CN102147408A - Method for testing anti-SmD1 antibody IgG and reagent device - Google Patents
Method for testing anti-SmD1 antibody IgG and reagent device Download PDFInfo
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Abstract
The invention provides a method for immunologically detecting an anti-SmD1 antibody IgG on the basis of an enzyme-linked immunosorbent assay principle and a reagent device , which relate to an analysis method used for enzyme-linked immunosorbent assay of the anti-SmD1 antibody IgG independently, individually and disposably, a reagent device and a matched reagent. In the method, a plurality of reagents required in the enzyme-linked immunosorbent assay of the anti-SmD1 antibody IgG can be contained in one analyzing device; and according to the method, relevant immunological tests can be conveniently conducted according to using requirements on the tested project, and better basis is provided for clinical practice.
Description
Technical field
The present patent application relates to a kind of method and reagent device of measuring anti-SmD1 IgG antibody, belongs to technical field of biological.
Background technology
Anti-Sm antibody is named with patient's name (Smith).The target antigen of anti-Sm antibody is positioned at one group in nucleus by on the molecule particle that nucleoprotein and RNA constituted.This histone is called as small nuclear ribonucleoprotein (snRNP).Uracil content enriches the UsnRNP so snRNP is otherwise known as among the micromolecular snRNP because this organizes.Wherein UsnRNP by forming spliced body, participates in the maturation of mRNA in cell.Can be called as Sm " common core " by the protein component of anti-S m antibody recognition among the UsnRNP.
Anti-Sm antibody is the serum marker antibody of the present SLE that generally acknowledges, but the positive rate in systemic loupus erythematosus (SLE) only is 20%~40%.The polypeptide of anti-Sm antibody identification has 9 kinds at least, and polypeptide D is main target antigen.Antigen SmD has three kinds of hypotypes, and SmD1, SmD2, SmD3 are the strongest to the SmD1 adhesion.The autoantibody of binding proteins specific polypeptide SmD1 is called anti-SmD1 antibody.SmD1 is a SLE high degree of specificity target antigen, and anti-SmD1 antibody positive rate can reach 70% in SLE patient, but not the immunological disease patient of SLE is 8.3%, and the normal healthy controls group in the research is all negative.Diagnosis not only has high degree of specificity to SmD1 antibody to SLE, and its susceptibility is higher than anti-Sm antibody.Can detect SmD1 antibody among the negative SLE patient of anti-Sm antibody. and in SmD1 negative antibody SLE patient, fail to detect Sm antibody, thereby this antibody test diagnosis is better than the Sm antibody test to SLE.Its detection method comprises following several:
One, immuno-precipitation
Immuno-precipitation is mainly used in the qualitative detection of antigen or antibody.Its principle is that soluble antigen and corresponding antibodies are under the situation that has electrolyte to exist, by the formed visible precipitate thing of proper proportion phenomenon.This method is to detect the goldstandard (gold standard) of anti-Sm antibody and anti-nRNP antibody, but this method and technology requires height, the application of radiation isotope, so be not widely adopted.And this law is single qualitative test, can not quantitative test, and background is easy-clear not, and the result is had a significant impact.
Two, immune double diffusion method
Immune double diffusion method specificity height, simple to operate, need not the antigen of specific apparatus or purifying, but its susceptibility is relatively low.
Three, indirect immunofluorescence
The general the method for using is carried out the examination of preliminary antibody.But its deficiency also is tangible:
(1) can't be when analysis result according to the non-specific identification of the size discrimination of molecular weight.
(2) operation relative complex needs the expensive fluorescent microscope of price, is difficult to promote at a lot of basic hospitals, also not too is applicable to the laboratory that specimen amount is more.
(3) background in the fluorometric assay is higher, and immunofluorence technic is used for quantitative measurement certain difficulty.
(4) result judges needs experienced professional, the objectivity deficiency of analysis result.
Four, Western blot:
After generally having passed through the examination of indirect immunofluorescence, confirm with Western blot.Its weak point is:
(1) qualitative and semi-quantitative analysis can only be carried out, the concrete amount of analyte can't be drawn.
(2) complex operation step, the test time spent is longer, carries out this in basic unit and detects relatively difficulty.
(3) sensitivity of Jian Ceing and specificity are still waiting to improve.
(4) simultaneously, owing to extract the whole process complexity of antigen, length consuming time, instrument and equipment requires high, and antigen is difficult to long preservation, also is unfavorable for clinical application.
(5) test findings is difficult for judging.
Five, enzyme linked immunosorbent assay
ELISA has advantages such as susceptibility height, running time weak point, but compare with other biological detection or immune detection, this ELISA detection method, technology, instrument or product still have more deficiency and make its application limited, and these deficiencies mainly comprise the following aspects:
(1) uses the special-purpose microwell plate in 12 * 8 types, 8 * 12 types or complete plate 96 holes as antigen or antibody sandwich articles for use and reaction vessel, can only be divided into 12 batches, 8 batches or whole plate in use and once use;
(2) the used reagent of quantitative measurement can reach 11 kinds, each detectable all will be come splendid attire with reagent bottle, and all need during a kind of reagent of every use to change in the micropore that imbibition nozzle is filled into microwell plate respectively, not only the reagent bottle kind is many, the operation of filling reagent is also very loaded down with trivial details, if do not use the full-automatic enzyme non-analysis meter, then all operation all will be carried out by hand, and the price of full-automatic enzyme non-analysis meter is very expensive, drops into bigger;
(3) each detection or each bar code that detects no reagent information, can only could understand or know the product batch number and the term of validity information of detectable by the sign of checking the kit external packing box, and the information of being known is not controlled in testing process, has very big randomness;
(4) detectable is open mode in testing process, causes the cross pollution between all ingredients easily and influences testing result;
(5) testing process when not adopting the full-automatic enzyme non-analysis meter to detect is manual operations, and the dosage of reagent or sample is not really accurate, and operating process is very loaded down with trivial details and complicated, and bust takes place easily, the inaccuracy of testing result and imprecision height;
(6) in the configuration of the quantity of test item reagent set and use and be item number * 96 person-portions, detect 10 projects if desired, then the configuration of reagent and use number must be 10 * 96 person-portions, if have only a duplicate samples need detect 10 different projects, also need to dispose the reagent of 10 * 96 person-portions.
Summary of the invention
In order to solve the weak point that exists in the existing method of anti-SmD1 IgG antibody analyzing and testing, the present patent application provides a kind of new detection method and reagent device and matched reagent.This method is sought a kind of simpler, accurate and effective methodology and reagent and is carried out detection by quantitative to satisfy the needs of clinical diagnosis based on the enzyme linked immunosorbent detection technology.
This method realizes the immune detection of anti-SmD1 IgG antibody based on the principle of enzyme linked immunosorbent detection, be a kind of independently, single part, disposable analytical approach, reagent device and the matched reagent that is used for the anti-SmD1 IgG antibody of enzyme linked immunosorbent detection, it can will resist the needed plurality of reagents of SmD1 IgG antibody enzyme linked immunosorbent detection to be contained on the analytical equipment, the immunology detection that can be correlated with according to the use needs of test item more easily by this method is for clinical practice provides better foundation.
The present patent application provides a kind of method of measuring anti-SmD1 IgG antibody, be to realize by the kit that the analytical reagent device and the matched reagent of enzyme linked immunosorbent detection are formed, this analytical reagent device comprises matrix that is provided with position, 8 holes and the handle that is positioned at matrix one end, it by special-purpose particular analysis instrument the various particular agent solution between each hole of reagent device is annotated and suction is abandoned, sample and reagent are reacted, measure the numerical value of the back solution color and luster that reacts then, finally obtain testing result by the numerical value of measuring is handled.
The method of the anti-SmD1 IgG antibody of described mensuration, be the to be measured anti-SmD1 IgG antibody in the testing sample and highly purified SmD1 antigen are reacted and to form first immune complex, the second antibody of this first immune complex and enzyme labeling is reacted and is formed second immune complex, the comparative analysis that develops the color of second complex compound that reaction is formed and chromogenic substrate, thus the content of anti-SmD1 IgG antibody to be measured obtained.
The method of the anti-SmD1 IgG antibody of described mensuration, wherein, described second antibody is the anti-human IgG antibody of horseradish peroxidase-labeled.
The present patent application also provides a kind of reagent device of measuring anti-SmD1 IgG antibody, be provided with position, 8 holes matrix, be positioned at the handle of matrix one end, and be used for the gentle component of matched reagent calibration object, Quality Control thing that euzymelinked immunosorbent assay (ELISA) detects the analytical reagent device of anti-SmD1 IgG antibody and respective numbers towards cleansing solution.
The reagent device of the anti-SmD1 IgG antibody of described mensuration, wherein, be pasted with the mark card of detectable bar code on the handle of described matrix one end, the numerical value of described bar code comprises every information that detects the sequence number of pairing test item code, detectable product batch number, the reagent term of validity, qualitative corrected value/quantitative measurement typical curve parameter, enzyme linked immunoassay type, reagent and analytical equipment.
In the reagent device of the anti-SmD1 IgG antibody of described mensuration, position, described hole comprises a reacting hole, a sample well, a dilution holes and five reagent wells, wherein,
1) sample well is contained and is held solution to be measured;
2) dilution holes is used for dilution of sample;
3) reacting hole is flat and has very high light source/light path permeability, when splendid attire colourless/absorbance to visible/ultraviolet/fluorescence during blank reagent solution levels off to zero, the hole endoperidium has the required highly purified SmD1 antigen of the anti-SmD1 IgG antibody of detection, this hole is used for containing appearance test sample and detectable, and with these samples and reagent generation enzyme linked immunoassay, be the container that enzyme linked immunoassay and color and luster show and detect;
4) each reagent wells is loaded with euzymelinked immunosorbent assay (ELISA) and detects the required a kind of reagent of anti-SmD1 IgG antibody, with sealing film the open peristoma of micropore is sealed behind the filling reagent.
The required reagent of institute's splendid attire enzyme linked immunosorbent detection comprises required immune response inhibitor/neutralizing agent/blocking agent/adsorbent, enzyme conjugates solution, chromogenic substrate solution, colour developing stop buffer, increased response agent/promoter, the diluted sample solution of enzyme linked immunoassay that detects anti-SmD1 IgG antibody in the reagent device of the anti-SmD1 IgG antibody of described mensuration, described reagent wells.
The reagent device of the anti-SmD1 IgG antibody of described mensuration, described sample well, reacting hole, dilution holes and reagent wells section shape comprise flat pattern, V-type or U type, or are the combination in any between flat pattern, V-type and the U type.
The method of the anti-SmD1 IgG antibody of described mensuration, described method comprises following step:
1) start: after opening instrument switch, instrument can automatically carry out a series of inspections, thereby prepares for the normal operation of instrument;
2) preparation of scrutiny program: configure corresponding solution on request, comprising: buffering cleansing solution, cleaning fluid, thimerosal and distilled water or deionized water, preparation finish and pack in the corresponding liquid jar;
3) flushing is checked:
4) preheating: after start, instrument can start heating schedule, and temperature is transferred to temperature to be checked;
5) connect machine with main frame: instrument can link to each other with main frame by the RS232 serial ports, handles thereby instrument operate as normal gained result is transferred to integrated system;
6) detection of anti-SmD1 IgG antibody: described detection comprises following step again:
I. open the package from one side that seal is arranged, take the analytical equipment of requirement, behind the deaeration that the sack envelope is tight;
Ii. check the substrate in the analytical equipment reagent wells, should be no color and luster and change, otherwise should discard;
Iii. add the undiluted sample of 50~100 μ L respectively in the sample well of each analytical equipment, the reagent of a lot number of every replacing should be got one of them analytical equipment and calibration object and carry out instrument calibration;
Iv. placing analytical equipment in the corresponding analytical equipment pallet, calibrates and detects according to operation instructions in the instrument;
V. in the analytical equipment pallet, put into the analytical equipment of corresponding quantity according to the quantity of required detection, and before the detection position, put into the analytical equipment that contains calibration object and Quality Control thing, instrument is discriminance analysis device bar code, Quality Control thing bar code and calibration object bar code automatically, selects row can be positioned " sample " hurdle or " detection " hurdle;
Vi. click beginning, the operation repertory scans each analytical equipment bar code, and to the Quality Control thing, calibration object and test sample are numbered;
Vii. operation detects table, and instrument moves automatically according to bar code information, and according to bar code, instrument can be selected the good typical curve of relative set, and program at first detects calibration object, comes curve default in the calibration instrument with this; Secondly the Quality Control thing is detected,, can be used for the detection of sample, begin the trace routine of sample at last if its testing result represents that then built-in curve is qualified in the scope that indicates;
Viii. dilution: the filling pin can be drawn sample automatically from sample well, punctured hole position sealing film is drawn dilution automatically and is carried out diluted sample at dilution holes, after action was finished, diluted sample can be moved to the time of one section program setting of reagent wells reaction by the filling pin, removes liquid afterwards;
Ix. washing: the filling pin can be drawn a certain amount of cleansing solution reagent wells is carried out removing liquid after three to five washings from corresponding flow container;
X. the anti-human IgG antibody that the broken reagent wells sealing film of the acupuncture of annotating is drawn a certain amount of horseradish peroxidase-labeled reacts to reagent wells, removes liquid after the time of one section program setting of reaction;
Xi. repeating step ix washing;
Xii. the broken reagent wells sealing film of the acupuncture of annotating is drawn a certain amount of enzyme reaction substrate carries out one section program setting to reagent wells time response;
Xiii. the broken reagent wells sealing film of the acupuncture of annotating is drawn a certain amount of stop buffer and is annotated to reagent wells, reads the OD value in 450nm in 10 minutes, if select the double wave regular way to measure, reference wavelength is 620nm~690nm;
7) testing result: when trace routine operation finishes, click the data transmission with main frame, instrument can send to main frame with operate as normal gained result automatically and transfer to the analysis of external data process software, generates report at last so that consult;
8) shutdown: after detecting end, before the instrument shutdown, must start cycles of washing, can avoid like this avoiding damaging instrument or causing testing result invalid from the residual salt crystallization in the liquid road in the solution, after washing was finished, instrument power source was closed automatically.
Described analytical reagent device be a kind of measure anti-SmD1 IgG antibody independently, single part, the disposable enzyme-linked immuno assay reagent device that is exclusively used in the particular analysis instrument.
Described detection method and the matched reagent that is used for anti-SmD1 IgG antibody enzyme linked immunological of the present patent application, wherein realize the analytical reagent device of anti-SmD1 IgG antibody immune detection, be a kind of independently, single part, the disposable enzyme linked immunosorbent detection reagent device that is exclusively used in the particular analysis instrument.
The method and the device of the anti-SmD1 IgG antibody of the described mensuration of the present patent application, inherited the high specificity that other detection methods had, highly sensitive, characteristics such as accuracy is good, cost is lower, request for utilization is not high, the operation is comparatively easy, the acquisition testing result time lacks, is widely used, solve many deficiencies of other detection methods, be embodied in the following aspects:
1. this detection method, its utilization enzyme-linked immuno assay principle, utilize specific analytical instrument, adopt the detection kit and the analytical reagent device of supporting special use, automatically realize the qualitative/quantitative measurement of anti-SmD1 IgG antibody, be a kind of brand-new, that be suitable for, practical, detect the scheme of anti-SmD1 IgG antibody efficiently, fast;
It is a kind of independently, single part detectable and analytical equipment, need not as general ELISA method, to use 12 * 8 types, 8 * 12 types or complete plate 96 hole special enzyme-linked immune microwell plates as antigen or antibody sandwich articles for use and reaction vessel, do not have the waste of reagent as long as there is a duplicate samples can carry out the detection of respective items purpose in use.If the quantity of sample surpasses a, use this reagent and analytical equipment to get final product by the actual sample number;
3. no matter be qualitative detection or detection by quantitative, it detects necessary reagent with each and is contained in the reagent wells position of an analytical reagent device, and detectable need not be come splendid attire with different reagent bottles respectively, not only operation is very easy, and be not easy to cause bust, thereby guarantee the correctness of testing result;
4. it all has a special-purpose bar code to each analytical reagent device, the numerical value of bar code comprises the information such as sequence number that detect pairing test item code, detectable product batch number, the reagent term of validity, quantitative measurement typical curve parameter, concrete enzyme linked immunoassay type, reagent and analytical equipment, can not arbitrarily be changed, strictness is controlled during use, especially when using above term of validity detectable, to be identified and stop and send examining report, thereby can guarantee the accuracy that detects;
5. it is effectively separated each detectable and seals, and can not cause the cross pollution between all ingredients and influences testing result;
6. it is a kind of analytical reagent device that is exclusively used in the particular analysis instrument, in testing process with full automatic accurate charger annotate detectable or sample, operation automation, dosage is accurate, the accuracy of testing result and precision height;
7. in the configuration of the quantity of test item reagent set and use, all use to be equipped with by reality and get final product, especially multinomial visual inspection are being surveyed, and are equipped with more in right amount, can not occur surpassing and dispose and operating position;
Description of drawings
Fig. 1 is the cross-sectional view of an embodiment of the reagent device of the anti-SmD1 IgG antibody of the described mensuration of the present patent application;
Fig. 2 is the top plan view of an embodiment of the reagent device of the anti-SmD1 IgG antibody of the described mensuration of the present patent application;
Fig. 3 is the cross-sectional view of another embodiment of the reagent device of the anti-SmD1 IgG antibody of the described mensuration of the present patent application;
Fig. 4 is the top plan view of another embodiment of the reagent device of the anti-SmD1 IgG antibody of the described mensuration of the present patent application;
Fig. 5 and Fig. 6 are the cross-sectional view of other embodiment of the reagent device of the anti-SmD1 IgG antibody of the described mensuration of the present patent application; Wherein, 1 is that sample well, 2,3,4,5,6 is that reagent wells, 7 is that reacting hole, 8 is that dilution holes, 9 is that handle, 10 is that sealing film, 11 is that matrix, 90 is a labeling.
Embodiment
Below in conjunction with concrete pick-up unit and implementation step described detection method of the present patent application and reagent device are further described, purpose is for the public better understands the described technical scheme of the present patent application, rather than to the restriction of described technical scheme.In fact, in spirit of the present invention, to the improvement of described method step, and to increase and decrease, replacement and the improvement of corresponding reagent apparatus structure all within the present patent application technical scheme required for protection.
Embodiment 1 detects indirect enzyme-linked immunosorbent detection method and the kit and the reagent device of anti-SmD1 IgG antibody
The present invention is based on technical a kind of simpler, the accurate and effective methodology of enzyme linked immunosorbent detection, and its ultimate principle that adopts is the indirect enzyme-linked immunosorbent method.To be adsorbed on the solid phase by highly purified SmD1 antigen, combine with antigen by the specific antibody of hatching in the human serum that makes dilution, the antibody that does not combine with solid phase is removed in washing, adds the anti-human immunoglobulin(HIg) enzyme connection thing with horseradish peroxidase-labeled, hatches.Remove unconjugated enzyme connection thing, add enzyme chromogen substrate.The color that produces is directly proportional with specific antibody concentration in the detection sample.This method mainly is to realize the immune detection of anti-SmD1 IgG antibody by analytical reagent device that is used for enzyme linked immunosorbent detection and matched reagent.By this kind of enzyme linked immunoassay method, use the analytical reagent device and the reagent of particular analysis instrument simultaneously, can be quick, make accurately and judge the needs that are used for clinical diagnosis.Its analytical equipment concrete structure is as follows:
Shown in Fig. 1-6, it is the described a kind of reagent device that anti-SmD1 IgG antibody enzyme linked immunosorbent detection is analyzed of measuring of the present patent application, comprise matrix 11, on described matrix 11, be provided with position, 1~8 hole (1,2,3,4,5,6,7,8), its mesopore position 1 is the sample well that is used for the splendid attire testing sample, and at the bottom of its bottom surface is " V " type groove, all the other positions, hole are reacting holes 7, dilution holes 8, reagent wells (2,3,4,5,6), described reacting hole 7 is to be used to receive test sample and detectable and as the reacting hole of the container of enzyme linked immunoassay and colorimetric, this reacting hole is the penetrating hole of light source/light path, be provided with handle 9 at described matrix one end, on described handle, be pasted with the labeling 90 that detects anti-SmD1 IgG antibody reagent information bar code.In the present embodiment, described label is the 2,3,4,5, the 6th, reagent wells, during use, these labels are to be loaded with in 2,3,4,5,6 reagent wells to detect required reagent, and its open peristoma can be square or circular, at the bottom of its bottom surface is " V " type groove, seal with sealing film 10 splendid attire reagent or vacant back, described label is 8 to be dilution holes, is used for dilution of sample, does not cover sealing film above.
Other reagent in the kit outside the analytical reagent device comprise: calibration object, Quality Control thing, buffering cleansing solution, cleaning fluid, thimerosal and distilled water or deionized water.
Making-the indirect method of embodiment 2 reagent devices or kit detects anti-SmD1 IgG antibody
As the testing sample container containing, add fluid sample with position, hole 1 in use, take during for detection;
, seal as emptying aperture with position, hole 2 with sealing film, standby for detecting;
, add and seal with sealing film after sample dilutes reagent as reagent container with position, hole 3, take during for detection;
As reagent container, adding stops sealing with sealing film behind the reagent with position, hole 4, uses during for detection;
, seal with sealing film behind anti-human IgG antibody's reagent of adding horseradish peroxidase-labeled as reagent container with position, hole 5, use during for detection;
, seal with sealing film behind the adding enzyme reaction substrate reagent as reagent container with position, hole 6, use during for detection;
As encrusting substance hole/reaction vessel/colorimetric hole, be coated with highly purified SmD1 antigen with position, hole 7, and, added at last and carry out absorbance measurement after enzyme reaction substrate is hatched as reaction vessel filling fluid sample and detectable and cleansing solution to be measured;
, use during as the diluted sample container with position, hole 8 for dilute sample;
Be pasted with the bar code 90 of anti-SmD1 IgG antibody detectable and analytical equipment information at handle 9, this bar code comprises the sequence number of test item code, detectable product batch number, the reagent term of validity, qualitative correction coefficient/detection by quantitative correction coefficient, enzyme linked immunoassay type, reagent and analytical equipment.
Prepare some analytical equipments according to the method described above, prepare reagent corresponding in addition, comprise calibration object, Quality Control thing, buffering cleansing solution, cleaning fluid, thimerosal and distilled water or deionized water etc.So promptly constitute complete anti-SmD1 IgG antibody and measured reagent constituents.With detecting pack into the external packing box of kit of the reagent device of anti-SmD1 IgG antibody and supporting use component, promptly make and detect anti-SmD1 IgG antibody kit.
The analysis operation flow process that embodiment 3 detects the anti-SmD1 IgG antibody of sample by the fully-automatic analyzer realization
Fully-automatic analyzer comprises an analytical equipment pallet, and it is to match with the shape of analytical equipment, and one has 30 positions can place for analytical equipment, is used for check and analysis.Comprise the integrated mechano-electronic structure of modular in addition, can realize the application of sample of robotization, dilution is hatched, washing and reading process.Each position independent quantitative is analyzed, and an electronic sensor monitoring instrument operation surplus having 200, guarantees result's accuracy.After the instrument operation, the analytical equipment pallet can turn to different positions voluntarily and carry out application of sample, and dilution is hatched, the step of washing and reading.
(1) start
After opening instrument switch, instrument can automatically carry out a series of inspections, thereby prepares for the normal operation of instrument.
(2) preparation of scrutiny program
Configure corresponding solution on request, comprising: buffering cleansing solution, cleaning fluid, thimerosal and distilled water or deionized water, preparation finish and pack in the corresponding liquid jar.
(3) flushing is checked
(4) preheating
After start, instrument can start heating schedule, and temperature is transferred to temperature to be checked.
(5) connect machine with main frame
Instrument can link to each other with main frame by the RS232 serial ports, handles thereby instrument operate as normal gained result is transferred to integrated system.
(6) detection of anti-SmD1 IgG antibody
1) open the package from one side that seal is arranged, take the analytical equipment of requirement, behind the deaeration that the sack envelope is tight;
2) substrate in the inspection analytical equipment reagent wells 6 should be no color and luster and changes, otherwise should discard;
3) add the undiluted sample of 50~100 μ L respectively in the sample well 1 of each analytical equipment, the reagent of a lot number of every replacing should be got one of them analytical equipment and calibration object and carry out instrument calibration;
4) place analytical equipment in the instrument in the corresponding analytical equipment pallet, calibrate (if being necessary) and detect according to operation instructions;
5) in the analytical equipment pallet, put into the analytical equipment of corresponding quantity according to the quantity of required detection, and before the detection position, put into the analytical equipment that contains calibration object and Quality Control thing, instrument is discriminance analysis device bar code, Quality Control thing bar code and calibration object bar code automatically, selects row can be positioned " sample " hurdle or " detection " hurdle;
6) click beginning, the operation repertory scans each analytical equipment bar code, and to the Quality Control thing, calibration object and test sample are numbered;
7) operation detects table, and instrument moves automatically according to bar code information, and according to bar code, instrument can be selected the good typical curve of relative set, and program at first detects calibration object, comes curve default in the calibration instrument with this; Secondly the Quality Control thing is detected,, can be used for the detection of sample if its testing result represents that then built-in curve is qualified in the scope that indicates; Begin the trace routine of sample at last;
8) dilution: the filling pin can be drawn sample automatically from sample well 1, punctured hole position sealing film 10 is drawn dilution automatically and is carried out diluted sample at dilution holes 8, after action is finished, diluted sample can be moved to the time of one section program setting of reagent wells 3 reactions by the filling pin, removes liquid afterwards;
9) washing: the filling pin can be drawn a certain amount of cleansing solution reagent wells 3 is carried out removing liquid after three to five washings from corresponding flow container;
10) broken reagent wells 5 sealing films of the filling acupuncture anti-human IgG antibody that draws a certain amount of horseradish peroxidase-labeled reacts to reagent wells 3, removes liquid after the time of one section program setting of reaction;
11) repeating step 9) washing;
12) broken reagent wells 6 sealing films of filling acupuncture are drawn a certain amount of enzyme reaction substrate to reagent wells 3 and are carried out the time response of one section program setting;
13) broken reagent wells 4 sealing films of filling acupuncture are drawn a certain amount of stop buffer and are annotated to reagent wells 3, read OD value in 450nm in 10 minutes, if select double wave regular way mensuration, reference wavelength is 620nm~690nm;
(7) testing result
When trace routine operation finishes, click the data transmission with main frame, instrument can send to main frame with operate as normal gained result automatically and transfer to the analysis of external data process software, generates report at last so that consult;
(8) shutdown
After detecting end, before the instrument shutdown, must start cycles of washing, can avoid like this avoiding damaging instrument or causing testing result invalid from the residual salt crystallization in the liquid road in the solution, after washing was finished, instrument power source was closed automatically.
The Quality Control of detection application, interpretation of result and the detection of embodiment 4 patient's samples
Adopt method of operating and the program of embodiment 3, using method is used embodiment 2 described kits, can be used for the anti-SmD1 IgG antibody level in the quantitative measurement human serum.
The Anti-Sm class belongs to antinuclear antibodies, finds anti-snRNPs (snRNPs, small nuclear ribonuleoproteinparticles) antibody in systemic loupus erythematosus (SLE) patient who is named as " Sm ", therefore gains the name in 1966.Its corresponding antigens is by U-RNA and common core albumen (being B, B, N, D1, D2, D3, E, F, G albumen composition).Anti-SmD1 only is found among the SLE patient, the characteristic property antibody that is considered to SLE, the diagnostic criteria that has been put into SLE, in SLE patient, positive rate is 20%-30%, its level not activity with the SLE disease is relevant, and also any clinical manifestation with SLE is not relevant, but Anti-SmD1 to early stage, atypical SLE or after treatment is alleviated the retrospective diagnosis of SLE have very great help.Therefore we can carry out clinical diagnosis according to the result who detects, and tentatively judge the situation that the patient is ill, finally make a definite diagnosis and should take all factors into consideration in conjunction with clinical manifestation or other diagnostic method/indexs.
Below be the analysis of testing result:
(1) reference value (term of reference)
Normal reference value: 0~25AU/mL; Detect the negative sample of some (having statistical significance), result's mean value adds 3 times of standard deviations (promptly
) be the upper limit of normal reference value.Advise that each laboratory according to actual conditions, sets up the normal reference value of oneself.
Clinical practice for convenience, we recommend:
Sample value<20AU/mL feminine gender
20AU/mL≤sample value≤30AU/mL is suspicious
Sample value>30AU/mL the positive
(2) explanation of testing result
The result explains: during sample value>30AU/mL, show that antibody concentration obviously raises, should make a definite diagnosis whether suffer from systemic loupus erythematosus in conjunction with clinical manifestation or other diagnostic method/indexs; During sample value<20AU/mL, show that the anti-SmD1 IgG antibody of body level do not have obvious rising; During 20AU/mL≤sample value≤30AU/mL, should detect again, if be suspicious still, 2-3 gathers pattern detection again after week.
Below be positive and negative quality controlled serum with the testing process duplicate detection of embodiment 3, the repeatability of check result obtains following result:
According to the present invention, can automatically carry out the detection of the anti-SmD1 IgG antibody of several samples simultaneously by identical analytic process, this just makes, and detection is more simplified, cost reduces, shorten detection time, be difficult for that cross pollution takes place, detecting operation carries out easily; And the high specificity that detects, highly sensitive, accuracy good.
Claims (9)
1. method of measuring anti-SmD1 IgG antibody, it is characterized in that: described method is to realize by the kit that the analytical reagent device of enzyme linked immunosorbent detection and matched reagent are formed, this analytical reagent device comprises matrix that is provided with position, 8 holes and the handle that is positioned at matrix one end, it by special-purpose particular analysis instrument the various particular agent solution between each hole of reagent device is annotated and suction is abandoned, sample and reagent are reacted, measure the numerical value of the back solution color and luster that reacts then, finally obtain testing result by the numerical value of measuring is handled.
2. the method for the anti-SmD1 IgG antibody of mensuration according to claim 1, it is characterized in that: described method is the to be measured anti-SmD1 IgG antibody in the testing sample and highly purified SmD1 antigen are reacted and to form first immune complex, the second antibody of this first immune complex and enzyme labeling is reacted and is formed second immune complex, the comparative analysis that develops the color of second complex compound that reaction is formed and chromogenic substrate, thus the content of anti-SmD1 IgG antibody to be measured obtained.
3. the method for the anti-SmD1 IgG antibody of mensuration according to claim 2 is characterized in that: described second antibody is the anti-human IgG antibody of horseradish peroxidase-labeled.
4. reagent device that is used to measure anti-SmD1 IgG antibody, it is characterized in that: described reagent device be provided with position, 8 holes matrix, be positioned at the handle of matrix one end, and be used for the gentle component of matched reagent calibration object, Quality Control thing that euzymelinked immunosorbent assay (ELISA) detects the analytical reagent device of anti-SmD1 IgG antibody and respective numbers towards cleansing solution.
5. the reagent device that is used to measure anti-SmD1 IgG antibody according to claim 4, it is characterized in that: be pasted with the mark card of detectable bar code on the handle of described matrix one end, the numerical value of described bar code comprises every information that detects the sequence number of pairing test item code, detectable product batch number, the reagent term of validity, qualitative corrected value/quantitative measurement typical curve parameter, enzyme linked immunoassay type, reagent and analytical equipment.
6. the reagent device that is used to measure anti-SmD1 IgG antibody according to claim 4 is characterized in that: position, described hole comprises a reacting hole, a sample well, a dilution holes and five reagent wells, wherein,
1) sample well is contained and is held solution to be measured;
2) dilution holes is used for dilution of sample;
3) reacting hole is flat and has very high light source/light path permeability, when splendid attire colourless/absorbance to visible/ultraviolet/fluorescence during blank reagent solution levels off to zero, the hole endoperidium has the required highly purified SmD1 antigen of the anti-SmD1 IgG antibody of detection, this hole is used for containing appearance test sample and detectable, and with these samples and reagent generation enzyme linked immunoassay, be the container that enzyme linked immunoassay and color and luster show and detect;
4) each reagent wells is loaded with euzymelinked immunosorbent assay (ELISA) and detects the required a kind of reagent of anti-SmD1 IgG antibody, with film the open peristoma of micropore is sealed behind the filling reagent.
7. the reagent device that is used to measure anti-SmD1 IgG antibody according to claim 6 is characterized in that: the required reagent of institute's splendid attire enzyme linked immunosorbent detection comprises required immune response inhibitor/neutralizing agent/blocking agent/adsorbent, enzyme conjugates solution, chromogenic substrate solution, colour developing stop buffer, increased response agent/promoter, the diluted sample solution of enzyme linked immunoassay that detects anti-SmD1 IgG antibody in the described reagent wells.
8. the reagent device that is used to measure anti-SmD1 IgG antibody according to claim 6, it is characterized in that: described sample well, reacting hole, dilution holes and reagent wells section shape comprise flat pattern, V-type or U type, or are the combination in any between flat pattern, V-type and the U type.
9. the method for the anti-SmD1 IgG antibody of mensuration according to claim 1, it is characterized in that: described method comprises following step:
1) start: after opening instrument switch, instrument can automatically carry out a series of inspections, thereby prepares for the normal operation of instrument;
2) preparation of scrutiny program: configure corresponding solution on request, comprising: buffering cleansing solution, cleaning fluid, thimerosal and distilled water or deionized water, preparation finish and pack in the corresponding liquid jar;
3) flushing is checked:
4) preheating: after start, instrument can start heating schedule, and temperature is transferred to temperature to be checked;
5) connect machine with main frame: instrument can link to each other with main frame by the RS232 serial ports, handles thereby instrument operate as normal gained result is transferred to integrated system;
6) detection of anti-SmD1 IgG antibody: described detection comprises following step again:
I. open the package from one side that seal is arranged, take the analytical equipment of requirement, behind the deaeration that the sack envelope is tight;
Ii. check the substrate in the analytical equipment reagent wells, should be no color and luster and change, otherwise should discard;
Iii. add the undiluted sample of 50~100 μ L respectively in the sample well of each analytical equipment, the reagent of a lot number of every replacing should be got one of them analytical equipment and calibration object and carry out instrument calibration;
Iv. placing analytical equipment in the corresponding analytical equipment pallet, calibrates and detects according to operation instructions in the instrument;
V. in the analytical equipment pallet, put into the analytical equipment of corresponding quantity according to the quantity of required detection, and before the detection position, put into the analytical equipment that contains calibration object and Quality Control thing, instrument is discriminance analysis device bar code, Quality Control thing bar code and calibration object bar code automatically, selects row can be positioned " sample " hurdle or " detection " hurdle;
Vi. click beginning, the operation repertory scans each analytical equipment bar code, and to the Quality Control thing, calibration object and test sample are numbered;
Vii. operation detects table, and instrument moves automatically according to bar code information, and according to bar code, instrument can be selected the good typical curve of relative set, and program at first detects calibration object, comes curve default in the calibration instrument with this; Secondly the Quality Control thing is detected,, can be used for the detection of sample, begin the trace routine of sample at last if its testing result represents that then built-in curve is qualified in the scope that indicates;
Viii. dilution: the filling pin can be drawn sample automatically from sample well, punctured hole position sealing film is drawn dilution automatically and is carried out diluted sample at dilution holes, after action was finished, diluted sample can be moved to the time of one section program setting of reagent wells reaction by the filling pin, removes liquid afterwards;
Ix. washing: the filling pin can be drawn a certain amount of cleansing solution reagent wells is carried out removing liquid after three to five washings from corresponding flow container;
X. the anti-human IgG antibody that the broken reagent wells sealing film of the acupuncture of annotating is drawn a certain amount of horseradish peroxidase-labeled reacts to reagent wells, removes liquid after the time of one section program setting of reaction;
Xi. repeating step ix washing;
Xii. the broken reagent wells sealing film of the acupuncture of annotating is drawn a certain amount of enzyme reaction substrate carries out one section program setting to reagent wells time response;
Xiii. the broken reagent wells sealing film of the acupuncture of annotating is drawn a certain amount of stop buffer and is annotated to reagent wells, reads the 0D value in 450nm in 10 minutes, if select the double wave regular way to measure, reference wavelength is 620nm~690nm;
7) testing result: when trace routine operation finishes, click the data transmission with main frame, instrument can send to main frame with operate as normal gained result automatically and transfer to the analysis of external data process software, generates report at last so that consult;
8) shutdown: after detecting end, before the instrument shutdown, must start cycles of washing, can avoid like this avoiding damaging instrument or causing testing result invalid from the residual salt crystallization in the liquid road in the solution, after washing was finished, instrument power source was closed automatically.
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