CN118033130B - Surfaces, microplates, assemblies, methods and applications for simultaneous detection of HIV, syphilis and hepatitis C - Google Patents

Abstract

The embodiment discloses a surface, a microplate, a component, a method and application for detecting AIDS, syphilis and hepatitis C. The surface is coated with LgBiT subunit and SmBit subunit labeled HIV-1p24 antibody, lgBiT subunit and SmBit subunit labeled treponema pallidum antibody and LgBiT subunit and SmBit subunit labeled hepatitis C virus core antibody. Based on the antibody with chemical markers on the coating surface, HIV, TP, HCV viruses in the same sample can be detected, the coating surface is not required to be washed, the operation steps are simple and convenient, and the method is suitable for a conventional full-automatic enzyme immunoassay process. Compared with ELISA method, the detection method using the coated surface has smaller error and higher efficiency.

Description

Surfaces, microplates, components, methods and applications for simultaneous detection of HIV, syphilis and hepatitis C

Technical Field

The present application relates to the technical field of detection of AIDS, syphilis and hepatitis C virus, and specifically to surfaces, microplates, components, methods and applications for simultaneous detection of AIDS, syphilis and hepatitis C.

Background technique

At present, AIDS, syphilis and hepatitis C are all infectious diseases with high harm in clinical practice. Common detection methods are mainly divided into two categories, namely serological detection and nucleic acid detection. Serological detection includes enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), colloidal gold immunoassay, Western Blot (WB), recombinant immunoblot (RIBA), Treponemapallidumparticle assay (TPPA), and toluidine red unheated serum test (TRUST). Nucleic acid detection is mainly based on real-time fluorescence quantitative polymerase chain reaction (PCR) technology. Through the above analysis, each detection method has its own shortcomings and cannot achieve accurate detection while reducing costs.

Summary of the invention

Based on this, the present application provides a coated surface, a microporous reaction plate, a coating method, a kit, a method for detecting in vitro samples and applications for simultaneously detecting AIDS, syphilis and hepatitis C.

In a first aspect, the embodiments disclose a coated surface, wherein the surface is coated with HIV-1p24 antibody labeled with LgBiT subunit, Treponema pallidum antibody labeled with LgBiT subunit, and hepatitis C virus core antibody labeled with LgBiT subunit.

Based on the chemically labeled antibodies on the coated surface, the three viruses HIV, TP, and HCV can be detected in the same sample without washing the coated surface. The operation steps are simple and suitable for conventional fully automatic enzyme immunoassay process. Moreover, compared with the ELISA method, the detection method using the coated surface has smaller errors and higher efficiency.

In some embodiments of the first aspect, the HIV-1p24 antibody labeled with the LgBiT subunit, the Treponema pallidum antibody labeled with the LgBiT subunit, and the HCV core antibody labeled with the LgBiT subunit are all coupled to fluorescent microspheres and then coated on the coating surface. In some embodiments, after the labeled antibody is labeled with the LgBit subunit, the HIV-1p24 antibody, Treponema pallidum antibody, and HCV core antibody are coupled with red, yellow, and blue fluorescent microspheres, respectively, to achieve staining of the three antibodies.

For example, the preparation of antibody solution and surface coating process include: reacting the antibody and fluorescent microspheres (containing LgBiT subunits) at a mass ratio of 4 to 6:300 in a reaction buffer with a pH of 8.0 to 8.5 for 1 to 2 hours to obtain antibodies coupled to fluorescent microspheres; using the antibodies coupled to fluorescent microspheres to coat 96-well plates: dilute the three antibodies coupled to fluorescent microspheres with a CBS coating solution at pH 9.6 to 6 μg/mL and add them to the enzyme-labeled wells, add 40 μL of solution to each well, and add a total of 120 μL to each well to bind to the solid phase carrier, and coat at room temperature, 4°C or 37°C overnight. Discard the coating solution (pat dry vigorously and then dry with dust-free paper), wash the plate 3 times with a 1×PBST washing solution at pH 7.2-7.4 (fill each well), 3 min to 5 min each time. Add 250 μL of blocking solution (1% BSA) to each well, seal at 37°C for 2 hours to dry, and seal for use.

In the second aspect, the embodiment discloses a microporous reaction plate. The microporous reaction plate includes a plate body having at least one open surface, and at least one micropore having the coated surface described in the first aspect formed inwardly on the open surface. The microporous reaction plate is various types of ELISA plates, cell reaction plates, 24-well plates, 48-well plates or 96-well plates. Among them, the 96-well plate is a 96-well opaque microporous plate made of polystyrene, which is white or black and opaque. Each plate has 96 micropores and can be disassembled.

In a third aspect, the embodiment discloses a surface coating method, which comprises simultaneously coupling HIV-1p24 antibody labeled with LgBiT subunit, Treponema pallidum antibody labeled with LgBiT subunit and hepatitis C virus core antibody labeled with LgBiT subunit to the surface.

In a fourth aspect, an embodiment discloses a coating method for a microporous reaction plate. The coating method comprises preparing a first antibody solution containing HIV-1p24 antibody labeled with LgBiT subunit, a second antibody solution containing Treponema pallidum antibody labeled with LgBiT subunit, and a third antibody solution containing hepatitis C virus core antibody labeled with LgBiT subunit; and simultaneously adding the first antibody solution, the second antibody solution, and the third antibody solution to the micropores of the microporous reaction plate as claimed in claim 2 for coating.

In some embodiments of the fourth aspect, the concentration of HIV-1p24 antibody labeled with LgBiT subunit in the first antibody solution is 1 to 10 μg/mL, optionally 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL or 10 μg/mL.

In some embodiments of the fourth aspect, the concentration of the Treponema pallidum antibody labeled with the LgBiT subunit in the second antibody solution is 1 to 10 μg/mL, optionally 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL or 10 μg/mL.

In some embodiments of the fourth aspect, the concentration of the hepatitis C virus core antibody labeled with the LgBiT subunit in the second antibody solution is 1 to 10 μg/mL, optionally 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL or 10 μg/mL.

In some embodiments of the fourth aspect, during the coating process, the volume ratio of the first antibody solution, the second antibody solution and the third antibody solution in the microwell is (1-5):(1-5):(1-5), optionally 1:1:1, 1:2:1, 1:2:2, 1:2:3, 1:2:4, 1:2:5, 1:3:1, 1:3:2, 1:3:3, 1:3:4, 1:3:5, 1:4:1, 1:4:2, 1:4:3, 1:4:4, 1:4:5, 1:5:1, 1:5:2, 1:5:3, 1:5:4, 1:5:5, 2:1:1 , 2:2:1, 2:3:1, 2:4:1, 2:5:1, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 3:1:1, 3:2:1, 3:3:1, 3:4:1, 3:5:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 4:1:1, 4:2:1, 4:3:1, 4:4:1, 4:5:1, 4:1:2, 4:1:3, 4:1:4, 4:1:5, 5:1:1, 5:2:1, 5:3:1, 5:4:1, 5:5:1, 5:1:2, 5:1:3, 5:1:4, or 5:1:5.

In a fifth aspect, an embodiment discloses a component for simultaneously detecting AIDS, syphilis and hepatitis C. The component comprises the coated surface described in the first aspect and/or the microporous reaction plate described in the second aspect.

In some embodiments of the fifth aspect, the kit further comprises a fluorescent reaction substrate reagent.

In some embodiments of the fifth aspect, the assembly further comprises a filter. The filter may cover the microwell, and the filter is used to form a fluorescence effect excitation wavelength corresponding to the coating groove in the same microwell.

In the sixth aspect, the embodiment discloses a method for detecting an in vitro sample containing at least one of AIDS, syphilis and hepatitis C. The method comprises: obtaining the coated surface described in the first aspect; contacting the in vitro sample with the coated surface; and detecting the in vitro sample according to the optical signal of the in vitro sample contacting the coated surface. The method can achieve high-throughput detection of serum samples for HIV, TP, and HCV. Not only is the detection method sensitive and has a wide linear range, but it can also be completed in just 30-70 minutes, greatly saving experimental time; no new equipment or devices are required, greatly reducing experimental costs; no washing steps are required, reducing the possibility of human errors.

In some embodiments of the fifth aspect, the method further comprises covering the microwell with filters corresponding to two colors other than the detection virus antibody staining. For example, when detecting HIV, yellow and blue filters are used to cover the surface to filter out the blue and yellow fluorescence stimulated by the detection of hepatitis C and syphilis. The red fluorescence is observed by naked eyes to qualitatively detect positive and negative.

In a sixth aspect, the embodiments disclose the use of the coated surface described in the first aspect, the microporous reaction plate described in the second aspect, or the kit described in the fourth aspect in the preparation of products for detecting AIDS, syphilis and/or hepatitis C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the coating surface structure provided in the embodiment.

FIG. 2 is a schematic diagram showing a micropore covered by a filter provided in an embodiment.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail in conjunction with the examples below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

Currently, there is no detection kit for the above three viruses constructed using lumit technology on the market. The coated surface, microporous reaction plate, coating method, kit, method and application of detecting in vitro samples for detecting AIDS, syphilis and hepatitis C simultaneously provided by the embodiments of the present application are superior to ELISA kits in terms of detection time and process.

In some embodiments, quantitative detection of three viruses, HIV, TP, and HCV, can be achieved. For example, an antibody coated with a LgBiT subunit is coated on a solid surface. When an in vitro sample is added, the antibody captures the corresponding antigen in the sample, and then a detection antibody labeled with a SmBit subunit is added. The antibody labeled with the SmBit subunit binds to the captured antigen, and the SmBit subunit combines with the LgBiT subunit of the coated antibody to form biological activity. A specific detection substrate is added to produce a fluorescent reaction, and a quantitative reaction can be performed by detecting the fluorescent reaction. Only after the detection antibody is added and combined with the captured antigen, the subunit of the detection antibody and the subunit of the coated antibody are combined, and the enzyme effect can react with the substrate to produce a fluorescent reaction. At this time, according to the virus to be detected, the corresponding detection antibody is added, and the presence of the corresponding virus can be detected, and the load of the corresponding virus can be quantitatively detected according to the fluorescent reaction.

In some embodiments, qualitative detection of three viruses, HIV, TP, and HCV, can be achieved. For example, HIV-1p24 antibodies are stained and labeled with red fluorescent microspheres, hepatitis C virus core antibodies are stained and labeled with blue fluorescent microspheres, and syphilis spirochetes antibodies are stained and labeled with yellow fluorescent microspheres. Then, antibodies labeled with three different fluorescent microspheres are coated in an enzyme-labeled plate, and in vitro samples are added so that the corresponding antigens therein are captured by antibodies. Then, three detection antibodies (antibodies labeled with corresponding SmBit subunits) are added, substrates are added, and incubated to produce a fluorescent effect. Since the coated antibodies are dyed with different colors of fluorescent microspheres in advance, different viruses correspond to different colors of fluorescence. As shown in Figures 1 and 2, taking HIV as an example, one of the enzyme-labeled wells is covered with yellow and blue filters. At this time, only the red light emitted by the antibody detecting HIV-1p24 can be observed. The positive and negative of the sample are qualitatively detected by observing the color of the enzyme-labeled well after the filter is covered, and the intensity of the positive reaction is judged according to the depth of the color. Finally, the intensity of luminescence is detected by an ELISA instrument to quantitatively detect the relationship between viral load and virus load. In order to exclude the influence of the fluorescence reaction on the detection of a single virus during the detection of the other two viruses, only a single detection antibody can be added to the sample in another ELISA well to detect the intensity of its fluorescence effect.

The following examples will be described in more detail, wherein the antibodies involved are:

Anti-HIV1 p24 antibody (ab63913), Anti-SynpHilin 1 antibody (ab217373), Anti-HCVsubtype 1bNS5B antibody [10D6] (ab100895), the manufacturer is abcam. LgBiT subunit and SmBit subunit are from Promega. Fluorescent microspheres, Beijing Biotech Biotechnology Co., Ltd.

Example 1

1380 serum samples were collected from high-risk groups in entertainment venues in a certain area for serological index testing, namely TP testing; the subjects have signed informed consent. Instruments and reagents used: fully automatic enzyme immunoassay analyzer; micropipette; 37°C constant temperature box; LumitTM immunoassay labeling kit and detection reagents. Serum samples: 1380 serum samples from high-risk groups in a certain area in 2023. Collection of serum samples: 5 ml of human whole blood was collected on an empty stomach in the early morning, placed in a vacuum negative pressure tube, without adding anticoagulant, and temporarily stored in a 4°C refrigerator for 2 hours.

Sample preparation: Take out the serum sample from the refrigerator, centrifuge at 3000r for 10min, and take the supernatant.

Steps:

1. Prepare the ELISA plate

2. Fluorescence staining

The coated antibodies for HIV virus detection were pre-stained with red fluorescent microspheres, the Treponema pallidum antibodies were stained with yellow fluorescent microspheres, and the hepatitis C core antibodies were stained with blue fluorescent microspheres.

3. Coating

Three LgBiT subunit labeled antibodies coupled with fluorescent microspheres were diluted to 6μg/mL with CBS coating solution at pH 9.6 and added to the enzyme-labeled wells. 40μL of solution was added to each well, and a total of 120μL was added to each well to bind to the solid phase carrier. Coating was carried out at room temperature, 4°C or 37°C overnight. Discard the coating solution (pat dry and then dry with dust-free paper), wash the plate 3 times with 1×PBST washing solution at pH 7.2-7.4 (fill each well), 3min to 5min each time. Add 250μL of blocking solution (1% BSA) to each well, block at 37°C for 2h, block with BSA blocking solution, dry, and seal for use.

4. Sample addition: Take appropriate amount of the sample to be tested, positive control, and negative control and add them to the corresponding microwells.

5. Add detection antibodies: Add SmBit subunit labeled antibodies and three detection antibodies at a concentration of 8 μg/mL for each antibody.

6. Color development: Add Lumit detection reagent to the corresponding area of the microwell and incubate for 30 minutes.

7. Determination: Use the corresponding filter to filter out the other two colors of fluorescence, leaving only one color of fluorescence, to qualitatively determine the positive result; and judge the strength of the positive result based on the depth of the solution color after passing through the filter. Use an ELISA reader with a single wavelength of 450nm or dual wavelengths of 450nm/630nm to determine the OD value of each well. (When using a single wavelength for determination, a blank control well must be used to adjust to zero) and record the results.

8. Qualitative detection: Take TP detection as an example. The coating process is as above. Add 120μL of patient sample to the coated enzyme-labeled wells, and then add three detection antibodies labeled with SmBit subunits, 40μL of each. Add Lumit detection reagent to the corresponding area of the microwell and incubate for 30 minutes. Cover with red and blue filters to observe whether the color of the solution is yellow to quantitatively detect the positivity of TP virus. For HIV virus detection, change the filter to yellow and blue. HCV and so on. Each well uses a combination of red and yellow, red and blue, and yellow and blue filters for filter detection.

9. Quantitative detection: Take the detection of TP as an example. The coating process is as above. Add 120μL of patient samples to the coated ELISA wells, and then add three detection antibodies labeled with SmBit subunits, 40μL of each. Add Lumit detection reagent to the corresponding area of the microwell and incubate for 30min. Use the ELISA wells filtered by all three color filters as the control group, cover the red and blue filters, and use the ELISA reader to measure the OD value of each well at a single wavelength of 450nm or dual wavelengths of 450nm/630nm. The OD value can be read directly on the ELISA reader. Use the ELISA wells filtered by all three color filters as the control group (when using a single wavelength for determination, the blank control well needs to be zeroed), and record the results. For HIV virus detection, the filters are replaced with yellow and blue. HCV and so on. Each well uses a combination of red and yellow, red and blue, and yellow and blue filters for filter detection.

10. Result determination: positive samples are screened out according to the preset reference value. The same well is considered double positive if it displays different colors after filtering light with different combinations of filters. For example, HIV and HCV are double positive, when the same well is filtered with yellow and blue filters and displays red fluorescence, and then covered with red and yellow filters and displays blue fluorescence.

Table 1 Example 1 Positive sample results

Number of HIV-positive samples 193 HIV positive rate 13.98% Number of HCV-positive samples 295 HCV positive sample rate 21.38% Number of TP positive samples 243 TP positive sample rate 17.61% HIV and HCV dual positivity rate 0 HCV and TP dual positive rate 5.51% HCV and TP dual positive rate 0

Calculation of double positive samples in Table 1: Double positive rate is the number of wells that show the corresponding color after covering with two filters/total number of samples tested. For example, taking TP and HCV double positive as an example, TP and HCV double positive means that the same well that shows yellow fluorescence after filtering with red and blue filters and then shows blue fluorescence after covering with red and yellow filters is TP and HCV double positive 71 as the numerator, and the total number of samples tested 1380 as the denominator.

The test results of Example 1 are shown in Table 1: 1380 sera, 193 micropores showed HIV positive, 295 micropores showed HCV positive, and 243 micropores showed TP positive. 76 micropores showed double positivity. In Example 1, different filter combinations were used to find that 76 samples could simultaneously show yellow fluorescence and blue fluorescence, indicating that the 76 samples were simultaneously detected to be positive for hepatitis C and TP viruses. Therefore, the method provided by the present application is not only suitable for simultaneous screening of a large number of HIV, HCV, and TP patients, but can also be used for initial screening in exploring the correlation between HIV and HCV and TP diseases, and for statistically analyzing the probability of having two or more diseases at the same time.

Comparative Example 1: Single Detection of HIV

1380 serum samples were collected from high-risk groups in entertainment venues in a certain area for serological index testing, i.e. HIV testing; the subjects have signed informed consent. Instruments and reagents used: fully automatic enzyme immunoassay analyzer; micropipette; 37°C constant temperature box; LumitTM immunoassay labeling kit and detection reagents. Serum samples: 1380 serum samples from high-risk groups in a certain area in 2023. Collection of serum samples: 5 ml of whole human blood was collected on an empty stomach in the early morning, placed in a vacuum negative pressure tube, without adding anticoagulant, and temporarily stored in a 4°C refrigerator for 2 hours.

Sample preparation: Take out the serum sample from the refrigerator, centrifuge at 3000r for 10min, and take the supernatant.

Steps:

1. Numbering: Take the required number of microporous strips and fix them on the bracket, and number them in sequence.

2. Sample addition: Take appropriate amount of the positive control and negative control samples to be tested and add them to the corresponding wells.

3. Color development: Add Lumit detection reagent and incubate for 30 minutes.

4. Determination: Use an ELISA reader with a single wavelength of 450nm or dual wavelengths of 450nm/630nm to determine the OD value of each well. (When using a single wavelength for determination, a blank control well must be used for zeroing) and record the results.

5. Result determination: screen out positive samples based on preset reference values.

Since this experiment uses a common 96-well plate, with 1 blank control, 1 negative control and two positive controls; therefore, each plate can only detect 92 samples. Therefore, there are 92 samples in a group, a total of 15 groups, or 15 plates. The screening results are shown in Table 1. There are 1380 sera and 206 positive samples. Therefore, the positive rate of HIV-Lumit single antigen detection is 14.93% (206/1380).

Comparative Example 2: Single Detection of HCV

1380 serum samples were collected from high-risk people in entertainment venues in a certain area for serological index testing, namely HCV testing; the subjects had signed informed consent.

Instruments and reagents used: fully automatic enzyme immunoassay analyzer; micropipette; 37°C incubator; LumitTM immunoassay labeling kit and detection reagents. Serum samples: 1,380 serum samples from high-risk populations in a certain area in 2023. Collection of serum samples: 5 ml of whole blood was collected in the early morning on an empty stomach, placed in a vacuum negative pressure tube, without anticoagulant, and stored in a 4°C refrigerator for 2 hours.

Sample preparation: Take out the serum sample from the refrigerator, centrifuge at 3000r for 10min, and take the supernatant.

Steps:

1. Numbering: Take the required number of microporous strips and fix them on the bracket, and number them in sequence.

2. Sample addition: Take appropriate amount of the positive control and negative control samples to be tested and add them to the corresponding wells.

3. Color development: Add Lumit detection reagent and incubate for 30 minutes.

4. Determination: Use an ELISA reader with a single wavelength of 450nm or dual wavelengths of 450nm/630nm to determine the OD value of each well. (When using a single wavelength for determination, a blank control well must be used for zeroing) and record the results.

5. Result determination: screen out positive samples based on preset reference values.

This experiment uses a common 96-well plate, with 1 blank control, 1 negative control and two positive controls; therefore, each plate can only detect 92 samples. Therefore, there are 92 samples in a group, a total of 15 groups, or 15 plates. The test results are shown in Table 1. Among the 1380 sera, 319 were positive samples, so the positive rate of HCV-LUMIT single antigen detection was 23.12% (319/1380).

Comparative Example 3: Single Detection of TP

1380 serum samples were collected from high-risk groups in entertainment venues in a certain area for serological index testing, namely TP testing; the subjects have signed informed consent. Instruments and reagents used: fully automatic enzyme immunoassay analyzer; micropipette; 37°C constant temperature box; LumitTM immunoassay labeling kit and detection reagents. Serum samples: 1380 serum samples from high-risk groups in a certain area in 2023. Collection of serum samples: 5 ml of human whole blood was collected on an empty stomach in the early morning, placed in a vacuum negative pressure tube, without adding anticoagulant, and temporarily stored in a 4°C refrigerator for 2 hours.

Sample preparation: Take out the serum sample from the refrigerator, centrifuge at 3000r for 10min, and take the supernatant.

Steps:

1. Numbering: Take the required number of microporous strips and fix them on the bracket, and number them in sequence.

2. Sample addition: Take appropriate amount of the positive control and negative control samples to be tested and add them to the corresponding wells.

3. Color development: Add Lumit detection reagent and incubate for 30 minutes.

4. Determination: Use an ELISA reader with a single wavelength of 450nm or dual wavelengths of 450nm/630nm to determine the OD value of each well. (When using a single wavelength for determination, a blank control well must be used for zeroing) and record the results.

5. Result determination: screen out positive samples based on preset reference values.

This test consists of 92 serum samples per group, with a total of 15 groups or 15 plates.

The results of blood testing by ELISA, colloidal gold and WB were used as gold standards, respectively, and the false positive rate, specificity and sensitivity of the test results of the comparative example and the comparative example were compared. The detection effects of the comparative example and the comparative example were obtained as shown in Table 2 below.

Table 2

In Table 2, sensitivity = number of true positives/(number of true positives + number of false negatives) × 100%; specificity = number of true negatives/(number of true negatives + number of false positives) × 100%.

Positive rate: Positive rate = number of positive samples/total number of samples × 100%

False positive rate: False positive rate = number of false positives / number of gold standard negatives × 100%

The OD value can be read on the microplate reader, and the microplate wells covered with three color filters are used as the control group. The OD value is proportional to the solution concentration. The concentration of the corresponding microwell can be calculated based on the OD value of each microwell for quantitative detection.

As can be seen from Table 2, the false positive rate of Example 1 is lower than that of Comparative Examples 1, 2 and 3, the sensitivity is higher than that of Comparative Examples 1, 2 and 3, and the specificity of Example 1 is higher than that of Comparative Example 2. It can be seen that the surface, microplate, assembly, method and application for simultaneous detection of AIDS, syphilis and hepatitis C provided by the present application have a lower false positive rate, higher sensitivity and specificity.

The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims (5)

1. An assembly for simultaneously detecting aids, syphilis and hepatitis c comprising:

A fluorogenic substrate reagent;

A filter comprising a red filter, a yellow filter, and a blue filter;

a detection antibody which is a SmBit subunit-labeled HIV-1p24 antibody, a SmBit subunit-labeled treponema pallidum antibody, and a SmBit subunit-labeled hepatitis c virus core antibody; and

A microporous reaction plate;

Wherein, the micropore reaction plate includes:

A plate body having at least one open surface;

at least one microwell formed in the open surface invagination having a coated surface coated simultaneously with a red fluorescent microsphere-labeled HIV-1p24 antibody containing LgBiT subunits, a yellow fluorescent microsphere-labeled treponema pallidum antibody containing LgBiT subunits, and a blue fluorescent microsphere-labeled hepatitis c virus core antibody containing LgBiT subunits;

The red filter is used for filtering red fluorescence excited when detecting the HIV in the same micropore, the yellow filter is used for filtering yellow fluorescence excited when detecting the syphilis in the same micropore, and the blue filter is used for filtering blue fluorescence excited when detecting the hepatitis C in the same micropore.

2. The assembly of claim 1, wherein the coating method of the micro-porous reaction plate comprises:

Preparing a first antibody solution of a red fluorescent microsphere marked HIV-1p24 antibody containing LgBiT subunits, a second antibody solution of a yellow fluorescent microsphere marked treponema pallidum antibody containing LgBiT subunits and a third antibody solution of a blue fluorescent microsphere marked hepatitis C virus core antibody containing LgBiT subunits;

The first antibody solution, the second antibody solution and the third antibody solution are simultaneously added to the microwells of the microwell reaction plate of claim 1 for coating.

3. The assembly of claim 2, wherein the concentration of the red fluorescent microsphere-labeled HIV-1p24 antibody containing LgBiT subunits in the first antibody solution is 1-10 μg/mL;

the concentration of the yellow fluorescent microsphere marked treponema pallidum antibody containing LgBiT subunits in the second antibody solution is 1-10 mug/mL;

The concentration of the blue fluorescent microsphere marked hepatitis C virus core antibody containing LgBiT subunits in the third antibody solution is 1-10 mug/mL;

in the coating process, the volume ratio of the first antibody solution to the second antibody solution to the third antibody solution in the micropores is (1-5): 1-5.

4. A method for detecting an in vitro sample containing at least one of aids, syphilis and hepatitis c for non-diagnostic purposes comprising:

obtaining the assembly of claim 1;

contacting the in vitro sample with a coated surface in the assembly;

Adding the detection antibody of claim 1;

adding the fluorogenic substrate reagent;

Covering the microwells of the module with at least one of the red filter for filtering red fluorescence excited when detecting aids virus in the same microwell, the yellow filter for filtering yellow fluorescence excited when detecting syphilis in the same microwell, and the blue filter for filtering blue fluorescence excited when detecting hepatitis c in the same microwell; and

Detecting the in vitro sample according to a fluorescent signal generated after the in vitro sample is contacted with the coated surface and a detection antibody;

Wherein, when detecting the HIV, the yellow filter and the blue filter are covered on the micropores;

When syphilis is detected, the red filter and the blue filter are covered on the micropores;

and when detecting the hepatitis C, covering the red filter and the yellow filter on the micropores.

5. Use of the module of claim 1 for the manufacture of a product for detecting aids, syphilis and/or hepatitis c.