CN103185795A - 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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- CN103185795A CN103185795A CN2011104539741A CN201110453974A CN103185795A CN 103185795 A CN103185795 A CN 103185795A CN 2011104539741 A CN2011104539741 A CN 2011104539741A CN 201110453974 A CN201110453974 A CN 201110453974A CN 103185795 A CN103185795 A CN 103185795A
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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 method thereof that detects Epstein-Barr virus antibody, belongs to clinical immunology detection technique field.
Background technology
Epstein-Barr virus (epstein-barr virus, EBV), claim again the nerpes vinrus hominis (Human herpesvirus4, HHV-4), its form is similar to other herpesvirals, is circle, diameter 180nm, basic structure contains nuclear sample thing, capsid and cyst membrane three parts.Cyst membrane is made up of the nuclear membrane of infection cell, and the membrane glycoprotein of encoding viral is arranged on it, have the lymphocytic Epstein-Barr virus acceptor of identification and with function such as Fusion of Cells, also have one deck albumen tunicle between this external cyst membrane and the capsid.
Epstein-Barr virus is a kind of nerpes vinrus hominis of the B of having a liking for cell, mainly invades the B cell, and people's bone-marrow-derived lymphocyte, pharynx epithelial cell and gland cell are had affinity.Think in the past and have only the B cell surface that the EBV acceptor is arranged, but recent findings some epithelial cell outside Blasius' duct, pharynx and uterine neck also has the EBV acceptor, so EBV can also infects epithelial.In case infect, EBV will hide for a long time in human body B cell, the sufferer will become lifelong carrier.Under the effect of certain some inducible factor of conditioned disjunction, the EBV genome in the latent infection cell is activated and expresses, and is converted into proliferative and infects.Infected by EBV and to cause or to infect diseases related with EBV and mainly contain infectious mononucleosis, african children lymphoma (being the Burkitt lymthoma), nasopharyngeal carcinoma.
The genome that is had EBV by the cell of virus infections, can produce various antigens, mainly contain: EBV nuclear antigen (EBNA), early antigen (EA), membranous antigen (MA), capsid antigen (VCA) and lymphocyte identification membranous antigen (lydma), can induce anti-EBNA antibody behind the human infection EBV, anti-EA antibody, anti-VCA antibody and anti-MA antibody.
Epstein-Barr virus capsid antigen IgG and IgM antibody positive be the serodiagnosis index of infectious mononucleosis normally.During convalescence of ebv infection, capsid antigen IgG antibody positive can exist throughout one's life; During the Epstein-Barr virus previous infection, EBV VCA IgG slightly raises; When research on chronic active Epstein-Barr virus infection was arranged, the EBV VCA IgG of high titre appearred.When ebv infection is in acute stage, the anti-capsid antigen antibody IgM of the Epstein-Barr virus positive; When recent infection or viral continuously active state, EBV VCA IgM raises, so early stage EBV VCA IgM checks it is one of reliable index of ebv infection.
Anti EB virus cAg antibody generally begins after 1 month to occur in morbidity, raises gradually, continues throughout one's life to exist.In the process of catching the monocyte increase disease, viral capsid antigen antibody produces the earliest, produce after the cAg antibody, and mainly be the IgG antibody-like, and the 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 simultaneously, as the useful indicators of nasopharyngeal carcinoma early detection, prognosis judgement and observation of curative effect.
The common methods of clinical detection Epstein-Barr virus antibody comprises golden mark method, indirect immunofluorescence, drips the method for golden immunoassay, enzyme linked immunosorbent assay, but these methods all exist weak point.
One, golden mark method
Gold mark method is a kind of novel immunolabelling technique that is applied to antigen-antibody with collaurum as the 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 fields such as immunology, histology, pathology and cell biology.
This 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 is to form antigen antibody complex after being combined with the antigen of specific antibody in microslide, continues and is combined with antigen antibody complex with fluorescence antibody, forms the antigen-antibody fluorescent composition.Under fluorescent microscope, determine the antibody that detects according to the luminous situation of compound.This method is because the fluorescence antibody that is combined on the antigen antibody complex increases, and the fluorescent brightness that sends is strong, thereby its susceptibility is strong.But its deficiency also is tangible:
(1) can't be according to the non-specific identification of the size discrimination of molecular weight when analysis result;
(2) operation relative complex needs the expensive fluorescent microscope of price, is difficult to promote at a lot of basic hospitals, also not too is applicable to the laboratory that specimen amount is more;
(3) background in the fluorometric assay is higher, and immunofluorence technic is used for quantitative measurement certain difficulty;
(4) result judges needs experienced professional, the objectivity deficiency of analysis result;
(5) can only carry out qualitative detection, can not quantitatively detect.
Three, drip golden immunoassay (dripping golden method)
Dripping golden immunoassay is a kind of simple and rapid dot immunobinding assay that grows up in the later stage eighties, its ultimate principle is to be carrier with the miillpore filter, be immune marker with the collaurum, transudation by film, antigen-antibody reaction is finished rapidly on film, and final positive findings punctation occurs at film.The characteristics of dripping golden method are easy and simple to handle, quick, can finish mensuration at short notice, and be single part of mensuration, also do not need any instrument and equipment.
But this method cost price costliness, poor specificity, 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 this method also exists following weak point:
(1) use the special-purpose microwell plate in 12 * 8 types, 6 * 8 types, 8 * 12 types or complete plate 96 holes as antigen coated apparatus and reaction vessel, can only be divided into 12 batches, 6 batches, 8 batches or whole plate in use and once use, can't carry out independently, single part detection;
(2) it is more to measure used reagent type, each detects reagent all will come splendid attire with reagent bottle, and all need during a kind of reagent of every use to change in the micropore that imbibition nozzle is filled into microwell plate respectively, not only the reagent bottle kind is many, and the operation of filling reagent is also very loaded down with trivial details;
(3) lack detecting the corresponding mark of information, can only could understand or know product batch number and the term of validity information that detects reagent by the sign of checking the kit external packing box, and the information of knowing is not controlled in testing process, has very big randomness;
(4) detect reagent and in testing process, be in open space, cause the cross pollution between all ingredients easily and influence the accuracy of testing result;
(5) testing process adopts manual operations more, and the dosage of reagent or sample is not really accurate, and operating process is very loaded down with trivial details and complicated, and bust takes place easily, and accuracy and the precision of testing result are relatively poor;
(6) quantity configuration and the use at the test item reagent set is item number * 48/96 person-portion, detect 10 projects if desired, then the configuration of reagent and use number must be 10 * 48/96 person-portions, if have only a sample need detect 10 different projects, also need to dispose the reagent of 10 * 48/96 person-portions, exist the shortcoming of not enough economical rationality.
Summary of the invention
Patented claim of the present invention namely be at present for above shortcomings part in the Epstein-Barr virus detection of antibodies, provide a kind of and can carry out reagent device and the detection method thereof that the separate single person-portion detects.
Patented claim of the present invention realizes the Epstein-Barr virus detection of antibodies 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 also provides the corresponding reagent device supporting with this method, this reagent device is contained in one independently, in the reagent device of porous structure, annotate, move to abandon and finish the detection step by substep with the needed plurality of reagents of Epstein-Barr virus antibody ELISA immune detection; Further, described reagent device can also carry out corresponding full-automatic the detection by the immunity analysis instrument of special use.
One of purpose of the present patent application provides a kind of reagent device that detects Epstein-Barr virus antibody, and this purpose is to realize by following technical scheme:
Specifically, the reagent device of the described detection Epstein-Barr virus of the present patent application antibody is strip, comprise the matrix with position, eight holes and the handle that is positioned at matrix one end, putting in order of position, eight holes begins to be followed successively by sample aperture near handle end, the assistant agent hole, the enzyme conjugates hole, the substrate hole, the stop buffer hole, the dilution hole, reacting hole and dilution holes, fill sample to be tested in the sample aperture, the assistant agent hole adds the auxiliary reagent use whenever necessary for detecting, fill enzyme conjugates solution in the enzyme conjugates hole, substrate fills substrate solution in the hole, stop buffer fills stop buffer in the hole, dilution fills dilution in the hole, reacting hole is flat and has high light penetrability, the hole endoperidium has the Epstein-Barr virus proteantigen, and as reaction vessel filling liquid sample and detection reagent and cleansing solution to be measured, carry out absorbance measurement behind the reaction terminating, dilution holes is used during for diluted sample as the sample cut-back tank.
Further, in the described reagent device, when detecting the Epstein-Barr virus IgG antibody, the assistant agent hole does not add any reagent, and when detecting the Epstein-Barr virus antibody IgM, adsorbent adds in the assistant agent hole or with dilution and adds in the dilution hole.
Further, in the described reagent device, be coated with sealing film on assistant agent hole, enzyme conjugates hole, substrate hole, stop buffer hole and the dilution hole, the sealing film mainly plays in the process of transporting and moving and prevents the effect that liquid overflows, in addition, also play effect dustproof anti-pollution and enhancing stability.
Further, be pasted with bar code on the described handle, this bar code identification has the relevant information of Epstein-Barr virus antibody test reagent and analytical equipment.
Further, described information comprises the sequence number of test item code, detection 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, described support column be positioned at the reagent device matrix below, be separated with position, more than one hole between between the adjacent supports post, the effect of support column is for the physical strength of strengthening matrix and balance.
Further, reacting hole is made of outer hole and endoporus in the described reagent device, the bottom in outer hole has bottom outlet, endoporus pass bottom outlet and with the bottom outlet wringing fit, leave the anti-overflow chamber between outer hole and the endoporus, the effect in anti-overflow chamber is in course of reaction, if liquid overflows, can stay in the chamber, prevent from polluting instrument and other reagent devices.
Further, 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 in the described reagent device.
Further, described Epstein-Barr virus capsid antigen comprises Epstein-Barr virus capsid antigen, VCA-18p albumen or the BFRF3 recombinant protein of purifying and deactivation, the Epstein-Barr virus cAg comprises Epstein-Barr virus cAg or the BKRF1 recombinant protein of purifying and deactivation, and eb early antigen comprises eb early antigen or the BMRF1 recombinant protein of purifying and deactivation.
Should be clear and definite be, in the present patent application, the size of each position, hole, shape are not limited in the described reagent device, for example the open peristoma of each position, 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 light penetrability preferably, it is flat that the bottom should be.
Further, the sample that adds in the described sample aperture is test serum or blood plasma, and the amount of adding is determined according to the size of sample aperture and the multiple of detection needs dilution.
Described assistant agent hole is the usefulness that adds auxiliary reagent when being necessary, for example adsorbent, neutralizing agent, inhibitor, damping fluid etc.As when detecting the Epstein-Barr virus antibody IgM, adsorbent adds in the assistant agent hole or with dilution and adds in the dilution hole, its Main Ingredients and Appearance is anti-human IgG antibody or people's staphylococcal protein A (SPA), to remove in the testing process other materials such as specific IgG and rheumatoid factor to the interference of testing result.
Add enzyme conjugates solution in the 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 the anti-human IgG of beta galactosidase mark or IgA or IgM or Ig (GAM) antibody.
The substrate solution Main Ingredients and Appearance that adds in the described substrate hole is TMB, OPD or ABTS.
TMB is as a kind of chromogen reagent of new type of safe, the benzidine derivative that progressively replaces strong carcinogenic substance biphenylamine and other carcinogenicity, be applied to fields such as clinical assay, forensic medical examination, criminal detection and environmental monitoring, especially aspect the clinical biochemical check, TMB has obtained to use widely in enzyme immunoassay (EIA) (EIA) and enzyme linked immunosorbent assay method (ELISA) as the new substrate of peroxidase.
The stop buffer Main Ingredients and Appearance that adds in the described stop buffer hole is that concentration is sulfuric acid or the hydrochloric acid solution of 0.1～0.5M.
The dilution Main Ingredients and Appearance that adds in the described dilution hole is phosphate buffer, Tris damping fluid, borate buffer solution or carbonate buffer solution, and contain components such as certain density bovine serum albumin(BSA), sodium azide, be used for interference and the increase stability of the nonspecific reaction of eliminating sample small-molecule substance.
Another purpose of the present patent application provides utilizes the mentioned reagent device to carry out Epstein-Barr virus detection of antibodies method, and described method comprises following step:
1) in described sample aperture, adds undiluted sample;
2) drawing dilution from the dilution hole adds in the dilution holes;
3) remove remaining liq in the dilution hole, from dilution holes, draw partially liq and go back to the dilution hole;
4) from sample aperture, draw sample and add in the dilution hole, sample is diluted;
5) imbitition adds in the dilution holes from the dilution hole, and sample is further diluted;
When 6) adding adsorbent in detecting Epstein-Barr virus antibody IgM and assistant agent hole, add the reacting hole from assistant agent hole imbitition, other situations then do not need this step;
7) imbitition adds in the reacting hole from dilution holes, under 25～37 ℃ the condition, hatches 30～60min;
8) with cleansing solution reacting hole is carried out removing liquid after 3～5 washings;
9) from the enzyme conjugates hole, draw enzyme conjugates solution and add in the reacting hole, under 25～37 ℃ the condition, remove liquid behind reaction 30～60min;
10) repeating step 8);
11) from the substrate hole, draw substrate solution and add in the reacting hole, under 25～37 ℃ the condition, reaction 10～20min;
12) draw stop buffer from the stop buffer hole and add in the reacting hole, read the OD value in 450nm in 1～10min, or select the double wave regular way to measure, wavelength coverage is at 620nm～690nm.
Further, described method comprises following step:
1) add undiluted sample in described sample aperture, volume is at least 1/4 of sample aperture capacity;
2) draw dilution from the dilution hole and add in the dilution holes, the amount of absorption is more than 7/10 of total amount of liquid in the hole;
3) remove remaining liq in the dilution hole, from dilution holes, draw partially liq according to the expection extension rate and go back to the dilution hole;
4) draw sample from sample aperture and add in the dilution hole, sample is diluted, extension rate is 1～10 times;
5) imbitition adds in the dilution holes from the dilution hole, and sample is further diluted, and extension rate is 1～100 times;
When 6) adding adsorbent in detecting Epstein-Barr virus antibody IgM and assistant agent hole, add the reacting hole from assistant agent hole imbitition, other situations then do not need this step;
7) imbitition adds in the reacting hole from dilution holes, under 25～37 ℃ the condition, hatches 30～60min;
8) with cleansing solution reacting hole is carried out removing liquid after 3～5 washings;
9) from the enzyme conjugates hole, draw enzyme conjugates solution and add in the reacting hole, under 25～37 ℃ the condition, remove liquid behind reaction 30～60min;
10) repeating step 8);
11) from the substrate hole, draw substrate solution and add in the reacting hole, under 25～37 ℃ the condition, reaction 10～20min;
12) draw stop buffer from the stop buffer hole and add in the reacting hole, read the OD value in 450nm in 1～10min, or select the double wave regular way to measure, wavelength coverage is at 620nm～690nm.
Further, described method comprises following step:
1) in described sample aperture, adds sample 80～150 μ L;
2) from the dilution hole, draw 360 μ L liquid in dilution holes with accurate charger;
3) remove remaining liq in the dilution hole, from dilution holes, draw 180 μ L liquid and go back to the dilution hole;
4) from sample aperture, draw 20 μ L liquid in the dilution hole with accurate charger, 10 times of dilutions of sample;
5) with accurate charger from the dilution hole imbitition 20 μ L in dilution holes, 100 times of dilutions of sample;
When 6) adding adsorbent in detecting Epstein-Barr virus antibody IgM and assistant agent hole, add the reacting hole from assistant agent hole imbitition 30 μ L with accurate charger, other situations then do not need this step;
7) in reacting hole, under 25～37 ℃, remove liquid after hatching 60min with accurate charger imbitition 100 μ L from the dilution hole;
8) with cleansing solution reacting hole is carried out removing liquid after 3～5 washings;
9) abzyme bond solution to the reacting hole of drawing 100 μ L horseradish peroxidase-labeled with accurate charger from the enzyme conjugates hole reacts, and under 25～37 ℃, removes liquid behind the reaction 30min;
10) repeating step 8;
11) the tmb substrate solution of drawing 100 μ L with accurate charger from the substrate hole reacts 10～15min to reacting hole;
12) stop buffer of drawing 100 μ L with accurate charger from the stop buffer hole is annotated to reacting hole, detects under 450nm and 630nm dual wavelength condition in 1～10min, reads the 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, and very strong pH surge capability is arranged again, is commonly used to the basal liquid of washing cell and cultivating as cell, and Tween-20 is a kind of non-ionic surfactant, cell growth can exert an influence, and Tween-20 has the effect of renaturation antigen, can improve specific recognition capability.
Reagent device and the method thereof of the described detection Epstein-Barr virus of the present patent application antibody, not only has high specificity that other detection methods have, highly sensitive, accuracy is good, cost is lower, request for utilization is not high, the operation is comparatively easy, obtain the advantage that the testing result time lacks, is widely used etc., and solved many deficiencies of other detection methods, be embodied in the following aspects:
1. described detection method is used the enzyme-linked immuno assay principle, utilize specific analytical instrument, adopt detection kit and the analytical reagent device of supporting special use, automatically realize Epstein-Barr virus antibody qualitative detection, be a kind of brand-new, that be suitable for, practical, detect the scheme of Epstein-Barr virus antibody efficiently, fast;
It is a kind of independently, single part detection reagent and analytical equipment, need not as general ELISA method, to use 12 * 8 types, 6 * 8 types, 8 * 12 types or complete plate 96 hole special enzyme-linked immune microwell plates as antigen coated articles for use and reaction vessel, there is not the waste of reagent as long as there is a sample can carry out the detection of respective items purpose in use, if the quantity of sample surpasses a, use this reagent and analytical equipment to get final product by the actual sample number;
3. it is contained in the essential reagent of each detection in the reagent wells position of an analytical reagent device, and need not come splendid attire with different reagent bottles respectively with detecting reagent, not only operation is very easy, and is not easy to cause bust, thereby guarantees the correctness of testing result;
4. it has a special-purpose bar code to each analytical reagent device, the numerical value of bar code comprise detect corresponding test item code, detect the reagent product batch number, the information such as sequence number of the reagent term of validity, qualitative correction coefficient, concrete enzyme linked immunoassay type, reagent and analytical equipment, can not arbitrarily be changed, strictness is controlled during use, especially when using above term of validity detection reagent, to be identified and stop and send examining report, thereby can guarantee the accuracy that detects;
5. it detects reagent with each and effectively separates and seal, and can not cause the cross pollution between all ingredients and influences testing result;
6. it is a kind of analytical reagent device that is exclusively used in the particular analysis instrument, in testing process, annotate with full automatic accurate charger and detect reagent, cleansing solution or sample, and operation automation, dosage is accurate, the accuracy of testing result and precision height;
7. in the configuration of the quantity of test item reagent set and use, all use to be equipped with by reality and get final product, especially multinomial visual inspection are being surveyed, and are equipped with more in right amount, can not occur surpassing and dispose and operating position.
Description of drawings
Fig. 1 is the cross-sectional view of an embodiment of the described reagent device of the present patent application;
Fig. 2 is the top plan view of the described embodiment of Fig. 1;
Fig. 3 is the cross-sectional view of another embodiment of the described reagent device of the present patent application;
Fig. 4 is the top plan view of reacting hole among another embodiment of the described reagent device of the present patent application;
Fig. 5 and Fig. 6 are the assembling synoptic diagram in endoporus and outer hole in the reacting hole in the described reagent device of Fig. 4;
Wherein, 10 is that matrix, 20 is that handle, 30 is that bar code, 40 is that sealing film, 50 is that support column, 11 is that sample aperture, 12 is that assistant agent hole, 13 is that enzyme conjugates hole, 14 is that substrate hole, 15 is that stop buffer hole, 16 is that dilution hole, 17 is that reacting hole, 18 is that dilution holes, 171 is that outer hole, 172 is that endoporus, 173 is that anti-overflow chamber, 174 is bottom outlet.
Embodiment
Below in conjunction with concrete pick-up unit and implementation step the described reagent device of the present patent application and detection method are further described, purpose is for the public better understands the described technical scheme of the present patent application, rather than to the restriction of described technical scheme.In fact, with identical or approximate principle, to the improvement that described reagent device carries out, comprise the change of its shape, size, used material, 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
Shown in Fig. 1-2, the reagent device of the described detection Epstein-Barr virus of the present patent application antibody is strip, comprise the matrix 10 with position, eight holes and the handle 20 that is positioned at matrix 10 1 ends, putting in order of position, eight holes begins to be followed successively by sample aperture 11 from nearly handle 10 ends, assistant agent hole 12, enzyme conjugates hole 13, substrate hole 14, stop buffer hole 15, dilution hole 16, reacting hole 17 and dilution holes 18, fill sample to be tested in the described sample aperture 11, assistant agent hole 12 adds auxiliary reagent and uses (adding adsorbent in the assistant agent hole when detecting IgM) for detecting when needing, add enzyme conjugates solution in the enzyme conjugates hole 13, add substrate solution in the substrate hole 14, stop buffer hole 15 adds stop buffer, add dilution in the dilution hole 16, reacting hole 17 is flat and has very high light penetrability, when splendid attire during colourless or blank reagent solution, to the absorbance of visible light or ultraviolet light or fluorescence close to zero, be coated with the Epstein-Barr virus proteantigen in the hole, and as reaction vessel filling liquid sample and detection reagent and cleansing solution to be measured, add at last and carry out absorbance measurement after enzyme reaction substrate is hatched, dilution holes 18 is used during for diluted sample as the sample cut-back tank.
Further, in the described reagent device, be coated with sealing film 40 on assistant agent hole 12, enzyme conjugates hole 13, substrate hole 14, stop buffer hole 15 and the dilution hole 16, sealing film 40 mainly plays in the process of transporting and moving, prevent the effect that liquid overflows, in addition, also play effect dustproof anti-pollution and increase stability.
Further, be pasted with bar code 30 on the described handle 20, these bar code 30 signs have the information of Epstein-Barr virus antibody test reagent and analytical equipment, and described information comprises the 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 reagent device among the embodiment one, also comprise several support columns 50 at described reagent device, described support column 50 be arranged in described reagent device matrix 10 below, be separated with position, more than one hole between between the adjacent supports post 50, the effect of support column 50 is for the physical strength of strengthening matrix and balance.
Embodiment three detects the reagent device three of Epstein-Barr virus antibody
Shown in Fig. 4-6, preferred embodiment for the reagent device of the described detection Epstein-Barr virus of the present patent application antibody, 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 of outer hole 171 and endoporus 172, the bottom in outer hole 171 has bottom outlet 174, endoporus 172 pass bottom outlet 174 and with bottom outlet 174 wringing fits, leave anti-overflow chamber 173 between outer hole 171 and the endoporus 172, the effect in anti-overflow chamber 173 is in course of reaction, if liquid overflows, can stay in the chamber, prevent from polluting instrument and other reagent devices.
Further, the cooperation fixed form in described endoporus and outer hole can also be that the outer wall with 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 in outer hole equates with the external diameter of endoporus hypomere, like this endoporus hypomere is inserted in the bottom outlet, the epimere of endoporus is stuck on the bottom outlet just, can play fastening effect equally.
The making of embodiment four reagent devices or kit-indirect method detects Epstein-Barr virus antibody
It is technical a kind of simpler that the present invention is based on enzyme linked immunosorbent detection, accurately, effective methodology, its ultimate principle that adopts is the indirect enzyme-linked immunosorbent method: will be adsorbed on the solid phase by the Epstein-Barr virus proteantigen, be combined with antigen by hatching the human serum or the specific antibody in the blood plasma that make dilution, the antibody of not being combined with solid phase is removed in washing, add the anti-human immunoglobulin(HIg) enzyme connection thing with horseradish peroxidase-labeled, hatch, remove unconjugated enzyme connection thing, add enzyme chromogen substrate, the color of generation is directly proportional with specific antibody concentration in the detection sample.This method mainly is by the analytical reagent device that is used for enzyme linked immunosorbent detection and the immune detection that matched reagent is realized Epstein-Barr virus antibody.By this kind of enzyme linked immunoassay method, use analytical reagent device and the reagent of particular analysis instrument simultaneously, can be quick, make accurately and judging for auxiliary clinical diagnosis.
As sample aperture, add liquid sample with position, hole 11 in use, take during for detection;
, detect and to add auxiliary reagent (adding adsorbent when detecting IgM) whenever necessary as the assistant agent hole with position, hole 12, seal with sealing film, take for detecting;
, add enzyme conjugates solution and seal with sealing film as the enzyme conjugates hole with position, hole 13, take during for detection;
, seal with sealing film behind the adding tmb substrate solution as the substrate hole with position, hole 14, use during for detection;
, seal with sealing film behind the adding stop buffer as the stop buffer hole with position, hole 15, use during for detection;
, seal with sealing film behind the adding sample dilution as the dilution hole with position, hole 16, use during for detection;
As encrusting substance hole/reacting hole/colorimetric hole, be coated with the Epstein-Barr virus proteantigen with position, hole 17, and as reaction vessel filling liquid sample and detection reagent and cleansing solution to be measured, added at last and carry out absorbance measurement after enzyme reaction substrate is hatched;
, use during for diluted sample as dilution holes with position, hole 18;
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 the 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 constituted complete Epstein-Barr virus antibody assay kit component.With detecting pack into the external packing box of kit of the reagent device of Epstein-Barr virus antibody and supporting use component, namely make and detect the Epstein-Barr virus antibody kit.
Embodiment five Epstein-Barr virus cAg IgG detection of antibodies methods
The present patent application provides a kind of method of utilizing the mentioned reagent device to carry out Epstein-Barr virus cAg IgG antibody test, and described method comprises following step:
1. in described sample aperture, add sample 80～150 μ L;
2. from the dilution hole, draw 360 μ L liquid in dilution holes with accurate charger;
3. remove the remaining liq in the dilution hole, from dilution holes, draw 180 μ L liquid and go back to the dilution hole;
4. from sample aperture, draw 20 μ L liquid in the dilution hole with accurate charger, 10 times of dilutions of sample;
With accurate charger from the dilution hole imbitition 20 μ L in dilution holes, 100 times of dilutions of sample;
6. in reacting hole, under 25 ℃, remove liquid after hatching 60min with accurate charger imbitition 100 μ L from the dilution hole;
7. with cleansing solution reacting hole is carried out removing liquid after 3～5 washings;
8. anti-human IgG antibody's enzyme conjugates solution to the reacting hole of drawing 100 μ L horseradish peroxidase-labeled with accurate charger from the enzyme conjugates hole reacts, and under 25～37 ℃, removes liquid behind the reaction 30min;
9. repeating step 7;
10. the tmb substrate solution of drawing 100 μ L with accurate charger from the substrate hole reacts 10～15min to reacting hole;
Annotate to reacting hole 11. from the stop buffer hole, draw the stop buffer of 100 μ L with accurate charger, under 450nm and 630nm dual wavelength condition, detect in 1～10min, read the OD value.
Embodiment six is by the analysis operation flow process of fully-automatic analyzer realization to Epstein-Barr virus cAg IgG antibody test in the sample
Further, the described detection method of the present patent application also comprises the corresponding fully-automatic analyzer that is used, this fully-automatic analyzer comprises an analytical equipment pallet, it is to match with the shape of analytical equipment, one has 30 positions can place for analytical equipment, for detection of analysis, comprise the integrated mechano-electronic structure of modular and software control system in addition, can realize robotization filling, dilute, hatch, washing, reading, analytic process.Each position independence qualitative analysis, assurance result's accuracy.After the instrument operation, the analytical equipment pallet can turn to different positions voluntarily and annotate, and dilution is hatched, 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, preparation finishes and packs in the corresponding liquid bottles;
(2) link to each other with external computing machine: instrument links to each other with external computing machine, handles thereby instrument operate as normal gained result is transferred to integrated system;
(3) start: after opening instrument switch, the working interface by external computing machine arranges the automatic or manual self check to instrument, thereby prepares for the normal operation of instrument;
(4) preheating: after start, instrument can start heating schedule, and temperature is transferred to temperature to be checked;
(5) flushing checks;
(6) Epstein-Barr virus cAg IgG detection of antibodies:
1) open the package from one side that seal is arranged, take the analytical reagent device of requirement, behind the deaeration that the sack envelope is tight;
2) solution in the inspection analytical reagent device tmb substrate hole should be no color and luster and changes, otherwise should discard;
3) add the undiluted sample of 50～120 μ L respectively in the sample aperture of each analytical reagent device, suggestion adds the sample of 100 μ L, and the reagent of a lot number of every replacing should be got one of them analytical reagent device and calibration object and carry out instrument calibration;
4) in the analytical equipment pallet, put into the analytical reagent device of corresponding quantity according to the quantity of required detection, click " beginning ", " scanning ", instrument is scanning analysis reagent device bar code, Quality Control thing bar code and calibration object bar code automatically, then sample is numbered, or by the outer strip code scanner sample bar code is scanned;
5) click " RUN ", instrument moves automatically according to bar code information, and according to bar code, program at first detects calibration object, by calibration, revises typical curve; Secondly the Quality Control thing is detected, if its testing result in the scope that indicates, represents then that the typical curve that detects meets the requirements, can be used for the detection of sample, begin the trace routine of sample at last;
6) dilution: filling acupuncture holes position sealing film is drawn dilution 360 μ L automatically in dilution holes, and remove remaining liq in the dilution hole, from dilution holes, draw dilution 180 μ L again and go back to the dilution hole, the filling pin is drawn sample 20 μ L automatically from sample aperture and is carried out the sample dilution to the dilution hole, from the dilution hole, draw 20 μ L again to dilution holes, after action is finished, moved to reacting hole and hatch 30～60min by the sample that the diluted pin of being annotated, usually instrument is set at 60min, removes liquid afterwards;
7) washing: flushing needle is drawn a certain amount of lavation buffer solution reagent wells is carried out removing liquid after three to five washings from corresponding liquid bottle;
8) sealing film anti-human IgG antibody's enzyme conjugates solution to the reacting hole of drawing 100 μ L horseradish peroxidase-labeled in the broken enzyme conjugates hole of filling acupuncture reacts, and removes liquid behind reaction 30～60min, and instrument is set at 30min usually;
9) repeating step 7);
10) the broken tmb substrate hole of filling acupuncture sealing film draw 100 μ L tmb substrate to reacting hole reaction 10～20min, instrument is set at 10min usually;
11) the sealing film stop buffer of drawing 100 μ L in the broken stop buffer hole of filling acupuncture is annotated to reacting hole, reads the OD value in 1～10min under the 450nm/630nm dual wavelength.
(7) testing result: when the trace routine operation finishes, can generate report at last in order to consult by the data processing software analysis on the computing machine;
(8) shutdown: after detection finishes, before the instrument shutdown, must start clean cycle, can avoid like this avoiding damaging instrument or causing testing result invalid from the residual salt crystallization in the liquid road in the solution, after cleaning was finished, instrument power source was closed automatically.
The Quality Control of the application of Epstein-Barr virus cAg IgG detection of antibodies, interpretation of result and detection in embodiment seven patient's samples
Adopt method of operating and the program of embodiment five, use embodiment four described kits, can be used for the Epstein-Barr virus cAg IgG antibody horizontal in qualitative detection human serum or the blood plasma.
Epstein-Barr virus extensively infects in the crowd, viral capsid antigen IgM antibody occurs, and cAg IgG antibody do not occur, and viral capsid antigen IgG antibody appears in prompting existing disease infection simultaneously and cAg IgG antibody is then pointed out previous infection.Can assist clinical diagnosis according to the result who detects, tentatively judge the situation that the patient is ill, finally make a definite diagnosis and to take all factors into consideration in conjunction with clinical manifestation or other diagnostic method/indexs.
Below be the analysis of testing result:
(1) reference value (term of reference)
Normal reference value: 0～1.0.
(2) explanation of testing result
If sample value＞1.0, the prompting antibody horizontal raises, but should make a definite diagnosis in conjunction with clinical manifestation or other diagnostic method/indexs.
Clinical practice for convenience, can be with reference to following judgment rule:
Sample value＜0.9 feminine gender
0.9≤sample value≤1.1 are suspicious
Sample value＞1.1 positives
When testing result when being suspicious, should detect again, if be suspicious still, need be gathered pattern detection again after 2～3 weeks.
Below be positive and negative quality controlled serum with the testing process duplicate detection of embodiment six, the repeatability of check result obtains following result:
Embodiment eight Epstein-Barr virus capsid antigen IgM detection of antibodies methods
The present patent application provides a kind of method of utilizing the mentioned reagent device to carry out Epstein-Barr virus capsid antigen IgM antibody test, and described method comprises following step:
1. in described sample aperture, add sample 80～150 μ L;
2. from the dilution hole, draw 370 μ L liquid in dilution holes with accurate charger;
3. remove the remaining liq in the dilution hole, from dilution holes, draw 180 μ L liquid and go back to the dilution hole;
4. from sample aperture, draw 20 μ L liquid in the dilution hole with accurate charger, 10 times of dilutions of sample;
With accurate charger from the dilution hole imbitition 20 μ L in dilution holes, 100 times of dilutions of sample;
6. in the time of in adsorbent adds the assistant agent hole, imbitition 30 μ L from the assistant agent hole when adsorbent adds in the dilution hole with dilution, then do not need this step in reacting hole with accurate charger;
7. in reacting hole, under 25～37 ℃, remove liquid after hatching 60min with accurate charger imbitition 100 μ L from the dilution hole;
8. with cleansing solution reacting hole is carried out removing liquid after 3～5 washings;
9. anti-human IgM antibody enzyme conjugates solution to the reacting hole of drawing 100 μ L horseradish peroxidase-labeled with accurate charger from the enzyme conjugates hole reacts, and under 25～37 ℃, removes liquid behind the reaction 30min;
10. repeating step 8;
To reacting hole, react 10～15min 11. from the substrate hole, draw the tmb substrate solution of 100 μ L with accurate charger;
Annotate to reacting hole 12. from the stop buffer hole, draw the stop buffer of 100 μ L with accurate charger, under 450nm and 630nm dual wavelength condition, detect in 1～10min, read the OD value.
Embodiment nine is by the analysis operation flow process of fully-automatic analyzer realization to Epstein-Barr virus capsid antigen IgM antibody test in the sample
Further, the described detection method of the present patent application also comprises the corresponding fully-automatic analyzer that is used, this fully-automatic analyzer comprises an analytical equipment pallet, it is to match with the shape of analytical equipment, one has 30 positions can place for analytical equipment, for detection of analysis, comprise the integrated mechano-electronic structure of modular and software control system in addition, can realize robotization filling, dilute, hatch, washing, reading, analytic process.Each position independence qualitative analysis, assurance result's accuracy.After the instrument operation, the analytical equipment pallet can turn to different positions voluntarily and annotate, and dilution is hatched, 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, preparation finishes and packs in the corresponding liquid bottles;
(2) link to each other with external computing machine: instrument links to each other with external computing machine, handles thereby instrument operate as normal gained result is transferred to integrated system;
(3) start: after opening instrument switch, the working interface by external computing machine arranges the automatic or manual self check to instrument, thereby prepares for the normal operation of instrument;
(4) preheating: after start, instrument can start heating schedule, and temperature is transferred to temperature to be checked;
(5) flushing checks;
(6) Epstein-Barr virus capsid antigen IgM detection of antibodies:
1) open the package from one side that seal is arranged, take the analytical reagent device of requirement, behind the deaeration that the sack envelope is tight;
2) solution in the inspection analytical reagent device tmb substrate hole should be no color and luster and changes, otherwise should discard;
3) add the undiluted sample of 50～120 μ L respectively in the sample aperture of each analytical reagent device, suggestion adds the sample of 100 μ L, and the reagent of a lot number of every replacing should be got one of them analytical reagent device and calibration object and carry out instrument calibration;
4) in the analytical equipment pallet, put into the analytical reagent device of corresponding quantity according to the quantity of required detection, click " beginning ", " scanning ", instrument is scanning analysis reagent device bar code, Quality Control thing bar code and calibration object bar code automatically, then sample is numbered, or by the outer strip code scanner sample bar code is scanned;
5) click " RUN ", instrument moves automatically according to bar code information, and according to bar code, program at first detects calibration object, by single-point calibration, sets up typical curve; Secondly the Quality Control thing is detected, if its testing result in the scope that indicates, represents then that the typical curve curve of setting up is qualified, can be used for the detection of sample, begin the trace routine of sample at last;
6) dilution: filling acupuncture holes position sealing film is drawn dilution 370 μ L automatically in dilution holes, and remove remaining liq 50 μ L in the dilution hole, from dilution holes, draw dilution 180 μ L again and go back to the dilution hole, the filling pin is drawn sample 20 μ L automatically from sample aperture and is carried out the sample dilution to the dilution hole, from the dilution hole, draw 20 μ L again to dilution holes, from the auxiliary reagent hole, draw 30 μ L adsorbents in addition in reacting hole, after action is finished, moved to reacting hole and hatch 30～60min by the sample that the diluted pin of being annotated, usually instrument is set at 60min, removes liquid afterwards;
7) washing: flushing needle is drawn a certain amount of lavation buffer solution reagent wells is carried out removing liquid after three to five washings from corresponding liquid bottle;
8) sealing film anti-human IgM antibody enzyme conjugates solution to the reacting hole of drawing 100 μ L horseradish peroxidase-labeled in the broken enzyme conjugates hole of filling acupuncture reacts, and removes liquid behind reaction 30～60min, and instrument is set at 30min usually;
9) repeating step 7);
10) the broken tmb substrate hole of filling acupuncture sealing film draw 100 μ L tmb substrate to reacting hole reaction 10～20min, instrument is set at 10min usually;
11) the sealing film stop buffer of drawing 100 μ L in the broken stop buffer hole of filling acupuncture is annotated to reacting hole, reads the OD value in 1～10min under the 450nm/630nm dual wavelength.
(7) testing result: when the trace routine operation finishes, can generate report at last in order to consult by the data processing software analysis on the computing machine;
(8) shutdown: after detection finishes, before the instrument shutdown, must start clean cycle, can avoid like this avoiding damaging instrument or causing testing result invalid from the residual salt crystallization in the liquid road in the solution, after cleaning was finished, instrument power source was closed automatically.
The Quality Control of the application of Epstein-Barr virus capsid antigen IgM detection of antibodies, interpretation of result and detection in embodiment ten patient's samples
Adopt method of operating and the program of embodiment eight, use embodiment four described kits, can be used for the Epstein-Barr virus capsid antigen IgM antibody horizontal in qualitative detection human serum or the blood plasma.
Epstein-Barr virus extensively infects in the crowd, and the patient 1gM type antibody can occur in the morbidity early stage blood serum, has 60～80% cases to be positive, and minority normal person and serum sickness patient are also contained this antibody.Can assist clinical diagnosis according to the result who detects, tentatively judge the situation that the patient is ill, finally make a definite diagnosis and to take all factors into consideration in conjunction with clinical manifestation or other diagnostic method/indexs.
Below be the analysis of testing result:
(1) reference value (term of reference)
Normal reference value: 0～1.0.
(2) explanation of testing result
If sample value＞1.0, the prompting antibody horizontal raises, but should make a definite diagnosis in conjunction with clinical manifestation or other diagnostic method/indexs.
Clinical practice for convenience, can be with reference to following judgment rule:
Sample value＜0.9 feminine gender
0.9≤sample value≤1.1 are suspicious
Sample value＞1.1 positives
When testing result when being suspicious, should detect again, if be suspicious still, need be gathered pattern detection again after 2～3 weeks.
Below be positive and negative quality controlled serum with the testing process duplicate detection of embodiment nine, the repeatability of check result obtains following result:
According to the present invention, can automatically carry out many increments Epstein-Barr virus detection of antibodies originally simultaneously by identical analytic process, and can be associated with other or unconnected project detects simultaneously, this just makes, and detection is more simplified, cost reduces, shorten detection time, be difficult for taking place cross pollution, detect processing ease carries out; And the high specificity that detects, highly sensitive, accuracy good.
Claims (10)
1. reagent device that detects Epstein-Barr virus antibody, it is characterized in that: described reagent device is strip, comprise the matrix with position, eight holes and the handle that is positioned at matrix one end, putting in order of position, eight holes begins to be followed successively by sample aperture near handle end, the assistant agent hole, the enzyme conjugates hole, the substrate hole, the stop buffer hole, the dilution hole, reacting hole and dilution holes, fill sample to be tested in the sample aperture, the assistant agent hole adds the auxiliary reagent use whenever necessary for detecting, fill enzyme conjugates solution in the enzyme conjugates hole, substrate fills substrate solution in the hole, stop buffer fills stop buffer in the hole, dilution fills dilution in the hole, reacting hole is flat and has high light penetrability, be coated with the Epstein-Barr virus proteantigen in the hole, and as reaction vessel filling liquid sample and detection reagent and cleansing solution to be measured, carry out absorbance measurement behind the reaction terminating, dilution holes is used during for diluted sample as the sample cut-back tank.
2. reagent device according to claim 1, it is characterized in that: in the described reagent device, when detecting the Epstein-Barr virus IgG antibody, do not add any reagent in the assistant agent hole, when detecting the Epstein-Barr virus antibody IgM, adsorbent adds in the assistant agent hole or with dilution and adds in the dilution hole.
3. reagent device according to claim 1 is characterized in that: in the described reagent device, be coated with sealing film on assistant agent hole, enzyme conjugates hole, substrate hole, stop buffer hole and the dilution hole.
4. according to each described reagent device of claim 1-3, it is characterized in that: described reagent device also comprises several support columns, described support column be arranged in described reagent device matrix below, be separated with position, more than one hole between between the adjacent supports post.
5. according to each described reagent device of claim 1-3, it is characterized in that: reacting hole is made of outer hole and endoporus in the described reagent device, and the bottom in outer hole has bottom outlet, endoporus pass bottom outlet and with the bottom outlet wringing fit, leave the anti-overflow chamber between outer hole and the endoporus.
6. according to each described reagent device of claim 1-3, it is characterized in that: 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 in the described reagent device.
7. reagent device according to claim 6, it is characterized in that: described Epstein-Barr virus capsid antigen comprises Epstein-Barr virus capsid antigen, VCA-18p albumen or the BFRF3 recombinant protein of purifying and deactivation, the Epstein-Barr virus cAg comprises Epstein-Barr virus cAg or the BKRF1 recombinant protein of purifying and deactivation, and eb early antigen comprises eb early antigen or the BMRF1 recombinant protein of purifying and deactivation.
8. the method for utilizing the described reagent device of claim 1-7 to carry out the Epstein-Barr virus antibody test is characterized in that: described method comprises following step:
1) in described sample aperture, adds undiluted sample;
2) drawing dilution from the dilution hole adds in the dilution holes;
3) remove remaining liq in the dilution hole, from dilution holes, draw partially liq and go back to the dilution hole;
4) from sample aperture, draw sample and add in the dilution hole, sample is diluted;
5) imbitition adds in the dilution holes from the dilution hole, and sample is further diluted;
When 6) adding adsorbent in detecting Epstein-Barr virus antibody IgM and assistant agent hole, add the reacting hole from assistant agent hole imbitition, other situations then do not need this step;
7) imbitition adds in the reacting hole from dilution holes, under 25～37 ℃ the condition, hatches 30～60min;
8) with cleansing solution reacting hole is carried out removing liquid after 3～5 washings;
9) from the enzyme conjugates hole, draw enzyme conjugates solution and add in the reacting hole, under 25～37 ℃ the condition, remove liquid behind reaction 30～60min;
10) repeating step 8);
11) from the substrate hole, draw substrate solution and add in the reacting hole, under 25～37 ℃ the condition, reaction 10～20min;
12) draw stop buffer from the stop buffer hole and add in the reacting hole, read the OD value in 450nm in 1～10min, or select the double wave regular way to measure, wavelength coverage is at 620nm～690nm.
9. method according to claim 8, it is characterized in that: described method comprises following step:
1) add undiluted sample in described sample aperture, volume is at least 1/4 of sample aperture capacity;
2) draw dilution from the dilution hole and add in the dilution holes, the amount of absorption is more than 7/10 of total amount of liquid in the hole;
3) remove remaining liq in the dilution hole, from dilution holes, draw partially liq according to the expection extension rate and go back to the dilution hole;
4) draw sample from sample aperture and add in the dilution hole, sample is diluted, extension rate is 1～10 times;
5) imbitition adds in the dilution holes from the dilution hole, and sample is further diluted, and extension rate is 1～100 times;
When 6) adding adsorbent in detecting Epstein-Barr virus antibody IgM and assistant agent hole, add the reacting hole from assistant agent hole imbitition, other situations then do not need this step;
7) imbitition adds in the reacting hole from dilution holes, under 25～37 ℃ the condition, hatches 30～60min;
8) with cleansing solution reacting hole is carried out removing liquid after 3～5 washings;
9) from the enzyme conjugates hole, draw enzyme conjugates solution and add in the reacting hole, under 25～37 ℃ the condition, remove liquid behind reaction 30～60min;
10) repeating step 8);
11) from the substrate hole, draw substrate solution and add in the reacting hole, under 25～37 ℃ the condition, reaction 10～20min;
12) draw stop buffer from the stop buffer hole and add in the reacting hole, read the OD value in 450nm in 1～10min, or select the double wave regular way to measure, wavelength coverage is at 620nm～690nm.
10. method according to claim 9, it is characterized in that: described method comprises following step:
1) in described sample aperture, adds sample 80～150 μ L;
2) from the dilution hole, draw 360 μ L liquid in dilution holes with accurate charger;
3) remove remaining liq in the dilution hole, from dilution holes, draw 180 μ L liquid and go back to the dilution hole;
4) from sample aperture, draw 20 μ L liquid in the dilution hole with accurate charger, 10 times of dilutions of sample;
5) with accurate charger from the dilution hole imbitition 20 μ L in dilution holes, 100 times of dilutions of sample;
When 6) adding adsorbent in detecting Epstein-Barr virus antibody IgM and assistant agent hole, add the reacting hole from assistant agent hole imbitition 30 μ L with accurate charger, other situations then do not need this step;
7) in reacting hole, under 25～37 ℃, remove liquid after hatching 60min with accurate charger imbitition 100 μ L from the dilution hole;
8) with cleansing solution reacting hole is carried out removing liquid after 3～5 washings;
9) anti-human IgM antibody enzyme conjugates solution to the reacting hole of drawing 100 μ L horseradish peroxidase-labeled with accurate charger from the enzyme conjugates hole reacts, and under 25～37 ℃, removes liquid behind the reaction 30min;
10) repeating step 8;
11) the tmb substrate solution of drawing 100 μ L with accurate charger from the substrate hole reacts 10～15min to reacting hole;
12) stop buffer of drawing 100 μ L with accurate charger from the stop buffer hole is annotated to reacting hole, detects under 450nm and 630nm dual wavelength condition in 1～10min, reads the OD value.
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