CN103185795B - Reagent device and method for detecting EB virus antibody - Google Patents
Reagent device and method for detecting EB virus antibody Download PDFInfo
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Abstract
The invention provides a reagent device and a method used for detecting EB virus antibodies. The reagent device has a long strip shape, and has a base body comprising 8 hole positions and a handle positioned on one end of the base body. From the end proximal to the handle, the 8 hole positions are sequentially a sample hole, an auxiliary agent hole, an enzyme conjugate hole, a substrate hole, a termination liquid hole, a dilution liquid hole, a reaction hole, and a dilution hole. According to the invention, based on a principle of enzyme-linked immunoassay, EB virus antibody is detected by using the reagent device. The method is an independent single-person analysis and detection method. The device can be used in cooperation with a corresponding specific analysis instrument. During a detection process, detection reagents or samples are injected by using a full-automatic precise dosing device. The device and the method have the advantages of automatic operation, precise dosing, high detection result accuracy, high detection result precision, and wide application prospect.
Description
Technical field
The present patent application relates to a kind of reagent device and the method thereof that detect Epstein-Barr virus antibody, belongs to clinical immunology detection technique field.
Background technology
Epstein-Barr virus (epstein-barr virus, EBV), also known as nerpes vinrus hominis (Human herpesvirus4, HHV-4), its form is similar to other herpesvirals, is circle, diameter 180nm, basic structure is containing core sample thing, capsid and cyst membrane three part.Cyst membrane is made up of the nuclear membrane of infection cell, it has the membrane glycoprotein of encoding viral, have identify lymphocytic Epstein-Barr virus acceptor and with the function such as Fusion of Cells, also have one deck albumen tunicle between this external cyst membrane and capsid.
Epstein-Barr virus is a kind of nerpes vinrus hominis addicted to B cell, mainly invades B cell, has affinity to the bone-marrow-derived lymphocyte of people, pharynx epithelial cell and gland cell.Think in the past and only have B cell surface to have EBV acceptor, but find that some epithelial cell outside Blasius' duct, pharynx and uterine neck also has EBV acceptor recently, therefore EBV can also infects epithelial.Once infect, EBV will hide in human body B cell for a long time, and sufferer will become lifelong carrier.Under the effect of certain some inducible factor of conditioned disjunction, the EBV genome in latent infected cells is activated and expresses, and is converted into proliferative and infects.Infected to cause or infect diseases related with EBV by EBV and mainly contain infectious mononucleosis, african children lymphoma (i.e. Burkitt lymthoma), nasopharyngeal carcinoma.
Be there is by the cell of virus infections the genome of EBV, various antigen can be produced, mainly contain: EBV nuclear antigen (EBNA), early antigen (EA), membranous antigen (MA), capsid antigen (VCA) and lymphocyte identification membranous antigen (lydma), anti-EBNA antibody can be induced after human infection EBV, anti-EA antibody, anti-VCA antibody and anti-MA antibody.
The serodiagnostic markers of Epstein-Barr virus capsid antigen IgG and the positive normally infectious mononucleosis of IgM antibody.During convalescence of ebv infection, capsid antigen IgG antibody is positive, can exist throughout one's life; During Epstein-Barr virus previous infection, EBV VCA IgG slightly raises; When having research on chronic active Epstein-Barr virus infection, there is the EBV VCA IgG of high titre.When ebv infection is in acute stage, the anti-capsid antigen antibody IgM of Epstein-Barr virus is positive; When recent infection or viral persistence active state, EBV VCA IgM raises, therefore early stage EBV VCA IgM checks it is one of reliable index of ebv infection.
Anti EB virus cAg antibody generally starts after 1 month to occur in morbidity, raises gradually, lifelong sustainable existence.In the process catching monocyte increase disease, viral capsid antigen antibody produces the earliest, produces after cAg antibody, mainly IgG antibody-like, and IgA antibody-like is mainly used in the serodiagnosis of nasopharyngeal carcinoma.
Eb early antigen IgG, IgM, IgA antibody are mainly used in the serodiagnosis of nasopharyngeal carcinoma.
Usually can detect two or more antibody clinically as required, as the useful indicators of nasopharyngeal carcinoma early detection, Index for diagnosis and observation of curative effect simultaneously.
The common methods of clinical detection Epstein-Barr virus antibody comprises the method for Gold standard, indirect immunofluorescence, a golden immunoassay, enzyme linked immunosorbent assay, but these methods all also exist weak point.
One, Gold standard
Gold standard is a kind of novel immunolabelling technique being applied to antigen-antibody using collaurum as tracer label thing.According to some physical behaviors of collaurum, as high electron density, grain size, shape and color reaction, add immunity and the biological characteristics of bond, collaurum has been widely used in the fields such as immunology, histology, pathology and cell biology.
The method is quick, susceptibility is high, but its poor specificity, false positive rate is higher, and can only do qualitative detection, can not be quantitative.
Two, indirect immunofluorescence
The ultimate principle of this method forms antigen antibody complex after being combined with the antigen of specific antibody in microslide, continues and use fluorescence antibody to be combined with antigen antibody complex, form antigen-antibody fluorescent composition.Under fluorescent microscope, the luminous situation according to compound determines detected antibody.The method increases due to the fluorescence antibody be combined on antigen antibody complex, and the fluorescent brightness sent is strong, and thus its susceptibility is strong.But its deficiency is also obvious:
(1) cannot according to the non-specific identification of the size discrimination of molecular weight when analysis result;
(2) operate relative complex, need price fluorescent microscope costly, be difficult to promote at a lot of basic hospital, be also not too applicable to the more laboratory of specimen amount;
(3) background in fluorometric assay is higher, and immunofluorence technic is used for quantitative measurement certain difficulty;
(4) result judges to need experienced professional, and the objectivity of analysis result is not enough;
(5) can only qualitative detection be carried out, can not quantitatively detect.
Three, golden immunoassay (dripping golden method) is dripped
Dripping golden immunoassay is the simple and rapid dot immunobinding assay of one that Later development gets up in the eighties, its ultimate principle take miillpore filter as carrier, take collaurum as immune marker, by the transudation of film, antigen-antibody reaction is completed rapidly on film, there is punctation in final positive findings on film.The feature of dripping golden method is easy and simple to handle, quick, can complete mensuration at short notice, and be single part of mensuration, also not need any instrument and equipment.
But the method cost price is expensive, poor specificity, and false positive rate is higher, and can only do qualitative detection, can not be quantitative.
Four, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used in detecting Epstein-Barr virus antibody, but the method also also exists following weak point:
(1) use 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate as antigen coated apparatus and reaction vessel, 12 batches, 6 batches, 8 batches can only be divided in use or whole plate once uses, cannot carry out independently, the detection of single part;
(2) reagent type used is measured more, each detects reagent and all carrys out splendid attire with reagent bottle, and all need to change imbibition nozzle when often using a kind of reagent to be filled into respectively in the micropore of microwell plate, not only reagent bottle kind is many, and the operation of filling reagent is also very loaded down with trivial details;
(3) the corresponding mark to Detection Information is lacked, can only by checking that the product batch number and term of validity information detecting reagent could be understood or know to the mark of kit external packing box, and the information known is not controlled in testing process, there is very large randomness;
(4) detect reagent in testing process, be in open space, easily cause the cross pollution between various reagent and affect the accuracy of testing result;
(5) the many employing manual operationss of testing process, the dosage of reagent or sample is not bery accurate, and operating process is very loaded down with trivial details and complicated, easily bust occurs, and accuracy and the precision of testing result are poor;
(6) in the quantity configuration and use of test item reagent set, item number × 48/96 person-portion is, if need detection 10 projects, then reagent configuration and use number must be 10 × 48/96 person-portions, if only have the project that a sample needs detection 10 different, also need the reagent of configuration 10 × 48/96 person-portion, there is the shortcoming of inadequate economical rationality.
Summary of the invention
Patented claim of the present invention be namely for current for above shortcomings part in the detection of Epstein-Barr virus antibody, a kind of reagent device and the detection method thereof that can carry out the detection of separate single person-portion are provided.
Patented claim of the present invention realizes the detection of Epstein-Barr virus antibody based on the principle of enzyme linked immunosorbent detection, be a kind of independently, single part, the disposable method of inspection; Further, the present patent application additionally provides the corresponding reagent device supporting to the method, plurality of reagents required for Epstein-Barr virus antibody ELISA immune detection is contained in one independently, in the reagent device of porous structure, annotated by substep, moved and abandoned detecting step by this reagent device; Further, described reagent device can also carry out corresponding fully-automated synthesis by special immunity analysis instrument.
One of object of the present patent application is to provide a kind of reagent device detecting Epstein-Barr virus antibody, and this object is realized by following technical scheme:
Specifically, the reagent device of the detection Epstein-Barr virus antibody described in the present patent application is strip, comprise the matrix with position, eight holes and the handle being positioned at matrix one end, putting in order of position, eight holes is followed successively by sample aperture near handle end, assistant agent hole, enzyme conjugates hole, substrate hole, stop fluid apertures, dilution fluid apertures, reacting hole and dilution holes, sample to be tested is filled in sample aperture, assistant agent hole adds auxiliary reagent whenever necessary for detection, enzyme conjugates solution is filled in enzyme conjugates hole, substrate fills substrate solution in hole, stop filling stop buffer in fluid apertures, dilution is filled in dilution fluid apertures, reacting hole is flat and has high light penetrability, hole endoperidium has Epstein-Barr virus proteantigen, and to annotate liquid sample to be measured and detect reagent and cleansing solution as reaction vessel, absorbance measurement is carried out after reaction terminating, dilution holes is as Sample Dilution container, during for diluted sample.
Further, in described reagent device, when detecting Epstein-Barr virus IgG antibody, assistant agent hole does not add any reagent, and when detecting Epstein-Barr virus antibody IgM, adsorbent adds in assistant agent hole or together with dilution and adds in dilution fluid apertures.
Further, in described reagent device, assistant agent hole, enzyme conjugates hole, substrate hole, termination fluid apertures and dilution fluid apertures are coated with sealing film, sealing film mainly plays and prevent the effect of overflow in the process of transport and movement, in addition, the effect that preventing dust pollutes and strengthens stability is also played.
Further, described handle is pasted with bar code, this bar code identification has the relevant information of Epstein-Barr virus antibody test reagent and analytical equipment.
Further, described information comprises test item code, detects the sequence number of reagent product batch number, the reagent term of validity, qualitative correction coefficient, enzyme linked immunoassay type, reagent and analytical equipment.
Further, described reagent device also comprises several support columns, and described support column is positioned at below reagent device matrix, is separated with position, more than one hole between adjacent supports post, and the effect of support column is physical strength in order to strengthen matrix and balance.
Further, in described reagent device, reacting hole is made up of exit orifice and endoporus, the bottom of exit orifice has bottom outlet, endoporus through bottom outlet and with bottom outlet wringing fit, leave anti-overflow chamber between exit orifice and endoporus, the effect in anti-overflow chamber is in course of reaction, if overflow, can stay in chamber, preventing pollution instrument and other reagent devices.
Further, in described reagent device, the Epstein-Barr virus proteantigen of the endoporus bag quilt of reacting hole comprises Epstein-Barr virus capsid antigen, Epstein-Barr virus cAg or eb early antigen.
Further, described Epstein-Barr virus capsid antigen comprises purifying and the Epstein-Barr virus capsid antigen of deactivation, VCA-18p albumen or BFRF3 recombinant protein, Epstein-Barr virus cAg comprises purifying and the Epstein-Barr virus cAg of deactivation or BKRF1 recombinant protein, and eb early antigen comprises purifying and the eb early antigen of deactivation or BMRF1 recombinant protein.
It is clearly understood that, in the present patent application, in described reagent device, the size of position, each hole, shape are not limited, such as the open peristoma of position, each hole can be square or circular, the shape of its section also can be square, " U " type or " V " type, but for reacting hole, in order to obtain good light penetrability, bottom should be flat.
Further, the sample added in described sample aperture is test serum or blood plasma, and the amount added is determined according to the multiple of the size of sample aperture and the dilution of detection needs.
Described assistant agent hole is the use adding auxiliary reagent when being necessary, such as adsorbent, neutralizing agent, inhibitor, damping fluid etc.As when detecting Epstein-Barr virus antibody IgM, adsorbent adds and to add in dilution fluid apertures in assistant agent hole or together with dilution, its Main Ingredients and Appearance is anti-human IgG antibodies or Human fetal cardiomyocytes albumin A (SPA), to remove in testing process other materials such as specific IgG and rheumatoid factor to the interference of testing result.
Enzyme conjugates solution is added in described enzyme conjugates hole, Main Ingredients and Appearance is anti-human igg or IgA or IgM or Ig (GAM) antibody of horseradish peroxidase-labeled, the anti-human igg of alkali phosphatase enzyme mark or IgA or IgM or Ig (GAM) antibody, the anti-human igg of glucose oxidase thing enzyme labeling or IgA or IgM or Ig (GAM) antibody, or beta galactosidase mark anti-human igg or IgA or IgM or Ig (GAM) antibody.
The substrate solution Main Ingredients and Appearance added in described substrate hole is TMB, OPD or ABTS.
TMB is as a kind of chromogen reagent of new type of safe, progressively replace the benzidine derivative of strong carcinogen biphenylamine and other carcinogenicity, be applied to the fields such as clinical assay, forensic medical examination, criminal detection and environmental monitoring, especially in biochemistry test, TMB, as the new substrate of peroxidase, obtains a wide range of applications in enzyme immunoassay (EIA) (EIA) and enzyme linked immunosorbent assay method (ELISA).
Sulfuric acid or the hydrochloric acid solution of the stop buffer Main Ingredients and Appearance added in described termination fluid apertures to be concentration be 0.1 ~ 0.5M.
The dilution Main Ingredients and Appearance added in described dilution fluid apertures is phosphate buffer, Tris damping fluid, borate buffer solution or carbonate buffer solution, and containing components such as certain density bovine serum albumin(BSA), sodium azide, for getting rid of the interference of the nonspecific reaction of sample small molecular material and increasing stability.
Another object of the present patent application is to provide the detection method utilizing mentioned reagent device to carry out Epstein-Barr virus antibody, and described method comprises following step:
1) in described sample aperture, undiluted sample is added;
2) from dilution fluid apertures, drawing dilution adds in dilution holes;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw partially liq return dilution fluid apertures;
4) from sample aperture, draw sample add in dilution fluid apertures, sample is diluted;
5) from dilution fluid apertures, imbitition adds in dilution holes, is further diluted by sample;
6) when detection Epstein-Barr virus antibody IgM and when adding adsorbent in assistant agent hole, add reacting hole from assistant agent hole imbitition, other situations then do not need this step;
7) from dilution holes, imbitition adds in reacting hole, under the condition of 25 ~ 37 DEG C, hatches 30 ~ 60min;
8) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
9) from enzyme conjugates hole, drawing enzyme conjugates solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, removes liquid after reaction 30 ~ 60min;
10) step 8 is repeated);
11) from substrate hole, drawing substrate solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, and reaction 10 ~ 20min;
12) from termination fluid apertures, drawing stop buffer adds in reacting hole, reads OD value in 1 ~ 10min in 450nm, or Double wavelength method measures, and wavelength coverage is at 620nm ~ 690nm.
Further, described method comprises following step:
1) in described sample aperture, add undiluted sample, volume is at least 1/4 of sample aperture capacity;
2) from dilution fluid apertures, drawing dilution adds in dilution holes, and the amount of absorption is more than 7/10 of total amount of liquid in hole;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw partially liq according to expection extension rate and return dilution fluid apertures;
4) from sample aperture, draw sample add in dilution fluid apertures, diluted by sample, extension rate is 1 ~ 10 times;
5) from dilution fluid apertures, imbitition adds in dilution holes, and further diluted by sample, extension rate is 1 ~ 100 times;
6) when detection Epstein-Barr virus antibody IgM and when adding adsorbent in assistant agent hole, add reacting hole from assistant agent hole imbitition, other situations then do not need this step;
7) from dilution holes, imbitition adds in reacting hole, under the condition of 25 ~ 37 DEG C, hatches 30 ~ 60min;
8) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
9) from enzyme conjugates hole, drawing enzyme conjugates solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, removes liquid after reaction 30 ~ 60min;
10) step 8 is repeated);
11) from substrate hole, drawing substrate solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, and reaction 10 ~ 20min;
12) from termination fluid apertures, drawing stop buffer adds in reacting hole, reads OD value in 1 ~ 10min in 450nm, or Double wavelength method measures, and wavelength coverage is at 620nm ~ 690nm.
Further, described method comprises following step:
1) in described sample aperture, add sample 80 ~ 150 μ L;
2) from dilution fluid apertures, 360 μ L liquid are drawn in dilution holes with accurate charger;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw 180 μ L liquid return dilution fluid apertures;
4) from sample aperture, 20 μ L liquid are drawn in dilution fluid apertures, the dilution of 10 times, sample with accurate charger;
5) with accurate charger from dilution fluid apertures imbitition 20 μ L in dilution holes, the dilution of 100 times, sample;
6) when detection Epstein-Barr virus antibody IgM and when adding adsorbent in assistant agent hole, add reacting hole with accurate charger from assistant agent hole imbitition 30 μ L, other situations then do not need this step;
7) in reacting hole, at 25 ~ 37 DEG C, after hatching 60min, liquid is removed with accurate charger imbitition 100 μ L from dilution fluid apertures;
8) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
9) react to reacting hole with the abzyme bond solution that 100 μ L horseradish peroxidase-labeled drawn by accurate charger from enzyme conjugates hole, at 25 ~ 37 DEG C, after reaction 30min, remove liquid;
10) step 8 is repeated;
11) to reacting hole, 10 ~ 15min is reacted with the tmb substrate solution that 100 μ L drawn by accurate charger from substrate hole;
12) annotate in reacting hole from the stop buffer stopping drawing fluid apertures 100 μ L with accurate charger, detects under 450nm and 630nm dual wavelength condition in 1 ~ 10min, read OD value.
Described cleansing solution comprises PBST solution, PBST solution refers to that PBS solution adds Tween-20, PBS solution refers to phosphate buffer (Phosphate Buffer Solution), osmotic pressure is similar to physiological condition, has again very strong pH surge capability, is commonly used to wash cell and the basal liquid as cell chulture, and Tween-20 is a kind of non-ionic surfactant, cell growth can have an impact, and Tween-20 has the effect of renaturation antigen, can improve specific recognition capability.
Reagent device and the method thereof of Epstein-Barr virus antibody is detected described in the present patent application, not only there is high specificity that other detection methods have, highly sensitive, accuracy is good, cost is lower, request for utilization is not high, operation is comparatively easy, obtain the advantage that the testing result time is short, be widely used etc., and solve many deficiencies of other detection methods, be embodied in the following aspects:
1. the detection method described in uses enzyme-linked immuno assay principle, utilize specific analytical instrument, adopt supporting special detection kit and analytical reagent device, automatically realize Epstein-Barr virus antibody qualitative detection, be a kind of completely newly, be suitable for, practical, efficiently, detect the scheme of Epstein-Barr virus antibody fast;
2. it be a kind of independently, the detection reagent of single part and analytical equipment, without the need to using 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole special enzyme-linked immune microwell plate as antigen coated articles for use and reaction vessel as general ELISA method, as long as have a sample can carry out the detection of respective items object in use and without the waste of reagent, if the quantity of sample exceedes portion, use this reagent and analytical equipment by actual sample number;
3. each is detected required reagent and is contained in the reagent wells position of an analytical reagent device by it, and detection reagent need not be carried out splendid attire with different reagent bottles respectively, not only operate very easy, and be not easy to cause bust, thus ensure the correctness of testing result;
4. it has a Special bar code to each analytical reagent device, the numerical value of bar code comprises test item code corresponding to detection, detects reagent product batch number, the reagent term of validity, qualitative correction coefficient, concrete enzyme linked immunoassay type, reagent and analytical equipment the information such as sequence number, can not arbitrarily be changed, strictly controlled during use, especially when use exceedes term of validity detection reagent, to be identified and stop and send examining report, thus the accuracy of detection can be guaranteed;
5. each detection reagent is effectively separated and seals by it, can not cause the cross pollution between various reagent and affect testing result;
6. it is a kind of analytical reagent device being exclusively used in particular analysis instrument, and annotate with full automatic accurate charger in testing process and detect reagent, cleansing solution or sample, operation automation, dosage is accurate, and accuracy and the precision of testing result are high;
7., in the quantity configuration and use of test item reagent set, all use needs to carry out being equipped with by reality, especially detecting entry, are equipped with more appropriate, there will not be super configuration and service condition.
Accompanying drawing explanation
Fig. 1 is the cross-sectional view of an embodiment of reagent device described in the present patent application;
Fig. 2 is the top plan view of embodiment described in Fig. 1;
Fig. 3 is the cross-sectional view of another embodiment of reagent device described in the present patent application;
Fig. 4 is the top plan view of reacting hole in another embodiment of reagent device described in the present patent application;
Fig. 5 and Fig. 6 is the assembling schematic diagram of endoporus and exit orifice in reacting hole in reagent device described in Fig. 4;
Wherein, 10 be matrix, 20 be handle, 30 be bar code, 40 be sealing film, 50 be support column, 11 be sample aperture, 12 be assistant agent hole, 13 be enzyme conjugates hole, 14 be substrate hole, 15 for stop fluid apertures, 16 for dilution fluid apertures, 17 be reacting hole, 18 be dilution holes, 171 be exit orifice, 172 be endoporus, 173 be anti-overflow chamber, 174 for bottom outlet.
Embodiment
Below in conjunction with concrete pick-up unit and implementation step, the reagent device described in the present patent application and detection method are conducted further description, object is in order to the public better understands technical scheme described in the present patent application instead of the restriction to described technical scheme.In fact, with identical or approximate principle, to the improvement that described reagent device carries out, comprise the change of its shape, size, material used, and the increase and decrease of corresponding construction or replacement, all within the present patent application technical scheme required for protection.
Embodiment one detects the reagent device one of Epstein-Barr virus antibody
As shown in Figure 1-2, the reagent device of the detection Epstein-Barr virus antibody described in the present patent application is strip, comprise the matrix 10 with position, eight holes and the handle 20 being positioned at matrix 10 one end, putting in order of position, eight holes is followed successively by sample aperture 11 from nearly handle 10 end, assistant agent hole 12, enzyme conjugates hole 13, substrate hole 14, stop fluid apertures 15, dilution fluid apertures 16, reacting hole 17 and dilution holes 18, sample to be tested is filled in described sample aperture 11, assistant agent hole 12 adds auxiliary reagent (as added adsorbent in assistant agent hole during detection IgM) when needing for detection, enzyme conjugates solution is added in enzyme conjugates hole 13, substrate solution is added in substrate hole 14, stop fluid apertures 15 and add stop buffer, dilution is added in dilution fluid apertures 16, reacting hole 17 is flat and has very high light penetrability, when splendid attire is colourless or Reagent blank solutions time, to the absorbance of visible ray or ultraviolet light or fluorescence close to zero, Epstein-Barr virus proteantigen has been coated with in hole, and to annotate liquid sample to be measured and detect reagent and cleansing solution as reaction vessel, finally add after enzyme reaction substrate is hatched and carry out absorbance measurement, dilution holes 18 is as Sample Dilution container, during for diluted sample.
Further, in described reagent device, assistant agent hole 12, enzyme conjugates hole 13, substrate hole 14, termination fluid apertures 15 and dilution fluid apertures 16 are coated with sealing film 40, sealing film 40 mainly play transport and movement process in, prevent the effect of overflow, in addition, the effect that preventing dust pollutes and increases stability is also played.
Further, described handle 20 is pasted with bar code 30, this bar code 30 mark has the information of Epstein-Barr virus antibody test reagent and analytical equipment, and described information comprises test item code, detects the sequence number of reagent product batch number, the reagent term of validity, qualitative correction coefficient, enzyme linked immunoassay type, reagent and analytical equipment.
Embodiment two detects the reagent device two of Epstein-Barr virus antibody
As shown in Figure 3, the reagent device of the detection Epstein-Barr virus antibody in the present embodiment, its basic structure is identical with the reagent device in embodiment one, several support columns 50 are also comprised at described reagent device, described support column 50 is arranged in below described reagent device matrix 10, be separated with position, more than one hole between adjacent supports post 50, the effect of support column 50 is physical strength in order to strengthen matrix and balance.
Embodiment three detects the reagent device three of Epstein-Barr virus antibody
As Figure 4-Figure 6, for detecting the preferred embodiment of the reagent device of Epstein-Barr virus antibody described in the present patent application, its basic structure is identical with embodiment one or embodiment two, difference is that reacting hole 17 is detachable structure, reacting hole 17 is made up of exit orifice 171 and endoporus 172, the bottom of exit orifice 171 has bottom outlet 174, endoporus 172 through bottom outlet 174 and with bottom outlet 174 wringing fit, anti-overflow chamber 173 is left between exit orifice 171 and endoporus 172, the effect in anti-overflow chamber 173 is in course of reaction, if overflow, can stay in chamber, preventing pollution instrument and other reagent devices.
Further, the cooperation fixed form of described endoporus and exit orifice can also be that the outer wall of endoporus is designed to two sections, the thickness of epimere outer wall is greater than the thickness of hypomere outer wall, the diameter of the bottom outlet of exit orifice is equal with the external diameter of endoporus hypomere, like this endoporus hypomere is inserted in bottom outlet, the epimere of endoporus is stuck on bottom outlet just, can play fastening effect equally.
Making-the indirect method of embodiment four reagent device or kit detects Epstein-Barr virus antibody
The present invention is simpler based on the technical one of enzyme linked immunosorbent detection, accurately, effective methodology, its ultimate principle adopted is Dot-ELISA: will be adsorbed in solid phase by Epstein-Barr virus proteantigen, by hatch make dilution human serum or blood plasma in specific antibody be combined with antigen, the antibody be not combined with solid phase is removed in washing, add the human immunoglobulins's enzyme connection thing by horseradish peroxidase-labeled, hatch, remove unconjugated enzyme connection thing, add enzyme chromogen substrate, the color produced is directly proportional to the specific antibody concentrations detected in sample.This method is mainly by realizing the immune detection of Epstein-Barr virus antibody for the analytical reagent device of enzyme linked immunosorbent detection and matched reagent.By this enzyme-linked immunoassay method, use analytical reagent device and the reagent of particular analysis instrument simultaneously, can be quick, judge accurately and diagnose for adjuvant clinical.
Using position, hole 11 as sample aperture, add liquid sample in use, take for during detection;
Using position, hole 12 as assistant agent hole, detect and add auxiliary reagent (as added adsorbent when detecting IgM) whenever necessary, with sealing film sealing, take for detection;
Using position, hole 13 as enzyme conjugates hole, add enzyme conjugates solution sealing film and seal, take for during detection;
Using position, hole 14 as substrate hole, seal with sealing film after adding tmb substrate solution, during for detection;
Using position, hole 15 as termination fluid apertures, seal with sealing film after adding stop buffer, during for detection;
Using position, hole 16 as dilution fluid apertures, seal with sealing film after adding Sample dilution, during for detection;
Using position, hole 17 as encrusting substance, hole/reacting hole/colorimetric hole, has been coated with Epstein-Barr virus proteantigen, and to annotate liquid sample to be measured and detect reagent and cleansing solution as reaction vessel, finally adds after enzyme reaction substrate is hatched and carries out absorbance measurement;
Using position, hole 18 as dilution holes, during for diluted sample;
Be pasted with the bar code 30 of Epstein-Barr virus antibody test reagent and analytical equipment information at handle 20, this bar code comprises test item code, detects the sequence number of reagent product batch number, the reagent term of validity, qualitative correction coefficient, enzyme linked immunoassay type, reagent and analytical equipment.
Prepare some analytical equipments according to the method described above, prepare corresponding calibration object and Quality Control thing in addition.So namely, constitute complete Epstein-Barr virus antibody assay kit component.By detecting the reagent device of Epstein-Barr virus antibody and supporting the use the external packing box of component loading kit, namely make and detect Epstein-Barr virus antibody kit.
The detection method of embodiment five Epstein-Barr virus cAg IgG antibody
The present patent application provides a kind of method utilizing mentioned reagent device to carry out the detection of Epstein-Barr virus cAg IgG antibody, and described method comprises following step:
1. in described sample aperture, add sample 80 ~ 150 μ L;
2. from dilution fluid apertures, draw 360 μ L liquid in dilution holes with accurate charger;
3. remove the remaining liq in dilution fluid apertures, from dilution holes, draw 180 μ L liquid return dilution fluid apertures;
4. from sample aperture, draw 20 μ L liquid in dilution fluid apertures, the dilution of 10 times, sample with accurate charger;
5. with accurate charger from dilution fluid apertures imbitition 20 μ L in dilution holes, the dilution of 100 times, sample;
6. in reacting hole, at 25 DEG C, after hatching 60min, remove liquid with accurate charger imbitition 100 μ L from dilution fluid apertures;
7. after 3 ~ 5 washings being carried out to reacting hole with cleansing solution, remove liquid;
8. react to reacting hole with anti-human IgG antibodies's enzyme conjugates solution that 100 μ L horseradish peroxidase-labeled drawn by accurate charger from enzyme conjugates hole, at 25 ~ 37 DEG C, after reaction 30min, remove liquid;
9. repeat step 7;
10. to reacting hole, react 10 ~ 15min with the tmb substrate solution that 100 μ L drawn by accurate charger from substrate hole;
11. annotate in reacting hole from the stop buffer stopping drawing fluid apertures 100 μ L with accurate charger, detects, read OD value in 1 ~ 10min under 450nm and 630nm dual wavelength condition.
Embodiment six realizes the analysis operation flow process detected Epstein-Barr virus cAg IgG antibody in sample by fully-automatic analyzer
Further, detection method described in the present patent application also comprise corresponding with the use of fully-automatic analyzer, this fully-automatic analyzer comprises an analytical equipment pallet, it overlaps with the matching form of analytical equipment, one has 30 positions can place for analytical equipment, for detecting analysis, comprising the integrated mechano-electronic structure of modular and software control system in addition, the filling of robotization can be realized, dilute, hatch, wash, reading, analytic process.The independence qualitative analysis of each position, ensures the accuracy of result.After instrument runs, analytical equipment pallet can turn to different positions voluntarily and annotate, and dilution, hatches, the step of washing and reading.
Concrete operation comprises following step:
(1) preparation of scrutiny program: configure corresponding solution on request, comprise lavation buffer solution, cleaning fluid or thimerosal, prepare in the corresponding liquid bottles of complete loading;
(2) be connected with external computing machine: instrument is connected with external computing machine, thus the acquired results that normally worked by instrument transfers to integrated system process;
(3) start shooting: after opening instrument switch, by the working interface of external computing machine to the self-inspection of instrument designing automatic or manual, thus prepare for the normal operation of instrument;
(4) preheating: after powering, instrument can start heating schedule, and temperature is adjusted to temperature to be checked;
(5) inspection is rinsed;
(6) detection of Epstein-Barr virus cAg IgG antibody:
1) from have seal while open the package, take the analytical reagent device of requirement, after deaeration, sack be tamping;
2) solution in Inspection and analysis reagent device tmb substrate hole, should be without color change, otherwise should discard;
3) in the sample aperture of each analytical reagent device, add the undiluted sample of 50 ~ 120 μ L respectively, suggestion adds the sample of 100 μ L, often changes the reagent of a lot number, should get one of them analytical reagent device and calibration object carries out instrument calibration;
4) in analytical equipment pallet, the analytical reagent device of corresponding quantity is put into according to the quantity of required detection, click " beginning ", " scanning ", instrument can automatically scanning analysis reagent device bar code, Quality Control thing bar code and calibration object bar code, then sample is numbered, or by outer strip code scanner, sample bar code is scanned;
5) click " RUN ", instrument runs automatically according to bar code information, and according to bar code, program is testing calibration product first, by calibration, revises typical curve; Secondly Quality Control thing is detected, if its testing result is in the scope indicated, then represents that the typical curve of detection meets the requirements, can be used for the detection of sample, finally start the trace routine of sample;
6) dilute: acupuncture holes position sealing film automatic sucking dilution 360 μ L is in dilution holes in filling, and the remaining liq removed in dilution fluid apertures, from dilution holes, draw dilution 180 μ L again return dilution fluid apertures, filling pin carries out Sample Dilution from sample aperture automatic sucking sample 20 μ L to dilution fluid apertures, 20 μ L are drawn to dilution holes again from dilution fluid apertures, after action completes, moved to reacting hole hatch 30 ~ 60min by the sample diluted pin of being annotated, usual instrument is set as 60min, removes liquid afterwards;
7) wash: flushing needle is drawn after a certain amount of lavation buffer solution carries out three to five washings to reagent wells and removed liquid from corresponding liquid bottle;
8) acupuncture of annotating is broken anti-human IgG antibodies's enzyme conjugates solution that enzyme conjugates hole sealing film draws 100 μ L horseradish peroxidase-labeled and is reacted to reacting hole, and remove liquid after reaction 30 ~ 60min, usual instrument is set as 30min;
9) step 7 is repeated);
10) acupuncture of annotating is broken tmb substrate hole sealing film and is drawn the tmb substrate of 100 μ L to reacting hole reaction 10 ~ 20min, and usual instrument is set as 10min;
11) acupuncture of annotating is broken termination fluid apertures sealing film and is drawn the stop buffer filling of 100 μ L to reacting hole, in 1 ~ 10min, read OD value under 450nm/630nm dual wavelength.
(7) testing result: when trace routine is run complete, by the data processing software analysis on computing machine, finally generates report to consult;
(8) shut down: detect after terminating, before instrument shutdown, must clean cycle be started, can avoid from the residual salt crystallization in fluid path in solution like this, avoid damaging instrument or causing testing result invalid, after having cleaned, instrument power source is closed automatically.
The Quality Control of the detection application of Epstein-Barr virus cAg IgG antibody in embodiment seven clinical samples, interpretation of result and detection
Adopt method of operating and the program of embodiment five, use the kit described in embodiment four, can be used for the Epstein-Barr virus cAg IgG antibody level in qualitative detection human serum or blood plasma.
Epstein-Barr virus extensively infects in crowd, occurs viral capsid antigen IgM antibody, and does not occur cAg IgG antibody, and prompting Current Infection, occurs that viral capsid antigen IgG antibody and cAg IgG antibody then point out previous infection simultaneously.Adjuvant clinical diagnosis can be carried out according to the result detected, tentatively judge the situation of patient, finally make a definite diagnosis and should consider in conjunction with clinical manifestation or other diagnostic method/indexs.
Be below the analysis of testing result:
(1) reference value (term of reference)
Normal reference value: 0 ~ 1.0.
(2) explanation of testing result
If sample value > 1.0, prompting antibody horizontal raises, but should make a definite diagnosis in conjunction with clinical manifestation or other diagnostic method/indexs.
Conveniently clinical practice, can with reference to following judgment rule:
Sample value < 0.9 is negative
0.9≤sample value≤1.1 are suspicious
Sample value > 1.1 is positive
When testing result is suspicious, should again detect, if be still suspicious, need after 2 ~ 3 weeks Resurvey pattern detection.
Be below that the testing process duplicate detection of embodiment six is positive and negative quality controlled serum, the repeatability of check result, obtains following result:
The detection method of embodiment eight Epstein-Barr virus capsid antigen IgM antibody
The present patent application provides a kind of method utilizing mentioned reagent device to carry out the detection of Epstein-Barr virus capsid antigen IgM antibody, and described method comprises following step:
1. in described sample aperture, add sample 80 ~ 150 μ L;
2. from dilution fluid apertures, draw 370 μ L liquid in dilution holes with accurate charger;
3. remove the remaining liq in dilution fluid apertures, from dilution holes, draw 180 μ L liquid return dilution fluid apertures;
4. from sample aperture, draw 20 μ L liquid in dilution fluid apertures, the dilution of 10 times, sample with accurate charger;
5. with accurate charger from dilution fluid apertures imbitition 20 μ L in dilution holes, the dilution of 100 times, sample;
6., when adsorbent adds in assistant agent hole, in reacting hole, when adsorbent adds in dilution fluid apertures together with dilution, then do not need this step with accurate charger imbitition 30 μ L from assistant agent hole;
7. in reacting hole, at 25 ~ 37 DEG C, after hatching 60min, remove liquid with accurate charger imbitition 100 μ L from dilution fluid apertures;
8. after 3 ~ 5 washings being carried out to reacting hole with cleansing solution, remove liquid;
9. react to reacting hole with anti-human IgM antibodies's enzyme conjugates solution that 100 μ L horseradish peroxidase-labeled drawn by accurate charger from enzyme conjugates hole, at 25 ~ 37 DEG C, after reaction 30min, remove liquid;
10. repeat step 8;
11. react 10 ~ 15min with the tmb substrate solution that 100 μ L drawn by accurate charger from substrate hole to reacting hole;
12. annotate in reacting hole from the stop buffer stopping drawing fluid apertures 100 μ L with accurate charger, detects, read OD value in 1 ~ 10min under 450nm and 630nm dual wavelength condition.
Embodiment nine realizes the analysis operation flow process detected Epstein-Barr virus capsid antigen IgM antibody in sample by fully-automatic analyzer
Further, detection method described in the present patent application also comprise corresponding with the use of fully-automatic analyzer, this fully-automatic analyzer comprises an analytical equipment pallet, it overlaps with the matching form of analytical equipment, one has 30 positions can place for analytical equipment, for detecting analysis, comprising the integrated mechano-electronic structure of modular and software control system in addition, the filling of robotization can be realized, dilute, hatch, wash, reading, analytic process.The independence qualitative analysis of each position, ensures the accuracy of result.After instrument runs, analytical equipment pallet can turn to different positions voluntarily and annotate, and dilution, hatches, the step of washing and reading.
Concrete operation comprises following step:
(1) preparation of scrutiny program: configure corresponding solution on request, comprise lavation buffer solution, cleaning fluid or thimerosal, prepare in the corresponding liquid bottles of complete loading;
(2) be connected with external computing machine: instrument is connected with external computing machine, thus the acquired results that normally worked by instrument transfers to integrated system process;
(3) start shooting: after opening instrument switch, by the working interface of external computing machine to the self-inspection of instrument designing automatic or manual, thus prepare for the normal operation of instrument;
(4) preheating: after powering, instrument can start heating schedule, and temperature is adjusted to temperature to be checked;
(5) inspection is rinsed;
(6) detection of Epstein-Barr virus capsid antigen IgM antibody:
1) from have seal while open the package, take the analytical reagent device of requirement, after deaeration, sack be tamping;
2) solution in Inspection and analysis reagent device tmb substrate hole, should be without color change, otherwise should discard;
3) in the sample aperture of each analytical reagent device, add the undiluted sample of 50 ~ 120 μ L respectively, suggestion adds the sample of 100 μ L, often changes the reagent of a lot number, should get one of them analytical reagent device and calibration object carries out instrument calibration;
4) in analytical equipment pallet, the analytical reagent device of corresponding quantity is put into according to the quantity of required detection, click " beginning ", " scanning ", instrument can automatically scanning analysis reagent device bar code, Quality Control thing bar code and calibration object bar code, then sample is numbered, or by outer strip code scanner, sample bar code is scanned;
5) click " RUN ", instrument runs automatically according to bar code information, and according to bar code, program is testing calibration product first, by single-point calibration, and Criterion curve; Secondly Quality Control thing is detected, if its testing result is in the scope indicated, then represents that the typical curve curve of foundation is qualified, can be used for the detection of sample, finally start the trace routine of sample;
6) dilute: acupuncture holes position sealing film automatic sucking dilution 370 μ L is in dilution holes in filling, and the remaining liq 50 μ L removed in dilution fluid apertures, from dilution holes, draw dilution 180 μ L again return dilution fluid apertures, filling pin carries out Sample Dilution from sample aperture automatic sucking sample 20 μ L to dilution fluid apertures, 20 μ L are drawn to dilution holes again from dilution fluid apertures, 30 μ L adsorbents are drawn in addition in reacting hole from auxiliary reagent hole, after action completes, moved to reacting hole hatch 30 ~ 60min by the sample diluted pin of being annotated, usual instrument is set as 60min, remove liquid afterwards,
7) wash: flushing needle is drawn after a certain amount of lavation buffer solution carries out three to five washings to reagent wells and removed liquid from corresponding liquid bottle;
8) acupuncture of annotating is broken anti-human IgM antibodies's enzyme conjugates solution that enzyme conjugates hole sealing film draws 100 μ L horseradish peroxidase-labeled and is reacted to reacting hole, and remove liquid after reaction 30 ~ 60min, usual instrument is set as 30min;
9) step 7 is repeated);
10) acupuncture of annotating is broken tmb substrate hole sealing film and is drawn the tmb substrate of 100 μ L to reacting hole reaction 10 ~ 20min, and usual instrument is set as 10min;
11) acupuncture of annotating is broken termination fluid apertures sealing film and is drawn the stop buffer filling of 100 μ L to reacting hole, in 1 ~ 10min, read OD value under 450nm/630nm dual wavelength.
(7) testing result: when trace routine is run complete, by the data processing software analysis on computing machine, finally generates report to consult;
(8) shut down: detect after terminating, before instrument shutdown, must clean cycle be started, can avoid from the residual salt crystallization in fluid path in solution like this, avoid damaging instrument or causing testing result invalid, after having cleaned, instrument power source is closed automatically.
The Quality Control of the detection application of Epstein-Barr virus capsid antigen IgM antibody in embodiment ten clinical samples, interpretation of result and detection
Adopt method of operating and the program of embodiment eight, use the kit described in embodiment four, can be used for the Epstein-Barr virus capsid antigen IgM antibody level in qualitative detection human serum or blood plasma.
Epstein-Barr virus extensively infects in crowd, and patient can occur 1gM type antibody in morbidity early stage blood serum, has 60 ~ 80% cases to be positive, and minority normal person and serum sickness patient are also containing this antibody.Adjuvant clinical diagnosis can be carried out according to the result detected, tentatively judge the situation of patient, finally make a definite diagnosis and should consider in conjunction with clinical manifestation or other diagnostic method/indexs.
Be below the analysis of testing result:
(1) reference value (term of reference)
Normal reference value: 0 ~ 1.0.
(2) explanation of testing result
If sample value > 1.0, prompting antibody horizontal raises, but should make a definite diagnosis in conjunction with clinical manifestation or other diagnostic method/indexs.
Conveniently clinical practice, can with reference to following judgment rule:
Sample value < 0.9 is negative
0.9≤sample value≤1.1 are suspicious
Sample value > 1.1 is positive
When testing result is suspicious, should again detect, if be still suspicious, need after 2 ~ 3 weeks Resurvey pattern detection.
Be below that the testing process duplicate detection of embodiment nine is positive and negative quality controlled serum, the repeatability of check result, obtains following result:
According to the present invention, automatically carry out the detection of many increments Epstein-Barr virus antibody originally by identical analytic process simultaneously, and can be associated with other or unconnected project detects simultaneously, this just makes, and detection more simplifies, cost reduces, detection time shortens, cross pollution not easily occurs, detect processing ease carries out; And detect high specificity, highly sensitive, accuracy good.
Claims (3)
1. detect a method for Epstein-Barr virus antibody, it is characterized in that, described method utilizes reagent device and has the fully-automatic analyzer overlapped with reagent device matching form carries out Epstein-Barr virus antibody test, described reagent device is strip, comprise the matrix with position, eight holes and the handle being positioned at matrix one end, putting in order of position, eight holes is followed successively by sample aperture near handle end, assistant agent hole, enzyme conjugates hole, substrate hole, stop fluid apertures, dilution fluid apertures, reacting hole and dilution holes, sample to be tested is filled in sample aperture, assistant agent hole adds auxiliary reagent whenever necessary for detection, enzyme conjugates solution is filled in enzyme conjugates hole, substrate fills substrate solution in hole, stop filling stop buffer in fluid apertures, dilution is filled in dilution fluid apertures, reacting hole is flat and has high light penetrability, hole endoperidium has Epstein-Barr virus proteantigen, and to annotate liquid sample to be measured and detect reagent and cleansing solution as reaction vessel, absorbance measurement is carried out after reaction terminating, dilution holes is as Sample Dilution container, during for diluted sample,
In described reagent device, assistant agent hole, enzyme conjugates hole, substrate hole, termination fluid apertures and dilution fluid apertures are coated with sealing film; Described reagent device also comprises several support columns, and described support column is arranged in below described reagent device matrix, is separated with position, more than one hole between adjacent supports post; In described reagent device, reacting hole is made up of exit orifice and endoporus, the bottom of exit orifice has bottom outlet, endoporus through bottom outlet and with bottom outlet wringing fit, the outer wall of endoporus is designed to two sections, the thickness of epimere outer wall is greater than the thickness of hypomere outer wall, and the bottom diameter of exit orifice is equal with endoporus hypomere external diameter, is inserted by endoporus hypomere in bottom outlet, the epimere of endoporus is stuck on bottom outlet just, leaves anti-overflow chamber between exit orifice and endoporus; In described reagent device, the Epstein-Barr virus proteantigen of the endoporus bag quilt of reacting hole comprises Epstein-Barr virus capsid antigen, Epstein-Barr virus cAg or eb early antigen; After described fully-automatic analyzer runs, reagent device pallet can turn to different positions voluntarily and annotate, and dilution, hatches, the step of washing and reading;
Described method comprises following step:
1) in described sample aperture, undiluted sample is added;
2) from dilution fluid apertures, drawing dilution adds in dilution holes;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw partially liq return dilution fluid apertures;
4) from sample aperture, draw sample add in dilution fluid apertures, sample is diluted;
5) from dilution fluid apertures, imbitition adds in dilution holes, is further diluted by sample;
6) when detection Epstein-Barr virus antibody IgM and when adding adsorbent in assistant agent hole, add reacting hole from assistant agent hole imbitition, other situations then do not need this step;
7) from dilution holes, imbitition adds in reacting hole, under the condition of 25 ~ 37 DEG C, hatches 30 ~ 60min;
8) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
9) from enzyme conjugates hole, drawing enzyme conjugates solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, removes liquid after reaction 30 ~ 60min;
10) step 8 is repeated);
11) from substrate hole, drawing substrate solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, and reaction 10 ~ 20min;
12) from termination fluid apertures, drawing stop buffer adds in reacting hole, OD value is read in 450nm in 1 ~ 10min, or Double wavelength method measures, detect under the condition of another wavelength of 620nm ~ 690nm in 450nm and wavelength coverage in 1 ~ 10min, read OD value.
2. method according to claim 1, is characterized in that, described method comprises following step:
1) in described sample aperture, add undiluted sample, volume is at least 1/4 of sample aperture capacity;
2) from dilution fluid apertures, drawing dilution adds in dilution holes, and the amount of absorption is more than 7/10 of total amount of liquid in hole;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw partially liq according to expection extension rate and return dilution fluid apertures;
4) from sample aperture, draw sample add in dilution fluid apertures, diluted by sample, extension rate is 1 ~ 10 times;
5) from dilution fluid apertures, imbitition adds in dilution holes, and further diluted by sample, extension rate is 1 ~ 100 times;
6) when detection Epstein-Barr virus antibody IgM and when adding adsorbent in assistant agent hole, add reacting hole from assistant agent hole imbitition, other situations then do not need this step;
7) from dilution holes, imbitition adds in reacting hole, under the condition of 25 ~ 37 DEG C, hatches 30 ~ 60min;
8) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
9) from enzyme conjugates hole, drawing enzyme conjugates solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, removes liquid after reaction 30 ~ 60min;
10) step 8 is repeated);
11) from substrate hole, drawing substrate solution adds in reacting hole, under the condition of 25 ~ 37 DEG C, and reaction 10 ~ 20min;
12) from termination fluid apertures, drawing stop buffer adds in reacting hole, OD value is read in 450nm in 1 ~ 10min, or Double wavelength method measures, detect under the condition of another wavelength of 620nm ~ 690nm in 450nm and wavelength coverage in 1 ~ 10min, read OD value.
3. method according to claim 1, is characterized in that, described method comprises following step:
1) in described sample aperture, add sample 80 ~ 150 μ L;
2) from dilution fluid apertures, 360 μ L liquid are drawn in dilution holes with accurate charger;
3) remove the remaining liq in dilution fluid apertures, from dilution holes, draw 180 μ L liquid return dilution fluid apertures;
4) from sample aperture, 20 μ L liquid are drawn in dilution fluid apertures, the dilution of 10 times, sample with accurate charger;
5) with accurate charger from dilution fluid apertures imbitition 20 μ L in dilution holes, the dilution of 100 times, sample;
6) when detection Epstein-Barr virus antibody IgM and when adding adsorbent in assistant agent hole, add reacting hole with accurate charger from assistant agent hole imbitition 30 μ L, other situations then do not need this step;
7) in reacting hole, at 25 ~ 37 DEG C, after hatching 60min, liquid is removed with accurate charger imbitition 100 μ L from dilution fluid apertures;
8) liquid is removed after 3 ~ 5 washings being carried out to reacting hole with cleansing solution;
9) react to reacting hole with anti-human IgM antibodies's enzyme conjugates solution that 100 μ L horseradish peroxidase-labeled drawn by accurate charger from enzyme conjugates hole, at 25 ~ 37 DEG C, after reaction 30min, remove liquid;
10) step 8 is repeated);
11) to reacting hole, 10 ~ 15min is reacted with the tmb substrate solution that 100 μ L drawn by accurate charger from substrate hole;
12) annotate in reacting hole from the stop buffer stopping drawing fluid apertures 100 μ L with accurate charger, detects under 450nm and 630nm dual wavelength condition in 1 ~ 10min, read OD value.
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