CN101021530A - Automatic channel resolution chemiluminescent multicomponent immunodetection system and analytical method - Google Patents

Abstract

An automatic channel-resolution chemiluminescence multi-component immunoassay system and analysis method, in the detection system, a test tube (6) of an incubation system is placed with magnetic beads (7) and a stirrer (8), and placed in a magnetic stirrer (9 ); the solution transmission system consists of a peristaltic pump (11), a connecting pipe (10) and a multi-position valve (5); the signal resolution acquisition system consists of a detection channel (12), a light gate (13), a magnet (14) and A chemiluminescence detector (15) is composed; the analysis method is to immobilize coated antibodies of various components on the surface of functionalized magnetic beads, and react with tracer antibodies labeled with alkaline phosphatase to form sandwich immune complexes ; Wash the unbound excess enzyme-labeled antibody with buffer solution, pass it into the chemiluminescent substrate solution, cover the detection channel with a light gate, and detect the concentration of components in different channels respectively. After the detection is completed, wash out the magnetic beads and enter 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

Automated channel resolution chemiluminescence multi-component immunoassay system and analysis method

1. Technical field

The invention is a channel-resolving multi-component chemiluminescence immunoassay technique, which relates to an immunoassay method for nearly simultaneously detecting multiple components in the same sample, especially a multi-component immunoassay combining chemiluminescence and flow analysis techniques and using magnetic beads as immunoreaction carriers. Component immunoassay method; the present invention also relates to an automatic detection instrument system for multi-component chemiluminescence immunoassay.

2. Background technology

Due to its high selectivity, low detection limit, good universality, and simple instrumentation, immunoassay has been widely used in the fields of clinical diagnosis, environmental monitoring, food safety, drug analysis, and microbiological testing. In practical applications, it is often necessary to determine the content of multiple components in complex samples. For example, in disease diagnosis, the joint analysis of multiple biomarkers provides a strong basis for diagnosis. Another example is in environmental monitoring, it is often necessary to simultaneously monitor the concentration indicators of various pesticides and herbicides in the environmental water body to comprehensively evaluate the level of environmental pollution.

To determine the content of multiple components in complex systems, traditionally, parallel single-component analysis is often used, that is, each analysis process only measures the content of one of the components, and the process is executed in parallel multiple times, and finally all the required components are obtained content. This analysis mode takes a long time, consumes a lot of reagents, and requires a lot of labor. The multi-component immunoassay technology that has emerged in recent years can detect multiple components simultaneously or nearly simultaneously in a single analysis process, and has the outstanding advantages of high analysis throughput, short time required, less sample consumption, and low analysis cost. The current multi-component immunoassay technology can be roughly divided into two categories. The first type is the multi-marker mode. score recognition. For example, in fluorescence and absorption photometry detection, it is identified by wavelength; in electrochemical method, it is identified by working potential; in time-resolved fluorescence method, it can be identified by both wavelength and decay time. A big problem with the multi-marker-based model is that different markers often have completely different optimal analysis conditions, such as the optimal pH values of horseradish peroxidase, alkaline phosphatase, and galactase, respectively. For 5-7, 8-10 and 6-8. Simply using a variety of markers in the same analysis system can only select a compromise condition among the optimal analysis conditions of different markers, which leads to a decrease in the analysis effect. The more markers, the more difficult the condition selection, so

At present, most analysis methods based on multi-marker mode do not measure more than two groups (Kricka LJ. Multianalyte testing. Clin. Chem. 1992; 38: 327). At the same time, although different markers can be distinguished by parameters such as wavelength or potential, their resolution efficiency is often limited, and the problem of signal overlap is sometimes very prominent (Goldman ER, Clapp AR, Anderson GP, Uyeda HT, Mauro JM, Medintz IL, et al . Multiplexed toxin analysis using four colors of quantum dot fluororeagents. Anal. Chem. 2004; 76: 684). The biggest problem with the multi-marker approach is also the limited number of tracers available. Although there are many detection methods of immunoassay, each method has high sensitivity and good stability, and only two or three tracers are widely used in practical work, which greatly limits the groups that can be detected simultaneously. Number of points (Mastichiadis C, Kakabakos SE, Christofidis I, Koupparis MA, Willetts C, Misiakos K. Simultaneous determination of pesticides using four-band disposable optical capillary immunosensor. Anal. Chem. 2002; 74: 6064). The second type of multi-component immunoassay technology is the spatial resolution mode, which is to immobilize the corresponding immune reagents of different components in different regions of the immunoreactor, so that the immune reactions of different components occur at different spatial positions of the reactor, and then array The detector is used for detection to realize the simultaneous detection of multiple components. This mode is mostly seen in the array method, and its energy conversion methods include optical (chemiluminescence, fluorescence, ultraviolet-visible absorption photometry) and electrochemical (amperometric, capacitive) methods. This mode usually requires expensive array detectors such as charge-coupled device (CCD) detectors and multi-channel electrochemical workstations (Fu ZF, Liu H, Ju HX. Flow-through multianalyte chemiluminescent immunosensing system with designed substratezone-resolved technique for sequential detection of tumor markers. Anal. Chem. 2006;78:6999).

The current commonly used immunoassay technology generally requires multiple times of sample addition, incubation, plate washing and reaction, and finally instrumental detection. Most of the operations are done manually, the workload is heavy, and the time required is more than 2 hours, which is not suitable for high-throughput rapid analysis. 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. 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 currently one of the most sensitive analytical techniques and is especially suitable for the detection of trace substances.

3. Contents of the invention

The object of the present invention is to provide an automatic channel resolution chemiluminescence multi-component immunoassay method based on the spatial resolution mode, with immunomagnetic beads as the carrier, combined with chemiluminescence detection and flow analysis technology; another object of the present invention It also provides a set of automatic channel resolution chemiluminescence multi-component immunoassay system.

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

An automatic channel resolution chemiluminescence multi-component immunoassay system is characterized in that the system is composed of an incubation system, a solution transmission system, a signal resolution acquisition system and a computer, wherein the incubation system consists of 1 to 6 built-in magnetic beads ( 7) and the glass test tube (6) of micro-stirrer (8) and magnetic force stirrer (9) form, and micro-glass test tube (6) is placed on the magnetic force stirrer (9); The solution transmission system consists of multi-channel peristaltic pump (11 ), connecting pipes (10) and 1 to 6 multi-position valves (5), the connecting pipes (10) are respectively connected to the three valve position inlets (1 ), (2), (3), the inlet (1) leads to the incubation system to transmit the magnetic bead suspension through the connecting tube (10), and the inlet (2) transmits the chemiluminescent substrate solution (19) through the connecting tube (10) , the inlet (3) transmits the washing buffer (18) through the connecting pipe (10); the signal resolution acquisition system consists of 1 to 6 detection channels (12), 1 to 6 tubular light gates (13), magnets (14) and Chemiluminescence detector (15) is formed, and the inlet of detection channel (12) is connected with the outlet (4) of multi-position valve (5) center, and connecting pipe leads out the raffinate in detection channel (12), and this raffinate is from raffinate The outlet (16) is discharged, the magnet (14) is placed above the detection channel (12), and the chemiluminescence detector (15) is placed below, and the solution delivery system and the chemiluminescence detector (15) are controlled by a computer (17).

The rotation speed and liquid flow rate of the above-mentioned multi-channel peristaltic pump (11) are automatically adjusted by a computer (17).

The above-mentioned multi-position valve (5) can switch between different flow paths by rotating the multi-position valve. The three inlets (1), (2) and (3) on the multi-position valve (5) are respectively connected to the The outlets (4) are connected, and the rotation of the multi-position valve (5) is automatically controlled by the computer (17).

The above-mentioned magnet (14) is used for intercepting the magnetic bead particles (7) in the suspension.

The above-mentioned tubular light gate (13) is movable, and is used to cover the detection channel (12), so as to realize the resolution of luminescent signals in different channels.

An automatic channel resolution chemiluminescence multi-component immunoassay method, the analysis steps are as follows:

① Add the sample, the antibody-coated magnetic beads (7) of the components to be tested, and the alkaline phosphatase-labeled antibody into the glass test tube (6) of the incubation system, and incubate with stirring at room temperature to form an immune sandwich complex;

② Switch the valve position port to the inlet (1), use the peristaltic pump (11) to flow the magnetic bead suspension through the connecting pipe (10) into the multi-position valve outlet (4) through the inlet (1) and then into the detection channel (12) , retain the magnetic beads (7) with the magnet (14), and the remaining raffinate is discharged from the raffinate outlet (16);

③ Switch the valve position to (3), use the peristaltic pump (11) to feed the washing buffer (18) through the connecting pipe (10), and wash away the unbound immune reagents adsorbed on the surface of the magnetic beads (7);

④ Switch the valve position to (2), pass the chemiluminescent substrate solution (19) through the connecting tube (10) with the peristaltic pump (11), remove the magnetic field of the magnetic beads (14), and trigger the chemiluminescent reaction;

⑤ detect the chemiluminescence in a single channel (12), and cover the remaining detection channels (12) with a tubular light gate (13) to obtain the concentration of the detected component in the single channel, and obtain the detected components of each channel in the same way. the concentration of the component;

⑥Switch the valve position to (3), use the peristaltic pump (11) to pass the washing buffer (18) through the connecting pipe (10), and discharge the magnetic beads (7) from the detection channel (10) to complete the entire detection process , and enter the next analysis cycle, uniformly collect the used magnetic beads (7), and treat them with regeneration buffer for reuse.

The washing buffer (18) in the above step ③ is 0.01M phosphate buffer, pH 7.4, containing 0.05% Tween-20.

The chemiluminescent substrate solution (19) described in the above step ④ is disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 ^3,7 ] decan}-4-yl)phenyl phosphate, CSPD.

The regeneration buffer described in the above step ⑥ is 0.1M glycine/hydrochloric acid buffer, pH 2.2.

The three inlets (1), (2) and (3) on the above-mentioned multi-position valve (5) can communicate with the central outlet (4) respectively by switching valve positions, so as to realize the switching of different flow paths. Above-mentioned connecting pipe (10) available inner diameter 0.8 millimeter polytetrafluoroethylene is made.

The immunoreaction carrier used in the above immunoassay system is an immunomagnetic bead, the surface of which is coated with the corresponding antibody of the analyte.

The entire detection system and the entire analysis process, including incubation, sample injection, washing, detection and discharge of magnetic beads, are automatically controlled by the computer, and the measured chemiluminescent signal values are also output from the computer.

The working principle of the detection system:

This detection system is based on the traditional spatial resolution mode, combined with flow analysis and channel resolution technology, it can measure multiple substances in one analysis process. The capture antibodies of different components are covalently bound to the surface of functionalized magnetic beads and used as immune reaction carriers; the magnetic beads immobilized with different antibodies are mixed with samples and corresponding enzyme-labeled antibodies, and incubated at room temperature with stirring to form sandwich immune complexes The magnetic bead suspension corresponding to different components is passed into the parallel detection channel, and then the magnetic beads are intercepted by a magnet; the unbound immunological reagent is washed away with the washing buffer; 4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[ ^3.3.1.13,7 ]decan}-4-yl)phenylphosphate, CSPD), produces strong chemiluminescence; chemiluminescence signal The resolution and collection of the light gate is completed with the help of the light gate, that is, when detecting the chemiluminescence of one of the channels, the tubular light gate is used to cover the other detection channels, and the content of each component can be obtained after recording the luminescence of each detection channel respectively; The washing buffer discharges the magnetic beads to enter the next measurement cycle; the discharged magnetic beads are collected uniformly and treated with the regeneration buffer to realize the repeated use of the immunomagnetic beads.

During the analysis process, all 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 valve. The whole process is programmed and automatically controlled by the computer, and the signal is also output from the computer.

The invention combines flow analysis technology and chemiluminescence detection, and proposes a channel-resolving 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 and detection only takes 18 minutes, which is not only one of the fastest multi-component immunoassay methods at present, but also much faster than ordinary single-component immunoassay method.

(3) The instrument equipment is simple and low in cost, and does not require expensive array detectors commonly used in multi-component immunoassays. Chemiluminescence detector composition.

(4) Antibody-coated magnetic beads can be regenerated repeatedly with regeneration buffer, which greatly saves expensive coated antibodies and further reduces the analysis cost compared with conventional immunoassay methods.

(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 automated channel-resolved multi-component chemiluminescent immunoassay detection system

1-incubation system, 2-chemiluminescent substrate, 3-washing buffer, 4-multi-position valve outlet, 5-multi-position valve, 6-glass test tube, 7-magnetic beads, 8-micro stirring Sub, 9-magnetic stirrer, 10-connecting tube, 11-peristaltic pump, 12-detection channel, 13-tubular light gate, 14-magnet, 15-chemiluminescence detector, 16-residue outlet, 17-computer, 18-washing buffer, 19-chemiluminescence substrate solution.

5. Specific implementation

Embodiment 1 further explains the three-component A, B, C detection system in conjunction with accompanying drawing 1:

The detection system is composed of an incubation system, a solution transmission system, a signal resolution acquisition system and a computer. For three-component detection, the incubation system consists of three glass test tubes with a diameter of 0.8 cm, a height of 2 cm, and a built-in micro-stirrer with a length of 0.5 cm and a magnetic stirrer. The glass test tubes are placed on the magnetic stirrer; the solution The transmission system consists of a multi-channel peristaltic pump with continuously adjustable speed, three multi-position valves with three inlets and one outlet, and several Teflon connecting pipes with an inner diameter of 0.8 mm. The inlet 1 of the multi-position valve leads to each component. Corresponding incubation system, inlet 2 leads to the chemiluminescence substrate solution, and inlet 3 leads to the washing buffer; the signal resolution acquisition system consists of three detection channels, three movable tubular light gates, a chemiluminescence detector and a magnet Composition, the detection channel is a quartz tube with an inner diameter of 1 mm and a length of 4 cm, which is placed under the magnet and above the chemiluminescence detector, and there is a movable black tubular light gate next to the quartz tube. All analysis systems are automatically controlled by computer.

Embodiment 2 Chemiluminescent three-component immunoassay method

The specific analysis process is shown in Table 1. All the analysis steps are programmed and automatically controlled by a computer, and the measured chemiluminescent signal is output from the computer. After the analysis steps are completed, the antibody-coated magnetic beads are collected uniformly, soaked in the glycine/hydrochloric acid buffer solution of pH 2.2 for 10 minutes, and washed 5 times with 0.01M phosphate buffer solution (pH 7.4) to achieve regeneration and reuse the goal of.

Table 1. Specific analysis process Step number 123456789 Valve position 1323 Steps: Add antibody-coated magnetic beads of components A, B, and C to three incubation test tubes, 20 microliters each of enzyme-labeled antibody and sample, stir and incubate; use a peristaltic pump to inject the magnetic bead suspension for detection The channel uses a magnet to intercept the magnetic beads, and the remaining residual liquid is discharged; the peristaltic pump is continuously fed into the washing buffer at a flow rate of 0.4mL/min to wash the unbound immunological reagents adsorbed on the surface of the magnetic beads; each detection channel is separated by a peristaltic pump Inject 30 microliters of chemiluminescence substrate, remove the magnetic field after the flow is stopped, and trigger the chemiluminescence reaction; move the tubular light gate to cover the detection channels of components B and C, and detect the chemiluminescence in the channel of component A; move the tubular light gate to cover The detection channels of components A and C detect the chemiluminescence in the channel of component B; move the tubular light gate to cover the detection channels of components A and B, and detect the chemiluminescence in the channel of component C; set the peristaltic pump at 1.0 The flow rate of mL/min is continuously fed into the washing buffer to expel the magnetic beads from the channel; enter the next analysis cycle process. Start time (minute:second) 00:0010:0010:1012:0017:0017:1017:2017:3018:00

Example 3

Taking three important tumor markers: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and cancer antigen 125 (CA125) as examples, the application of this automated channel-resolution chemiluminescent multicomponent immunoassay method is illustrated.

The immune reaction carrier is carboxyl-activated magnetic beads, which are respectively coated with mouse monoclonal AFP antibody, mouse monoclonal CEA antibody and mouse monoclonal CA 125 antibody, and the residual active sites are blocked with bovine serum albumin. The tracer antibodies were alkaline phosphatase-labeled goat polyclonal AFP, CEA and CA 125 antibodies.

As shown in the process shown in Table 1, add 20 microliters of samples to each of the three test tubes of the incubation system, and add 20 microliters of AFP, CEA and CA 125 antibody-coated magnetic beads and enzyme-labeled antibodies respectively, and stir at room temperature. Incubate for 10 minutes. Switch the multi-position valve to valve position 1, and pass the magnetic bead suspension into the three detection channels. After the magnetic beads are trapped by the magnet, the residual liquid flows out from the channel. Switch the multi-position valve to valve position 3, and continuously inject washing buffer at a flow rate of 0.4 mL/min to wash the magnetic beads for 1 minute and 50 seconds to wash away unbound immunological reagents. Switch the multi-position valve to valve position 2, pass 30 microliters of chemiluminescent substrate into the three channels respectively, remove the magnetic field, and initiate the chemiluminescent reaction. After reacting for 5 minutes, cover the detection channel corresponding to CEA and CA 125 with a light gate, detect the chemiluminescence in the AFP channel, and obtain the concentration of AFP; cover the detection channel corresponding to CEA and AFP with a light gate, and detect the chemiluminescence in the CA 125 channel. The concentration of CA 125 is obtained by emitting light; the detection channel corresponding to AFP and CA 125 is blocked by a light gate, and the chemiluminescence in the CEA channel is detected to obtain the concentration of CEA. Switch the valve position to valve position 3, continuously inject the washing solution at a flow rate of 1.0 mL/min for 0.5 min, and discharge the magnetic beads from the channel. So far, the whole analysis process is completed. After the discharged magnetic beads were collected uniformly, they were soaked in glycine/HCl buffer solution with pH 2.2 for 10 minutes, and washed 5 times with 0.01 M phosphate buffer solution (pH 7.4) to achieve the purpose of regeneration and reuse.

Claims (9)

1. automatic channel resolution chemiluminescent multicomponent immunodetection system, it is characterized in that this system is made of incubation system, solution transmission system, signal resolution acquisition system and computing machine, wherein the incubation system is made up of with magnetic stirring apparatus (9) the glass test tube (6) of 1 to 6 built-in magnetic bead (7) and miniature stirrer (8), and micro glass test tube (6) is placed on the magnetic stirring apparatus (9); The solution transmission system is made up of multi-channel peristaltic pump (11), connecting pipe (10) and 1 to 6 multi-position valve (5), connecting pipe (10) is connected respectively to three valve position inlets (1), (2), (3) of multi-position valve (5) through multi-channel peristaltic pump (11), inlet (1) leads to incubation system transmissions bead suspension by connecting pipe (10), inlet (2) is by connecting pipe (10) transmission chemical luminous substrate liquid (19), and inlet (3) is by connecting pipe (10) transmission dcq buffer liquid (18); The signal resolution acquisition system is made up of with chemiluminescence detector (15) 1 to 6 sense channel (12), 1 to 6 tubulose optical gate (13), magnet (14), the inlet of sense channel (12) is connected with the outlet (4) at multi-position valve (5) center, connecting pipe is derived the raffinate in the sense channel (12), this raffinate is discharged from raffinate outlet (16), the top of sense channel (12) is placed magnet (14) below and is placed chemiluminescence detector (15), and wherein solution transmission system and chemiluminescence detector (15) are by computing machine (17) control automatically.

2. detection system according to claim 1 is characterized in that the rotating speed of described multi-channel peristaltic pump (11) and flow rate of liquid are to automatically adjust by computing machine (17).

3. detection system according to claim 1, it is characterized in that described multi-position valve (5) can realize the switching of different streams by rotating multi-position valve, three inlets (1) on the multi-position valve (5), (2), (3) communicate with the multi-position valve outlet (4) at center respectively, and the rotation of multi-position valve (5) is controlled by computing machine (17).

4. detection system according to claim 1 is characterized in that described magnet (14) is used for holding back suspending liquid magnetic bead particles (7).

5. detection system according to claim 1 is characterized in that described tubulose optical gate (13) is movably, is used for occlusion detection passage (12), realizes the resolution of luminous signal in the different passages.

6. automatic channel resolution chemiluminescent multicomponent immune analysis method, its analytical procedure is as follows:

1. the antibody sandwich magnetic bead (7) with sample, component to be measured joins in the glass test tube (6) of incubation system with alkali phosphatase enzyme mark antibody, stirs incubation under room temperature, forms immune sandwich complex;

2. the valve position mouth is switched to inlet (1), with peristaltic pump (11) bead suspension is flowed into multi-position valve outlet (4) through the inlet (1) of connecting pipe (10) by multi-position valve (5) and feed sense channel (12) again, hold back magnetic bead (7) with magnet (14), the residue raffinate is discharged from raffinate outlet (16);

3. the valve position mouth is switched to (3), feed dcq buffer liquid (18) through connecting pipe (10), clean the unconjugated immunoreagent of magnetic bead (7) surface adsorption with peristaltic pump (11);

4. the valve position mouth is switched to (2), feed chemical luminous substrate liquid (19) through connecting pipe (10), remove the magnetic field of magnetic bead (14), cause chemiluminescence reaction with peristaltic pump (11);

5. detect the chemiluminescence in the single passage (12), mobile tubulose optical gate (13) covers all the other sense channels (12), draws the concentration of institute's detected components in this single passage, and in kind obtains the concentration of each passage institute detected components;

6. the valve position mouth is switched to (3), feed dcq buffer liquid (18) with peristaltic pump (11) through connecting pipe (10), magnetic bead (7) is discharged from sense channel (12), finish full testing process, and enter next analysis cycle, the unified used magnetic bead (7) of collecting is handled to reuse with the regeneration damping fluid.

7. analytical approach according to claim 6 is characterized in that at the dcq buffer liquid (18) described in the step (3) be the 0.01M phosphate buffer, and pH7.4 contains 0.05% Tween-20.

8. analytical approach according to claim 6 is characterized in that at the chemical luminous substrate liquid (19) of step described in 4. be disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2 '-(5 '-chloro) tricyclo[3.3.1.1 ^3,7] decan}-4-yl) phenyl phosphate, CSPD.

9. analytical approach according to claim 6 is characterized in that at the regeneration damping fluid of step described in 6. be 0.1M glycocoll/hydrochloride buffer, and pH 2.2.