A quadruple immune complex and its application and immunoassay kit

Abstract

The present invention provides a quadruple immune complex, its application, and an immunoassay kit, relating to the field of molecular biology. The quadruple immune complex comprises a biotin-labeled nucleic acid molecule, a biotin-labeled detector, and streptavidin magnetic beads. The surface of the streptavidin magnetic beads has a streptavidin binding site. The biotin-labeled detector forms a biotin-labeled immune complex after recognizing and binding to the analyte. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site. When the analyte content is high, more biotin-labeled immune complexes bind to the streptavidin binding site, resulting in a lower amount of biotin-labeled nucleic acid molecules bound 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, thereby achieving qualitative or quantitative detection of the analyte.

Description

Technical Field

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

[0002] Currently, the primary detection methods for biomarkers such as small molecule antigens, hormones, and proteins are enzyme-linked immunosorbent assays (ELISAs) and chemiluminescence (CLIA) methods. Traditional CLIA and ELISA methods directly label antibodies with enzymes, and use the enzyme's substrate to generate a colorimetric or luminescent signal. This approach has several drawbacks: poor enzyme stability and significant steric hindrance lead to low detection sensitivity. Summary of the Invention

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

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

[0005] In the 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 a streptavidin binding site. The biotin-labeled detector forms a biotin-labeled immune complex after recognizing and binding to the detected object. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site.

[0006] Optionally, the nucleic acid molecule is a double-stranded DNA molecule comprising a template strand and a coding strand, and the sequences are:

[0007] Template chain: 5 ， -CAGTTTACTAGTGCCATTTGTTCAGTG-3 ， (SEQ ID NO. 1);

[0008] Coding chain: 5 ， -GTCAAATGATCACGGTAAACAAGTCAC-3 ， (SEQ ID NO. 2).

[0009] Optionally, the subject to be detected is hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen, and the detection subject is the corresponding hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody; or, the subject to be detected is hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody, and the detection subject is the corresponding hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen.

[0010] Optionally, the binding ratio of biotin to streptavidin magnetic beads is 1:4.

[0011] In a second aspect, the present invention provides an immunoassay kit comprising the immune complex as described in any one of the above items.

[0012] Optionally, the process of detecting the analyte by the immunoassay kit includes: incubating the biotin-labeled detection agent with the analyte to form a biotin-labeled immune complex;

[0013] Biotin-labeled nucleic acid molecules and streptavidin magnetic beads are added for incubation, and the biotin-labeled nucleic acid molecules and biotin-labeled immune complexes competitively bind to the streptavidin binding site;

[0014] The amount of biotin-labeled nucleic acid molecules bound to the streptavidin binding site is detected.

[0015] 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.

[0016] 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 crRNA complementary to the coding strand, DNA endonuclease, Mg 2+ and reaction buffer.

[0017] Optionally, the process of detecting the object by the immunoassay kit further includes: before detecting the content of the biotin-labeled nucleic acid molecules bound to the streptavidin binding site, amplifying the number of the biotin-labeled nucleic acid molecules bound to the streptavidin binding site.

[0018] In a third aspect, the present invention provides use of the immune complex described in any one of the above items in the preparation of a drug for detecting hepatitis B, pregnancy, gestational trophoblastic disease or tumor.

[0019] The beneficial effects of the quadruple immune complex and its application and immunoassay kit of the present invention are as follows: the immune complex comprises a biotin-labeled nucleic acid molecule, a biotin-labeled detection body and streptavidin magnetic beads; the molecular weight of biotin is 244.31 Da, and biotin is a small molecule with a molecular weight far less than 1000 Da; the small molecule biotin can stably label detection bodies such as nucleic acid molecules, antibodies or antigens; the labeling operation is easy to implement and the labeling efficiency is high; the high affinity between biotin and streptavidin ensures the 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 When the content of the analyte is high, more biotin-labeled immune complexes are bound to the streptavidin binding sites, resulting in a smaller amount of biotin-labeled nucleic acid molecules bound to the streptavidin binding sites on the surface of the magnetic beads; on the contrary, when the content of the analyte is low, less biotin-labeled immune complexes are bound to the streptavidin binding sites, resulting in a larger amount of biotin-labeled nucleic acid molecules bound to the streptavidin binding sites 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, and qualitative or quantitative detection of the analyte is achieved. It can also detect biomarkers such as antigens, antibodies, hormones or proteins, and establish a new labeled immunodiagnostic method. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the connection relationship after the quadruple immune complex recognizes and binds to the analyte according to an embodiment of the present invention;

[0021] Figure 2 Schematic diagram of the complementary pairing of double-stranded DNA molecules and crRNA sequences according to an embodiment of the present invention;

[0022] Figure 3 Schematic diagram of CRISPR signal values ​​for different groups of CRISPR reactions in Example 1;

[0023] Figure 4 Schematic diagram of the CRISPR signal value of the CRISPR reaction of the double-stranded DNA molecule and crRNA in Example 2. DETAILED DESCRIPTION

[0024] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as being limited to the embodiments described herein. Instead, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the present invention description are only for the purpose of describing specific embodiments and are not intended to limit the present invention;

[0026] The term "including" and its variations used in this document are open inclusions, that is, "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 other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts of "first" and "second" mentioned in the present invention are used to distinguish different objects, rather than to describe a specific order or a primary and secondary relationship. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, "multiple" means two or more.

[0027] This embodiment provides a quadruple immune complex, its application, and an immunoassay kit.

[0028] like Figure 1 As shown, an immune complex provided by an embodiment of the present invention includes a biotin-labeled nucleic acid molecule, a biotin-labeled detection body and streptavidin magnetic beads. The surface of the streptavidin magnetic beads has a streptavidin binding site. The biotin-labeled detection body forms a biotin-labeled immune complex after recognizing and binding to the detection body. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site.

[0029] In this embodiment, the immune complex includes a biotin-labeled nucleic acid molecule, a biotin-labeled detection body, 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 is a small molecule with a molecular weight far less than 1000 Da. Small molecule biotin can stably label detection bodies such as nucleic acid molecules, antibodies, or antigens. The labeling operation is easy to implement and the labeling efficiency is high. The high affinity of biotin (Biotin) and streptavidin (SA) ensures the 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 each other. At the streptavidin binding site, when the content of the analyte is high, more biotin-labeled immune complexes are bound to the streptavidin binding site, resulting in a smaller amount of biotin-labeled nucleic acid molecules bound to the streptavidin binding site on the surface of the magnetic beads; on the contrary, when the content of the analyte is low, less biotin-labeled immune complexes are bound to the streptavidin binding site, resulting in a larger amount of biotin-labeled nucleic acid molecules bound 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, and qualitative or quantitative detection of the analyte is achieved. It can also be used to detect biomarkers such as antigens, antibodies, hormones or proteins, thereby establishing a new labeled immunodiagnostic method.

[0030] Specifically, the biotin in the biotin-labeled nucleic acid molecule and the biotin-labeled detection body can be selected from one component or two different components.

[0031] Optionally, the nucleic acid molecule is a double-stranded DNA molecule comprising a template strand and a coding strand, and the sequences are:

[0032] Template chain: 5 ， -CAGTTTACTAGTGCCATTTGTTCAGTG-3 ， (SEQ ID NO. 1);

[0033] Coding chain: 5 ， -GTCAAATGATCACGGTAAACAAGTCAC-3 ， (SEQ ID NO. 2).

[0034] In this optional embodiment, the double-stranded DNA molecules have shorter chains, and the short DNA molecules have less steric hindrance and are more easily bound to the streptavidin binding sites on the surface of the magnetic beads, which is beneficial to improving the detection sensitivity and linear detection range.

[0035] Specifically, double-stranded DNA molecules can be replaced with single-stranded DNA molecules or RNA-type molecules to achieve the same purpose.

[0036] Optionally, the subject to be detected is hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen, and the detection subject is the corresponding hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody; or, the subject to be detected is hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody, and the detection subject is the corresponding hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen.

[0037] In this optional embodiment, the quadruple immune complex can detect not only a series of biomarkers such as hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen, but also the biotin-labeled antigen or antibody can be replaced with a biotin-labeled nucleic acid aptamer to achieve the purpose of detecting biomarkers such as small molecule antigens, antibodies, proteins or hormones.

[0038] Specifically, a series of biomarkers such as human chorionic gonadotropin, alpha-fetoprotein, carcinoembryonic antigen, etc. When used to detect other biomarkers, it is only necessary to replace the biotin-labeled hepatitis B surface antibody in the reaction system with other corresponding biotin-labeled antibodies. In other words, 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. A variety of different detection purposes can be achieved by simply replacing the biotin-labeled antigen or antibody, without having to replace most of the other components. It can be achieved by simply replacing the biotin-labeled detection body.

[0039] Specifically, human chorionic gonadotropin is mainly produced by gestational trophoblasts, and pregnancy, gestational trophoblastic disease, germ cell tumors or other malignant tumors such as lung, adrenal and liver tumors can all produce human chorionic gonadotropin.

[0040] Alternatively, as Figure 1 As shown, the binding ratio of biotin to streptavidin magnetic beads is 1:4.

[0041] In this optional embodiment, the 1:4 binding ratio of biotin to streptavidin can achieve cascade amplification of biological signals.

[0042] Specifically, the biological signal amplification effect of the CRISPR system can be as high as 1,000 times or more, further improving the detection sensitivity of the quadruple immune complex.

[0043] like Figure 1As shown, an immunoassay kit provided by an embodiment of the present invention includes the quadruple immune complex as described in any one of the above items.

[0044] Optionally, the process of detecting the analyte by the immunoassay kit includes: incubating the biotin-labeled detection agent with the analyte to form a biotin-labeled immune complex;

[0045] Biotin-labeled nucleic acid molecules and streptavidin magnetic beads are added for incubation, and the biotin-labeled nucleic acid molecules and biotin-labeled immune complexes competitively bind to the streptavidin binding site;

[0046] The amount of biotin-labeled nucleic acid molecules bound to the streptavidin binding site is detected.

[0047] In this embodiment, the biotin-labeled detection body is incubated with the detected body, and the detection body and the detected body are combined 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 detected body is relatively high, more biotin-labeled immune complexes are bound to the streptavidin binding site, resulting in a smaller amount of biotin-labeled nucleic acid molecules bound to the streptavidin binding site on the surface of the magnetic beads; on the contrary, when the content of the detected body is relatively low, less biotin-labeled immune complexes are bound to the streptavidin binding site, resulting in a larger amount of biotin-labeled nucleic acid molecules bound 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 detected body is indirectly reflected, thereby realizing qualitative or quantitative detection of the detected body, and can realize the detection of biomarkers such as antigens, antibodies, hormones or proteins, thereby establishing a new labeled immunodiagnostic method.

[0048] 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.

[0049] In this optional embodiment, the fluorescent dye labeling of nucleic acid molecules refers to the biotin-labeled nucleic acid molecules bound to the streptavidin binding sites on the surface of the magnetic beads, or the biotin-labeled nucleic acid molecules bound to the streptavidin binding sites on the surface of the magnetic beads activate the CRISPR system in the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reaction solution to generate a fluorescent signal. The final fluorescent signal is detected by a fluorescence detector, and the amount of nucleic acid molecules on the surface of the magnetic beads can be detected, thereby achieving the purpose of indirectly detecting biomarkers such as antigens, antibodies, hormones or proteins.

[0050] 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 crRNA complementary to the coding strand, DNA endonuclease, Mg 2+ and reaction buffer.

[0051] Specifically, the fluorescent group is bound to the base TTTA of the template strand of the double-stranded DNA molecule.

[0052] In this optional embodiment, the nucleic acid molecule is a double-stranded DNA molecule including a template chain and a coding chain. The double-stranded DNA molecule has a specially designed sequence. The CRISPR reaction solution includes crRNA that is complementary to the coding chain. Only when the crRNA sequence is a corresponding sequence can a fluorescent signal be generated, thereby further improving the detection specificity.

[0053] Specifically, the crRNA sequence is 5 ， -UAAUUUCUACUAAUGGUAGAU-CUAGUGCCAUUUGUUCAGUG-3 ， (SEQ ID NO. 3). Figure 2 As shown, the double-stranded DNA molecule is complementary to the crRNA sequence; wherein, there is a hairpin loop on the crRNA sequence.

[0054] Specifically, crRNA is produced by transcribing the CRISPR array. Each crRNA molecule contains a guide sequence that targets a specific viral or plasmid DNA. This sequence enables the crRNA to complement and pair with the invading foreign DNA.

[0055] 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 detectable fluorescent signal.

[0056] In order to effectively achieve the cis and trans cutting activities of CRISPR, the CRISPR reaction solution must contain the following components: LbCas12a enzyme, crRNA, Mg 2+ , single-stranded DNA reporter molecule (ssReporter), target DNA.

[0057] Specifically, the ssReporter plays a crucial role in the CRISPR-Cas12a detection system. It is typically a fluorescently labeled single-stranded DNA fragment, modified with a fluorescent group (such as FAM) at its 5' end and a quencher (such as BHQ1) at its 3' end. During the reaction, if the target DNA is present, the Cas12a protein is activated and non-specifically cleaves the surrounding single-stranded DNA, including the ssReporter. This cleavage separates the fluorescent group and the quencher, resulting in the release of a fluorescent signal, indicating the presence of the target DNA.

[0058] Specifically, the CRISPR reaction solution also contains CRISPR buffer. The main components of the CRISPR buffer include salt ions, buffers, energy sources and other auxiliary components; salt ions, such as NaCl and KCl, 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 essential for the activity of Cas enzymes because they are involved in the catalytic process of DNA cleavage reactions; buffers, such as HEPES or Tris, are used to maintain the pH value of the solution to ensure that it is within an appropriate range (usually 7.0-8.0) to ensure the optimal activity of the CRISPR-Cas system; energy sources, such as ATP, some CRISPR-Cas systems (such as Cas12a) require ATP to activate their nuclease activity; other auxiliary components, such as BSA (bovine serum albumin), can stabilize proteins and reduce nonspecific binding.

[0059] Optionally, the process of detecting the object by the immunoassay kit further includes: before detecting the content of the biotin-labeled nucleic acid molecules bound to the streptavidin binding site, amplifying the number of the biotin-labeled nucleic acid molecules bound to the streptavidin binding site.

[0060] In this optional embodiment, the purpose of improving detection sensitivity and specificity is achieved by further amplifying the number of nucleic acid molecules by isothermal amplification of biotin-labeled nucleic acid molecules, including LAMP (loop-mediated isothermal amplification technology), RPA (recombinase polymerase isothermal amplification technology) or PCR (polymerase chain reaction) and other methods.

[0061] The embodiments of the present invention provide use of the quadruple immune complex as described in any one of the above items in the preparation of a drug for detecting hepatitis B, pregnancy, gestational trophoblastic disease or tumor.

[0062] The present invention is further described below with reference to specific embodiments.

[0063] Example 1: Immunodetection of hepatitis B surface antigen positive samples.

[0064] Prepare hepatitis B surface antigen-positive samples of different concentrations, take sterile water, hepatitis B negative control group and hepatitis B positive control group, and the groups are shown in Table 1.

[0065] Table 1. Groups of test subjects for immunoassay

[0066]

[0067] The process of testing the subject includes the following steps:

[0068] 1. Take the capture antibody-coated microplate and add 20uL of samples from different groups to each of the 8 wells according to the concentration distribution scheme in Table 1.

[0069] 2. Place in a 37°C water bath and incubate for 10 minutes.

[0070] 3. Take out the microplate, pour out the liquid in the wells, add 200uL of cleaning solution to each well, soak for 15 seconds and pour out, repeat 3 times.

[0071] 4. Add 100 μL of biotinylated HBsAg antibody to the wells. The biotinylated HBsAg antibody can be a single-biotinylated monoclonal anti-HBs antibody (mouse) or a double-biotinylated monoclonal anti-HBs antibody. Dissolve the biotinylated HBsAg antibody in 100 mmol / L phosphate buffer at a concentration of 0.5 mg / L, adjusted to pH 7.5. Double biotinylation refers to the process of linking two biotin molecules via a polyethylene glycol (PEG) chain to form a specific chemical compound. Incubate in a 37°C water bath for 10 minutes. The biotinylated HBsAg antibody will bind to the HBsAg to form a biotinylated immune complex.

[0072] 5. Take out the microplate, pour out the liquid in the wells, add 200uL of cleaning solution to each well, soak for 15 seconds and pour out, repeat 3 times.

[0073] 6. Add 5 μL of biotin-labeled nucleic acid probe (DNA-Biotin) tag and 50 μL of streptavidin-coated magnetic beads to each well and incubate for 10 minutes. The biotin-labeled nucleic acid probe (DNA-Biotin) tag and the biotin-labeled immune complex competitively bind to the streptavidin binding site.

[0074] 7. Place the microplate on the magnetic stand. The magnetic beads will accumulate at the bottom within 10 seconds. Discard the supernatant, add 100uL of sterile enzyme-free water, soak for 15 seconds, and repeat 3 times.

[0075] 8. Add 20uL of CRISPR reaction solution to each well and immediately test on the instrument to detect the content of the biotin-labeled nucleic acid probe (DNA-Biotin) tag bound to the streptavidin binding site and monitor the changes in the fluorescence signal value, such as Figure 3 The CRISPR reaction solution formula is as follows: Add 5 μL of Lb Cas12a enzyme, 5 μL of crRNA, 20 μL of CRISPR buffer, and 5 μL of ssReporter to a 0.2 mL centrifuge tube, and then add 160 μL of sterile, enzyme-free water to bring the total volume to 200 μL.

[0076] like Figure 3 As shown, sterile water and negative control wells failed to activate the CRISPR system (signal value 0), while the positive control wells and sample wells with different concentrations all activated the CRISPR system normally and produced detectable signal values. The signal value is negatively correlated with the concentration of the analyte in serum, and the CRISPR signal value can indirectly indicate the content of the analyte.

[0077] Furthermore, as sample concentration increases, the CRISPR signal gradually decreases, approaching zero, indicating good linearity within a certain range. When sample concentration exceeds a certain threshold, the CRISPR signal stabilizes and no longer changes significantly with increasing sample concentration. This may be due to saturation of the CRISPR system or interference from other components in the sample. By comparing the signal values ​​of samples of varying concentrations, a standard curve can be established for quantitative detection of unknown samples.

[0078] 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.

[0079] like Figure 2 As shown, the sequences of the double-stranded DNA molecules including the template strand and the coding strand are:

[0080] Template chain: 5 ， -CAGTTTACTAGTGCCATTTGTTCAGTG-3 ， (SEQ ID NO. 1);

[0081] Coding chain: 5 ， -GTCAAATGATCACGGTAAACAAGTCAC-3 ， (SEQ ID NO. 2).

[0082] The crRNA sequence is 5 ， -UAAUUUCUACUAAUGGUAGAU-CUAGUGCCAUUUGUUCAGUG-3 ，(SEQ ID NO. 3).

[0083] Design the formula of CRISPR reaction solution: add 5uL Lb Cas12a enzyme, 5uL crRNA, 20uL CRISPR buffer, 5uL ssReporter and 5uL double-stranded DNA molecules to a 0.2mL centrifuge tube, and then add 160uL sterile enzyme-free water to make the total volume reach 200uL.

[0084] The CRISPR reaction solution was gently shaken and mixed, then incubated on ice for 5 minutes. 200uL of the CRISPR reaction solution was divided into 20uL per well and transferred to a 0.1ml PCR reaction plate. The biological signal generated was detected by the machine. Figure 4 shown.

[0085] like Figure 4 As shown, the double-stranded DNA molecules including the template chain and the coding chain and the corresponding crRNA can activate the related protein Lb Cas12a of the CRISPR system and produce an obvious fluorescent signal. From the 0th second when the experiment starts, the fluorescence signal intensity is recorded every 30 seconds until 40 cycles are completed. By observing the change in the signal, the fluorescence signal quickly reaches a plateau within 5 minutes and remains stable for the next 5 to 40 minutes. This stable plateau provides an ideal observation window for subsequent biological signal detection. In addition, 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 is always close to zero, which shows that the background signal of the reaction system is very low, ensuring higher detection sensitivity and detection specificity, and the accuracy and reliability of the results are better.

[0086] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. An immune complex, characterized in that The invention comprises a biotin-labeled nucleic acid molecule, a biotin-labeled detection body, and streptavidin magnetic beads. The surface of the streptavidin magnetic beads has a streptavidin binding site. The biotin-labeled detection body forms a biotin-labeled immune complex after recognizing and binding to the detected body. The biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site. The nucleic acid molecule is a double-stranded DNA molecule including a template chain and a coding chain, and the sequences are respectively: Template chain: 5 ， -CAGTTTACTAGTGCCATTTGTTCAGTG-3 ， (SEQ ID NO. 1); Coding chain: 5 ， -GTCAAATGATCACGGTAAACAAGTCAC-3 ， (SEQ ID NO. 2).

2. The immune complex according to claim 1, characterized in that The detected object is hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen, and the detected object is the corresponding hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody; or, The detected object is hepatitis B virus antibody, anti-human chorionic gonadotropin antibody, anti-human alpha-fetoprotein antibody or carcinoembryonic antigen antibody, and the detected object is the corresponding hepatitis B virus antigen, human chorionic gonadotropin, alpha-fetoprotein or carcinoembryonic antigen.

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

4.

4. An immunoassay kit, characterized in that: The method comprises the immune complex according to any one of claims 1 to 3.

5. The immunoassay kit according to claim 4, characterized in that The process of detecting the analyte by the immunoassay kit includes: incubating the biotin-labeled test substance with the analyte to form a biotin-labeled immune complex; adding a biotin-labeled nucleic acid molecule and streptavidin magnetic beads for incubation, wherein the biotin-labeled nucleic acid molecule and the biotin-labeled immune complex competitively bind to the streptavidin binding site; The amount of the biotin-labeled nucleic acid molecule bound to the streptavidin binding site is detected.

6. The immunoassay kit according to claim 5, characterized in that 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.

7. The immunoassay kit according to claim 6, characterized in that The nucleic acid molecule is a double-stranded DNA molecule including a template strand and a coding strand, and the CRISPR reaction solution includes crRNA complementary to the coding strand, DNA endonuclease, Mg 2+ and reaction buffer.

8. The immunoassay kit according to claim 5, characterized in that The process of detecting the test object by the immunoassay kit further includes: before detecting the content of the biotin-labeled nucleic acid molecules bound to the streptavidin binding site, amplifying the amount of the biotin-labeled nucleic acid molecules bound to the streptavidin binding site.

9. Use of the immune complex according to any one of claims 1 to 3 in the preparation of a drug for detecting hepatitis B, pregnancy, gestational trophoblastic disease or tumor.

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