CN102147409B - A kind of method measuring Anti-nucleosome antibodies IgG and reagent device - Google Patents
A kind of method measuring Anti-nucleosome antibodies IgG and reagent device Download PDFInfo
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Abstract
The present invention provides method and the reagent device thereof of the immune detection that a kind of principle based on enzyme linked immunosorbent detection realizes Anti-nucleosome antibodies IgG, is a kind of independent of, single part, disposable the analysis method of enzyme linked immunosorbent detection Anti-nucleosome antibodies IgG, reagent device and matched reagent。Plurality of reagents required for Anti-nucleosome antibodies IgG enzyme linked immunosorbent detection can be contained on an analytical equipment by it, can use, according to detection purpose, the immunology detection needing to carry out being correlated with more easily by this method, provide better foundation for clinical practice。
Description
Technical field
The present application relates to a kind of method measuring Anti-nucleosome antibodies IgG and reagent device, belong to technical field of biological。
Background technology
Systemic lupus erythematosus (sle) (SLE) is that one involves whole body connective tissue, makes the chronic inflammation autoimmune disease that multi viscera is undermined。This disease is that one involves multiple system, with chronic nonsuppurative inflammation for feature, the diversified connective tissue disease of complicated clinical manifestation, the cause of disease is still not exclusively clearly, it is generally acknowledged and be likely to belong to multifactor property, namely relevant with inherited genetic factors, viral infection, endocrine factors, medicine factor, environmental factors etc.。
In SLE occurs, nucleosome is that the immunogenicity of main autoantigen and this nucleosome is very strong, drives helper T cell to carry out autoimmune response, and induction produces Anti-nucleosome antibodies (AnuA)。Natural nucleosome and nucleosome substructure (H2A-H2B) DNA are only worked by AnuA, and do not react with DNA therein or histone, and the formation of AnuA is again prior to Anti-hCG action and anti-histone antibody。In recent years, although domestic that AnuA research is still few, but the diagnostic value of SLE has been become a hot issue by, and AnuA is high to the clinical diagnosis sensitivity of SLE. and high specificity is one of SLE diagnostic flag antibody。It can occur in each different times of disease, and SLE early diagnosis and curative effect early and closely related with the activeness of disease are dynamically observed significant by time of appearance。The positive rate of AnuA is significantly larger than other antibody, and result confirmation nucleosome is the autoantigen of SLE is the root of Multiple Antibodies。Also the diagnosis improving SLE is had certain clinical meaning and using value by the detection of prompting AnuA, SLE medical diagnosis on disease is treated Observation On The Prognosis and has great importance。
So far had many different technology to have been used for detection AnuA, including LE test cell line, immuno-precipitation, immunoblotting, indirect immunofluorescence, elisa method, but these methods all also exist weak point；
One, LE test cell line
Due to LE test cell line also exist sensitivity difference, specificity strong, can not quantitatively, operating cost time, technical factor affect the limitation such as big, thus as far back as kahn in 1987 just to whether also needing to reexamine LE cell statement into question。1994, it was out-of-date test that the practical parameter of U.S. clinical pathologist association (ASCP)/measurement result committee more clearly proposes LE cytoscopy, and it is replaced that it should be had more deterministic immunological method。
Two, immuno-precipitation
It is mainly used in the qualitative detection of antigen or antibody。Its principle refers to that soluble antigen and corresponding antibodies are having electrolyte to deposit in case, by the visible precipitate thing phenomenon that proper proportion is formed。This law is single qualitative test, it is impossible to quantitative analysis；And background not easy-clear, result is had a significant impact。
Three, immunoblotting
Immunoblotting is to move on on film by protein transduction, then utilizes antibody to detect。To known expressing protein, available corresponding antibodies detects as primary antibodie, the expression product to new gene, can pass through to merge the antibody test of part。It is disadvantageous in that:
(1) quantitative and semi-quantitative analysis can only be carried out, it is impossible to draw the amount that analyte is concrete。
(2) complex operation step, the test used time is longer。
(3) sensitivity detected need to improve。
Four, indirect immunofluorescence
The ultimate principle of this method is, after being combined with the antigen in cutting into slices with specific antibody, to continue with indirect fluorescent antibody, be combined with antigen antibody complex above, forms antigen-antibody fluorescent composition。Under fluorescence microscope, determine, according to the luminous situation of complex, the antigen detected。The method is evaluated: the anti-fluorescein antibody owing to being combined on antigen antibody complex increases, and the fluorescent brightness sent is strong, thus its sensitivity is strong。But its deficiency is also apparent from:
(1) cannot according to the non-specific identification of the size discrimination of molecular weight when analyzing result。
(2) operation relative complex, it is necessary to price fluorescence microscope costly, is difficult to promote at a lot of basic hospitals, is also poorly suitable for the laboratory that specimen amount is more。
(3) background in fluoremetry is higher, and immunofluorence technic has certain difficulty for quantitative assay。
(4) result judges to need experienced professional, and the objectivity analyzing result is not enough。
Five, enzyme linked immunosorbent assay
Now with the most widely enzyme linked immunosorbent assay (ELISA) detect AnuA。Comparing with other biological detection or immune detection, this ELISA detection method, technology, instrument or product still have more deficiency and make it apply limited, and these deficiencies mainly include the following aspects:
(1) use 12 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate to be coated articles for use and reaction vessel as antigen or antibody, 12 batches, 8 batches or imposite first use can only be divided in use；
(2) reagent used by quantitative assay can up to 11 kinds, each detectable will contain with reagent bottle, and often use be required for during a kind of reagent change imbibition nozzle be filled into respectively in the micropore of microwell plate, not only reagent bottle kind is many, the operation of filling reagent is also extremely loaded down with trivial details, if not using ELISA Auto Analyze System, then all operations will carry out by hand, and the price of ELISA Auto Analyze System is sufficiently expensive, put into bigger；
(3) each detection or every time detection without the bar code of reagent information, just will appreciate that or know product batch number and the effect duration information of detectable only by the mark checking test kit external packing box, and the information known is uncontrolled in detection process, there is very big randomness；
(4) detectable is open mode in detection process, it is easy to cause the cross-contamination between various reagent to affect testing result；
(5) the detection process when being provided without ELISA Auto Analyze System and detecting is manual operations, and the dosage of reagent or sample is not bery accurate, and operating process is extremely loaded down with trivial details and complicated, it is easy to occur bust, the inaccuracy of testing result and imprecision high；
(6) in the quantity configuration of detection project reagent set and use, it is item number × 96 person-portion, if needing 10 projects of detection, then reagent configuration and use number must be 10 × 96 person-portions, if only a sample needs 10 different projects of detection, it is also desirable to the reagent of configuration 10 × 96 person-portions。
Summary of the invention
The weak point existed in the existing method of detection to solve Anti-nucleosome antibodies IgG to analyze, the present patent application provides a kind of new detection method and reagent device and matched reagent。The method, based on enzyme linked immunosorbent detection technology, seeks a kind of simpler, accurate and effective methodology and reagent to carry out detection by quantitative to meet the needs of clinical diagnosis。
This method realizes the immune detection of Anti-nucleosome antibodies IgG based on the principle of enzyme linked immunosorbent detection, it is a kind of independent of, single part, disposable the analysis method of enzyme linked immunosorbent detection Anti-nucleosome antibodies IgG, reagent device and matched reagent, plurality of reagents required for Anti-nucleosome antibodies IgG enzyme linked immunosorbent detection can be contained on an analytical equipment by it, can use, according to detection purpose, the immunology detection needing to carry out being correlated with more easily by this method, provide better foundation for clinical practice。
The present patent application provides a kind of method measuring Anti-nucleosome antibodies IgG, be the analytical reagent device by enzyme linked immunosorbent detection and matched reagent composition test kit realize, this analytical reagent device includes being provided with the matrix of position, 8 holes and being positioned at the handle of matrix one end, it by special particular analysis instrument the various particular agent solution between each hole of reagent device is filled and suction is abandoned, sample and reagent is made to react, then measure the numerical value of solution color after reacting, obtain testing result eventually through the numerical value measured is processed。
The described method measuring Anti-nucleosome antibodies IgG, it is the Anti-nucleosome antibodies IgG to be measured in testing sample is reacted with highly purified nucleosome antigen and forms the first immune complex, the second antibody of this first immune complex and enzyme labelling reacts formation the second immune complex, the second complex reaction formed carries out visual comparison's analysis with chromogenic substrate, thus obtaining the content of Anti-nucleosome antibodies IgG to be measured。
The described method measuring Anti-nucleosome antibodies IgG, wherein, described second antibody is the anti-human IgG antibodies of horseradish peroxidase-labeled。
The present patent application also provides for a kind of reagent device measuring Anti-nucleosome antibodies IgG, it is provided with the matrix of position, 8 holes, is positioned at the handle of matrix one end, and detect the analytical reagent device of Anti-nucleosome antibodies IgG and the component of the matched reagent calibration object of respective numbers, Quality Control thing and buffer solution for euzymelinked immunosorbent assay (ELISA)。
The described reagent device measuring Anti-nucleosome antibodies IgG, wherein, being pasted with the mark note of detectable bar code on the handle of described matrix one end, the numerical value of described bar code comprises the information of the serial number of the corresponding detection item code of each detection, detectable product batch number, reagent effect duration, qualitative corrected value/quantitative assay standard curve parameter, enzyme linked immunoassay type, reagent and analytical equipment。
In the described reagent device measuring Anti-nucleosome antibodies IgG, position, described hole includes 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 for the dilution of sample；
3) reacting hole is flat and has significantly high light source/light path permeability, when contain colourless/Reagent blank solutions time the absorbance of visible/ultraviolet/fluorescence is leveled off to zero, hole endoperidium has the highly purified nucleosome antigen needed for detection Anti-nucleosome antibodies IgG, this hole is used for containing appearance detection sample and detectable, and with these samples and reagent generation enzyme linked immunoassay, be enzyme linked immunoassay and container that color and luster shows and detects；
4) each reagent wells is loaded with a kind of reagent needed for euzymelinked immunosorbent assay (ELISA) detection Anti-nucleosome antibodies IgG, the open peristoma of micropore is closed with sealing film after filling reagent。
The described reagent device measuring Anti-nucleosome antibodies IgG, in described reagent wells, the reagent needed for contained enzyme linked immunosorbent detection includes the immunoreation inhibitor/nertralizer/blocker/adsorbent needed for detecting the enzyme linked immunoassay of Anti-nucleosome antibodies IgG, enzyme conjugates solution, Chromogenic Substrate Solution, colour developing stop buffer, increased response agent/accelerator, sample diluent solution。
The described reagent device measuring Anti-nucleosome antibodies IgG, described sample well, reacting hole, dilution holes and reagent wells section shape include flat pattern, V-type or U-shaped, or be flat pattern, V-type and U-shaped between combination in any。
The described method measuring Anti-nucleosome antibodies IgG, described method comprises the following steps that
1) start: after opening instrument switch, instrument can automatically carry out a series of inspection, thus preparing for the properly functioning of instrument；
2) preparation of program is checked: the good corresponding solution of configuration on request, including: buffer solution, cleanout fluid, disinfectant solution and distilled water or deionized water, prepare in the corresponding liquid tank of complete loading；
3) inspection is rinsed:
4) preheating: after powering, instrument can start heating schedule, and temperature is adjusted to temperature to be checked；
5) connect machine with main frame: instrument by RS232 serial ports can and main frame be connected, thus transferring to integrated system to process instrument normal operation acquired results；
6) detection of Anti-nucleosome antibodies IgG: described detection comprises the following steps that again
I. from have seal while opening the package, take the analytical equipment of requirement, after getting rid of air, bag mouth be tamping；
Ii. the substrate in Inspection and analysis device reagent wells, should be without color change, otherwise should discard；
Iii. in the sample well of each analytical equipment, add 50 respectively～the 100 undiluted samples of μ L, often change the reagent of a lot number, one of them analytical equipment should be taken and calibration object carries out instrument calibration；
Iv. place analytical equipment in corresponding analytical equipment pallet, be calibrated according to operation instructions and detect in instrument；
V. in analytical equipment pallet, corresponding number of analytical equipment is put into according to the quantity of required detection, and put into the analytical equipment containing calibration object and Quality Control thing before detection position, instrument can automatically discriminatory analysis device bar code, Quality Control thing bar code and calibration object bar code, select row can be positioned " sample " hurdle or " detection " hurdle；
Vi. click and start, run project table, scan each analytical equipment bar code, Quality Control thing, calibration object and detection sample are numbered；
Vii. running detection table, instrument runs automatically according to bar code information, and according to bar code, instrument can select the standard curve that relative set is good, and program is testing calibration product first, carrys out curve default in calibration instrument with this；Secondly Quality Control thing is detected, if its testing result is in the scope indicated, then it represents that built-in curve is qualified, can be used for the detection of sample, finally starts the detection program of sample；
Viii. dilution: filling pin can from sample well automatic sucking sample, punctured hole position sealing film automatic sucking diluent carries out diluted sample at dilution holes, after action completes, the sample being diluted can be moved to the time of reagent wells one section of program setting of reaction by filling pin, removes liquid afterwards；
Ix. washing: filling pin can be drawn from corresponding flow container after reagent wells is carried out three to five washings by a certain amount of cleaning mixture and remove liquid；
X. the anti-human IgG antibodies that a certain amount of horseradish peroxidase-labeled drawn by the broken reagent wells sealing film of filling acupuncture reacts to reagent wells, removes liquid after the time of one section of program setting of reaction；
Xi. step ix washing is repeated；
Xii. the broken reagent wells sealing film a certain amount of enzyme reaction substrate of absorption of filling acupuncture carries out the time response of one section of program setting to reagent wells；
Xiii. the broken reagent wells sealing film of filling acupuncture draws the filling of a certain amount of stop buffer to reagent wells, reads OD value in 450nm in 10 minutes, if Double wavelength method measures, reference wavelength is 620nm～690nm；
7) testing result: when detection program is run complete, click the data with main frame and transmit, normal operation acquired results can be sent to main frame and transfer to external data to process software analysis by instrument automatically, ultimately produces report to consult；
8) shutdown: after detection terminates, before instrument shuts down, it is necessary to starts cycles of washing, so can avoid from the residual salt crystallization in fluid path in solution, it is to avoid damaging instrument or cause that testing result is invalid, after having washed, instrument power source is automatically switched off。
Described analytical reagent device is a kind of part independent, single measuring Anti-nucleosome antibodies IgG, the disposable enzyme immunoassay reagent device being exclusively used in particular analysis instrument。
Described in the present patent application for the detection method of Anti-nucleosome antibodies IgG enzyme linked immunological and matched reagent, wherein realize the analytical reagent device of Anti-nucleosome antibodies IgG immune detection, be a kind of enzyme linked immunosorbent detection reagent device being exclusively used in particular analysis instrument independent, single part, disposable。
Method and the device of Anti-nucleosome antibodies IgG is measured described in the present patent application, inherit high specificity that other detection methods have, highly sensitive, accuracy is good, less costly, instructions for use is not high, it is relatively simple to operate, obtain the features such as the testing result time is short, be widely used, solve many deficiencies of other detection methods, be embodied in the following aspects:
1. this detection method, it uses enzyme immunoassay principle, utilize specific analytical tool, adopt supporting special detection kit and analytical reagent device, it is automatically obtained the qualitative/quantitative mensuration of Anti-nucleosome antibodies IgG, is a kind of brand-new, that be suitable for, practical, scheme detecting Anti-nucleosome antibodies IgG efficient, quick；
2. it is detectable and the analytical equipment of a kind of independent, single part, without using 12 × 8 types, 8 × 12 types or complete plate 96 hole special enzyme-linked immune microwell plate to be coated articles for use and reaction vessel as antigen or antibody as general ELISA method, as long as there being a sample can carry out respective items purpose detection in use without the waste of reagent。If the quantity of sample exceedes portion, use this reagent and analytical equipment by actual sample number；
3. no matter it is qualitative detection or detection by quantitative, necessary for each detection reagent is contained in the reagent wells position of an analytical reagent device by it, and detectable need not be contained with different reagent bottle respectively, not only operate extremely easy, and cannot be easily caused bust, thus ensure the correctness of testing result；
4. each analytical reagent device is had a Special bar code by it, the numerical value of bar code comprises the information such as the serial number of the corresponding detection item code of detection, detectable product batch number, reagent effect duration, quantitative assay standard curve parameter, concrete enzyme linked immunoassay type, reagent and analytical equipment, can not arbitrarily be changed, during use carefully controlled, particularly in when using more than effect duration detectable, identified and prevention are sent examining report, such that it is able to guarantee the accuracy of detection；
5. each detectable is carried out effectively separating and sealing by it, and the cross-contamination between various reagent will not be caused to affect testing result；
6. it is a kind of analytical reagent device being exclusively used in particular analysis instrument, fills detectable or sample with full automatic accurate charger, operate automatization in detection process, and dosage is accurate, and the accuracy of testing result and precision are high；
7. in the quantity configuration of detection project reagent set and use, all are undertaken being equipped with by actually used needs, detect particularly in entry, are equipped with more appropriate, do not have super configuration and service condition；
Accompanying drawing explanation
Fig. 1 is the cross-sectional view of an embodiment of the reagent device measuring Anti-nucleosome antibodies IgG described in the present patent application；
Fig. 2 is the top plan view of an embodiment of the reagent device measuring Anti-nucleosome antibodies IgG described in the present patent application；
Fig. 3 is the cross-sectional view of another embodiment of the reagent device measuring Anti-nucleosome antibodies IgG described in the present patent application；
Fig. 4 is the top plan view of another embodiment of the reagent device measuring Anti-nucleosome antibodies IgG described in the present patent application；
Fig. 5 and Fig. 6 is the cross-sectional view of the other embodiments of the reagent device measuring Anti-nucleosome antibodies IgG described in the present patent application；
Wherein, 1 be sample well, 2,3,4,5,6 be reagent wells, 7 be reacting hole, 8 be dilution holes, 9 be handle, 10 be sealing film, 11 be matrix, 90 for labeling。
Detailed description of the invention
Detection method described in the present patent application and reagent device are conducted further description with implementing step below in conjunction with concrete detecting device, in order that the public is better understood from the technical scheme described in the present patent application rather than the restriction to described technical scheme。It is true that in spirit of the invention, the improvement to described method step, and to the increase and decrease of corresponding reagent apparatus structure, replacement and improvement all within the present patent application technical scheme required for protection。
Embodiment 1 detects the indirect enzyme-linked immunosorbent detection method of Anti-nucleosome antibodies IgG and test kit and reagent device
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 adopted is Dot-ELISA。Will by highly purified nucleosome Antigen adsorption in solid phase, be combined with antigen by hatching the specific antibody in the human serum making dilution, washing remove not with the antibody of solid phase binding, human immunoglobulins's enzyme of additions horseradish peroxidase-labeled joins thing, hatches。Remove unconjugated enzyme connection thing, add enzyme chromogen substrate。The color produced is directly proportional to the specific antibody concentrations in detection sample。This method is mainly by the immune detection realizing Anti-nucleosome antibodies IgG 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 the needs for clinical diagnosis accurately。Its analytical equipment concrete structure is as follows:
As shown in figures 1 to 6, it it is a kind of reagent device measuring the analysis of Anti-nucleosome antibodies IgG enzyme linked immunosorbent detection described in the present patent application, including matrix 11, described matrix 11 is provided with position, 1～8 hole (1, 2, 3, 4, 5, 6, 7, 8), its mesopore position 1 is the sample well for containing testing sample, and its bottom surface is " V " type groove-bottom, all the other positions, hole are reacting holes 7, dilution holes 8, reagent wells (2, 3, 4, 5, 6), described reacting hole 7 is for receiving detection sample and detectable the reacting hole as enzyme linked immunoassay and the container of colorimetric, this reacting hole is the through-hole of light source/light path, it is provided with handle 9 in described matrix one end, described handle is pasted with the labeling 90 of detection Anti-nucleosome antibodies IgG reagent information bar code。In the present embodiment, it is described that to be numbered 2,3,4,5,6 be reagent wells, during use, these are numbered in 2,3,4,5,6 reagent wells and have been loaded with the reagent that detection is required, and its open peristoma can be square or circular, and its bottom surface is " V " type groove-bottom, seal with sealing film 10 after containing reagent or being vacant, described to be numbered 8 be dilution holes, for the dilution of sample, is not covered with sealing film above。
Other reagent in test kit outside analytical reagent device include: calibration object, Quality Control thing, buffer solution, cleanout fluid, disinfectant solution and distilled water or deionized water。
The making of embodiment 2 reagent device or test kit-indirect method detection Anti-nucleosome antibodies IgG
Using position, hole 1 as testing sample container containing, add fluid sample in use, take during for detection；
Using position, hole 2 as emptying aperture, seal with sealing film, standby for detection；
Using position, hole 3 as reagent container, seal with sealing film after adding Sample Dilution reagent, take during for detection；
Using position, hole 4 as reagent container, add and seal with sealing film after terminating reagent, during for detection；
Using position, hole 5 as reagent container, seal with sealing film after adding anti-human IgG antibodies's reagent of horseradish peroxidase-labeled, during for detection；
Using position, hole 6 as reagent container, seal with sealing film after adding enzyme reaction substrate reagent, during for detection；
Using position, hole 7 as being coated thing hole/reaction vessel/colorimetric hole, it is coated with highly purified nucleosome antigen, and has filled fluid sample to be measured and detectable and cleaning mixture as reaction vessel, be eventually adding after enzyme reaction substrate is hatched and carry out absorbance measurement；
Using position, hole 8 as diluted sample container, during for dilute sample；
Be pasted with the bar code 90 of Anti-nucleosome antibodies IgG detectable and analytical equipment information at handle 9, this bar code includes the serial number of detection item code, detectable product batch number, reagent effect duration, 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, additionally prepare corresponding reagent, including calibration object, Quality Control thing, buffer solution, cleanout fluid, disinfectant solution and distilled water or deionized water etc.。So namely constitute complete Anti-nucleosome antibodies IgG and measure reagent constituents。By the reagent device of detection Anti-nucleosome antibodies IgG and the external packing box supporting the use component loading test kit, namely make detection Anti-nucleosome antibodies IgG test kit。
Embodiment 3 realizes the analysis operation flow process to sample Anti-nucleosome antibodies IgG detection by fully-automatic analyzer
Fully-automatic analyzer includes an analytical equipment pallet, and it is that the matching form with analytical equipment overlaps, and one has 30 positions is available for analytical equipment placement, is used for detecting analysis。Additionally include the integrated mechano-electronic structure of modular, it may be achieved the application of sample of automatization, dilution, hatch, wash and reading process。Each position independent quantitative is analyzed, and has more than 200 electronic sensor monitoring instruments to run, it is ensured that the accuracy of result。After instrument runs, analytical equipment pallet can turn to different positions voluntarily and carry out application of sample, dilution, hatches, the step of washing and reading。
(1) start
After opening instrument switch, instrument can automatically carry out a series of inspection, thus preparing for the properly functioning of instrument。
(2) preparation of program is checked
The good corresponding solution of configuration on request, including: buffer solution, cleanout fluid, disinfectant solution and distilled water or deionized water, prepare in the corresponding liquid tank of complete loading。
(3) inspection is rinsed
(4) preheating
After powering, instrument can start heating schedule, and temperature is adjusted to temperature to be checked。
(5) machine is connected with main frame
Instrument can be connected with main frame by RS232 serial ports, thus transferring to integrated system to process instrument normal operation acquired results。
(6) detection of Anti-nucleosome antibodies IgG
1) from have seal while opening the package, take the analytical equipment of requirement, after getting rid of air, bag mouth be tamping；
2) substrate in Inspection and analysis device reagent wells 6, should be without color change, otherwise should discard；
3) in the sample well 1 of each analytical equipment, add 50 respectively～the 100 undiluted samples of μ L, often change the reagent of a lot number, one of them analytical equipment should be taken and calibration object carries out instrument calibration；
4) place analytical equipment and in corresponding analytical equipment pallet, be calibrated (if being necessary) and detection according to operation instructions in instrument；
5) in analytical equipment pallet, corresponding number of analytical equipment is put into according to the quantity of required detection, and put into the analytical equipment containing calibration object and Quality Control thing before detection position, instrument can automatically discriminatory analysis device bar code, Quality Control thing bar code and calibration object bar code, select row can be positioned " sample " hurdle or " detection " hurdle；
6) click starts, and runs project table, scans each analytical equipment bar code, and Quality Control thing, calibration object and detection sample are numbered；
7) running detection table, instrument runs automatically according to bar code information, and according to bar code, instrument can select the standard curve that relative set is good, and program is testing calibration product first, carrys out curve default in calibration instrument with this；Secondly Quality Control thing is detected, if its testing result is in the scope indicated, then it represents that built-in curve is qualified, can be used for the detection of sample；Finally start the detection program of sample；
8) dilution: filling pin can from sample well 1 automatic sucking sample, punctured hole position sealing film 10, automatic sucking diluent carries out diluted sample at dilution holes 8, after action completes, the sample being diluted can be moved to reagent wells 3 by filling pin and react the time of one section of program setting, removes liquid afterwards；
9) washing: filling pin can be drawn from corresponding flow container after reagent wells 3 is carried out three to five washings by a certain amount of cleaning mixture and remove liquid；
10) anti-human IgG antibodies that a certain amount of horseradish peroxidase-labeled drawn by broken reagent wells 5 sealing film of filling acupuncture reacts to reagent wells 3, removes liquid after the time of one section of program setting of reaction；
11) step 9 is repeated) washing；
12) the broken reagent wells 6 sealing film a certain amount of enzyme reaction substrate of absorption of filling acupuncture carries out the time response of one section of program setting to reagent wells 3；
13) broken reagent wells 4 sealing film of filling acupuncture draws the filling of a certain amount of stop buffer to reagent wells 3, reads OD value in 450nm in 10 minutes, if Double wavelength method measures, reference wavelength is 620nm～690nm；
(7) testing result
When detection program is run complete, clicking the data with main frame and transmit, normal operation acquired results can be sent to main frame and transfer to external data to process software analysis by instrument automatically, ultimately produces report to consult；
(8) shutdown
After detection terminates, before instrument shuts down, it is necessary to start cycles of washing, so can avoid from the residual salt crystallization in fluid path in solution, it is to avoid damaging instrument or cause that testing result is invalid, after having washed, instrument power source is automatically switched off。
The Quality Control of detection application, interpretation of result and the detection of embodiment 4 Patient Sample A
Adopting operational approach and the program of embodiment 3, using method uses the test kit described in embodiment 2, can be used for the Anti-nucleosome antibodies IgG level in quantitative assay human serum。
Anti-nucleosome antibodies is considered a kind of SLE diagnosis marker。Activeness SLE patient and the inactivity SLE of 62% almost 100% can detect this kind of antibody (detecting that in inactivity SLE patient the probability of anti-dsDNA antibody is only 3.3%) in the patient。Occur because relatively more Zao than anti-dsDNA antibody, so Anti-nucleosome antibodies is considered the SLE early sign thing worsened。Therefore we can carry out clinical diagnosis according to the result of detection, tentatively judges the situation of patient, finally makes a definite diagnosis and should consider in conjunction with clinical manifestation or other diagnostic method/indexs。
It is below the analysis of testing result:
(1) reference value (term of reference)
Normal reference value: 0～25AU/mL；The negative sample of detection some (having statistical significance), (namely the meansigma methods of result adds 3 times of standard deviations) for the upper limit of normal reference value。Advise that each laboratory is according to practical situation, sets up the normal reference value of oneself。
In order to facilitate clinical practice, we prefer that:
Sample value < 20AU/mL is negative
20AU/mL≤sample value≤30AU/mL is suspicious
Sample value > 30AU/mL is positive
(2) explanation of testing result
Result is explained: during sample value > 30AU/mL, it was shown that antibody concentration is significantly raised, should in conjunction with clinical manifestation or other diagnostic method/indexs make a definite diagnosis whether suffer from systemic lupus erythematosus (sle)；During sample value < 20AU/mL, it was shown that body Anti-nucleosome antibodies IgG level is without obvious rising；During 20AU/mL≤sample value≤30AU/mL, should again detect, if being still suspicious, Resurvey pattern detection after 2-3 week。
The following is positive for the detection process duplicate detection of embodiment 3 and negative quality controlled serum, check the repeatability of result, it is thus achieved that following result:
According to the present invention, can automatically being carried out the detection of the Anti-nucleosome antibodies IgG of several samples by identical analysis process, this just makes detection more simplify, cost reduces, the detection time shortens, be not susceptible to cross-contamination, detection processing ease carries out simultaneously；And detection high specificity, highly sensitive, accuracy good。
Claims (1)
1. the enzyme linked immunosorbent detection analytical reagent device purposes in formation determination Anti-nucleosome antibodies IgG test kit, it is characterized in that: the test kit that described mensuration is the analytical reagent device by enzyme linked immunosorbent detection and matched reagent composition realizes, by special particular analysis instrument the various particular agent solution between each hole of analytical reagent device are filled and suction is abandoned, sample and reagent is made to react, then the numerical value of solution color after reacting is measured, testing result is obtained eventually through the numerical value measured is processed, described mensuration is to be reacted with highly purified nucleosome antigen by the Anti-nucleosome antibodies IgG to be measured in testing sample and form the first immune complex, the second antibody of this first immune complex and enzyme labelling reacts formation the second immune complex, the second immune complex reaction formed carries out visual comparison's analysis with chromogenic substrate, thus obtaining the content of Anti-nucleosome antibodies IgG to be measured, the second antibody of described enzyme labelling is the anti-human IgG antibodies of horseradish peroxidase-labeled；
Described test kit includes the matched reagent calibration object of analytical reagent device and the respective numbers detecting Anti-nucleosome antibodies IgG for euzymelinked immunosorbent assay (ELISA), the component of Quality Control thing and buffer solution, described analytical reagent device is provided with the matrix of position, 8 holes and is positioned at the hands handle of matrix one end, the handle of described matrix one end is pasted with the mark note of detectable bar code, the numerical value of described bar code comprises the detection item code that each detection is corresponding, detectable product batch number, reagent effect duration, qualitative corrected value/quantitative assay standard curve parameter, enzyme linked immunoassay type, the information of the serial number of reagent and analytical reagent device；
Position, 8 holes on described matrix is arranged in a row, and position, 8 holes respectively is:
Position, hole 1 is sample well, as testing sample container containing, adds fluid sample in use, takes during for detection；
Position, hole 2 is reagent wells, as emptying aperture, seals with sealing film, standby for detection；
Position, hole 3 is reagent wells, as reagent container, seals with sealing film after adding Sample Dilution reagent, takes during for detection；
Position, hole 4 is reagent wells, as reagent container, adds and seals with sealing film after terminating reagent, during for detection；
Position, hole 5 is reagent wells, as reagent container, seals with sealing film after adding anti-human IgG antibodies's reagent of horseradish peroxidase-labeled, during for detection；
Position, hole 6 is reagent wells, as reagent container, seals with sealing film after adding enzyme reaction substrate reagent, during for detection；
Position, hole 7 is reacting hole, as being coated thing hole/reaction vessel/colorimetric hole, has been coated with the nucleosome antigen of purification, and has filled fluid sample to be measured and detectable and cleaning mixture as reaction vessel, be eventually adding after enzyme reaction substrate is hatched and carry out absorbance measurement；Reacting hole is flat and has significantly high light source/light path permeability, when contain colourless/Reagent blank solutions time the absorbance of visible/ultraviolet/fluorescence is leveled off to zero, hole endoperidium has the highly purified nucleosome antigen needed for detection Anti-nucleosome antibodies IgG, this hole is used for containing appearance detection sample and detectable, and with these samples and reagent generation enzyme linked immunoassay, be enzyme linked immunoassay and container that color and luster shows and detects；
Position, hole 8 is dilution holes, is not covered with sealing film above, as diluted sample container, during for dilute sample；
With thin film, the open peristoma of micropore is closed after each reagent wells filling reagent；
Described sample well, reacting hole, dilution holes and reagent wells section shape include flat pattern, V-type or U-shaped, or be flat pattern, V-type and U-shaped between combination in any；
Described mensuration comprises the following steps that
1) start: after opening instrument switch, instrument can automatically carry out a series of inspection, thus preparing for the properly functioning of instrument；
2) preparation of program is checked: the good corresponding solution of configuration on request, including: buffer solution, cleanout fluid, disinfectant solution and distilled water or deionized water, prepare in the corresponding liquid tank of complete loading；
3) inspection is rinsed;
4) preheating: after powering, instrument can start heating schedule, and temperature is adjusted to temperature to be checked；
5) connect machine with main frame: instrument by RS232 serial ports can and main frame be connected, thus transferring to integrated system to process instrument normal operation acquired results；
6) detection of Anti-nucleosome antibodies IgG: described detection comprises the following steps that again
I. from have seal while opening the package, take the analytical reagent device of requirement, after getting rid of air, bag mouth be tamping；
Ii. the substrate in Inspection and analysis reagent device reagent wells, should be without color change, otherwise should discard；
Iii. in the sample well of each analytical reagent device, add 50 respectively～the 100 undiluted samples of μ L, often change the reagent of a lot number, one of them analytical reagent device should be taken and calibration object carries out instrument calibration；
Iv. place analytical reagent device in corresponding analytical reagent device pallet, be calibrated according to operation instructions and detect in instrument；
V. in analytical reagent device pallet, corresponding number of analytical reagent device is put into according to the quantity of required detection, and put into the analytical reagent device containing calibration object and Quality Control thing before detection position, instrument can automatically discriminatory analysis reagent device bar code, Quality Control thing bar code and calibration object bar code, select row can be positioned " sample " hurdle or " detection " hurdle；
Vi. click and start, run project table, scan each analytical reagent device bar code, Quality Control thing, calibration object and detection sample are numbered；
Vii. running detection table, instrument runs automatically according to bar code information, and according to bar code, instrument can select the standard curve that relative set is good, and program is testing calibration product first, carrys out curve default in calibration instrument with this；Secondly Quality Control thing is detected, if its testing result is in the scope indicated, then it represents that built-in curve is qualified, can be used for the detection of sample, finally starts the detection program of sample；
Viii. dilution: filling pin can from sample well automatic sucking sample, punctured hole position sealing film automatic sucking diluent carries out diluted sample at dilution holes, after action completes, the sample being diluted can be moved to the time of reacting hole one section of program setting of reaction by filling pin, removes liquid afterwards；
Ix. washing: filling pin can be drawn from corresponding flow container after reacting hole is carried out three to five washings by a certain amount of cleaning mixture and remove liquid；
X. the anti-human IgG antibodies that a certain amount of horseradish peroxidase-labeled drawn by the broken reagent wells sealing film of filling acupuncture reacts to reacting hole, removes liquid after the time of one section of program setting of reaction；
Xi. step ix washing is repeated；
Xii. the broken reagent wells sealing film a certain amount of enzyme reaction substrate of absorption of filling acupuncture carries out the time response of one section of program setting to reacting hole；
Xiii. the broken reagent wells sealing film of filling acupuncture draws the filling of a certain amount of stop buffer to reacting hole, reads OD value in 450nm in 10 minutes, if Double wavelength method measures, reference wavelength is 620nm ~ 690nm；
7) testing result: when detection program is run complete, click the data with main frame and transmit, normal operation acquired results can be sent to main frame and transfer to external data to process software analysis by instrument automatically, ultimately produces report to consult；
8) shutdown: after detection terminates, before instrument shuts down, it is necessary to starts cycles of washing, so can avoid from the residual salt crystallization in fluid path in solution, it is to avoid damaging instrument or cause that testing result is invalid, after having washed, instrument power source is automatically switched off。
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