CN107290515A - High flux immunoassay device - Google Patents

Abstract

The invention provides a high-throughput immunoassay device. The immunoassay device includes: a detection core member, a detection reaction area is provided on the outer wall near one end of the detection core member; and a housing member, a sample inlet is provided at one end of the housing member, and Multiple pairs of pouring holes are provided on the side wall, multiple pairs of sealing members are provided on the inner wall of the shell member, and water absorbing members are provided between each pair of sealing members. When assembled, the test core member is inserted into the housing member such that the one end of the test core member is adjacent to the one end of the housing member. After the assembly is completed, a plurality of independent cavity components are formed between the outer wall of the detection core component, the inner wall of the housing component and the sealing component, and each of the cavity components is connected to a pair of the perfusion holes respectively. connected.

Description

High-throughput immunoassay device

technical field

The invention relates to an immunological detection device in the technical field of biological immunology detection, which is a fast and high-throughput detection device for simultaneously detecting the content concentrations of various substances to be tested from a sample.

Background technique

With the development of immunoassay technology, high-throughput, accurate quantification, and portable and easy-to-operate features have become the basic requirements for detection products. Compared with traditional ELISA detection products, this product has the advantages of simple operation, small demand for samples, and multiple indicators at the same time. Advantages such as detection; Compared with colloidal gold and other products, it has the advantages of high detection throughput and more accurate quantification. This product can be suitable for both manual and machine reading.

Contents of the invention

The invention provides an immune detection device with simple operation and capable of quantitative detection of multiple indicators.

The immunoassay device comprises: a detection core component (1), a detection reaction zone (5) is provided on the outer wall near one end of the detection core component (1); and a shell component (2), on which the shell component (2) ) is provided with a sample inlet (16), on the side wall of the housing member (2) is provided with many pairs of perfusion holes (6), and the inner wall of the housing member (2) is provided with many pairs of sealing members ( 9), a water-absorbing member (8) is arranged between each pair of sealing members (9). When assembled, the detection core member (1) is inserted into the housing member (2) such that the one end of the detection core member (1) is adjacent to the one end of the housing member (2). After the assembly is completed, a plurality of independent cavity components are formed between the outer wall of the detection core component (1), the inner wall of the housing component (2) and the sealing component (9), each of the cavity components The components communicate with a pair of perfusion holes (6) respectively.

Preferably, the detection reaction area (5) includes a plurality of reaction subregions, the plurality of reaction subregions are evenly distributed along the circumferential interval of the detection core member (1), and the plurality of reaction subregions can be detected according to the The same or different chemical components need to be administered simultaneously to capture the object to be detected or to react chemically with the object to be detected and present a signal that can be recorded.

Preferably, five independent cavity members are formed between the outer wall of the detection core member (1), the inner wall of the housing member (2) and the sealing member (9).

Preferably, the other end of the detection core member (1) is provided with a handle member (3), and the detection core member (1) is pushed into the housing member by holding the handle member (3) during operation (2) or out of the housing member (2).

Preferably, the other end of the housing member (2) is provided with a housing handle (10), and the housing member (2) is stabilized by holding the housing handle (10) during operation.

Preferably, the high-throughput immunoassay device further includes a perfusion hole cap (7), and the perfusion hole cap (7) is used to close the perfusion hole (6).

Preferably, the high-throughput immunoassay device further includes a concentration comparison ruler (18), and the concentration comparison ruler (18) is arranged on the outer wall of the housing member (2), and is used as a detection signal and to read the Reference for concentration values.

The immunological detection device provided by the present invention utilizes the principle of immunology to realize multi-indicator integrated high-throughput detection, which has the characteristics of a chip; the required detection sample is less, at least 100 microliters; the operation is simple; the sensitivity is high, and it can be accurate Quantitative; results can be read either by instrumental analysis or by visual comparison.

Description of drawings

Fig. 1 is a schematic cross-sectional view showing a detection device according to a specific embodiment of the present invention.

FIG. 2 is a schematic perspective view showing the detection core member in FIG. 1 .

Fig. 3 is a schematic perspective view showing the shell member in Fig. 1

detailed description

A specific embodiment of the present invention will be described in detail below in conjunction with the accompanying drawings.

As shown in FIG. 1 , the detection device includes a detection core member 1 and a casing member 2 .

The detection core member 1 is cylindrical, for example, has a shape similar to the plunger core of a syringe, and can be made of materials such as polystyrene, polyvinyl chloride, polyethylene, etc., preferably made of polystyrene, and is opaque in light color, preferably milky white .

A detection reaction zone 5 is provided on the outer wall close to one end of the detection core member 1. The detection reaction zone 5 may include a plurality of reaction partitions, which are evenly distributed along the circumferential direction of the detection core member 1. Each reaction partition can be simultaneously administered with the same or different chemical components, which can capture the substance to be tested or undergo a chemical reaction and present a signal that can be recorded. The reaction of the tested substance.

An indicator line 4 surrounding the detection core member 1 is provided at a position adjacent to the detection reaction zone 5 to indicate the position of the detection reaction zone 5 in the device. The indicator line 4 may be located upstream or downstream of the detection reaction zone 5 in the advancing direction of the detection core member 1 . The indicator line 4 shown in FIG. 2 is located upstream of the detection reaction zone 5 .

The other end of the detection core member 1 may be provided with a handle member 3, which is convenient for the operator to hold and push or pull.

The shell member 2 is cylindrical and can be made of common transparent plastic materials. Multiple pairs of pouring holes 6 are provided on the shell member 2 . Each filling hole 6 is closed by a filling hole cap 7 .

Multiple pairs of annular sealing members 9 are provided on the inner sidewall of the shell member 2 , and the multiple pairs of sealing members 9 are distributed at intervals along the length direction of the shell member 2 . The specific number of sealing members 9 depends on the number of cavities described later. The sealing member 9 may be a rubber ring. A water-absorbing member 8 is arranged between each pair of sealing members 9, and the water-absorbing member 8 contains water-absorbing material. The number of water absorbing members 8 corresponds to the logarithm of sealing members 9 .

One end of the housing member 2 is provided with a sample inlet 16 , and the sample inlet 16 is closed by a sample inlet cap 17 .

The other end of the housing member 2 may be provided with a housing handle 10, and when the detection core member 1 is pushed or pulled, the operator holds the handle 10 to firmly hold the housing member 2, thereby facilitating smooth operation. Push or pull out the detection core member 1 .

When assembling, insert one end of the detection reaction zone 5 near the detection core member 1 into the housing member 2, and the other end is exposed outside the housing member 2, so that the handle member 3 of the detection core member 1 is closer than the outer shell of the housing member 2. The handle 16 is further outside, so that it is convenient for the user to perform the pushing or pulling operation of the detection core member 1 .

After the detection core component 1 and the shell component 2 are assembled, the outer wall of the detection core component 1 is closely attached to the sealing member 9, so that a plurality of independent annular cavity. The exact number of annular cavities depends on the specific immunoassay protocol. Each annular cavity is connected with a pair of perfusion holes 6 respectively, and the same or different solutions are perfused into each annular cavity through the perfusion holes 6 according to detection requirements.

As shown in Figure 2, the device of this embodiment forms a total of five cavities. For the convenience of the following description, starting from one end of the sample inlet 16 in the detection device, they are called the reaction chamber 11 and the first washing liquid chamber 12 in sequence. , a detection chamber 13, a second wash solution chamber 14 and a substrate chamber 15. Through the respective perfusion holes 6, the same or different solutions can be perfused into the above-mentioned cavities according to the detection requirements, and the perfusion hole caps 7 are covered after the perfusion. Correspondingly, as shown in FIGS. 2 and 3 , five pairs of sealing members 9 are provided in this embodiment.

When using the above detection device to detect samples, the operator holds the shell handle 16 with one hand, and the detection core handle 3 with the other hand, immerses the sample inlet 16 into the sample, and then pulls the detection core member 1 until the indicator line 4 moves to the position of the sealing member 9 of the reaction chamber 11, at this time the sample to be tested is sucked into the reaction chamber 11.

After the sample to be tested is sucked up, the direction of the detection device is adjusted so that the sample inlet 16 is vertically upward, the sample to be tested is in contact with the detection reaction zone 5, and the same or different chemical substances applied on the detection reaction zone 5 begin to "combine" or interact with the detection reaction zone 5. The reaction of the test substance, for example, different capture antibodies coated on the detection reaction zone 5 start to "capture" the corresponding target objects in the sample to be tested, and the same or different capture antibodies coated on the detection reaction zone 5 start to "capture". "The corresponding targets in the samples to be tested can be gently shaken during the reaction process.

After the reaction is completed, the detection core member 1 is further pulled, so that the detection reaction zone 5 passes through a water-absorbing member 8, so that the remaining sample liquid is sucked dry. Next, pull the detection core member 1 to make the detection reaction area 5 enter the first washing liquid chamber 12, wash the detection reaction area 5 in the first washing liquid chamber 12, and continue to pull the detection core member 1 through a water-absorbing member 8, so that Residual wash solution was blotted dry. Then, the detection core member 1 enters the detection chamber 13, and the entire detection device can be shaken slightly during the whole reaction process. In this process, if the principle of the double sandwich method is used to detect the sample to be tested, since the detection reaction zone 5 has captured the target sample to be tested in the sample, the corresponding HRP-labeled detection antibodies in the detection chamber 13 can respectively Combine with the substance to be tested. If the principle of competition method is used to detect the target substance, since the detection reaction zone 5 has captured the target substance in the sample, the corresponding HRP-labeled conjugates in the detection chamber 13 can interact with the untargeted substances on the capture antibody. Empty "binding sites" occupied by the analytes bind.

After the reaction is completed, the detection core member 1 is further pulled to make the detection reaction area 5 enter the second washing liquid chamber 14 for the second washing, and then pass through a water-absorbing member 8 to absorb the remaining detection liquid. Next, the detection reaction zone 5 enters the substrate chamber 15. If there is a target substance in the sample, the chemical substance applied on the detection reaction zone 5 will interact with the substance to be detected at this time to present a signal that can be recorded and quantified, for example , the antibody coated on the detection reaction zone 5 captures the target in the sample, and the target is combined with the detection antibody and is immobilized in the detection reaction zone 5, and the HRP enzyme catalyzed substrate labeled on the detection antibody is in the detection reaction zone 5 Formation of dark spots, the depth of dark spots is positively or negatively correlated with the target to be tested.

In order to read the test results more quickly and conveniently, a concentration comparison ruler 18 can be set on the housing member 2 as a reference, and the concentration of the target object to be tested can be read by referring to the concentration comparison ruler 18 according to the coloring depth of the substrate. The concentration comparison ruler 18 can be formed in the following manner: use standard samples of different concentrations to record the color state of the substance to be tested at each concentration, draw a gradient concentration ruler diagram through, for example, a computer, and finally form a concentration comparison ruler after debugging. And it is printed on the casing member 2 of the detection device.

Example

The following takes the detection of ectopic pregnancy as an example to further describe in detail the practical application of the detection device of the present invention.

According to the existing technology and the research data of our laboratory, this embodiment selects human chorionic gonadotropin (β-HCG), progesterone (P), estradiol (E2) and vascular endothelial growth factor (VEGF) at the same time As a joint inspection index of ectopic pregnancy.

(1) Coating of capture antibody

Four partitions are set in the detection reaction zone 5 located at one end of the detection core member 1, which are respectively coated with capture antibodies against β-HCG, P, E2 and VEGF, with 0.05M carbonate buffer (specific formula: 0.014M Na ₂ CO ₃ , 0.035M NaHCO ₃ , pH 9.6) Dilute the monoclonal antibodies against β-HCG, P, E2 and VEGF to a concentration of 10ug/ml respectively, drop them in the corresponding partitions, spot 10ul of each antibody, and Adsorb for 16 hours at 4°C and 80% humidity, and then immerse the entire detection reaction zone 5 in 0.01M TBS solution containing 2% BSA (specific formula: 0.01M Tris, 0.15M NaCl, pH 7.5) to seal 2 hours, then dry for later use.

(2) Perfusion of various buffer solutions

After the coating of the capture antibody is completed, the detection core member 1 is inserted into the housing member 2, and an annular cavity is formed between the detection core member 1 and the sealing ring (sealing member 9) on the housing member 2. From the top of the detection core member 1 In the direction of the detection core handle 3 are the reaction chamber 11 , the first washing liquid chamber 12 , the detection chamber 13 , the second washing liquid chamber 14 and the substrate chamber 15 . Each cavity is perfused with corresponding liquid through respective perfusion holes 6 . 0.05M TBST (specific formula: 0.05M Tris, 0.15M NaCl, 0.05% Tween-20, pH 7.4) was perfused into the first washing liquid chamber 12 and the second washing liquid chamber 14 as the washing liquid. The liquid perfused in the detection chamber 13 is HRP-labeled anti-β-HCG detection antibody working solution, HRP-labeled anti-β-HCG detection antibody working solution, HRP-labeled E2 coupling complex working solution and HRP-labeled P-coupling complex The above working solution is prepared by mixing HRP-labeled anti-β-HCG detection antibody, HRP-labeled anti-β-HCG detection antibody, HRP-labeled E2-coupled complex and HRP-labeled P-coupled complex with 0.05M TBS buffer solution (specific formulation: 0.05M Tris, 0.15M NaCl, pH 7.4) was adjusted to a concentration of 1mg/ml, and a final concentration of 1% bovine serum albumin (BSA) was added as a protective agent and final concentration 1‰ of Proclin300 prepared as a preservative. The substrate cavity 15 is perfused with a precipitation-type one-component TMB solution (manufactured by Fuinde Technology (Wuhan) Co., Ltd., product number is ELS0010P). After perfusion, cover the perfusion hole cap 7 on the perfusion hole 6, and cover the sample inlet cap 17 on the sample inlet 16. So far, the ectopic pregnancy immunoassay device is assembled and stored at 4°C for future use.

(3) Preparation of Concentration Comparison Ruler

Detect the standard concentration samples of β-HCG, P, E2 and VEGF at different concentrations, record the color state at each concentration, and measure the different analytes corresponding to different color depths (β-HCG, P, E2 and VEGF) Using a computer to draw a gradient concentration scale, after debugging, a concentration comparison scale is finally formed, which is printed on the shell member 2 of the detection device for detecting ectopic pregnancy.

(4) Detection of ectopic pregnancy and normal pregnancy blood samples

The Department of Obstetrics and Gynecology of Xiangfan Central Hospital assisted in the collection of serum from patients with normal pregnancy and confirmed ectopic pregnancy in the late period. The pregnancy time was 40±5 days, and 40 serum samples were collected from each patient. The substances to be tested (β-HCG, P, E2 and VEGF) in these serums have all been determined by radioimmunoassay, and the details are shown in Table 1.

Remove the inlet cap of the detection device, immerse the inlet into the sample to be tested, draw 100ul of the blood sample to be tested from the inlet 16, pull the detection core member 1 until its indicator line 4 moves to the sealing ring of the reaction chamber 11 At this time, the sample to be tested is sucked into the reaction chamber 11 .

After absorbing the sample to be tested, adjust the direction of the detection device so that the sample inlet 16 is vertically upward, and the different capture antibodies on the detection reaction zone 5 start to "capture" the corresponding target objects in the sample liquid to be tested, and the reaction time is 5 minutes , the whole reaction process can gently shake the whole detection device.

After the reaction is completed, the detection core member 1 is pulled to the detection reaction area 5 and enters the first washing liquid chamber 12. During this process, the detection reaction area 5 passes through a water-absorbing member 8, and the remaining sample liquid is blotted dry and enters the first washing liquid chamber 12. After washing the liquid chamber 12, stay in the first washing liquid chamber 12 for 2 minutes to soak and wash. Next, the detection reaction area 5 passes through the water-absorbing member 8, and the residual lotion is blotted dry, and then enters the detection chamber 13, where the reaction takes 5 minutes. The entire detection device can be shaken gently during the entire reaction process. During this process, if the double-sandwich method is used to detect the analyte and the detection area has captured the analyte in the sample, then each HRP-labeled detection antibody in the detection chamber 13 will bind to the analyte respectively. If the principle of the competition method is used to detect the analyte, and the detection area has captured the analyte in the sample, then each HRP-labeled conjugate in the detection chamber 13 can only interact with the capture antibody that is not occupied by the target analyte. empty "binding sites" for binding.

After the reaction in the detection chamber 13 is finished, continue to pull the detection core member 1 to make the detection reaction area 5 enter the second washing liquid chamber 14. During this process, the detection reaction area 5 passes through the water-absorbing member 8, and the residual detection liquid is sucked dry. After soaking and washing for 2 minutes in the second washing liquid chamber 14. Afterwards, it moves through the water-absorbing member 8, and the residual lotion is sucked dry. Then enter the fifth chamber 15 and react in the substrate chamber 15 for 5 minutes. If there is a target in the sample and the target is suitable for detection by the double-antibody sandwich method, then the target antibody on the detection reaction zone captures the target in the sample, and the target is combined with the detection antibody to be fixed in the detection zone, and the detection antibody is labeled with HRP enzyme, HRP enzyme catalyzes the substrate to form dark spots on the detection reaction zone 5, and the depth of dark spots is positively or negatively correlated with the concentration of the target to be tested; if there is a target in the sample and the target is suitable for the competition method Detection, then the antibody on the detection reaction zone captures the target in the sample, the HRP-labeled target conjugate binds to the site on the capture antibody that is not bound by the free target, and the HRP enzyme catalyzes the substrate to form in the detection reaction zone 5 Deposited stains, the depth of the deposited stains are negatively correlated with the concentration of the target to be detected. Read out the concentration of the target substance to be tested according to the coloring depth of the substrate and the concentration ratio ruler.

Table 1 Test results of ectopic pregnancy detection device

(Remarks: The critical standard is formulated based on the existing technology and the test data of our laboratory. The samples selected by this laboratory are the blood of about 40 days (40d±5d) of ectopic pregnancy and normal pregnancy.)

The technical solutions of the present invention have been described in detail above in conjunction with specific implementations and examples, but the present invention is not limited thereto. On the premise of realizing the object of the present invention, those skilled in the art can make various changes and modifications to the present invention.

Claims (7)

1. A high-throughput immunoassay device, comprising: A detection core member (1), a detection reaction zone (5) is provided on the outer wall near one end of the detection core member (1); and A casing member (2), a sample inlet (16) is provided at one end of the casing member (2), and a plurality of pairs of perfusion holes (6) are arranged on the side wall of the casing member (2), the casing The inner wall of the member (2) is provided with multiple pairs of sealing members (9), and a water-absorbing member (8) is arranged between each pair of the sealing members (9), When assembled, the detection core member (1) is inserted into the housing member (2), so that the one end of the detection core member (1) is adjacent to the one end of the housing member (2), After the assembly is completed, a plurality of independent cavity components are formed between the outer wall of the detection core component (1), the inner wall of the housing component (2) and the sealing component (9), each of the cavity components The components communicate with a pair of perfusion holes (6) respectively. 2. The high-throughput immunoassay device according to claim 1, characterized in that, the detection reaction zone (5) includes a plurality of reaction zones, and the multiple reaction zones are along the detection core member (1) The circumferential intervals are evenly distributed, and the multiple reaction zones can simultaneously apply the same or different chemical components according to the needs of immunoassays, for capturing the object to be tested or chemically reacting with the object to be tested and presenting can be recorded signal of. 3. The high-throughput immunoassay device according to claim 1, characterized in that, between the outer wall of the detection core member (1), the inner wall of the housing member (2) and the sealing member (9) Five independent cavity components are formed between them. 4. The high-throughput immunoassay device according to claim 1, characterized in that, the other end of the detection core member (1) is provided with a handle member (3), and is operated by holding the handle member (3) ) to push the detection core member (1) into the housing member (2) or pull it out from the housing member (2). 5. The high-throughput immunoassay device according to claim 1, characterized in that, the other end of the housing member (2) is provided with a housing handle (10), which can be operated by holding the housing handle (10) To stabilize the shell member (2). 6. The high-throughput immunoassay device according to claim 1, characterized in that it further comprises a perfusion hole cap (7), and the perfusion hole cap (7) is used to close the perfusion hole (6). 7. The high-throughput immunoassay device according to claim 1, characterized in that, it also comprises a concentration comparison ruler (18), and the concentration comparison ruler (18) is arranged on the outer wall of the housing member (2) for use in It is used as a reference for detecting signals and reading the concentration value of the analyte.