CN1908663A - Substrate regional band resolution chemical luminescent multi-component immunity analytical method and detection system thereof - Google Patents

Abstract

A new multi-component immunoassay method and detection system based on substrate zone resolution chemiluminescence, including a solution delivery system, an immunoreactor, a chemiluminescence detector and a computer; wherein the delivery system consists of a multi-channel peristaltic pump (6), Connecting pipe (7) and multi-position selector valve (8) form. This method is to immobilize the coated antibodies of multiple components in the immunoreactor, and after passing the sample and enzyme-labeled antibodies into the immunoreactor, if the two components are labeled with horseradish peroxidase and alkaline phosphatase respectively , forming two sandwich immune complexes. After washing the unbound excess antibody, pass through the chemiluminescent substrate zone of horseradish peroxidase, the washing buffer zone and the chemiluminescent substrate zone of alkaline phosphatase in sequence, and the components are determined by the signal intensity concentration. After the detection is completed, the renewable reactor enters the next analysis process. The method has the advantages of fast speed, low cost, good reproducibility and high sensitivity, and is suitable for environmental monitoring, clinical diagnosis, food safety and other fields.

Description

Substrate zone resolution chemiluminescence multicomponent immunoassay method and detection system

1. Technical field

The invention is a substrate zone resolution chemiluminescence technique, which relates to a simultaneous immunoassay method for multiple components in the same sample, especially a multi-component immunoassay method combined with chemiluminescence and flow analysis techniques; Detection system for two-component chemiluminescent immunoassays.

2. Background technology

As a highly selective and sensitive analytical method, immunoassay has been widely used in clinical diagnosis, environmental monitoring, food safety and other fields. In the field of practical application, it is often necessary to determine the content of multiple components in complex samples. For example, in tumor diagnosis, the joint analysis of multiple tumor markers provides a strong basis for diagnosis.

At present, in order to realize the combined analysis of multiple components in complex samples, the mode of multiple parallel single-component analysis is often used, that is, only one component is analyzed each time, and the analysis process is performed multiple times, and finally the results of all components are obtained. content. This mode is time-consuming, labor-intensive, and analytically expensive. In order to overcome these shortcomings, some multi-component immunoassay methods have appeared in recent years, mainly including array method and multi-label method. The array method is based on the principle of separating the immune reaction areas, that is, different components are detected in different array areas, but the method is complicated and requires expensive array detectors, such as multi-channel electrochemical workstations and CCDs. At present, domestic manufacturers have launched multi-component chemiluminescence immunoassay analyzers using CCD array detectors, but due to the high cost of the instruments, their products are difficult to be popularized. The principle of multi-labeling method is to label different components of immunological reagents with different markers, and obtain the content of each component by detecting the signals of different markers. The detection methods include electrochemical method, fluorescence method, photometric method and radiation method. In electrochemical detection methods, different components are usually distinguished by working potential; in fluorescence and photometry, different components are usually distinguished by wavelength and decay time. At present, the multi-labeling method cannot be detected by chemiluminescence, because chemiluminescence, as a detection technique that does not consider the wavelength, cannot distinguish the signals from the markers corresponding to different components by wavelength. Moreover, in the multi-labeling method, for different markers of different components, the optimal analysis conditions are often very different. Simple joint use of multiple markers often faces the problem of incompatibility of various marker analysis conditions, making The analysis effect is greatly reduced.

At present, the whole process of commonly used immunoassay technology requires multiple times of sample addition, incubation, plate washing and reaction, and finally instrumental detection. The operation process is cumbersome, labor-intensive, and takes more than 2 hours, which is not suitable for high-throughput rapid analysis. Combining flow analysis, immunomagnetic beads and electroluminescence technology, Roche Diagnostics GmbH of the United States has developed a commercialized Elecsys automatic immune analyzer, which has realized automatic and rapid immune analysis and has been applied in clinical practice. However, the price of the instrument and supporting kits is very expensive, and institutions with insufficient financial resources cannot afford it. Moreover, the instrument is also in a single-component analysis mode, and multiple parallel determinations are still required for complex samples.

Chemiluminescence analysis is an analytical technique that has developed rapidly in recent years. Its instruments are cheap, easy to operate, and environmentally friendly. It is also one of the most sensitive analytical techniques and is especially suitable for the detection of trace substances. Flow analysis technology has the advantages of good reproducibility, high degree of automation, and fast analysis speed, and is one of the most effective means to achieve high-throughput analysis. The combination of these two technologies with immunoassay has become a research hotspot in the field of immunoassay in recent years, and has made remarkable achievements.

3. Contents of the invention

The purpose of the present invention is to provide a multi-component immunoassay method based on substrate zone resolution technology and automatic high-throughput based on multi-labeling method, combined with flow analysis technology and chemiluminescence detection; Another object is to provide a set of automatic flow-through two-component substrate zone resolution chemiluminescence immunoassay detection system.

The purpose of the present invention is to realize by following technical scheme:

A flow-through substrate zone resolution multi-component chemiluminescence immunoassay detection system is characterized in that the system includes a solution delivery system, an immunoreactor, a chemiluminescence detector and a computer; the solution delivery system consists of a multi-channel peristaltic pump (6 ), connecting pipes (7) and multi-position directional valves (8), five connecting pipes (7) are respectively connected to five valve positions of multi-position directional valves (8) through multi-channel peristaltic pumps (6) (1), (2), (4), (5), inlet (1) transfers sample S, inlet (5) transfers regeneration buffer RB, inlet (4) transfers wash buffer WB, inlet (3), ( 2) Transfer two chemiluminescent substrates _S1 and _S2 respectively; the outlet at the center of the multi-position selector valve (8) is connected to the inlet of the immunoreactor through the connecting pipe (7), and the outlet of the immunoreactor is connected through the connecting pipe ( 7) The waste liquid is discharged, the immunoreactor is placed on the chemiluminescence detector, and the solution delivery system and the chemiluminescence detector are both connected to the computer.

The rotational speed and liquid flow rate of the above-mentioned multi-channel peristaltic pump (6) are adjustable.

The internal diameter of above-mentioned connecting pipe (7) can be made with the polytetrafluoroethylene of 0.8mm.

The above-mentioned multi-position selector valve (8) can realize the switching of different flow paths by rotating the multi-position selector valve, and the five valve position ports (1), (2), (3), ( 4), (5) communicate with the exit of the center respectively.

The above immunoreactor is an aldehyde-activated UltraBind membrane coated with various antibodies.

The above regeneration buffer is 0.1M amino acid/hydrochloric acid buffer, pH 2.0.

The above washing buffer is 0.01M phosphate buffer, pH7.4, containing 0.05% Tween-20.

A substrate zone resolution chemical reflection multi-component immunoassay method, the analysis steps are as follows:

(1) First switch the valve position port of the multi-position selector valve (8) to (1), and pass the sample to be tested containing antigen 1 and antigen 2 through the connecting pipe (7) with horseradish peroxidase and The tracer antibody 1 and antibody 2 labeled with alkaline phosphatase are passed into the immunoreactor, incubated at room temperature, and an enzyme-labeled immune sandwich complex of two components is formed in the immunoreactor;

(2) Then switch the valve position to (4), pass through the connecting tube (7) 1.0mL/min washing buffer WB to wash away unbound immunological reagents, after rinsing, switch the valve position to (3) , through the connecting tube (7) into the substrate zone S ₁ , horseradish peroxidase catalyzes the substrate zone to generate strong chemiluminescence, and records the luminescent signal to obtain the concentration of a component;

(3) Switch the valve position port to (4), and feed the washing buffer WB through the connecting pipe (7) to form a buffer zone;

(4) Switch the valve position port to (2), and then pass into the substrate zone S2 through the connecting tube (7). The concentration of points;

(5) After the measurement is completed, switch the valve position to (5) and (4) in turn, and pass through the regeneration buffer RB and the washing buffer WB successively for two cycles, so that the immune sandwich complex can be dissociated, and the immunoreactor Regenerate to enter the next measurement cycle.

The composition of the flow-through multi-component chemiluminescence immunoassay detection system:

The structure of the detection system is shown in Figure 1, and it includes four parts: the first part is the solution transmission system, which uses a multi-channel peristaltic pump (6) as the mass transfer power, and several polytetrafluoroethylene tubes Act as a mass transfer connecting pipe (7), control the direction of liquid flow with a multi-position selector valve (8), and inject different solutions into the immunoreactor; the second part is the immunoreactor, in which the corresponding components of various components are fixed. Coating antibody; the third part is a chemiluminescent detector for collecting luminescent signals; the fourth part is a computer control system.

The working principle of the detection system:

This detection system combines the traditional multi-label technology with the substrate zone resolution technology, and can measure multiple substances in one analysis process. The immune response is the traditional double-antibody sandwich method. Coating antibodies corresponding to various analytes (antigens) are immobilized in the immunoreactor. As shown in Figure 2, taking two antigens to be tested as an example, the antibodies of the two tested substances were immobilized in the reactor, and the tracer antibodies were labeled with horseradish peroxidase and alkaline phosphatase respectively; A complex sample of these two antigens is mixed with two enzyme-labeled antibodies and passed into an immunoreactor where a two-component enzyme-labeled immunosandwich complex is formed; the unbound immunoassay is then washed away by washing buffer. Reagent: after washing, first pass into the substrate zone S1 (a mixed solution of luminol, hydrogen peroxide and p-iodophenol), which is catalyzed by horseradish peroxidase to generate strong chemiluminescence, and record the luminescent signal , to obtain the content of a component; after washing the immunoreactor with the washing buffer zone WB, it is passed into the substrate zone S2 (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2'-( 5'-chloro)tricyclo[3.3.1.1 ^3,7 ]decan}-4-yl)phenylphosphate, CSPD), this zone produces strong chemiluminescence after encountering alkaline phosphatase, and another component can be obtained by recording the luminescent signal content.

After the measurement is completed, two cycles of regeneration buffer and washing buffer are passed in sequence to dissociate the immune sandwich complex and regenerate the immunoreactor to enter the next cycle of measurement.

During the analysis process, all the solutions are transferred into the analysis system by the connecting pipe on the peristaltic pump, and the switching of different flow paths is realized by turning the multi-position selector valve, and the whole process is programmed and automatically controlled by the computer.

The measurement principle of this detection system:

When there are two kinds of antigenic substances to be measured in the sample to be tested, the two kinds of antigenic substances and the corresponding immobilized coating antibodies on the reactor and the two enzyme-labeled antibodies added form horseradish peroxidase and Two immune complexes labeled with alkaline phosphatase. When the luminol-hydrogen peroxide-p-iodophenol-based horseradish peroxidase chemiluminescent substrate and the CSPD-based alkaline phosphatase chemiluminescent substrate are sequentially introduced with the substrate zone resolution technique, the The luminescence signals of the two components are obtained, so as to convert the concentrations of the two components.

Compared with the prior art, the present invention has the following characteristics:

The invention combines flow analysis technology and chemiluminescence detection, and proposes a substrate zone resolution multi-component immunoassay system, which detects multiple substances in one process. Compared with other multi-component immunoassay methods, it has the following characteristics:

(1) The operation is simple, the entire analysis process is completed in the flow system, and the computer is used for programmed automatic control, with very few manual operations and no skilled operators are required.

(2) The analysis time is short, and the whole process including sample addition, incubation, washing, detection and regeneration only takes 35 minutes, which is not only one of the fastest multi-component immunoassay methods at present, but also much faster than ordinary single-component immunoassay methods Immunoassay method.

(3) The equipment is simple and low in cost, and does not require the expensive array detector commonly used in multi-component immunoassay. device composition.

(4) The immunoreactor can be repeatedly regenerated by passing through the regeneration buffer. Compared with the conventional immunoassay method, the expensive coated antibody is greatly saved, and the analysis cost is further reduced.

(5) Since its detection mode is an extremely sensitive enzyme-catalyzed chemiluminescent reaction, this method can detect samples with extremely low concentrations, meeting most of the analysis requirements.

(6) Due to the use of automated flow analysis technology, the individual differences caused by the different operating methods and habits of operators are greatly reduced, and the reproducibility of the method is greatly improved compared with the traditional manual operation method, which is conducive to formulating relevant standard.

4. Description of drawings

Figure 1 Schematic diagram of the flow-through two-component chemiluminescence immunoassay detection system

1, 2, 3, 4, 5 are multi-position selector valve valve position inlets, 6-multi-channel peristaltic pump; 7-connecting pipe; 8-multi-position selector valve; S: sample; RB: regeneration buffer; WB : wash buffer; S1: substrate 1; S2: substrate 2

Figure 2 Schematic diagram of flow-through two-component chemiluminescent immunoassay principle

5. Specific implementation

Example 1 In conjunction with accompanying drawing 1, the flow-through two-component chemiluminescence immunoassay detection system is further described:

The flow-through substrate zone resolution multi-component chemiluminescence immunoassay detection system includes a solution delivery system, an immunoreactor, a chemiluminescence detector and a computer. The solution transmission system consists of a multi-channel peristaltic pump 6, a PTFE connecting pipe 7 with an inner diameter of 0.8mm and a multi-position selector valve 8. The five connecting pipes 7 are respectively connected to the multi-position selector valve through the multi-channel peristaltic pump 6. The five valve positions of valve 8 are ports 1, 2, 4, and 5. Inlet 1 transfers sample S, inlet 5 transfers regeneration buffer RB, inlet 4 transfers washing buffer WB, and inlets 3 and 2 transfer two chemiluminescent substrates respectively S ₁ and S ₂ ; one end of each connecting pipe 7 is connected with the outlet of the selector valve 8 center, the outlet is connected with the inlet of the immunoreactor, and another connecting pipe 7 leads out the waste liquid of the immunoreactor, and the immunoreactor is set On top of the chemiluminescence detector, both the solution delivery system and the chemiluminescence detector are connected with a computer.

Embodiment 2 chemiluminescence and component immunoassay method:

The specific analysis process is shown in Table 1, and all the analysis steps are programmed and automatically controlled by a computer. The regeneration buffer RB is 0.1M amino acid/hydrochloric acid buffer, pH2.0; the washing buffer WB is 0.01M phosphate buffer containing 0.05% Tween-20, pH7.4; the immunoreactor is coated with various antibodies Aldehyde-activated UltraBind membrane; S ₁ is a mixed solution of 0.5mmol/l luminol, 3mmol/l hydrogen peroxide and 0.5mmol/l p-iodophenol and S ₂ is a CSPD solution. Table 1 specific analysis process step number valve position step Start time (minutes) 1234567891011 11434254541 Inject the mixture of the sample and the two enzyme-labeled antibodies into the immunoreactor to stop the flow, incubate at room temperature and pass through the washing buffer at a flow rate of 1.0mL/min to wash the immunoreactor into the chemiluminescence substrate of horseradish peroxidase, Stop the flow, collect chemiluminescence data, obtain a component concentration, pass through the washing buffer at a flow rate of 1.0mL/min to wash the immunoreactor and inject the chemiluminescence substrate of alkaline phosphatase, stop the flow, collect chemiluminescence data, To obtain another component concentration, pass through the regeneration buffer at a flow rate of 0.5mL/min to the regeneration immunoreactor, pass through the washing buffer at a flow rate of 0.5mL/min, adjust the immune reactor, and pass through the regeneration buffer at the same flow rate for the second time Regulate the immunoreactor into the next analysis cycle by feeding the wash buffer at the same flow rate for the second time 00:0000:3020:3022:0026:3027:0031:3033:0033:3034:3035:00

Example 3

Taking two important tumor markers: carcinoembryonic antigen (CEA) and cancer antigen 125 (CA 125) as examples, the application of the flow-type two-component chemiluminescent immunoassay system is illustrated.

The immunoreactor is an aldehyde-activated UltraBind membrane coated with mouse monoclonal anti-CEA and mouse monoclonal anti-CA 125. The remaining active sites are blocked with bovine serum albumin and fixed in a 50 microliter flow cell. The tracer antibody to CEA was mouse monoclonal anti-CEA labeled with alkaline phosphatase, and the tracer antibody to CA 125 was mouse monoclonal anti-CA 125 labeled with horseradish peroxidase.

As shown in the process shown in Table 1, switch the valve to position 1, pass a mixture of 25 microliters of sample, 12.5 microliters of CEA tracer antibody and 12.5 microliters of CA 125 tracer antibody into the flow cell where the immunoreactor is fixed, Incubate statically for 20 minutes. Then switch the valve position to 4 and flush the immunoreactor for 1.5 minutes. After washing, switch the valve position to 3, inject horseradish peroxidase substrate, stop the flow, and collect the corresponding luminescent signal of CA 125 at the fourth minute. Then switch the valve position back to 4 and rinse for half a minute. Then switch the valve position to 2, inject the alkaline phosphatase substrate, stop the flow for 4 minutes, and detect the corresponding chemiluminescent signal of CEA. After the detection is completed, switch the valve between positions 5 and 4, repeatedly pass the regeneration buffer and washing buffer, and regenerate the immunoreactor for two cycles within 3.5 minutes to complete the entire analysis process. The standard curves of CEA and CA 125 were obtained by signal determination of a series of concentration samples. The concentration of these two tumor markers in clinical blood samples was obtained by using the standard curve method.

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 five valve position mouths (1), (2), (3), (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) are transmitted 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, five the valve position mouths (1) on the multidigit selection valve, (2), (3), (4), (5) 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 (1), 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 (4), feed not binding immunoassay reagent of dcq buffer liquid WB flush away by connecting pipe (7), after rinsing well, the valve position mouth is switched to (3), feed substrate regional band S1 by connecting pipe (7), 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 (4), feed dcq buffer liquid WB, form buffering liquid zone band by connecting pipe (7);

(4) the valve position mouth is switched to (2), 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 (5) and (4), 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 next mensuration circulation.

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