Quadruple immune complex, application thereof and immunodetection kit

Abstract

The invention provides a quadruple immune complex, application thereof and an immunodetection kit, and relates to the field of molecular biology. The quadruple immune complex comprises a biotin-labeled nucleic acid molecule, a biotin-labeled detection body and a streptavidin magnetic bead, the surface of the streptavidin magnetic bead is provided with a streptavidin binding site, and the biotin-labeled detection body forms the biotin-labeled immune complex after identifying and binding a detected body. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to a streptavidin binding site. When the content of the detected body is high, the biotin-labeled immune complex combined with the streptavidin binding site is high in quantity, so that the quantity of the biotin-labeled nucleic acid molecules combined with the streptavidin binding site on the surface of the magnetic bead is low, and the quantity of the detected body is indirectly reflected by detecting the quantity of the nucleic acid molecules on the surface of the magnetic bead; the qualitative or quantitative detection of the detected body is realized.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular, to a quadruple immune complex, its application, and an immunoassay kit. Background Art

[0002] At present, for the detection of biomarkers such as small molecule antigens, hormones, and proteins, the main detection methods used are enzyme-linked immunosorbent assay and chemiluminescence in labeled immunoassays. In traditional labeled immunoassays represented by chemiluminescence and enzyme-linked immunosorbent assay, enzymes are directly labeled on antibodies, and the corresponding substrates of the enzymes are used to generate color or luminescence signals. There are some disadvantages in this method: the stability of enzyme molecules is poor and the steric hindrance is large, resulting in low detection sensitivity. Summary of the Invention

[0003] The problem to be solved by the present invention is how to solve the problem of low detection sensitivity of biomarker detection means.

[0004] To solve the above problems, the present invention provides a quadruple immune complex, its application, and an immunoassay kit.

[0005] In a first aspect, the present invention provides an immune complex, comprising a biotin-labeled nucleic acid molecule, a biotin-labeled detector, and streptavidin magnetic beads. The surface of the streptavidin magnetic beads has streptavidin binding sites. After the biotin-labeled detector recognizes and binds to the analyte, a biotin-labeled immune complex is formed. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding sites.

[0006] Optionally, the nucleic acid molecule is a double-stranded DNA molecule comprising a template strand and a coding strand, and the sequences are respectively: Template strand: 5 ， -CAGTTTACTAGTGCCATTTGTTCAGTG-3 ， (SEQ ID NO.1); Coding strand: 5 ， -GTCAAATGATCACGGTAAACAAGTCAC-3 ， (SEQ ID NO.2).

[0007] Optionally, the analyte is hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein, or carcinoembryonic antigen, and the detector is the corresponding hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody, or carcinoembryonic antigen antibody; or, the analyte is hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody, or carcinoembryonic antigen antibody, and the detector is the corresponding hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein, or carcinoembryonic antigen.

[0008] Optionally, the binding ratio of biotin to streptavidin magnetic beads is 1:4.

[0009] In a second aspect, the present invention provides an immunoassay kit, comprising the immune complex described in any one of the above.

[0010] Optionally, the process of the immunoassay kit for detecting the analyte includes: incubating the biotin-labeled analyte with the analyte to form a biotin-labeled immune complex; adding a biotin-labeled nucleic acid molecule and streptavidin magnetic beads for incubation, and the biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site; detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding site.

[0011] Optionally, the immunoassay kit further comprises a nucleic acid molecule fluorescent dye or a CRISPR reaction solution for detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding site.

[0012] Optionally, the nucleic acid molecule is a double-stranded DNA molecule comprising a template strand and a coding strand, and the CRISPR reaction solution comprises a crRNA complementary to the coding strand, a DNA endonuclease, Mg 2+ and a reaction buffer.

[0013] Optionally, the process of the immunoassay kit for detecting the analyte further includes: before detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding site, amplifying the number of the biotin-labeled nucleic acid molecules bound to the streptavidin binding site.

[0014] In a third aspect, the present invention provides the use of the immune complex described in any one of the above in the preparation of a drug for detecting hepatitis B, pregnancy, gestational trophoblastic disease or tumor.

[0015] The beneficial effects of a quadruplex immune complex, its application and an immunoassay kit of the present invention are as follows: The immune complex includes a biotin-labeled nucleic acid molecule, a biotin-labeled analyte, and streptavidin magnetic beads. The molecular weight of biotin is 244.31 Da, and biotin belongs to small molecules with a molecular weight much less than 1000 Da. The small molecule biotin can be stably labeled with nucleic acid molecules, antibodies or antigens and other analytes. The labeling operation is easy to achieve and the labeling efficiency is high. The high affinity between biotin and streptavidin ensures good stability of the quadruplex immune complex and improves the detection sensitivity. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site. When the content of the analyte is relatively high, more biotin-labeled immune complexes bind to the streptavidin binding site, resulting in a smaller amount of biotin-labeled nucleic acid molecules binding to the streptavidin binding site on the surface of the magnetic beads. On the contrary, when the content of the analyte is relatively low, fewer biotin-labeled immune complexes bind to the streptavidin binding site, resulting in a larger amount of biotin-labeled nucleic acid molecules binding to the streptavidin binding site on the surface of the magnetic beads. Therefore, by detecting the amount of nucleic acid molecules on the surface of the magnetic beads, the amount of the analyte is indirectly reflected, realizing qualitative or quantitative detection of the analyte, and it is possible to detect biomarkers such as antigens, antibodies, hormones or proteins, thus establishing a new labeled immunoassay method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the connection relationship after the quadruplex immune complex of the embodiment of the present invention recognizes and binds to the analyte; Figure 2 Schematic diagram of complementary pairing between a double-stranded DNA molecule and a crRNA sequence of the embodiment of the present invention; Figure 3 Schematic diagram of CRISPR signal values of CRISPR reactions of different groups in Example 1; Figure 4 Schematic diagram of CRISPR signal values of CRISPR reactions of double-stranded DNA molecules and crRNAs in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments described herein. On the contrary, these embodiments are provided to more thoroughly and completely understand the present invention. It should be understood that the drawings and embodiments of the present invention are only for exemplary purposes and are not used to limit the protection scope of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention; As used herein, the term "comprising" and its variations are open-ended, i.e., "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiment". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts such as "first" and "second" mentioned in the present invention are used to distinguish different objects and are not used to describe a specific order or primary-secondary relationship. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0019] This embodiment provides a quadruplex immune complex, its application and an immunoassay kit.

[0020] As Figure 1 shown, an immune complex provided by an embodiment of the present invention includes a biotin-labeled nucleic acid molecule, a biotin-labeled detector, and streptavidin magnetic beads. The surface of the streptavidin magnetic beads has streptavidin binding sites. After the biotin-labeled detector recognizes and binds to the analyte, a biotin-labeled immune complex is formed. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding sites.

[0021] In this embodiment, the immune complex includes a biotin-labeled nucleic acid molecule, a biotin-labeled detector, and streptavidin magnetic beads. The streptavidin magnetic beads are formed by covalently binding magnetic beads and streptavidin. The immune complex is also called a quadruple immune complex. The molecular weight of biotin is 244.31 Da. Biotin belongs to small molecules with a molecular weight much less than 1000 Da. The small molecule biotin can be stably labeled with nucleic acid molecules, antibodies, antigens or other detectors. The labeling operation is easy to achieve and the labeling efficiency is high. The high affinity between biotin (Biotin) and streptavidin (SA) ensures good stability of the quadruple immune complex and improves the detection sensitivity. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site. When the content of the analyte is relatively high, more biotin-labeled immune complexes bind to the streptavidin binding site, resulting in a smaller amount of biotin-labeled nucleic acid molecules binding to the streptavidin binding site on the surface of the magnetic beads. On the contrary, when the content of the analyte is relatively low, fewer biotin-labeled immune complexes bind to the streptavidin binding site, resulting in a larger amount of biotin-labeled nucleic acid molecules binding to the streptavidin binding site on the surface of the magnetic beads. Therefore, by detecting the amount of nucleic acid molecules on the surface of the magnetic beads, the amount of the analyte is indirectly reflected, realizing qualitative or quantitative detection of the analyte, and it is possible to detect biological markers such as antigens, antibodies, hormones or proteins, establishing a new labeled immunoassay method.

[0022] Specifically, the biotin in the biotin-labeled nucleic acid molecule and the biotin-labeled detector can be selected as one component or two different components.

[0023] Optionally, the nucleic acid molecule is a double-stranded DNA molecule including a template strand and a coding strand, and the sequences are respectively: Template strand: 5 ， -CAGTTTACTAGTGCCATTTGTTCAGTG-3 ， (SEQ ID NO.1); Coding strand: 5 ， -GTCAAATGATCACGGTAAACAAGTCAC-3 ， (SEQ ID NO.2).

[0024] In this optional embodiment, the double-stranded DNA molecule has a short chain. The short DNA molecule has a small steric hindrance and is more likely to bind to the streptavidin binding site on the surface of the magnetic beads, which is beneficial to improving the detection sensitivity and the linear detection range.

[0025] Specifically, the double-stranded DNA molecule can be replaced with a single-stranded DNA molecule or an RNA molecule to achieve the same purpose.

[0026] Optionally, the analyte is hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen, and the detector is the corresponding hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody; or, the analyte is hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody, and the detector is the corresponding hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen.

[0027] In this optional embodiment, the analytes that can be detected by the tetrameric immune complex can not only be a series of biomarkers such as hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen, but also replace the biotin-labeled antigen or antibody with a biotin-labeled nucleic acid aptamer to achieve the purpose of detecting small molecule antigens, antibodies, proteins or hormones and other biomarkers.

[0028] Specifically, a series of biomarkers such as human chorionic gonadotropin, alpha-fetoprotein, carcinoembryonic antigen, etc. When used to detect other biomarkers, only need to replace the biotin-labeled hepatitis B surface antibody in the reaction system with other corresponding biotin-labeled antibodies. That is to say, when replaced with a biotin-labeled antigen, it can be used to detect antibodies; when replaced with a biotin-labeled anti-human chorionic gonadotropin antibody, it can be used to detect human chorionic gonadotropin in human serum or urine; when replaced with an anti-human alpha-fetoprotein antibody, it can be used to detect alpha-fetoprotein in human serum or body fluids. By simply replacing the biotin-labeled antigen or antibody, various different detection purposes can be achieved, and it is completely unnecessary to replace most of the other components, and only need to replace the biotin-labeled detector to achieve.

[0029] Specifically, human chorionic gonadotropin is mainly produced by gestational trophoblasts, and pregnancy, gestational trophoblastic diseases, germ cell tumors or other malignant tumors such as lung, adrenal and liver tumors can all produce human chorionic gonadotropin.

[0030] Optionally, as Figure 1 shown, the binding ratio of biotin to streptavidin magnetic beads is 1:4.

[0031] In this optional embodiment, the 1:4 binding ratio of biotin and streptavidin can achieve the cascade amplification effect of biological signals.

[0032] Specifically, the biological signal amplification effect of the combined CRISPR system can be up to more than 1000 times, further improving the detection sensitivity of the tetrameric immune complex.

[0033] As Figure 1 shown, an immunoassay kit provided by an embodiment of the present invention includes the tetrameric immune complex described in any one of the above.

[0034] Optionally, the process of the immunoassay kit for detecting the analyte includes: incubating the biotin-labeled analyte with the analyte to form a biotin-labeled immune complex; Adding a biotin-labeled nucleic acid molecule and streptavidin magnetic beads for incubation, and the biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site; Detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding site.

[0035] In this embodiment, the biotin-labeled analyte is incubated with the analyte, and the analyte binds to the analyte to form a biotin-labeled immune complex; the biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site. When the content of the analyte is relatively high, more biotin-labeled immune complexes bind to the streptavidin binding site, resulting in a relatively small amount of biotin-labeled nucleic acid molecules binding to the streptavidin binding site on the surface of the magnetic beads; on the contrary, when the content of the analyte is relatively low, fewer biotin-labeled immune complexes bind to the streptavidin binding site, resulting in a relatively large amount of biotin-labeled nucleic acid molecules binding to the streptavidin binding site on the surface of the magnetic beads. Thus, by detecting the amount of nucleic acid molecules on the surface of the magnetic beads, the amount of the analyte is indirectly reflected, realizing qualitative or quantitative detection of the analyte, and it is possible to detect biological markers such as antigens, antibodies, hormones or proteins, and a new labeled immunoassay method is established.

[0036] Optionally, the immunoassay kit further includes a nucleic acid molecule fluorescent dye or a CRISPR reaction solution for detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding site.

[0037] In this optional embodiment, the nucleic acid molecule fluorescent dye is labeled on the biotin-labeled nucleic acid molecule bound to the streptavidin binding site on the surface of the magnetic beads, or the biotin-labeled nucleic acid molecule bound to the streptavidin binding site on the surface of the magnetic beads activates the CRISPR system in the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reaction solution to generate a fluorescent signal. By detecting the final fluorescent signal with a fluorescence detector, the amount of nucleic acid molecules on the surface of the magnetic beads can be detected, and the purpose of indirectly detecting biological markers such as antigens, antibodies, hormones or proteins can be achieved.

[0038] Optionally, the nucleic acid molecule is a double-stranded DNA molecule including a template strand and a coding strand, and the CRISPR reaction solution includes a crRNA complementary to the coding strand, a DNA endonuclease, Mg 2+and reaction buffer.

[0039] Specifically, the fluorophore is bound to the base TTTA of the template strand of the double-stranded DNA molecule.

[0040] In this alternative embodiment, the nucleic acid molecule is a double-stranded DNA molecule comprising a template strand and a coding strand. The double-stranded DNA molecule has a specially designed sequence. The CRISPR reaction solution includes a crRNA that is complementary to the coding strand. Only when the crRNA sequence is the corresponding sequence can a fluorescence signal be generated, further improving the detection specificity.

[0041] Specifically, the crRNA sequence is 5 ， -UAAUUUCUACUAAUGGUAGAU-CUAGUGCCAUUUGUUCAGUG-3 ， (SEQ ID NO.3). As Figure 2 shown, the double-stranded DNA molecule is complementary to the crRNA sequence; among them, there is a hairpin loop on the crRNA sequence.

[0042] Specifically, crRNA is generated by transcribing the CRISPR array. Each crRNA molecule contains a guide sequence targeting a specific viral or plasmid DNA, and this sequence enables the crRNA to be complementary to the invading foreign DNA.

[0043] In the CRISPR-Cas system, by binding crRNA to the Cas9 protein, the DNA molecule and its corresponding CrRNA successfully activate the CRISPR reaction and generate a fluorescence signal for detection.

[0044] To effectively achieve the cis- and trans-cleavage activities of CRISPR, the CRISPR reaction solution must contain the following components: LbCas12a enzyme, crRNA, Mg 2+ , single-stranded DNA reporter (ssReporter), target DNA.

[0045] Specifically, in the CRISPR-Cas12a detection system, ssReporter plays a crucial role. It is usually a single-stranded DNA with a fluorescent label, with a fluorophore (such as FAM) modified at the 5' end and a quencher group (such as BHQ1) modified at the 3' end. During the reaction, if the target DNA is present, the Cas12a protein will be activated, and then non-specifically cleave the surrounding single-stranded DNA, including ssreporter. This cleavage will separate the fluorophore and the quencher group, resulting in the release of a fluorescence signal, thereby indicating the presence of the target DNA.

[0046] Specifically, the CRISPR reaction solution also contains a CRISPR buffer, and the main components of the CRISPR buffer include salt ions, buffers, energy sources, and other auxiliary components; salt ions, such as NaCl and KCl, which help maintain the ionic strength of the solution and promote the stability of proteins and DNA. In addition, certain divalent cations (such as MgCl2) are crucial for the activity of Cas enzymes because they participate in the catalytic process of DNA cleavage reactions; buffers, such as HEPES or Tris, which are used to maintain the pH value of the solution and ensure that it is within an appropriate range (usually 7.0 - 8.0) to guarantee the optimal activity of the CRISPR-Cas system; energy sources, such as ATP, and certain CRISPR-Cas systems (such as Cas12a) require ATP to activate their nuclease activity; other auxiliary components, such as BSA (bovine serum albumin), which can stabilize proteins and reduce non-specific binding.

[0047] Optionally, the process of the immunoassay kit for detecting the analyte further includes: before detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding site, amplifying the number of biotin-labeled nucleic acid molecules bound to the streptavidin binding site.

[0048] In this optional embodiment, by performing isothermal amplification on the biotin-labeled nucleic acid molecule, including methods such as LAMP (Loop-mediated Isothermal Amplification), RPA (Recombinase Polymerase Isothermal Amplification), or PCR (Polymerase Chain Reaction), etc., to further amplify the number of nucleic acid molecules to achieve the purpose of improving detection sensitivity and specificity.

[0049] The application of the tetrameric immune complex according to any one of the above embodiments provided by the present invention in the preparation of drugs for detecting hepatitis B, pregnancy, gestational trophoblastic disease, or tumors.

[0050] The present invention will be further described below in conjunction with specific embodiments.

[0051] Example 1, immunoassay for hepatitis B surface antigen positive samples.

[0052] Prepare hepatitis B surface antigen positive samples with different concentrations, and take sterile water, hepatitis B negative control group, and hepatitis B positive control group. The groups are shown in Table 1.

[0053] Table 1 Group table of analytes for immunoassay

[0054] The process of detecting the analyte includes the following steps: 1. Take a microplate coated with capture antibody, and according to the concentration distribution scheme in Table 1, add 20 μL of samples from different groups to 8 wells respectively.

[0055] 2. Incubate in a 37°C water bath for 10 minutes.

[0056] 3. Take out the microplate, pour out the liquid in the wells, add 200 μL of washing solution to each well, soak for 15 seconds and then pour out, repeat 3 times.

[0057] 4. Add 100 μL of biotinylated hepatitis B surface antibody to the wells. The biotinylated hepatitis B surface antibody can be a mono-biotinylated monoclonal anti-HBs antibody (mouse) or a di-biotinylated monoclonal anti-HBs antibody. The biotinylated hepatitis B surface antibody is dissolved in 100 mmol / L phosphate buffer, and the concentration of the biotinylated hepatitis B surface antibody is 0.5 mg / L, adjust the pH to 7.5. Di-biotinylation means that two biotin molecules are linked together through a polyethylene glycol (PEG) chain to form a specific chemical compound. Incubate in a 37°C water bath for 10 minutes. After the biotinylated hepatitis B surface antibody binds to the hepatitis B surface antigen, a biotinylated immune complex is formed.

[0058] 5. Take out the microplate, pour out the liquid in the wells, add 200 μL of washing solution to each well, soak for 15 seconds and then pour out, repeat 3 times.

[0059] 6. Add 5 μL of biotinylated nucleic acid probe (DNA-Biotin) label and 50 μL of streptavidin-coated magnetic beads to each well and incubate for 10 minutes. The biotinylated nucleic acid probe (DNA-Biotin) label and the biotinylated immune complex competitively bind to the streptavidin binding site.

[0060] 7. Place the microplate on a magnetic rack. The magnetic beads are enriched at the bottom within 10 seconds. Discard the supernatant, add 100 μL of sterile and enzyme-free water, soak for 15 seconds, repeat 3 times.

[0061] 8. Add 20 μL of CRISPR reaction solution to each well, immediately load it onto the machine for detection, detect the content of the biotinylated nucleic acid probe (DNA-Biotin) label that binds to the streptavidin binding site, and monitor the change in the fluorescence signal value, as Figure 3 shown. The formulation of the CRISPR reaction solution: Add 5 μL of Lb Cas12a enzyme, 5 μL of crRNA, 20 μL of CRISPR buffer, 5 μL of ssReporter to a 0.2 mL centrifuge tube, and then add 160 μL of sterile and enzyme-free water to make the total volume reach 200 μL.

[0062] As Figure 3As shown, sterile water and the negative control wells failed to activate the CRISPR system (signal value was 0), while the positive control wells and the sample wells with different concentrations could normally activate the CRISPR system and generate detectable signal values. The size of the signal value was negatively correlated with the concentration of the analyte in the serum, and the CRISPR signal value could indirectly indicate the content of the analyte.

[0063] In addition, as the sample concentration increased, the CRISPR signal value gradually decreased and approached 0, indicating that the method had a good linear relationship within a certain range. When the sample concentration exceeded a certain threshold, the CRISPR signal value tended to be stable and did not change significantly with the increase of the sample concentration, which might be due to the saturation of the CRISPR system or the interference of other components in the sample. By comparing the signal values of samples with different concentrations, a standard curve could also be established for the quantitative detection of unknown samples.

[0064] Example 2: Verify whether a double-stranded DNA molecule including a template strand and a coding strand and the corresponding crRNA can activate the CRISPR system.

[0065] As Figure 2 shown, the sequences of the double-stranded DNA molecule including the template strand and the coding strand were respectively: Template strand: 5 ， -CAGTTTACTAGTGCCATTTGTTCAGTG-3 ， (SEQ ID NO.1); Coding strand: 5 ， -GTCAAATGATCACGGTAAACAAGTCAC-3 ， (SEQ ID NO.2).

[0066] The crRNA sequence was 5 ， -UAAUUUCUACUAAUGGUAGAU-CUAGUGCCAUUUGUUCAGUG-3 ， (SEQ ID NO.3).

[0067] Design the formula of the CRISPR reaction solution: Add 5 μL of Lb Cas12a enzyme, 5 μL of crRNA, 20 μL of CRISPR buffer, 5 μL of ssReporter, and 5 μL of the double-stranded DNA molecule into a 0.2 mL centrifuge tube, and then add 160 μL of sterile and enzyme-free water to make the total volume reach 200 μL.

[0068] After gently shaking and mixing the CRISPR reaction solution, incubate it on ice for 5 minutes. Divide 200 μL of the CRISPR reaction solution into 20 μL per well and transfer it to a 0.1 ml PCR reaction plate, and then detect the generated biological signal on the machine asFigure 4 as shown

[0069] As Figure 4 shown, a double-stranded DNA molecule including a template strand and a coding strand and the corresponding crRNA can activate the related protein Lb Cas12a of the CRISPR system and generate an obvious fluorescence signal. Starting from the 0th second of the experiment, the fluorescence signal intensity was recorded every 30 seconds until 40 cycles were completed. By observing the change of the signal, the fluorescence signal quickly reached a plateau within 5 minutes and remained stable in the next 5 to 40 minutes. This stable plateau provides an ideal observation window for subsequent biological signal detection. In addition, in the experimental group with sterile water as the control set in Example 1, the results show that the fluorescence signal level of the experimental group with sterile water as the control was always close to zero, indicating that the background signal of this reaction system is very low, ensuring high detection sensitivity and detection specificity, and the accuracy and reliability of the results are both good.

[0070] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will all fall within the protection scope of the present invention.

Claims

1. An immune complex, characterized in that, It includes a biotin-labeled nucleic acid molecule, a biotin-labeled detector, and streptavidin magnetic beads. The surface of the streptavidin magnetic beads has streptavidin binding sites. After the biotin-labeled detector recognizes and binds to the analyte, a biotin-labeled immune complex is formed. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding sites.

2. The immune complex according to claim 1, wherein The nucleic acid molecule is a double-stranded DNA molecule including a template strand and a coding strand, and the sequences are respectively: Template strand: 5 ， -CAGTTTACTAGTGCCATTTGTTCAGTG-3 ， (SEQ ID NO.1); Coding strand: 5 ， -GTCAAATGATCACGGTAAACAAGTCAC-3 ， (SEQ ID NO.2).

3. The immune complex according to claim 1, wherein The analyte is hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen, and the detector is the corresponding hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody; or, The analyte is hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody, and the detector is the corresponding hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen.

4. The immune complex according to claim 1, characterized in that, The binding ratio of the biotin to the streptavidin magnetic beads is 1:

4.

5. An immunoassay kit, characterized in that, It includes the immune complex according to any one of claims 1-4.

6. The immunoassay kit according to claim 5, characterized in that, The process of the immune detection kit for detecting the analyte includes: incubating the biotin-labeled detector with the analyte to form a biotin-labeled immune complex; adding the biotin-labeled nucleic acid molecule and the streptavidin magnetic beads for incubation, and the biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding sites; detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding sites.

7. The immunoassay kit according to claim 6, wherein, The immune detection kit further includes a nucleic acid molecule fluorescent dye or a CRISPR reaction solution for detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding sites.

8. The immunoassay kit according to claim 7, wherein The nucleic acid molecule is a double-stranded DNA molecule comprising a template strand and a coding strand, and the CRISPR reaction solution comprises a crRNA complementary to the coding strand, a DNA endonuclease, Mg 2+ and a reaction buffer.

9. The immunoassay kit according to claim 6, wherein The process of the immune detection kit for detecting the analyte further includes: before detecting the content of the biotin-labeled nucleic acid molecule bound to the streptavidin binding sites, amplifying the number of the biotin-labeled nucleic acid molecules bound to the streptavidin binding sites.

10. Use of the immune complex according to any one of claims 1-4 in the preparation of a drug for detecting hepatitis, pregnancy, gestational trophoblastic disease or tumor.

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