add sample 80 ~ 150 μ L;
2. from dilution fluid apertures, draw 360 μ L liquid in dilution holes with accurate charger;
3. remove the remaining liq in dilution fluid apertures, from dilution holes, draw 180 μ L liquid return dilution fluid apertures;
4. from sample aperture, draw 20 μ L liquid in dilution fluid apertures, the dilution of 10 times, sample with accurate charger;
5. with accurate charger from dilution fluid apertures imbitition 20 μ L in dilution holes, the dilution of 100 times, sample;
6. in reacting hole, at 25 DEG C, after hatching 60min, remove liquid with accurate charger imbitition 100 μ L from dilution fluid apertures;
7. after 3 ~ 5 washings being carried out to reacting hole with cleansing solution, remove liquid;
8. react to reacting hole with anti-human IgG antibodies's enzyme conjugates solution that 100 μ L horseradish peroxidase-labeled drawn by accurate charger from enzyme conjugates hole, at 25 DEG C, after reaction 30min, remove liquid;
9. repeat step 7;
10. to reacting hole, react 10 ~ 15min with the tmb substrate solution that 100 μ L drawn by accurate charger from substrate hole;
11. annotate in reacting hole from the stop buffer stopping drawing fluid apertures 100 μ L with accurate charger, detects, read OD value in 1 ~ 10min under 450nm and 630nm dual wavelength condition.
Embodiment six realizes the analysis operation flow process detected U1-snRNP IgG antibody anti-in sample by fully-automatic analyzer
Further, detection method described in the present patent application also comprise corresponding with the use of fully-automatic analyzer, this fully-automatic analyzer comprises an analytical equipment pallet, it overlaps with the matching form of analytical equipment, one has 30 positions can place for analytical equipment, for detecting analysis, comprising the integrated mechano-electronic structure of modular and software control system in addition, the filling of robotization can be realized, dilute, hatch, wash, reading, analytic process.Each position independent quantitative is analyzed, and ensures the accuracy of result.After instrument runs, analytical equipment pallet can turn to different positions voluntarily and annotate, and dilution, hatches, the step of washing and reading.
Concrete operation comprises following step:
(1) preparation of scrutiny program: configure corresponding solution on request, comprise lavation buffer solution, cleaning fluid or thimerosal, prepare in the corresponding liquid bottles of complete loading;
(2) be connected with external computing machine: instrument is connected with external computing machine, thus the acquired results that normally worked by instrument transfers to integrated system process;
(3) start shooting: after opening instrument switch, by the working interface of external computing machine to the self-inspection of instrument designing automatic or manual, thus prepare for the normal operation of instrument;
(4) preheating: after powering, instrument can start heating schedule, and temperature is adjusted to temperature to be checked;
(5) inspection is rinsed;
(6) detection of anti-U1-snRNP IgG antibody:
1) from have seal while open the package, take the analytical reagent device of requirement, after deaeration, sack be tamping;
2) solution in Inspection and analysis reagent device tmb substrate hole, should be without color change, otherwise should discard;
3) in the sample aperture of each analytical reagent device, add the undiluted sample of 50 ~ 120 μ L respectively, suggestion adds the sample of 100 μ L, often changes the reagent of a lot number, should get one of them analytical reagent device and calibration object carries out instrument calibration;
4) in analytical equipment pallet, the analytical reagent device of corresponding quantity is put into according to the quantity of required detection, click " beginning ", " scanning ", instrument can automatically scanning analysis reagent device bar code, Quality Control thing bar code and calibration object bar code, then sample is numbered, or by outer strip code scanner, sample bar code is scanned;
5) click " RUN ", instrument runs automatically according to bar code information, and according to bar code, program is testing calibration product first, by calibration, revises typical curve; Secondly Quality Control thing is detected, if its testing result is in the scope indicated, then represents that the typical curve of detection meets the requirements, can be used for the detection of sample, finally start the trace routine of sample;
6) dilute: acupuncture holes position sealing film automatic sucking dilution 360 μ L is in dilution holes in filling, and the remaining liq removed in dilution fluid apertures, from dilution holes, draw dilution 180 μ L again return dilution fluid apertures, filling pin carries out Sample Dilution from sample aperture automatic sucking sample 20 μ L to dilution fluid apertures, 20 μ L are drawn to dilution holes again from dilution fluid apertures, after action completes, moved to reacting hole hatch 30 ~ 60min by the sample diluted pin of being annotated, usual instrument is set as 60min, removes liquid afterwards;
7) wash: flushing needle is drawn after a certain amount of lavation buffer solution carries out three to five washings to reagent wells and removed liquid from corresponding liquid bottle;
8) acupuncture of annotating is broken anti-human IgG antibodies's enzyme conjugates solution that enzyme conjugates hole sealing film draws 100 μ L horseradish peroxidase-labeled and is reacted to reacting hole, and remove liquid after reaction 30 ~ 60min, usual instrument is set as 30min;
9) step 7 is repeated);
10) acupuncture of annotating is broken tmb substrate hole sealing film and is drawn the tmb substrate solution of 100 μ L to reacting hole reaction 10 ~ 20min, and usual instrument is set as 10min;
11) acupuncture of annotating is broken termination fluid apertures sealing film and is drawn the stop buffer filling of 100 μ L to reacting hole, in 1 ~ 10min, read OD value under 450nm/630nm dual wavelength.
(7) testing result: when trace routine is run complete, by the data processing software analysis on computing machine, finally generates report to consult;
(8) shut down: detect after terminating, before instrument shutdown, must clean cycle be started, can avoid from the residual salt crystallization in fluid path in solution like this, avoid damaging instrument or causing testing result invalid, after having cleaned, instrument power source is closed automatically.
The Quality Control of the detection application of anti-U1-snRNP IgG antibody in embodiment seven clinical samples, interpretation of result and detection
Adopt method of operating and the program of embodiment five, use the kit described in embodiment four, can be used for the anti-U1-snRNP IgG antibody level in quantitative measurement human serum or blood plasma.
MCTD age of onset was from 4 years old to 80 years old, and Most patients occurs symptom about 30-40 year, and women is common, accounts for 80%.The anti-U1-snRNP antibody of high titre can be detected in its patients serum.Adjuvant clinical diagnosis can be carried out according to the result detected, tentatively judge the situation of patient, finally make a definite diagnosis and should consider in conjunction with clinical manifestation or other diagnostic method/indexs.
Be below the analysis of testing result:
(1) reference value (term of reference)
Negative reference value: < 10AU/mL;
Suitable term of reference can be set up according to actual conditions in each laboratory.
(2) explanation of testing result
If pattern detection value > is 10AU/mL, point out anti-U1-snRNP antibody horizontal to raise, should diagnose in conjunction with clinical or other diagnostic method/indexs.
Be below that the testing process duplicate detection of embodiment six is positive and negative quality controlled serum, the repeatability of check result, obtains following result:
According to the present invention, automatically carry out the detection of many increments anti-U1-snRNP antibody originally by identical analytic process simultaneously, and can be associated with other or unconnected project detects simultaneously, this just makes, and detection more simplifies, cost reduces, detection time shortens, cross pollution not easily occurs, detect processing ease carries out; And detect high specificity, highly sensitive, accuracy good.
Claims (3)
1. detect a detection method for anti-U1-snRNP antibody, it is characterized in that, described method utilizes reagent device and has the fully-automatic analyzer overlapped with reagent device matching form carries out anti-U1-snRNP antibody test, described reagent device is strip, comprise the matrix with position, eight holes and the handle being positioned at matrix one end, putting in order of position, eight holes is followed successively by sample aperture near handle end, assistant agent hole, enzyme conjugates hole, substrate hole, stop fluid apertures, dilution fluid apertures, reacting hole and dilution holes, sample to be tested is filled in sample aperture, assistant agent hole adds auxiliary reagent whenever necessary for detection, enzyme conjugates solution is filled in enzyme conjugates hole, substrate fills substrate solution in hole, stop filling stop buffer in fluid apertures, dilution is filled in dilution fluid apertures, reacting hole is flat and has high light penetrability, hole endoperidium has U1-snRNP antigen, and to annotate liquid sample to be measured and detect reagent and cleansing solution as reaction vessel, absorbance measurement is carried out after reaction terminating, dilution holes is as Sample Dilution container, during for diluted sample,
In described reagent device, assistant agent hole, enzyme conjugates hole, substrate hole, termination fluid apertures and dilution fluid apertures are coated with sealing film; Described reagent device also comprises several support columns, and described support column is arranged in below described reagent device matrix, is separated with position, more than one hole between adjacent supports post; In described reagent device, reacting hole is made up of exit orifice and endoporus, the bottom of exit orifice has bottom outlet, endoporus through bottom outlet and with bottom outlet wringing fit, the outer wall of endoporus is designed to two sections, the thickness of epimere outer wall is greater than the thickness of hypomere outer wall, and the bottom diameter of exit orifice is equal with endoporus hypomere external diameter, is inserted by endoporus hypomere in bottom outlet, the epimere of endoporus is stuck on bottom outlet just, leaves anti-overflow chamber between exit orifice and endoporus; In described reagent device, the antigen of the endoporus bag quilt of reacting hole comprises natural U1-snRNP or the U1-snRNP of restructuring; After described fully-automatic analyzer runs, reagent device pallet can turn to different positions voluntarily and annotate, and dilution, hatches, the step of washing and reading;
Described method comprises following step:
1) in described sample aperture, undiluted sample is added;
2) from dilution fluid apertures, drawing dilution adds in dilution holes;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw partially liq return dilution fluid apertures;
4) from sample aperture, draw sample add in dilution fluid apertures, sample is diluted;
5) from dilution fluid apertures, imbitition adds in dilution holes, is further diluted by sample;
6) from dilution holes, imbitition adds in reacting hole, under the condition of 25 ~ 37 DEG C, hatches 30 ~ 60min;
7) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
8) from enzyme conjugates hole, drawing enzyme conjugates solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, removes liquid after reaction 30 ~ 60min;
9) step 7 is repeated);
10) from substrate hole, drawing substrate solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, and reaction 10 ~ 20min;
11) from termination fluid apertures, drawing stop buffer adds in reacting hole, OD value is read in 450nm in 1 ~ 10min, or Double wavelength method measures, detect under the condition of another wavelength of 620nm ~ 690nm in 450nm and wavelength coverage in 1 ~ 10min, read OD value.
2. method according to claim 1, is characterized in that, described method comprises following step:
1) in described sample aperture, add undiluted sample, volume is at least 1/4 of sample aperture capacity;
2) from dilution fluid apertures, drawing dilution adds in dilution holes, and the amount of absorption is more than 7/10 of total amount of liquid in hole;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw partially liq according to expection extension rate and return dilution fluid apertures;
4) from sample aperture, draw sample add in dilution fluid apertures, diluted by sample, extension rate is 1 ~ 10 times;
5) from dilution fluid apertures, imbitition adds in dilution holes, and further diluted by sample, extension rate is 1 ~ 100 times;
6) from dilution holes, imbitition adds in reacting hole, under the condition of 25 ~ 37 DEG C, hatches 30 ~ 60min;
7) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
8) from enzyme conjugates hole, drawing enzyme conjugates solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, removes liquid after reaction 30 ~ 60min;
9) step 7 is repeated);
10) from substrate hole, drawing substrate solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, and reaction 10 ~ 20min;
11) from termination fluid apertures, drawing stop buffer adds in reacting hole, OD value is read in 450nm in 1 ~ 10min, or Double wavelength method measures, detect under the condition of another wavelength of 620nm ~ 690nm in 450nm and wavelength coverage in 1 ~ 10min, read OD value.
3. method according to claim 1, is characterized in that, described method comprises following step:
1) in described sample aperture, add sample 80 ~ 150 μ L;
2) from dilution fluid apertures, 360 μ L liquid are drawn in dilution holes with accurate charger;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw 180 μ L liquid return dilution fluid apertures;
4) from sample aperture, 20 μ L liquid are drawn in dilution fluid apertures, the dilution of 10 times, sample with accurate charger;
5) with accurate charger from dilution fluid apertures imbitition 20 μ L in dilution holes, the dilution of 100 times, sample;
6) in reacting hole, at 25 ~ 37 DEG C, after hatching 60min, liquid is removed with accurate charger imbitition 100 μ L from dilution fluid apertures;
7) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
8) react to reacting hole with the abzyme bond solution that 100 μ L horseradish peroxidase-labeled drawn by accurate charger from enzyme conjugates hole, at 25 ~ 37 DEG C, after reaction 30min, remove liquid;
9) step 7 is repeated);
10) to reacting hole, 10 ~ 15min is reacted with the tmb substrate solution that 100 μ L drawn by accurate charger from substrate hole;
11) annotate in reacting hole from the stop buffer stopping drawing fluid apertures 100 μ L with accurate charger, detects under 450nm and 630nm dual wavelength condition in 1 ~ 10min, read OD value.
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