CN1908663B - Substrate regional band resolution chemical luminescent multi-component immunity analytical method and detection system thereof - Google Patents
Substrate regional band resolution chemical luminescent multi-component immunity analytical method and detection system thereof Download PDFInfo
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Abstract
The multi-constituent immunity analysis and detection system comprises: a solution transmission system with a multi-channel peristaltic pump (6) and a connection pipe (7) and a multi-position selection valve (8), an immunity reactor, a chemiluminescence detector, and a computer, wherein the analysis method comprises: fixing the packed antibody in the reactor, adding sample and enzyme-mark antibody; marking with HRP and alkaline phosphates to form two types of sandwich composites; clearing, crossing in turns the HRP chemiluminescence substrate area, cleaning buffer liquid area and the alkaline phosphates chemiluminescence substrate area. This invention has fast speed and high sensitivity for wide application.
Description
One, technical field
The present invention is the substrate regional band resolution chemical luminescent technology, the multi-component immunity analytical method of immune analysis method, especially chemiluminescence and Flow Analysis Technique coupling when relating to various ingredients in the same sample; Present technique also relates to the detection system that is used for the two-component chemilumineschent immunoassay.
Two, background technology
Immunoassay has obtained increasingly extensive application as a kind of high selectivity and high-sensitivity analysis method in fields such as clinical diagnosis, environmental monitoring, food securities.In practical application area, often need to measure the content of various ingredients in the complex sample, as in diagnosing tumor, the Conjoint Analysis of kinds of tumors mark provides strong foundation for diagnosis.
At present, in order to realize the Conjoint Analysis of various ingredients in the complex sample, the patterns that adopt repeatedly parallel single component to analyze are only analyzed wherein a kind of component more promptly at every turn, and repeatedly execution analysis process finally obtains the content of all components.That this pattern expends time in is long, labor capacity is big, analysis cost is high.In order to overcome these shortcomings, some multi-component immunity analytical methods have appearred in recent years, mainly comprise Array Method and multiple labeling method.Array Method is based on the principle that the immune response zone separates, and promptly detects different components at different array regions, but this method apparatus is comparatively complicated, and needs expensive array detector, as multi-channel electrochemical workstation and CCD.At present domestic existing producer releases the multicomponent chemical luminescence immunoassay instrument that uses the ccd array detecting device, but because instrument cost is too high, its product is difficult to obtain promote.The multiple labeling ratio juris is to come the immunoreagent of mark different component with different labels, obtains each components contents by the signal that detects different labels, and its detection method comprises electrochemical process, fluorescence method, photometry and radioactive method.In electrochemical assay, how to differentiate different component with operating potential; In fluorescence and photometry, usually to differentiate wavelength and die-away time different component.At present, the multiple labeling method still can not be used chemiluminescence detection, because chemiluminescence is as a kind of detection technique of not considering wavelength, can't differentiate the signal that the label of different component correspondence sends by wavelength.And in the multiple labeling method, for the different labels of different component, its optimum analysis condition often has very big difference, simply unites and uses multiple label, often faces the incompatible problem of various analysis of markers conditions, makes analytical effect have a greatly reduced quality.
Current, the overall process of common immunoassays technology needs repeatedly application of sample, incubation, washes plate and reaction, carries out instrument detecting at last.Operating process is comparatively loaded down with trivial details, and labor capacity is big, and required time is many more than 2 hours, is not suitable for the high flux express-analysis.U.S. Roche Diagnostics GmbH company develops commercial Elecsys automatic lmunoassays analyzer in conjunction with flow analysis, immunomagnetic beads and electroluminescent technology, has realized robotization tachysynthesis analysis, has obtained application clinically.But this instrument and matched reagent box price are very expensive, and the mechanism of financial resources deficiency is difficult to burden.And this instrument also is the single component analytical model, still needs repeatedly replicate determination for complex sample.
Chemiluminescence analysis is fast-developing in recent years analytical technology, and its instrument is cheap, and is easy and simple to handle, environmental friendliness, and be one of at present the sensitiveest analytical technology, be particularly suitable for the detection of trace materials.Flow Analysis Technique has favorable reproducibility, the automaticity height, and advantage such as analysis speed is fast is to realize one of the most effective means of high throughput analysis.The coupling of these two kinds of technology and immunoassay is an immunoassay field research focus in recent years, has obtained the achievement that attracts people's attention.
Three, summary of the invention
The objective of the invention is:,,, provide a kind of based on substrate regional band resolution technology and the high-throughout multi-component immunity analytical method of robotization with chemiluminescence detection in conjunction with Flow Analysis Technique based on the multiple labeling method; Order of the present invention another also be provide one the cover robotization flow type bi-component substrate regional band resolution chemical luminescent immunoassay detection system.
The objective of the invention is to realize by following technical scheme:
A kind of flow type substrate regional band resolution multicomponent chemical luminescence immunoassay detection system is characterized in that this system comprises solution transmission system, immune reactor, chemiluminescence detector and computing machine; The solution transmission system is made up of multi-channel peristaltic pump (6), connecting pipe (7) and multidigit selection valve (8), five connecting pipes (7) are connected respectively to the first valve position mouth, the second valve position mouth, the 3rd valve position mouth, the 4th valve position mouth, the 5th valve position mouth of multidigit selection valve (8) through multi-channel peristaltic pump (6), sample S is failed in the first valve position oral instructions, the defeated regeneration of the 5th valve position oral instructions damping fluid RB, dcq buffer liquid WB is failed in the 4th valve position oral instructions, and the 3rd valve position mouth, the second valve position mouth transmit two chemical luminous substrate S respectively
1And S
2The outlet at multidigit selection valve (8) center is connected with the inlet of immune reactor by connecting pipe (7), the outlet of immune reactor is discharged waste liquid by connecting pipe (7), immune reactor places on the chemiluminescence detector, and the solution transmission system all is connected with computing machine with chemiluminescence detector.
The rotating speed and the flow rate of liquid of above-mentioned multi-channel peristaltic pump (6) are adjustable.
The internal diameter of above-mentioned connecting pipe (7) can be made with the teflon of 0.8mm.
Above-mentioned multidigit selection valve (8) can realize the switching of different streams by rotation multidigit selection valve, and the first valve position mouth on the multidigit selection valve, the second valve position mouth, the 3rd valve position mouth, the 4th valve position mouth, the 5th valve position mouth communicate with the outlet at center respectively.
The UltraBind film that above-mentioned immune reactor is activated by the aldehyde radical of multiple antibody for bag.
Above-mentioned regeneration damping fluid is 0.1M amino acid/hydrochloride buffer, pH2.0.
Above-mentioned dcq buffer liquid is the 0.01M phosphate buffer, and pH7.4 contains 0.05% Tween-20.
The reflective multi-component immunity analytical method of a kind of substrate regional band resolution chemistry, its analytical procedure is as follows:
(1) elder generation switches to the first valve position mouth with the valve position mouth of multidigit selection valve (8), to contain the testing sample of antigen 1 and antigen 2 and carry out the tracer antibody 1 and the antibody 2 of mark respectively with horseradish peroxidase and alkaline phosphatase by connecting pipe (7), feed immune reactor, the room temperature incubation, the enzyme-labeled immunity sandwich complex of two kinds of components of formation in immune reactor;
(2) then the valve position mouth is switched to the 4th valve position mouth, feed not binding immunoassay reagent of 1.0mL/min dcq buffer liquid WB flush away, after rinsing well, the valve position mouth is switched to the 3rd valve position mouth, feed substrate regional band S by connecting pipe (7) by connecting pipe (7)
1, the horseradish peroxidase enzyme catalytic substrate regional band produces strong chemiluminescence, and the record luminous signal obtains a kind of component concentrations;
(3) the valve position mouth is switched to the 4th valve position mouth, feed dcq buffer liquid WB, form buffering liquid zone band by connecting pipe (7);
(4) the valve position mouth is switched to the second valve position mouth, feed substrate regional band S2 again by connecting pipe (7), produce strong luminescence after this district's band is met alkaline phosphatase, the record luminous signal can obtain another component concentrations;
(5) after mensuration is finished, successively the valve position mouth is switched to the 5th valve position mouth and the 4th valve position mouth, successively feed regeneration damping fluid RB and two circulations of dcq buffer liquid WB, can make immune sandwich complex dissociate, immune reactor regeneration is to enter the next circulation of measuring.
The formation of flow type multicomponent chemical luminescence immunoassay detection system:
The structure of this detection system as shown in Figure 1, comprise four parts altogether: first part is the solution transmission system, this transmission system is served as mass transfer power with a multi-channel peristaltic pump (6), serve as mass transfer connecting pipe (7) with some polyfluortetraethylene pipes, with a multidigit selection valve (8) control liquid flow path direction, different solutions is injected immune reactor; Second part is immune reactor, wherein fixed the coated antibody of various ingredients correspondence; The 3rd part is chemiluminescence detector, is used to gather luminous signal; The 4th part is computer control system.
The principle of work of this detection system:
This detection system can be measured multiple material to traditional multiple labeling technology and the coupling of substrate regional band resolution technology in an analysis process.Immune response is traditional double antibody sandwich method.In immune reactor, fix the pairing coated antibody of multiple determinand (antigen).As shown in Figure 2, be example with two kinds of determined antigens, in reactor, fix the antibody of two kinds of determinands, tracer antibody carries out mark with horseradish peroxidase and alkaline phosphatase respectively; At first the complex sample that contains these two kinds of antigens is mixed with two kinds of enzyme labelled antibodies and feed immune reactor, in immune reactor, form the enzyme-labeled immunity sandwich complex of two kinds of components; Feed not binding immunoassay reagent of dcq buffer liquid flush away then; After rinsing well, feed earlier substrate regional band S1 (luminol, hydrogen peroxide with to the iodophenol mixed solution), this district's band is produced strong chemiluminescence by horseradish peroxidase enzyme catalytic, writes down luminous signal, obtains a kind of components contents; Behind dcq buffer liquid zone band WB cleaning immune reactor, feed substrate regional band S2 (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2 '-(5 '-chloro) tricyclo[3.3.1.1 again
3,7] decan}-4-yl) phenylphosphate, CSPD), the generation extensive chemical was luminous after this district's band was met alkaline phosphatase, and the record luminous signal can obtain another component concentration.
After mensuration is finished, feed regeneration damping fluid and two circulations of dcq buffer liquid successively, can make immune sandwich complex dissociate, immune reactor regeneration is to enter the next circulation of measuring.
In the analytic process, all solution enter analytic system by the connecting pipe on peristaltic pump transmission, realize the switching of different streams by rotating the multidigit selection valve, and overall process is carried out sequencing by computing machine and controlled automatically.
The measuring principle of this detection system:
When having two kinds of antigenic substances intending mensuration in the testing sample, corresponding immobilization coated antibody on these two kinds of antigenic substances and the reactor, and two kinds of enzyme labelled antibodies that add form two kinds of immune complexs of horseradish peroxidase and alkali phosphatase enzyme mark respectively.When feeding successively with the substrate regional band resolution technology based on luminol-hydrogen peroxide-to the horseradish peroxidase chemical luminous substrate of iodophenol and based on the alkaline phosphatase chemical luminous substrate of CSPD, can obtain the luminous signal of two kinds of components successively, thereby converse two kinds of component concentrations.
The present invention compared with prior art has following characteristics:
The present invention has proposed substrate regional band resolution multi-component immunity analytical system in conjunction with Flow Analysis Technique and chemiluminescence detection, detects multiple material in a flow process.With respect to other multi-component immunity analytical methods, have following characteristics:
(1) simple to operate, the total analysis process is all finished in current system, carries out sequencing with computing machine and controls automatically, and manual operations is few, need not skilled operating personnel.
(2) lack analysis time, overall process comprises that application of sample, incubation, flushing, detection and regeneration only need 35 minutes, are not only at present one of multi-component immunity analytical method the most fast, also greatly faster than common single component immune analysis method.
(3) instrument and equipment is simple, and is with low cost, need not the array detector of costliness commonly used in the multi-component immunity analytical, and whole analytic system is made up of peristaltic pump, multidigit selection valve, polyfluortetraethylene pipe and the chemiluminescence detector of low value.
(4) immune reactor can carry out the repeated regeneration use by feeding the regeneration damping fluid, compares with the immune analysis method of routine, has saved expensive coated antibody greatly, has further reduced analysis cost.
(5) because its detecting pattern is extremely sensitive enzymatic chemiluminescence reaction, and this method can be measured the sample of extremely low concentration, satisfy most analyze demands.
(6) owing to adopted the Flow Analysis Technique of robotization, feasible operation technique by operating personnel greatly reduces with the different individual differences that cause of custom, and the reappearance of method greatly improves than traditional manual manipulation method, helps formulating relevant criterion.
Four, description of drawings
The structural representation of Fig. 1 flow type two-component chemilumineschent immunoassay detection system
1,2,3,4,5 is multidigit selection valve valve position inlet, the 6-multi-channel peristaltic pump; The 7-connecting pipe; 8-multidigit selection valve; S: sample; RB: regeneration damping fluid; WB: dcq buffer liquid; S1: substrate 1;
S2: substrate 2
Fig. 2 flow type two-component chemilumineschent immunoassay principle schematic
Five, embodiment
Embodiment 11 pair of flow type two-component chemilumineschent immunoassay detection system in conjunction with the accompanying drawings is described further:
Flow type substrate regional band resolution multicomponent chemical luminescence immunoassay detection system comprises solution transmission system, immune reactor, chemiluminescence detector and computing machine.The solution transmission system is that the connecting pipe 7 and the multidigit selection valve 8 of the teflon of 0.8mm formed by multi-channel peristaltic pump 6, internal diameter, five connecting pipes 7 are connected respectively to five valve position mouths 1,2,4,5 of multidigit selection valve 8 through multi-channel peristaltic pump 6, inlet 1 transmission sample S, inlet 5 transmission regeneration damping fluid RB, inlet 4 transmission dcq buffer liquid WB, inlet 3,2 transmits two chemical luminous substrate S respectively
1And S
2One end of every connecting pipe 7 is connected with the outlet at selection valve 8 centers, outlet is connected with the inlet of immune reactor, another root connecting pipe 7 is derived the waste liquid of immune reactor, immune reactor places on the chemiluminescence detector, and the solution transmission system all is connected with computing machine with chemiluminescence detector.
The concrete analysis process is as shown in table 1, and all analytical procedures are carried out sequencing by computing machine and controlled automatically.Regeneration damping fluid RB is 0.1M amino acid/hydrochloride buffer, and pH 2.0; Dcq buffer liquid WB is the 0.01M phosphate buffer that contains 0.05% Tween-20, and pH 7.4; The UltraBind film that immune reactor is activated by the aldehyde radical of multiple antibody for bag; S
1For 0.5mmol/1 luminol, 3mmol/1 hydrogen peroxide and 0.5mmol/l to iodophenol mixed solution and S
2Be CSPD solution.
Embodiment 3
With two kinds of important tumor markers: carcinomebryonic antigen (CEA) is an example with cancer antigen 125 (CA 125), and the application of this flow-type two-component chemilumineschent immunoassay system is described.
Immune reactor is the UltraBind film of aldehyde radical activation, and bag, is fixed in the flow cell of 50 microlitres with bovine serum albumin(BSA) sealing residual activity site by mouse monoclonal anti CEA and mouse monoclonal anti-CA 125.The tracer antibody of CEA is the mouse monoclonal anti CEA of alkali phosphatase enzyme mark, and the tracer antibody of CA 125 is the mouse monoclonal anti-CA 125 of horseradish peroxidase-labeled.
Flow process as shown in table 1 switches to position 1 to valve, feeds the potpourri of 25 microlitre samples, 12.5 microlitre CEA tracer antibodies and 12.5 microlitre CA, 125 tracer antibodies in the flow cell of having fixed immune reactor, static incubation 20 minutes.Then valve position is switched to 4, flushing immune reactor 1.5 minutes.Clean the back valve position is switched to 3, inject the horseradish peroxidase substrate, arrhea, in the time of the 4th minute, gather CA 125 corresponding luminous signals.Valve position switchback 4, wash half a minute again.Then valve position is switched to 2, inject alkaline phosphatase substrate, arrheaed 4 minutes, detect the corresponding chemiluminescence signal of CEA.After detection is finished, valve is switched at 5 and 4 interdigits, feed regeneration damping fluid and dcq buffer liquid repeatedly, whole analytic processes are finished in two circulations of regeneration immune reactor in 3.5 minutes.By the signal measuring of a series of concentration samples, obtain the typical curve of CEA and CA 125.Utilize calibration curve method again, obtain the concentration of these two kinds of tumor markerses in the clinical blood sample.
Claims (7)
1. a flow type substrate regional band resolution multicomponent chemical luminescence immunoassay detection system is characterized in that this system comprises solution transmission system, immune reactor, chemiluminescence detector and computing machine; Wherein the solution transmission system is made up of multi-channel peristaltic pump (6), connecting pipe (7) and multidigit selection valve (8), five connecting pipes (7) are connected respectively to the first valve position mouth, the second valve position mouth, the 3rd valve position mouth, the 4th valve position mouth, the 5th valve position mouth of multidigit selection valve (8) through multi-channel peristaltic pump (6), sample S is failed in the first valve position oral instructions, the defeated regeneration of the 5th valve position oral instructions damping fluid RB, dcq buffer liquid WB is failed in the 4th valve position oral instructions, and the 3rd valve position mouth, the second valve position mouth transmit two chemical luminous substrate S respectively
1And S
2The outlet at selection valve (8) center is connected with the inlet of immune reactor by connecting pipe (7), the outlet of immune reactor is discharged waste liquid by connecting pipe (7), immune reactor places on the chemiluminescence detector, and the solution transmission system all is connected with computing machine with chemiluminescence detector.
2. detection system according to claim 1 is characterized in that the rotating speed of described multi-channel peristaltic pump (6) and flow rate of liquid are adjustable.
3. detection system according to claim 1, it is characterized in that described multidigit selection valve (8) can realize the switching of different streams by rotation multidigit selection valve, the first valve position mouth on the multidigit selection valve, the second valve position mouth, the 3rd valve position mouth, the 4th valve position mouth, the 5th valve position mouth communicate with the outlet at center respectively.
4. detection system according to claim 1 is characterized in that the UltraBind film that described immune reactor is activated by the aldehyde radical of multiple antibody for bag.
5. detection system according to claim 1 is characterized in that described regeneration damping fluid is 0.1M amino acid/hydrochloride buffer, and pH 2.0.
6. detection system according to claim 1 is characterized in that described dcq buffer liquid is the 0.01M phosphate buffer, and pH 7.4, contain 0.05% Tween-20.
7. the reflective multi-component immunity analytical method of substrate regional band resolution chemistry, its analytical procedure is as follows:
(1) at first the valve position mouth of multidigit selection valve (8) is switched to the first valve position mouth, to contain the testing sample of antigen 1 and antigen 2 and carry out the tracer antibody 1 and the antibody 2 of mark respectively with horseradish peroxidase and alkaline phosphatase by connecting pipe (7), feed immune reactor, the room temperature incubation, the enzyme-labeled immunity sandwich complex of two kinds of components of formation in immune reactor;
(2) then the valve position mouth is switched to the 4th valve position mouth, feed not binding immunoassay reagent of dcq buffer liquid WB flush away, after rinsing well, the valve position mouth is switched to the 3rd valve position mouth, feed substrate regional band S by connecting pipe (7) by connecting pipe (7)
1, the horseradish peroxidase enzyme catalytic substrate regional band produces strong chemiluminescence, and the record luminous signal obtains a kind of component concentrations;
(3) the valve position mouth is switched to the 4th valve position mouth, feed dcq buffer liquid WB, form buffering liquid zone band by connecting pipe (7);
(4) the valve position mouth is switched to the second valve position mouth, feed substrate regional band S2 again by connecting pipe (7), produce strong luminescence after this district's band is met alkaline phosphatase, the record luminous signal can obtain another component concentrations;
(5) after mensuration is finished, successively the valve position mouth is switched to the 5th valve position mouth and the 4th valve position mouth, successively feed regeneration damping fluid RB and two circulations of dcq buffer liquid WB, can make immune sandwich complex dissociate, immune reactor regeneration is to enter the next circulation of measuring.
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| CN101021530B (en) * | 2007-03-26 | 2011-08-31 | 南京大学 | Automatic channel resolution chemiluminescent multicomponent immunodetection system and analytical method |
| CN101672841B (en) * | 2008-09-09 | 2013-05-08 | 北京万德高科技发展有限公司 | Detection instrument and detection method for biological sample |
| CN103018441A (en) * | 2012-12-31 | 2013-04-03 | 西南大学 | Multicomponent immunoassay method based on time-resolved chemiluminescence |
| CN108008132B (en) * | 2017-12-04 | 2020-05-08 | 北京惠中医疗器械有限公司 | Kit for combined detection of ovarian cancer tumor markers HE4 and CA125 and preparation method and application thereof |
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|---|---|---|---|---|
| US5714388A (en) * | 1996-08-14 | 1998-02-03 | Bayer Corporation | Apparatus and method for detecting chemiluminescent light |
| CN2636230Y (en) * | 2003-08-21 | 2004-08-25 | 郝书顺 | Enzymatic lighting immunity analysis instrument |
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2006
- 2006-08-16 CN CN2006100413349A patent/CN1908663B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5714388A (en) * | 1996-08-14 | 1998-02-03 | Bayer Corporation | Apparatus and method for detecting chemiluminescent light |
| CN2636230Y (en) * | 2003-08-21 | 2004-08-25 | 郝书顺 | Enzymatic lighting immunity analysis instrument |
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| CN1908663A (en) | 2007-02-07 |
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