CN1138981C - Immunodetection method with rich information and its special detection board - Google Patents
Immunodetection method with rich information and its special detection board Download PDFInfo
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- CN1138981C CN1138981C CNB001207989A CN00120798A CN1138981C CN 1138981 C CN1138981 C CN 1138981C CN B001207989 A CNB001207989 A CN B001207989A CN 00120798 A CN00120798 A CN 00120798A CN 1138981 C CN1138981 C CN 1138981C
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Abstract
The present invention discloses a novel antibody or antigen immune detecting method and a special detecting board thereof. The novel antibody or antigen immune detecting method is a detecting method which can simultaneously detect various antigens or antibodies in a compact, sensitive, quick and timely way. The novel antibody or antigen immune detecting method is characterized in that two or more different specific antibodies and/or antigens are added to and immobilized on the same microwell plate according to a certain order and a hole arranging mode, and an immune microwell plate is obtained; then, a specimen of a patient to be detected is added to the immune microwell plate to react and be analyzed. By the one-time detection of the immune microwell plate, different information of the detected specimen can be obtained. The problems that a disease needs to be detected and diagnosed many times when the disease has many pathogenic factors and the treatment is delayed are solved.
Description
Technical field
The present invention relates to a kind of novel and disease detection, medical diagnosis is relevant, also can be the immunologic detection method and the dedicated detector board thereof of antigen or antibody in the antigen relevant or the detection of antibodies method, particularly biomedicine with various biology, zoology and botany, farming and animal husbandry.
Technical background
In immunology diagnosis, an experimental system, as enzyme linked immunosorbent assay, radioimmunoassay experiment and immunofluorescence detection technique etc. once can only detect a kind of antigen or antibody (antibody also claims immunoglobulin (Ig)) usually.And a kind of disease or clinical symptoms can be caused by multiple paathogenic factor, as virus hepatitis, but have many identical clinical manifestations.A kind of disease also can show different clinical symptoms, and promptly different diseases or clinical symptoms also can be by causing with a kind of paathogenic factor.Therefore, even a kind of disease also must detect respectively with different antigen-antibody detection systems sometimes simultaneously.So existing detection technique is time-consuming, effort, complex operation, cost height.Yet, when detecting a kind of antigen or antibody, can only prove that this antigen or antibody are relevant with this disease, but can not get rid of the existence of other relevant paathogenic factor.Particularly when obtaining negative result, the doctor be because of can't judging which kind of paathogenic factor is this disease be due to, and can't determine therapeutic scheme.In order to clarify a diagnosis, also must do further inspection, can make a definite diagnosis up to the testing result that obtains, diagnose untimely and delay treatment with reason.Patient Ze Yin can not get timely, correct treatment, and incur loss through delay or the treatment opportunity of letting sth. slip by, and regret to the very end of one's life, even loss of life.For non-common disease, because the incidence of disease is low, it is few to detect sample, so hospital, often need run up to sample concentrated detection the after certain quantity for reducing cost.And middle or small level hospital even common disease also are like this.Thereby, existing detection technique cost height, test results report is untimely.Some part small-middle hospital from far-off regions then abandons carrying out this type of test item at all.
Summary of the invention
The objective of the invention is to provide a kind of easy sensitivity, fast in time, can once detect multiple antigen and/or detection of antibodies method and dedicated detector board thereof simultaneously.1. comprise the bag quilt in order to reach the technical scheme that purpose of the present invention takes, mark, add sample to be checked, stream is washed or is embathed, add label, stream is washed or is embathed, add the substrate display result, steps such as analysis, it is characterized in that, its bag is that two or more not homospecific antibody and/or antigen are coated on respectively in the different micropores of same microwell plate or on other solid-phase matrix by step, so that detect multiple different antigen and antibody simultaneously, used microwell plate is the microwell plate of band microflute, microfluidic circuit by set on the microwell plate makes sealing, rinsing and sample, the interpolation of label and substrate reagent waits each step all can finish by single job.
Feature of the present invention also is: bag is to adopt biomacromolecule immobilization treatment technology, with antigen, antibody respectively immobilization by glass, or silicon, or metal, or (biomacromolecule immobilization treatment technology has detailed introduction in " the biomacromolecule immobilization technology and the application " of the Jiang Zhonghua work that chemical publishing house published in 1998) of finishing on the microwell plate made of solid-phase matrix materials such as macromolecular organic compound such as plastics, its concrete operations step is:
1) microwell plate is improved the conventional pre-service of its absorption property;
2) add encrusting substance: be dissolved in two or more not homospecific antibody and/or antigen in the damping fluid respectively, with artificial or full-automatic microarray point sample preparation system, be added to respectively in the different micropores, only wrap by a kind of antigen or antibody in 1 hole, on same microwell plate, can be coated with antigen and also can be coated with antibody simultaneously, bag can be had a plurality of (claim that they are repeating hole, or multiple hole) by the hole of same antigen or antibody, its arrangement can be continuous, also can be discontinuous; If when detecting corresponding antigen and antibody simultaneously, can adopt subregion bag quilt;
3) damping fluid stream is washed or is embathed, to remove uncured antigen or antibody;
4) add confining liquid, normal temperature was hatched 20-360 minute, with the sealing nonspecific binding site; But the microwell plate of lid is being arranged, then after sealing the formation microfluidic circuit, undertaken by microfluidic circuit;
If adopt existing 96 holes or 384 holes etc. plastic microporous is firm and hard and execute, antigen and antibody can be dissolved in respectively in the carbonate buffer solution more than the pH8.5 and be directly used in the bag quilt.
Feature of the present invention also is: in same detection system, when carrying out the common detection of corresponding antigen and antibody simultaneously, can adopt following any scheme to avoid detecting error:
1) antigen and antibody sandwich (are seen Fig. 1,2,3) on the high density microwell plate, with the method for direct mark thing to be checked such as enzyme, fluorescein and biotin.
2) utilize an antigen molecule that the characteristic of a plurality of identical with different specific antigen determinants is arranged, in label, choose different peptide sections and the different antigenic determinants of the same antigen molecule of identification or the antibody pairing of different peptide sections of antigen molecule and use.
3) when used microwell plate when being simple and easy subregion microwell plate (but uncovered is seen Fig. 4), respectively with antigen coated in antigen coated district, with antibody sandwich in the antibody sandwich district; When labelling thing,, add the antibody labeling thing of specific antigen to antibody sandwich district (Ab district) according to the antiantibody of subregion to antigen coated district (Ag district) adding mark; But sealing, embathe, add two bags of operation steps such as sample to be detected, adding substrate and stop buffer in the operation is distinguished and can be carried out jointly.
4) when being the private partition microwell plate, used microwell plate (sees Fig. 3, Fig. 5, Fig. 6), respectively with antigen coated in antigen coated district, with antibody sandwich in the antibody sandwich district, each bag is distinguished complete and independent microfluidic circuit is all arranged, and adds sample to be checked, stream wash, label thing, substrate and stop buffer etc. respectively by subregion; When adding label,, add the antibody labeling thing of specific antigen to antibody sandwich district (Ab district) to the antiantibody of antigen coated district (Ag district) adding mark.
5) when being dedicated detector board, used microwell plate (sees Fig. 7, Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12, Figure 13), with antigen and antibody subregion bag quilt, by the multi-path diverting valve on the dedicated detector board, in antigen coated district (Ag district) and antibody sandwich district (Ab district) but between form and can be communicated with and the microfluidic circuit of can be separate mobile modulation, when adding label, make the antiantibody label of adding enter antigen coated district along microfluidic circuit; The antibody labeling thing of specific antigen can only enter the antibody sandwich district.
6) scheme 2 can be respectively merges with scheme 3 or scheme 4, scheme 5 and uses.
Feature of the present invention also is: when employed micropore strip microflute, can adopt following method to carry out qualitative, quantitative test: because of on same microwell plate, the bag of same antigen or antibody can be had two or more by the quantity in hole, when the homologue in the sample to be checked on the trough of belt microwell plate, when crossing the micropore of each duplicate packages quilt by orifice flow, by constantly combination, and constantly from specimen fluids, removed, therefore, the binding capacity in each multiple hole, to reduce gradually according to the front and back order of each repeating hole arrangement.According to having or not of each hole reaction, can carry out qualitative analysis; When carrying out quantitative test, increase the micropore quantity of duplicate packages quilt, according to the variation of the depth of each repeating hole product color of front and back, fluorescence intensity, activity, the luminous power of biological or chemical with have or not, can draw substrate concentration to be checked-reacting hole quantity and strong and weak change curve by Computer Analysis, contrast sample volume, quantitative analysis results be can obtain, and dilution standard thing or encrusting substance in advance do not needed.
Feature of the present invention also is: used microwell plate is that Chinese patent application number is the described microwell plate of 00209973.X, i.e. high density microwell plate, and every square centimeter has 4 holes or more than 4 holes, hole depth 0-2 millimeter has between the hole or do not have microflute to link to each other.Microwell plate can have lid or uncovered, is respectively equipped with inlet opening 6, the fluid hole 7 that communicates with microflute 5 on micropore plate body 1 or plate lid 2.
Dedicated detector board of the present invention, comprise plate body 1, microwell plate lid 2, micropore 4, the microflute 5 that links to each other with micropore 4, it is characterized in that, on microwell plate lid 2, be respectively equipped with the inlet opening 6 that communicates with microflute 5, fluid hole 7 and hyperchannel diverting valve 8, the valve opening 17 of this hyperchannel diverting valve 8 circumferentially is divided into two districts by spool 18 edges: A district and B district, spool 18 centers have a bottom to have the sample well 14 of pipeline 15, and the sidewall of valve opening 17 is provided with the pipeline 11 that communicates with Ag district, antigen coated district, the pipeline 12 and 16 that communicates with Ab district, antibody sandwich district, the pipeline 13 that communicates with fluid hole 7.
Above two described microwell plate and dedicated detector board, it is characterized in that, between plate body 1 and microwell plate lid 2, can be added with sealing gasket 3.
The opening of microwell plate that the present invention is used and the inlet opening of dedicated detector board 6, fluid hole 7 and sample well 14 also can be on plate body 1.
This method has the following advantages: 1 contains much information.Existing ELISA method normally all only adds same species specific antigen or antibody in all micropores of same microwell plate (as 96 or 384 orifice plates), so a plate once can only detect a kind of antigen or antibody.What the present invention was different with enzyme linked immunosorbent assay (being called for short ELISA) method is that sample to be checked can obtain the result whether all antigens (or antibody) in the immune microwell plate exist by once immune micropore detection.When having solved a kind of many paathogenic factors of disease, need do repeatedly or multiple detection diagnosis the problem of delay treatment; 2. can detect corresponding antigen-antibody simultaneously.In same microwell plate, can carry out the common detection of (as detecting the antibody of hepatitis B surface antigen and anti-hepatitis B table antigen) of corresponding antigen and antibody simultaneously.The present invention has solved the problem that above-mentioned enzyme-linked immunosorbent assay and immunodotting hybrid method can't detect corresponding antigen-antibody simultaneously by six kinds of different schemes; 3. fast in time, cost is low.A patient only needs an immune microwell array plate, can be with arriving with inspection, and examining report is timely.Therefore, solved to saving reagent, the sample that reducing cost needs to wait for the accumulation some is done detection again, so that the problem delayed of test results report time; 4. highly sensitive.The present technique method adopts special-purpose microwell plate or check-out console to make solid-phase matrix, and each micropore has been equivalent to solidify the affinity chromatography matrices of antigen or antibody, and immune microwell plate then is equivalent to different affinity chromatography matrices segmentations is cascaded.When the antigen in the detected sample or antibody out-of-date by orifice flow in immune microwell plate, can with different antibodies in the solid phase or antigen specific bond (both filtration, affinity chromatography, separate and concentrated effectiveness) respectively; No longer mixed by the sample liquid after the special absorption of insolubilized antibody (or antigen) different in each hole of immune microwell array plate simultaneously, avoided detected material (antigen or antibody) because of diffusion repeatedly, the diluted influence of balance with sample stoste.In addition, the end of microwell plate and hole wall all wrap quilt cover and reaction surface, so to compare package amount many with the immunodotting hybrid method.Technology such as the present invention concentrates owing to separation and purification, immunoaffinity chromatography with protein reach and check and analysis method combine together, so sensitivity is higher relatively.Therefore with existing immunodotting hybridization, enzyme-linked immunosorbent assay detection method ratio, have highly sensitive characteristics; 5. easy and simple to handle.In order to improve method of operating, the microwell plate that the present invention preferentially selects for use is that Chinese patent application number is described microwell plate of 00209973.X and dedicated detector board of the present invention.Be characterized in having between each micropore microflute to connect, after adding a cover to microwell plate, microflute is sealed into the microtubule that two ends communicate with micropore.With antigen and antibody subregion bag quilt, these structures and hyperchannel diverting valve, the common microfluidic circuit of forming such as the inlet opening that microwell plate covers, fluid hole and sample well.By this present invention will be not synantigen, antibody forming system mixing, concentrate, separation, reaction and immune complex separate with unreacted free label, operations such as cleaning concentrate on to be carried out in the same immune microwell plate, makes it organically to combine together.Different with the ELISA detection method with immunodotting is need not add by the hole, or microwell plate is all immersed reactant liquor.In check and analysis, no matter will detect how many kinds of antigen or antibody, each experimental procedure only needing to carry out single job with this law, can finish the reaction of cleaning, sealing and sample to be checked and all each immune micropores etc.Therefore compare with the immunodotting hybridization technique with existing enzyme linked immunosorbent assay, operation steps is simple; 6. sample size is few.As adopt special-purpose high density microwell plate to carry out immune micropore check and analysis, sample is along microfluidic circuit each hole of progressively flowing through, so with immunodotting hybridization, enzyme-linked immunosorbent assay detection method ratio, required sample size is few, can solve the puzzlement that is brought because of the difficult acquisition of sample; 7. saving reagent easily is automated operation.This method concentrates on tens of kinds even thousands of kinds of antigen and/or antibody test analysis on the microwell plate.In the enforcement, the interpolation of sample, label and substrate and sealing, stream such as wash at all operations, all carry out simultaneously through inlet opening or the common microfluidic circuit of forming such as sample well, microtubule, hyperchannel diverting valve and fluid hole, changed the enzyme linked immunosorbent assay method and pursued the mode of operation that the hole is cleaned and the immunodotting hybrid method embathes in bigger container, can save reagent in a large number, be easy to realize full-automatic operation; 8. can be used for qualitative and quantitative analysis.According to each hole fluorescence, collaurum deposition, luminous having or not in the immune microwell plate, or the generation of quality product whether, control antibodies and/or the antigen arrangement position in immune microwell plate, can determine in this disease association paathogenic factor, there be (positive) in any or which kind paathogenic factor (pathogen or its antibody) in sample to be detected, there be not (feminine gender) in which, directly makes qualitative analysis.Aspect quantitative test, the present invention has solved the problem of quantitative test by increase the multiple hole number of the parallel bag quilt of same antigen and/or antibody on microwell plate; 9. kept existing immunological detection method good reproducibility, excellent characteristics such as accuracy height, good reliability.
Description of drawings Fig. 1 is a microwell plate plate body structure synoptic diagram:
Shown in the figure 1 be plate body, 4 for micropore, 5 is the microflute that links to each other with micropore 4, be located at microwell plate cover communicate with microflute 5 on 2 inlet opening 6, fluid hole 7 arranged; Fig. 2 is microwell plate lid structural representation:
2 are the plate lid shown in the figure, and inlet opening 6, fluid hole 7 communicate with microflute 5 on the plate body 1.Plate lid 2 can be local transparent, and transmitting plate lid 2 can be seen the micropore 4 that is located on the plate body 1; Fig. 3 is the microwell plate sectional view:
Shown in the figure for plate body 1, plate lid 2, sealing gasket 3, micropore 4, microflute 5, have the inlet opening 6 of O-ring seal; Fig. 4 is simple and easy subregion microwell plate structural representation:
The bag of being distinguished Ag district, antibody for the bag of plate body 1, micropore 4, antigen shown in the figure is distinguished Ab district and reaction tank 19.Fig. 5 is a private partition microwell plate structural representation:
The bag of being distinguished Ag district, antibody for the bag of plate body 1, micropore 4, microflute 5, antigen shown in the figure is distinguished Ab district, two and is distinguished inlet opening 6 that (Ab district) communicate, distinguished the fluid hole 7 that the Ab district communicates with the bag that the bag of antigen is distinguished Ag district, antibody with the bag that the bag of antigen is distinguished (Ag district), antibody respectively; Fig. 6 is private partition microwell plate lid structural representation
The bag of being distinguished Ag district and antibody for the bag of plate lid 2, the inlet opening 6 that communicates with microflute 5 on the plate body 1 respectively, antigen shown in the figure is distinguished the public fluid hole 7 in Ab district.Plate lid 2 is local transparent, and transmitting plate lid 2 can be seen the micropore 4 that is located on the plate body 1; Fig. 7 is a dedicated detector board plate body structure synoptic diagram
The bag of being distinguished Ag district, antibody for the bag of plate body 1, micropore 4, microflute 5, antigen shown in the figure is distinguished Ab district, the inlet opening 6 that communicates with the antigen coated Ag of district district, is distinguished fluid hole 7 that the Ab district communicates, hyperchannel diverting valve 8, the valve opening 17 of hyperchannel diverting valve 8, the sample well 14 that has pipeline 15 and communicate with it with the bag of antibody; The pipeline 13 that valve opening 17 sidewalls are provided with the pipeline 11 that communicates with antigen coated district Ag district, the pipeline 12 and 16 that communicates with Ab district, antibody sandwich district, communicate with fluid hole 7; Fig. 8 is the structural relation synoptic diagram of hyperchannel diverting valve and microfluidic circuit:
The pipeline 11 that is plate body 1 shown in the figure, communicates with Ag district, antigen coated district and valve opening 17; The pipeline 12 and 16 that communicates with Ab district, antibody sandwich district and valve opening 17; Pipeline 13 communicates with fluid hole 7; Fig. 9 is the skeleton view of hyperchannel diverting valve:
For to cover multi-path diverting valve 8 and each passage of seeing thereof, show the structural relation of multi-path diverting valve 8 and plate lid 2 and plate body 1 shown in the figure from the check-out console plate.Have sample well 14 on the check-out console plate lid 2; Spool 18 is divided into A district and B district with valve opening 17; The break-make relation and the liquid flow path direction synoptic diagram of the microfluidic circuit when Figure 10 is operating mode I; The break-make relation and the liquid flow path direction synoptic diagram of the microfluidic circuit when Figure 11 is operating mode II; Figure 12 is the front view of hyperchannel diverting valve spool:
Be spool 18, the sample well 14 that communicates with pipeline 15, the pipeline 15 that can communicate shown in the figure with pipeline 16; Figure 13 is the vertical view of hyperchannel diverting valve spool:
The sample well 14 that communicates for spool 18, with pipeline 15 shown in the figure, be connected the pipeline 15 of sample well 14 and pipeline 16; Figure 14 is immune microwell array method of operating and detection principle schematic:
shown in the figure is a solid phase carrier; -Ag is the curing antigen A g that is solidificated on the solid phase carrier; -Ab is the curing antibody Ab that is solidificated on the solid phase carrier; + expression adds and combination.
Embodiment
Concrete experimental implementation step of the present invention is as follows:
1. bag quilt: use known various biomacromolecule immobilization technology, with multiple antibody and/or antigen respectively immobilization (or claim bag by) in the different micropores of microwell plate; The poly-D-lysine of all available 10-200 mcg/ml of the microwell plate of various materials is handled, and room temperature 20-360 minute or 4 ℃ of placements are spent the night.The polystyrene plastics microwell plate can be handled with isotope irradiation, the concentrated sulphuric acid, nitric acid dousing, or in polystyrene material, adds the macromolecular material that has aldehyde radical, amino, hydroxyl or sulfydryl isoreactivity group of proper proportion, to improve its adsorptive power.Or the microwell plate made from the various materials sold of merchant.Envelope antigen and antibody are dissolved in respectively in the damping fluid of pH7-10, and the bag of antigen, antibody is the 0.1-2000 mcg/ml by concentration; With artificial or full-automatic microarray point sample system, every hole adds 0.1-5ul, or is as the criterion to fill it up with.Nonspecific binding site residual in the micropore can seal with the damping fluid that contains bovine serum albumin(BSA), skim milk or Normal animal serum, promptly obtain bag thus by different antibodies and/or antigen, one-time detection can obtain two or more different specific antibodies and/or detection of antigens result's immune microwell plate simultaneously.This moment, immune microwell plate can directly use, or airing put below 10 ℃ preserve standby;
2. mark: use known biomolecular labeling technology, use mark samples to be checked such as biotin, enzyme, fluorescein, collaurum and isotope respectively, known antigens, antibody, antiantibody, staphylococcal protein A or Avidin and streptavidin etc. obtain corresponding label.
3. add sample to be checked: mark or unlabelled sample to be checked are added in the immune microwell plate, make it and immobilised not synantigen of each Kong Zhongyi and/or antibody, carry out immunological response; In the test sample if any the antibody and the antigen of correspondence, can with the antigen and the antibodies of bag quilt, in conjunction with antibody and antigen promptly be adsorbed, unconjugated and irrelevant antigen and antibody flow out immune microwell plate.
4. stream is washed or is embathed: by the microfluidic circuit of microwell plate, and residual unconjugated irrelevant antigen and antibody in the tested sample, the available washing lotions such as phosphate buffer that contain Tween 20, the stream flush away removes; (as detecting, promptly adopt the method for mark sample to be checked, connect step 7 under its operation with direct method.With things of marking such as biotins, also adopt directly (bridging) method, connect step 5 under its operation.As adopt the uncovered microwell plate, also can adopt the method for embathing); Simple and easy microwell plate is with the method for embathing.
5. adding label: if sample to be detected adds and reacts through the specific antigen of marks such as fluorescein, isotope, collaurum, enzyme or biotin and/or the label of antibody through the mark reason; As in same detection system, detecting antibody and antigen simultaneously, as previously mentioned, according to selected microwell plate and the bag by the difference of mode, can be undertaken by following different schemes respectively: a. selects the simple and easy microwell plate of uncovered for use, see Fig. 4, subregion bag quilt adds the label of antiantibody respectively to antigen coated district (Ag district), to the antibody labeling thing of antibody sandwich district (Ab district) adding specific antigen (can carry out jointly but seal, embathe, add operation steps such as sample to be detected).B. select the private partition microwell plate for use, see Fig. 3,5,6, by wrapping by the difference of subregion, through inlet opening 6, to antigen coated district (Ag district) and antibody sandwich district (Ab district) thing (add sample to be checked, stream step such as wash also carry out respectively) of labelling respectively, flow out immune microwell plate through common fluid hole 7; C. select dedicated detector board for use, see Fig. 7,8,9,10,11,12,13, with antigen and antibody subregion bag quilt, by the multi-path diverting valve 8 on the dedicated detector board, in antigen coated district (Ag district) and antibody sandwich district (Ab district) but between form and can be communicated with and the microfluidic circuit of can be separate mobile modulation; Respectively through inlet opening 6 and sample well 14, to antigen coated district (Ag district) and antibody sandwich district (Ab district) thing of labelling respectively, make the antiantibody label of adding enter antigen coated district (Ag district) simultaneously along microfluidic circuit; The antibody labeling thing of specific antigen can only enter the antibody sandwich district; Unconjugated labelled antigen and/or antibody flow out immune microwell plate through common fluid hole 7
6. stream is washed or is embathed: the label of residual unconjugated antigen and/or antibody etc. in the tested sample, and the available washing lotions such as phosphate buffer that contain Tween 20, stream is washed or is embathed removal;
7. adding substrate, display result: various enzyme labeling things need to add different zymolytes through inlet opening 6, observe the biochemiluminescence phenomenon then; As observe dye-forming reaction, also need add stop buffer or stream is washed cessation reaction; But result's Direct observation of fluorescein, colloid gold label thing; Isotopic label then need be used the autoradiography observations;
8. interpretation of result: according to each hole fluorescence, collaurum deposition, luminous having or not in the immune microwell plate, or the generation of quality product whether, control antibodies and/or the antigen arrangement position in immune microwell plate, can determine in this disease association paathogenic factor, there be (positive) in any or which kind paathogenic factor (pathogen or its antibody) in sample to be detected, there be not (feminine gender) in which, promptly obtains simultaneously and whole envelope antigens and/or the relevant qualitative conclusion of antibody.According to the depth of each multiple hole product color of front and back, strong and weak variation such as fluorescence intensity, activity, biological or chemical are luminous is carried out quantitative test by computing machine.
The aforesaid operations step, different according to its encrusting substance, detected material, label in force with the display result method, multiple array mode can be arranged, as shown in figure 14, they are respectively:
1. direct method: in immune microwell array plate (but envelope antigen Ag and/or antibody A b), adding is through sample to be checked (containing antigen, the antibody) 10-300ul of marks such as fluorescein, collaurum or isotope, reacted 5-120 minute, phosphate or Tris damping fluid (are looked the difference of label, can add different scaling agents such as 0.1-0.5% polysorbas20) stream washes or embathes 0.5-15 minute to remove unconjugated part, then according to the difference of label, with fluorescence, collaurum deposition or the radioactive intensity in each holes of various different observation of use instrument such as scanner.Fluorescence, collaurum deposition or the both positive hole of radioactive micropore are arranged, represent to have in the tested sample corresponding with it antigen or antibody.Direct method sensitivity is lower, and available biotin---Avidin (or streptavidin) the system thing that serves as a mark is to improve its detection sensitivity (seeing the bridging method).
2. indirect method: in the immune microwell plate of envelope antigen (Ag), add 30-300ul sample to be checked (containing antibody), room temperature reaction 1-90 minute, phosphate or Tris damping fluid (containing the 0.1-0.5% polysorbas20) stream is washed or was embathed 0.5-15 minute, to remove unreacted free antibodies, add the 30-100ul enzyme again (as horseradish peroxidase, alkaline phosphatases etc.) antiantibody of mark is (as sheep, rabbit, the immunoglobulin (Ig) of mouse-anti people or other animal) or staphylococcal protein A, react after 1-180 minute, stream is washed or is embathed and adds substrate (as the mixed liquor of diaminobenzidine and hydrogen peroxide) 50-120ul, lucifuge, the colour developing back adds stop buffer (as 2N equivalent sulfuric acid) 50-120 microlitre, or the stream flush away removes the substrate cessation reaction, observe the dye-forming reaction in each hole, the both positive hole, hole that dye-forming reaction is arranged, representing has the antibody corresponding with this antigen in the tested sample.
3. sandwich method: at the immune microwell plate of coated antibody (Ab) (as bag by the anti-mouse inhomogeneity of rabbit, subclass, the antibody of type and hypotype) in, add 30-200ul sample to be checked, (as contain the Hybridoma Cell Culture liquid of mouse monoclonal antibody, at this as antigen) react, damping fluid such as phosphate or Tris (containing the 0.1-0.5% polysorbas20) stream is washed or was embathed 0.5-15 minute, to remove unreacted free antigen, the antibody (species specificity) that adds the anti-mouse immuning ball protein of rabbit of 30-100ul collaurum (also can be other label) mark again reacts, the antibody that perhaps adds marks such as 30-100ul enzyme reacts, after stream is washed or is embathed, (with the luminescence method is example to add the substrate of enzyme again, as chemical illuminating reagents such as luminols, belonging to the merchant and sell reagent) 50-120ul reacts, the luminescence-producing reaction in each hole is observed in lucifuge or darkroom.Positive hole had both represented that the antigen corresponding with this hole antibody was arranged in the tested sample.
4. competition law (is example with the fluorescence method): with 30-100ul sample to be checked and fluorescein (as Cy3, Cy5, FITC, texas Red, rhodamine) etc. the known antigens of mark or antibody mix, add then in the immune microwell plate (bag is by Ag or Ab), the immobilization antibody (Ab) or the antigen (Ag) of bag quilt in the known antigens of antigen to be checked or antibody and mark or the antibody competition binding immunoassay microwell plate, phosphate buffer stream is washed or was embathed 0.5-15 minute, to remove unreacted free antigen or antibody, with the different fluorescence result of observation of use instrument such as laser or CCD microarray scanner record.Also can mark antigen to be checked or antibody in the enforcement, and known antigen/or antibody respectively with different fluorescein-labelled realizations of color with it, both so-called double-tagging, or multiple labeling experiment.
5. bridging method: in above-mentioned immune microwell array plate (operation steps with aforementioned indirect method is an example), add 30-200ul sample to be checked (containing antibody), reacted 1-90 minute, damping fluid such as Tris or phosphate stream is washed or was embathed 0.5-15 minute, to remove unconjugated free antibodies in the sample, the anti-human immunoglobulin(HIg) antibody of animal that adds marks such as 30-100ul biotin again, reacted 1-180 minute, damping fluid stream is washed or is embathed 0.5-15 minute to remove unconjugated biotin labeled free antibodies, the affinity element (antibiotin) or the streptavidin that add marks such as 30-100ul enzyme or fluorescein then react, after stream is washed or is embathed, add substrate 50-120ul, lucifuge, the colour developing back adds stop buffer (as 2 equivalent sulfuric acid) 50-120 microlitre or stream is washed cessation reaction, observes each hole reaction result.In aforesaid method of operating 1-4, its label all can adopt the bridging method to carry out.Promptly all adopt biotin mark substance markers antigen, antibody, add the affinity element or the streptavidin of marks such as enzyme, collaurum, isotope, fluorescein then, improve the sensitivity that detects to use its amplification.
In the enforcement, in same detection system, usually need carry out the detection of (as detecting the antibody of hepatitis B surface antigen and anti-hepatitis B surface antigen) of corresponding antigen and antibody simultaneously, to judge patient's immunity and Infection Status and infectiousness.The simplest and the most direct solution is a direct method, but because of sensitivity low, and less employing.Indirect method, sandwich method or competition law sensitivity, because of the operator need not mark sample to be checked, thus rapid and simple, but single method is difficult to realize the corresponding antigen and the common detection of antibody.Unite use as two or more method, then, can't implement because of intersecting in conjunction with influencing interpretation of result (being the detection error that preamble is proposed).With embodiment I is example (see for details and hereinafter reach Figure 14).Adopt indirect method (survey antibody), need in micropore, to wrap, add anti-human immunoglobulin(HIg) antibody labeling thing and show reaction result by various hepatitis virus antigen; Detection of antigen then must adopt sandwich method, and the Kong Zhongxu coated antibody need add the specific antibody label of related antigen during display result.Be not difficult to find out that by Figure 14 in sandwich method, the antigen-specific antibodies label of adding also can combine with the envelope antigen in the indirect method.Because result's display packing identical (all using enzyme) as label, be because of having in the sample due to the antibody to be checked so can't distinguish color that micropore presents, or due to the antigen-specific antibodies label combines with intersecting of envelope antigen.As the thing that makes marks with fluorescein, though adopt double label method or multiple labeling method can solve the problem that this result can't distinguish, fluorescent marker reagent costs an arm and a leg, and interpretation of result must be used expensive exact instrument, so the cost height.As adopt competition law to survey antigen and antibody, then need add the label of antigen and antibody simultaneously.Be not difficult to find out that by Figure 14 labelled antigen in the label and antibody can interosculate, or with the bag quilt antigen and antibody, and corresponding antibody in the sample or the combination of antigen intersection, and influence the analysis of testing result, and being head it off, the present invention has adopted following scheme:
1. use the directly method of mark thing to be checked such as enzyme, fluorescein and biotin, preferred bridging method.Its advantage is only to need mark sample to be detected, but the marking operation complexity, influence factor is many, and operator's workload is bigger, and the time cycle is long.Because artifical influence factor is more, the result of different experiments chamber is difficult to comparison.
2. utilize an antigen molecule that the characteristic of a plurality of identical with different specific antigen determinants is arranged, in label, choose different peptide sections and the different antigenic determinants of the same antigen molecule of identification or the antibody pairing of different peptide sections of antigen molecule and use.For example: hepatitis B surface antigen (HBsAg) has a, d, y, w, five antigenic determinants of r, and a belongs to specific common antigenic determinant, and d and y, w and r are respectively two pairs of inferior determinants, can form 4 basic hypotype adw, adr, ayw, ayr.Bag by the time, optional anti-a antibody is encrusting substance and anti-d and y or w and r antibody labeling thing and is matched, and detects hepatitis B surface antigen with sandwich method; Select a antigen to do encrusting substance,, survey the antibody of people's anti-hepatitis B surface antigen with indirect method with anti-human immunoglobulin(HIg) label pairing.But some antigen molecule has only a kind of antigenic determinant (just having only a kind of serotype as hepatitis A HAV), can't solve with scheme 2 separately.Available such scheme 1 or following proposal 3 or 4 or 5 or 6 solve.
3. with uncovered simple and easy subregion microwell plate (see figure 4), respectively with antigen coated in antigen coated district, with antibody sandwich in the antibody sandwich district.When labelling thing,, add the antibody labeling thing of specific antigen to the antibody sandwich district according to the antiantibody of subregion to antigen coated district's adding mark; But steps such as sealing, embathe, add sample to be detected, adding substrate and stop buffer can be carried out jointly in the operation.
4. with private partition microwell plate (lid is arranged) (seeing Fig. 3,5,6), respectively antigen is distinguished at different bags with antibody sandwich, each bag is distinguished all complete and independent microfluidic circuit, press subregion through different inlet opening 6, add sample to be checked, stream wash, label thing (add the antiantibody of mark to antigen coated district, add the antibody labeling thing of antigentic specificity to the antibody sandwich district), substrate and stop buffer simultaneously respectively; Shown in Fig. 3,5,6, the antiantibody liquid of mark enters the Ag district through inlet opening 6, is flowed out by fluid hole 7; The antibody labeling thing of antigentic specificity through another inlet opening 6, enters the Ab district, is flowed out by fluid hole 7;
5. use dedicated detector board, with antigen and antibody subregion bag quilt, by the multi-path diverting valve 8 on the dedicated detector board, but between antigen coated district and antibody sandwich district, form the microfluidic circuit of the modulation of connection and individual flow, make the antiantibody label of adding enter antigen coated district along microfluidic circuit; The antibody labeling thing of specific antigen can only enter the antibody sandwich district, finally is implemented in the purpose that detects corresponding antigen-antibody in the same detection system simultaneously; Shown in Fig. 7-13.Antigen and antibody are coated on same Ag district and Ab district on the microwell plate by subregion, are provided with multi-path diverting valve 8 between two bags are by the district.Microwell plate covers except that leaving inlet opening 6 and fluid hole 7, also has sample well 14 on the multi-path diverting valve.Use the present invention that two kinds of operating modes are arranged, shown in Fig. 8,9,10,11:
Operating mode I (see figure 10): in sealing, when sample to be checked and substrate are washed, added to stream, multi-path diverting valve 8 is in the position of operating mode I, be that pipeline 11 communicates with pipeline 12, pipeline 11 is obstructed with pipeline 13, sample well 14 and pipeline 15 are obstructed with pipeline 16, this moment, liquid was through inlet opening 6, Ag district, pipeline 11, pipeline 12, Ab district, at last by fluid hole 7 outflows;
Operating mode II (seeing Figure 11): when adding label, multi-path diverting valve 8 is in the position of operating mode II, be that pipeline 11 communicates with pipeline 13, sample well 14 and pipeline 15 communicate with pipeline 16, but pipeline 11 is obstructed with pipeline 12, and the antibody labeling thing of the antiviral antigen of adding enters the Ab district through sample well 14 and pipeline 15 and pipeline 16, flow out by fluid hole 7 at last, therefore have no chance to meet or mix and can't directly combine with the hepatitis virus antigen that indirect method (Ag district) is wrapped quilt.And the antiantibody label that adds is through inlet opening 6, Ag district, pipeline 11, pipeline 13, at last by fluid hole 7 outflows; Hatch and flow after flush away removes unconjugated label finishing, multi-path diverting valve 8 can be in any position among operating mode I or the operating mode II, finishes whole subsequent operations.See Fig. 7,8,9,10,11,12,13.
6. scheme 2 can be respectively merges with scheme 3 or scheme 4, scheme 5 and uses.
In the such scheme, the preferred Chinese patent application of used microwell plate number is described microwell plate of 00209973.X and dedicated detector board of the present invention.
In actual detected is analyzed, also often must carry out quantitative test to the content of antigen and antibody, but the absolute light density value that is obtained between the different experiments chamber is different, and the direct optical density value in each hole conclusion that obviously can lead to errors relatively is particularly between different laboratories.The method of enzyme linked immunosorbent assay normally on same immune microwell plate, is reserved one group of immunity micropore, adds variable concentrations or dilution standard items, and the preparation standard curve with test result of samples to be checked in contrast, calculates its concentration or content.When checking matter concentration or content exceed sensing range, need dilution in advance usually.The present invention not only can carry out qualitative analysis, also can carry out quantitative test.Solution is: the multiple hole number that increases the parallel bag quilt of same antigen and/or antibody on microwell plate, when the homologue in the sample to be checked by orifice flow when crossing the multiple hole of each parallel bag quilt, by constantly combination, and constantly from specimen fluids, removed, therefore, the binding capacity in each multiple hole will reduce gradually according to the front and back order of each multiple hole arrangement.So, add label etc., the depth according to each multiple hole product color of front and back, the variation of the power that fluorescence intensity, activity, biological or chemical are luminous, can draw substrate concentration to be checked-reacting hole quantity and strong and weak change curve by Computer Analysis, contrast sample volume can obtain quantitative analysis results.
The present invention is described further below in conjunction with embodiment.
Scheme I: in the diagnosis of communicable disease, often need to detect antibody, survey antigen to understand its infectiousness to understand patient's anti-infectious immunity power.With the virus hepatitis is example.The virus that causes virus hepatitis has each Hepatitis virus such as first, second, third, fourth, penta, heptan, and every kind of virus can have a kind of or several serotypes, promptly produces not homospecific antibody.At first that virus hepatitis is various different specific antigens and corresponding antibody is kind of heterogeneity surplus in the of totally 20, by artificial or full-automatic microarray point sample preparation system, by antigen coated district and antibody sandwich district, be added in different the bag in the micropore of being distinguished, respectively so that detect antigen and antibody simultaneously.Using known biomacromolecule immobilization technology to carry out immobilization respectively microwell plate handles, has promptly obtained to wrap quilt the antigen of each Hepatitis virus and the immune microwell plate of antibody.Concrete operations step in force comprises:
1. the adsorbability of microwell plate is handled, and is slightly different with the difference of selected materials, and every kind of its concrete disposal route of material and condition all have multiple, this paper at this with " biomacromolecule immobilization technology and application, Jiang Zhonghua etc.,, chemical publishing house in 1998 " be cited as the main reference document.According to the present invention includes: the poly-D-lysine of all available 10-100 mcg/ml of the microwell plate of various materials is handled, and room temperature 20-360 minute or 4 ℃ of placements are spent the night.The polystyrene plastics microwell plate can be handled with isotope irradiation, the concentrated sulphuric acid, nitric acid dousing, or in polystyrene material, adds the macromolecular material that has aldehyde radical, amino, hydroxyl or sulfydryl isoreactivity group of proper proportion, to improve its adsorptive power.Or directly with discussing the microwell plate that the solid-phase matrix material sold is made.
2. antigen and antibody are dissolved in respectively in the damping fluid of pH7-10, the volume of antigen that every hole adds or antibody sandwich liquid can be from not enough picoliter to the number microlitre.The concentration of antigen, antibody is the 0.5-200 mcg/ml, add volume with the density of quantity, kind and the microwell plate micropore of the detection antigen of every microwell plate and/or antibody and the degree of depth and decide, the concentration of antigen, antibody is then successively decreased with the descending difference of its molecular weight.With artificial or full-automatic microarray point sample system, be added to respectively in the different micropores, only wrap by a kind of antigen or antibody in 1 hole.In the routine operation, every hole adds 2-5ul, or is as the criterion to fill it up with the hole.
3. phosphate buffer stream is washed 0.5-5 minute to remove uncured antigen or antibody.Nonspecific binding site residual in the micropore can seal with the damping fluid that contains bovine serum albumin(BSA), skim milk or Normal animal serum, confining liquid (phosphate buffer+3-10% sheep blood serum) 200ul adds through sample well, normal temperature was hatched 180 minutes, had promptly obtained to wrap quilt the antigen of each Hepatitis virus and the immune microwell plate of antibody thus;
4. when carrying out as with plastic microporous plates such as 96 existing holes or 384 holes, antigen and antibody can be dissolved in respectively in the carbonate buffer solution more than the pH9 and be directly used in the bag quilt.
Immune microwell plate through aforesaid operations obtains can directly use.But also airing, put preserve below 10 ℃ standby.
In the actual detected, can operate by following scheme respectively according to bag by the difference of mode:
1. subregion bag (being used direct method) not: in above-mentioned immune microwell plate, adding through the sample to be checked of marks such as fluorescein, collaurum or isotope (as serum, tissue fluid, secretion, excreta, tissue extract etc., contain antigen or antibody) 100ul, react after 60-120 minute, stream is washed to remove unconjugated part, then according to the difference of label, with fluorescence, collaurum deposition or the radioactivity in each holes of various different observation of use instrument such as scanner.Fluorescence, collaurum deposition or the both positive hole of radioactive micropore are arranged, represent to have in the tested sample corresponding with it antigen or antibody.As present positive reaction in wrapping by the micropore of hepatitis B and hepatitis C antigen (survey antibody), other each hole presents negative reaction, just show that this patient infection crosses hepatitis B and hepatitis C virus, and it is had certain immunity, and other various nothing infects history, does not also have immunity.As present positive reaction in wrapping by the micropore of hepatitis B and c-hepatitis antibody (survey antigen), other each hole presents negative reaction, shows that then this patient has infected hepatitis B and hepatitis C virus, may have certain infectiousness, and other various nothing infects or do not have infectiousness.The result of other immune microwell plate judges with identical or similar method.Can use the bridging method for improving sensitivity, be about to the sample biotin labeling, use the Avidin or the streptavidin display result of marks such as fluorescein, collaurum or isotope again.
2. the subregion bag is by (indirect method and sandwich method and with): in the immune microwell plate of hepatitis (simple and easy or private partition plate), add sample to be checked, reaction, unreacted free antigen of flush away and antibody, the anti-human immunoglobulin(HIg) of animal (or staphylococcal protein A of mark such as enzyme) (indirect method) that adds mark such as enzyme respectively in antigen coated district, the specific antibody that adds anti-each Hepatitis virus of marks such as enzyme in the antibody sandwich district reacts (sandwich method), stream is washed or is embathed the back and adds substrate (as the mixed liquor of chemical illuminating reagent or diaminobenzidine and hydrogen peroxide etc.), and luminous result is observed in the dark place.Or lucifuge, the colour developing back adds stop buffer (as 2N equivalent sulfuric acid), or eccysis substrate cessation reaction, observes the dye-forming reaction in each hole, and the hole that dye-forming reaction is arranged is positive hole, and representing has corresponding antigen or antibody in the tested sample.
3. use dedicated detector board: the multi-path diverting valve is transferred to operating mode I, and sample to be checked adds in the hepatitis immunity microwell plate through liquid filling hole, and reaction, stream flush away remove unreacted free antigen and antibody.The multi-path diverting valve is transferred to operating mode II, and antigen coated district adds the anti-human immunoglobulin(HIg) of animal (or staphylococcal protein A of mark such as enzyme) (indirect method) of mark such as enzymes through liquid filling hole 6; (sandwich method) reacts through the antibody that sample well 14 adds anti-each Hepatitis virus of marks such as enzyme in the antibody sandwich district, stream is washed back multi-path diverting valve and is replied operating mode I, add substrate (as the mixed liquor of chemical illuminating reagent or diaminobenzidine and hydrogen peroxide), luminous result is observed in the dark place.Or lucifuge, the colour developing back adds stop buffer (as 2N equivalent sulfuric acid), or eccysis substrate cessation reaction, observes the dye-forming reaction in each hole, and the both positive hole, hole of dye-forming reaction is arranged, and representing has the antibody corresponding with this antigen in the tested sample.
Scheme II: with human major histocompatibility antigen (HLA) is example.I class antigen (HLA-A, HLA-B, HLA-C genetic locus), its A site has 74 kinds, kind surplus the B site has 80, kind surplus the C site has 10; II class antigen DR site has 53 kinds, and DQ has 9 kinds, and DP has 6 kinds, and D antigen (not determining type) has 26 kinds; Surplus III class antigen has 10 more than the kind, amount to about 300 surplus kind.The various combination of these antigens, constitute and produced the individual difference of major histocompatibility antigen, playing a decisive role in the graft-rejection in the human organ transplants, is to carry out histoorgan before the organ transplant to join type, selects donor and the required main reference foundation of acceptor.In the enforcement, at first each site is amounted to the different specific antibody of kind surplus in the of nearly 300,, be added in respectively in the different micropores with artificial or full-automatic microarray point sample preparation system, carry out immobilization and handle, promptly obtain to can be used for detecting the immune microwell plate of human major histocompatibility antigen (HLA).
In above-mentioned immune microwell plate, add with biotin labeled sample to be checked (histocyte homogenate or histocyte extract etc., containing antigen) 30-500ul reacts, and stream is washed to remove unconjugated part, adds Avidin or streptavidin with marks such as fluoresceins.Observe the fluorescence intensity in each hole with scanner.Corresponding with it histocompatibility antigen is represented to have in the tested sample in the both positive hole of micropore that fluorescence is arranged.
Scheme III: with different biological various growth factors and acceptors is example, at present known human growth factor have approximately 65 kinds, associated receptor have 100 approximately surplus kind, the quantity number of growth factor that other animal is known and acceptor is slightly different.At first with the different specific antibody of various growth factors and acceptor, press the difference of species, with artificial or full-automatic microarray point sample preparation system, be added in respectively in the different micropores, carry out immobilization and handle, promptly obtain to can be used for detecting human or the various growth factors of various different plant species and the immune microwell plate of acceptor respectively.Add then and react with biotin labeled sample to be checked (serum, cells in vitro nutrient solution, histocyte homogenate or histocyte extract etc. contain antigen), stream is washed to remove unconjugated part, adds Avidin or streptavidin with marks such as enzymes.The stream flush away removes unconjugated label, adds luminous substrate, with the luminous intensity in each holes of observation such as scanner.The both positive hole of luminous micropore is arranged, represent to have in the tested sample corresponding with it cell factor or acceptor.As the bag by the time each composition all wrap by the multiple hole in 4-10 hole, according to the luminous power in each multiple hole, front and back, can carry out quantitative test.
Scheme IV: the diagnostic detection with tumour is an example, and generation development known and tumour at present and relevant gene and protein product thereof such as treatment and prognosis have tens of classes, several thousand kinds.At first the specific antibody of various protein moleculars that will be relevant with various tumours with artificial or full-automatic microarray point sample preparation system, is added in respectively in the different micropores, carries out immobilization and handles, i.e. acquisition can be used for the immune microwell plate of the diagnostic detection of tumour.Add then with biotin labeled sample to be checked (serum, cells in vitro nutrient solution, biopsy tumor tissue cell homogenates or histocyte extract etc., contain antigen) react, stream is washed to remove unconjugated part, adds Avidin or streptavidin with marks such as enzymes.The stream flush away removes unconjugated label and adds luminous substrate, with the luminous intensity in each holes of observation such as scanner.The both positive hole of luminous micropore is arranged, represent to have in the tested sample corresponding with it tumor associated antigen.Some tumor associated antigen also can occur in normal or other nonneoplastic tissue or during disease, but aspect amount difference to some extent, therefore often need do quantitative test.As bag by the time each composition all wrap by the multiple hole in 4-10 hole, according to answering the luminous power in hole before and after each, can carry out quantitative test.
Plan V: monoclonal antibody is a most frequently used biological reagent in the various biomedical checks, in the Study of Monoclonal Antibodies process, the somatotype of the monoclonal antibody progressive type, hypotype, class and the subclass that produce is identified it is a very important job.Normally adopt immune double diffusion method.And adopting double antibody sandwich method of the present invention, association colloid gold or enzyme labeling technology are then simple.Be about to the antiantibody of anti-inhomogeneity, subclass, type and hypotype antibody, the difference of pressing species is with artificial or full-automatic microarray point sample preparation system, be added in respectively in the different micropores, carry out immobilization and handle, promptly obtain can be used for the immune microwell plate that antibody typing is identified.
Identify in the immune microwell plate at the mouse antibodies somatotype, add 30-50ul mouse monoclonal antibody Hybridoma Cell Culture liquid and (contain mouse monoclonal antibody, as antigen) react, stream is washed to remove unreacted free antigen, adding the rabbit of colloid gold label or the antibody (species specificity) of goat-anti mouse immuning ball protein again reacts, the antibody that perhaps adds marks such as enzyme reacts, and then (with the luminescence method is example to add the substrate of enzyme, as chemical illuminating reagents such as luminols, belong to the merchant and sell reagent) react, the luminescence-producing reaction in each hole is observed in lucifuge or darkroom.Positive hole had both represented that the antibody classification corresponding with this hole antibody arranged in the tested sample.
The present invention is a kind of immunology diagnosis and detection method one immune microwell plate detection technique of antigen or antibody of novel concept.It is in a certain order a kind of or form, with several, tens, even the antigen of several thousand or higher quantity kind and/or antibody add in the hole of high density microwell plate, high density row is listed in and makes immune microwell plate together, react with patient's sample to be checked, can once obtain the biomedical detection technique of all each hole known antigens in the immune microwell plate and/or detection of antibodies result's high flux biological information.It once can to several, tens kinds, even paathogenic factor such as the antigen of several thousand kinds or higher quantity and/or antibody is carried out parallel check and analysis simultaneously.Its most outstanding advantage is, only needs a small amount of patient specimen, can obtain the information of several even thousands of kinds of diseases related by one-time detection.So immune microwell plate detection technique of the present invention have contain much information, in time, quick, easy and simple to handle and be easy to realize advantage such as full-automatic operation, production cost be low.In addition, this method normally concentrates on a number millimeters thick with tens kinds even thousands of kinds of test items, carry out on the microwell plate that the area less than is 20 square centimeters, significantly reduced the consumption of various experiment consumptive materials and test sample, therefore the handling problem that can reduce or partly solve infectiousness dirt and harmful chemical.Help protecting environment, reduce pollution environment.
Claims (10)
1. high information quantity immunologic detection method, comprise the bag quilt, mark, add sample to be checked, stream is washed or is embathed, add label, stream is washed or is embathed, add substrate, display result, steps such as analysis, it is characterized in that, its bag is that two or more not homospecific antibody and/or antigen are coated on respectively in the different micropores of same microwell plate or on other solid-phase matrix by step, so that detect multiple different antigen and antibody simultaneously, used microwell plate is the microwell plate of band microflute, microfluidic circuit by set on the microwell plate makes sealing, rinsing and sample, the interpolation of label and substrate reagent waits each step all can finish by single job.
2. a kind of high information quantity immunologic detection method according to claim 1, it is characterized in that bag is to adopt biomacromolecule immobilization treatment technology, with antigen, antibody respectively immobilization by glass, or silicon, or metal, or finish on the microwell plate made of solid-phase matrix materials such as macromolecular organic compound such as plastics, its concrete operations step is:
1) microwell plate is improved the conventional pre-service of its absorption property;
2) add encrusting substance: be dissolved in two or more different specific antibodies and/or antigen in the damping fluid respectively, with artificial or full-automatic microarray point sample preparation system, be added to respectively in the different micropores, 1 hole is only added or is wrapped by a kind of antigen or antibody, on same microwell plate, can be coated with antigen and also can be coated with antibody simultaneously, bag can be had a plurality of by the hole of same antigen or antibody, its arrangement can be continuous, also can be discontinuous; If when detecting corresponding antigen and antibody simultaneously, can adopt subregion bag quilt;
3) damping fluid stream is washed or is embathed, to remove not immobilised antigen or antibody;
4) add confining liquid, normal temperature was hatched 20-360 minute, with the sealing nonspecific binding site; But the microwell plate of lid is being arranged, then after sealing the formation microfluidic circuit, undertaken by microfluidic circuit;
If adopt existing 96 holes or 384 holes etc. plastic microporous is firm and hard and execute, antigen and antibody can be dissolved in respectively in the carbonate buffer solution more than the pH8.5 and be directly used in the bag quilt;
3. a kind of high information quantity immunologic detection method according to claim 1 is characterized in that: in same detection system, when carrying out the common detection of corresponding antigen and antibody simultaneously, can adopt following any one scheme to avoid detecting error:
1) with antigen and antibody sandwich on the high density microwell plate, with the direct method of mark thing to be checked such as enzyme, fluorescein and biotin;
2) utilize an antigen molecule that the characteristic of a plurality of identical with different specific antigen determinants is arranged, in label, choose different peptide sections and the different antigenic determinants of the same antigen molecule of identification or the antibody pairing of different peptide sections of antigen molecule and use;
3) when used microwell plate is simple and easy subregion microwell plate, respectively with antigen coated in antigen coated district, with antibody sandwich in the antibody sandwich district; When labelling thing,, add the antibody labeling thing of specific antigen to the antibody sandwich district according to the antiantibody of subregion to antigen coated district's adding mark; But seal, embathe, add sample to be detected in the operation, two bags of operation steps such as adding substrate and stop buffer are distinguished jointly carries out.
4) when used microwell plate is the private partition microwell plate, respectively with antigen coated in antigen coated district, in the antibody sandwich district, each bag is distinguished complete and independent microfluidic circuit is all arranged with antibody sandwich, adds sample to be checked, stream wash, label thing, substrate and stop buffer etc. respectively by subregion; But when labelling thing,, add the antibody labeling thing of specific antigen to the antibody sandwich district to the antiantibody of antigen coated district's adding mark;
5) when used microwell plate is dedicated detector board,,, can be communicated with and the microfluidic circuit of can be separate mobile modulation but between antigen coated district and antibody sandwich district, form by the multi-path diverting valve on the dedicated detector board with antigen and antibody subregion bag quilt; When adding label, make the antiantibody label of adding enter antigen coated district along microfluidic circuit; The antibody labeling thing of specific antigen can only enter the antibody sandwich district;
6) scheme 2 can be respectively merges with scheme 3 or scheme 4, scheme 5 and uses.
4. according to claim 1 or 2 or 3 described a kind of high information quantity immunologic detection methods, it is characterized in that: when employed micropore strip microflute, it is qualitative to adopt following method to carry out, quantitative test: because of on same microwell plate, the bag of same antigen or antibody can be had two or more by hole number, when the homologue in the sample to be checked on the trough of belt microwell plate, when crossing the micropore of each duplicate packages quilt by orifice flow, by constantly combination, and constantly from specimen fluids, removed, therefore, the binding capacity in each multiple hole, will be according to the front and back order of each repeating hole arrangement, reduce gradually,, carry out qualitative analysis according to having or not of each hole reaction; When carrying out quantitative test, increase the micropore quantity of duplicate packages quilt, according to the variation of the depth of each repeating hole product color of front and back, fluorescence intensity, activity, the luminous power of biological or chemical with have or not, can draw substrate concentration to be checked-reacting hole quantity and strong and weak change curve by Computer Analysis, contrast sample volume, quantitative analysis results be can obtain, and dilution standard thing or encrusting substance in advance do not needed.
5. a kind of immunologic detection method according to claim 1, it is characterized in that, used microwell plate is the high density microwell plate, every square centimeter has 4 holes or more than 4 holes, hole depth 0-2 millimeter, have between the hole or do not have microflute to link to each other, microwell plate can have lid or uncovered, is respectively equipped with inlet opening (6), the fluid hole (7) that communicates with microflute (5) on micropore plate body (1) or microwell plate lid (2).
6. dedicated detector board that is used for the described immunologic detection method of claim 1, comprise plate body (1), microwell plate lid (2), micropore (4), the microflute (5) that links to each other with micropore (4), it is characterized in that, on microwell plate lid (2), be respectively equipped with the inlet opening (6) that communicates with microflute (5), fluid hole (7) and hyperchannel diverting valve (8), the valve opening (17) of this hyperchannel diverting valve (8) circumferentially is divided into two districts by spool (18) edge: A district and B district, spool (18) center has a bottom to have the sample well of pipeline (15) (14), and the sidewall of valve opening (17) is provided with the pipeline (11) that communicates with antigen coated district (Ag district), the pipeline (12) and (16) that communicate with antibody sandwich district (Ab district), the pipeline (13) that communicates with fluid hole (7).
7. immunologic detection method according to claim 5 is characterized in that, used microwell plate can be added with sealing gasket (3) between plate body (1) and microwell plate lid (2).
8. dedicated detector board according to claim 6 is characterized in that, can be added with sealing gasket (3) between plate body (1) and microwell plate lid (2).
9. according to claim 6 or 8 described dedicated detector board, it is characterized in that the opening of inlet opening (6), fluid hole (7) and sample well (14) also can be on plate body (1).
10. immunologic detection method according to claim 7 is characterized in that, the opening of the inlet opening of used microwell plate (6), fluid hole (7) and sample well (14) also can be on plate body (1).
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| CN102818897A (en) * | 2012-07-31 | 2012-12-12 | 白仲虎 | Detection device |
| WO2021219819A1 (en) * | 2020-04-29 | 2021-11-04 | Lumiradx Uk Ltd | Antibody/antigen detection |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100414296C (en) * | 2002-06-14 | 2008-08-27 | 赵翀 | Embedded high-pass three-dimensional biological detecting technique and agent box |
| CN1313622C (en) * | 2002-08-02 | 2007-05-02 | 赵翀 | High-flux cell biological chip testing technology and reagent case |
| CN101526520B (en) * | 2008-03-07 | 2013-04-03 | 国家纳米科学中心 | Method and device for biological sample detection |
| WO2019068804A1 (en) * | 2017-10-04 | 2019-04-11 | Unisensor | Device for the on-demand optical reading of a removable solid carrier for detecting and/or quantifying analytes present in a sample |
| CN108375572A (en) * | 2018-02-26 | 2018-08-07 | 深圳市生强科技有限公司 | Detection method and device based on chemoluminescence method |
| CN109870582B (en) * | 2019-02-27 | 2020-07-10 | 华中科技大学 | Multi-target magnetic immunochemistry luminescence microfluidic chip detection platform and method |
| CN116679049A (en) * | 2023-02-27 | 2023-09-01 | 科赫生物科技（北京）有限公司 | Immunodetection method |
-
2000
- 2000-07-14 CN CNB001207989A patent/CN1138981C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102818897A (en) * | 2012-07-31 | 2012-12-12 | 白仲虎 | Detection device |
| WO2021219819A1 (en) * | 2020-04-29 | 2021-11-04 | Lumiradx Uk Ltd | Antibody/antigen detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1320820A (en) | 2001-11-07 |
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