An ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification

Abstract

The present invention discloses an ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification in the field of medical detection technology, comprising the following steps: 1. immune binding; 2. enzymatic cleavage and release to release a hairpin DNA single strand; 3. complementary and enzymatic ligation to cyclize the circular DNA single strand by adding T4 ligase; 4. rolling circle amplification; and 6. detection, quantification and localization by qPCR and in situ oligonucleotide fluorescent labeling detection methods. The present invention uses the hairpin structure on the oligonucleotide DNA chain coupled to the antibody to protect the complementary sequence from generating nonspecific circular DNA single strands, and combines the rolling circle amplification technique to amplify the detection signal, thereby suppressing the intensity of the detection background signal while enhancing the specificity of the detection signal, further improving the sensitivity of the detection.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and in particular is an ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification. Background Art

[0002] Immunoassays (IAs) are based on the specific binding of antigens and antibodies, using markers (enzymes, fluorescent molecules, radioisotopes, etc.) to convert the binding event into a detectable signal. Due to the specificity and sensitivity of the antigen-antibody reaction, IAs are widely used in various fields of medical testing. Any substance can be detected using an immunoassay as long as a corresponding specific antibody is available. However, with the advancement of medical testing technology, various problems with immunoassays have also emerged. The sensitivity of traditional immunoassays is limited by the efficiency of antigen-antibody binding and the signal amplification mechanism. Since each antibody carries only a limited number of labeled molecules, and nonspecific binding during the detection process can mask low-concentration signals, resulting in insufficient sensitivity. For example, the detection limit of ELISA is usually at the pg / mL level, which makes it difficult to detect trace biomarkers (such as early tumor markers and low-abundance cytokines). In addition, due to the high concentration of the target, the marker is quickly depleted, exceeding the linear detection range. The readout method also relies on visual interpretation or simple optical equipment, with low resolution. Some immunoassay methods (such as colloidal gold test strips) can only provide qualitative or semi-quantitative results, which makes it difficult to accurately cover targets with a wide concentration range. This has created a dilemma that current immunoassay methods need to be improved.

[0003] Rolling circle amplification (RCA) is an isothermal nucleic acid amplification technique that utilizes a circular DNA template and a DNA polymerase with strand-displacing activity (such as Phi29) to continuously synthesize long, repeating single-stranded DNA (ssDNA) sequences. Its signal amplification mechanism relies on the generation of several thousand bases of ssDNA per circular template, which serves as a signal carrier. Multiple probe binding sites can be designed on the signal carrier, achieving geometric signal amplification.

[0004] The existing proximity immunoassay method using rolling circle amplification for signal amplification has become an option for high-sensitivity immunomedical testing. It is based on the proximity effect triggered by the sandwich immune complex formed by the antibody and antigen. The added two-terminal single-stranded DNA is complementary to the oligonucleotide DNA probe coupled to the antibody to form a circular structure, which is then connected by polymerase to form a circular DNA template. Rolling circle amplification is used to amplify the binding site signal on the template, achieving high-sensitivity detection with a detection limit as low as fM level, as described in the academic paper "Protein detection using proximity-dependent DNA ligation assays PLA system".

[0005] However, even when amplifying the immune binding signal through rolling circle amplification, PLA systems still face problems such as difficulty in reducing the detection background, affecting the detection limit, a high probability of nonspecific binding, which reduces the reliability of the detection signal, and even produces false positive signals. Therefore, it is necessary to propose an ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification that can suppress the detection background signal intensity, protect the oligonucleotide DNA chain from nonspecific binding, thereby improving the sensitivity of the immunoassay, widening the detection limit range, and realizing automation. Summary of the Invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide an ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification, which protects the complementary sequence by the hairpin loop structure on the oligonucleotide DNA chain coupled to the antibody to avoid the generation of nonspecific circular DNA single strands, and combines the rolling circle amplification technology to amplify the detection signal, thereby suppressing the detection background signal intensity while enhancing the specificity of the detection signal, further improving the sensitivity of the detection and facilitating the realization of automated detection.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows: an ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification, comprising the following steps:

[0008] Step 1: immune binding, adding a first DNA antibody-coupled probe and a second DNA antibody-coupled probe to the sample to bind to the antigen to form an immune complex;

[0009] Step 2: Enzymatic cleavage and release: Use a branched endonuclease to cut the 5' end branch of the DNA hairpin structure of the first DNA antibody-coupled probe and the second DNA antibody-coupled probe, releasing a single strand of hairpin DNA that is complementary to the root of the oligonucleotide DNA chain of the other coupled probe;

[0010] Step 3: Complementation and enzyme ligation. Under the influence of the proximity effect formed by antibody immunobinding, the oligonucleotide DNA chains of the first DNA antibody-coupled probe and the second DNA antibody-coupled probe complement each other, forming a circular DNA single strand with two gaps that are complementary to the oligonucleotide DNA chains of the two coupled probes, and the ends of the single strands at the gaps are adjacent. T4 ligase is added to circularize the circular DNA single strand.

[0011] Step 4: Rolling circle amplification, adding phi29 polymerase to perform rolling circle amplification of circular DNA single strands;

[0012] Step 5: Detection: Analyze the antibody content by detecting the repeated specific sequence on the rolling circle amplification product through qPCR, and perform in situ fluorescence imaging in tissue cells by detecting the repeated specific sequence on the rolling circle amplification product through in situ oligonucleotide fluorescence labeling detection method.

[0013] The basic approach works as follows: The oligonucleotide strands of the first and second DNA antibody-conjugated probes are designed with a hairpin structure, with the loop containing complementary sequences. When the two antibody probes simultaneously bind to the same antigen (forming an "immune sandwich complex"), spatial proximity causes the hairpin to be cleaved by a branched endonuclease, releasing the complementary strands. The released complementary strands form a circular DNA template through the proximity effect, which is then circularized using T4 ligase. This ensures that only the specifically bound antigen triggers the formation of the circular template, preventing nonspecific amplification.

[0014] The circularized DNA template undergoes RCA under the action of phi29 polymerase, generating long ssDNA chains containing thousands of repeating units, achieving exponential signal amplification. qPCR enables precise detection of trace antigens by quantitatively analyzing repeat sequences within the amplified product. In situ fluorescence imaging directly labels the amplified product in situ within tissues or cells, preserving spatial information and making it suitable for pathological analysis.

[0015] The beneficial effects of the basic scheme are: 1. The linear amplification ability of RCA combined with the proximity-dependent template generation mechanism converts single antigen binding into long-chain DNA signal carriers, with a detection limit of up to fg / mL, which is more than 1000 times more sensitive than traditional ELISA.

[0016] 2. The double antibody sandwich method ensures target selectivity and achieves antibody-antigen specific recognition. The proximity-dependent template DNA circularization reaction is initiated only when two antibody probes bind to the antigen simultaneously, doubly verifying the presence of the target and achieving double assurance of high specificity.

[0017] 3. Ultra-sensitive quantification through qPCR is suitable for detecting trace markers in body fluids (serum and urine). In situ imaging of fluorescently labeled RCA products can locate the distribution of antigens in cells or tissues, which can be used for cancer pathological staging or infection lesion analysis, adapting to flexible use in various scenarios.

[0018] 4. The present invention does not require expensive labeled antibodies (such as enzyme-labeled antibodies) and reduces costs through universal fluorescent probes. In addition, the RCA reaction is an isothermal amplification and does not require a thermal cycler. It is suitable for scenarios with limited resources and improves the cost-effectiveness of the immunoassay method.

[0019] Furthermore, the antibody proteins on the first DNA antibody-coupled probe and the second DNA antibody-coupled probe can bind to the antigen to form a sandwich immune complex.

[0020] The beneficial effects of the basic scheme are: 1. Through the double antibody sandwich method (that is, two antibody probes simultaneously bind to the antigen to form a sandwich structure), high selectivity for the target antigen can be ensured, the possibility of non-specific binding is reduced, and the accuracy of the test results is improved.

[0021] 2. The formation of the sandwich immune complex allows the oligonucleotide chains on the two antibody probes to approach each other, making it easier to form a circular DNA template in the subsequent enzymatic release, complementation, and enzyme ligation steps. This proximity effect promotes the occurrence of the rolling circle amplification (RCA) reaction, thereby achieving significant signal amplification.

[0022] Furthermore, a single-stranded DNA is coupled to the second DNA antibody-coupled probe. The single-stranded DNA is a hairpin loop structure and has a 3'-5' end sequence.

[0023] The basic approach offers the following benefits: 1. The hairpin loop structure allows the single-stranded DNA to self-close when not digested by enzymes, preventing nonspecific hybridization and amplification. Upon antibody-antigen binding, specific enzyme cleavage opens the hairpin loop, releasing complementary DNA sequences that hybridize to form a circular DNA template. The precise design of the hairpin loop ensures efficient hybridization and circularization, thereby improving the signal amplification efficiency of rolling circle amplification (RCA).

[0024] 2. The hairpin loop design simplifies the operation steps. No additional DNA template preparation steps are required before rolling circle amplification, as the hairpin loop structure can be directly used for hybridization and circularization after being opened. This reduces operational complexity and improves detection efficiency.

[0025] Furthermore, the first DNA antibody-coupled probe is coupled with a portion of double-stranded DNA, wherein one of the DNA single strands is a hairpin loop structure and has a 3'-5' end sequence.

[0026] The basic scheme's beneficial effect is that the 3'-5' sequence design of the hairpin loop structure enhances hybridization specificity. Only when the first DNA antibody-conjugated probe binds to the antigen and the hairpin loop is properly opened can the sequence complementary to the other DNA single strand be released. This proximity dependence ensures that only specifically bound antigen triggers the subsequent rolling circle amplification reaction.

[0027] Furthermore, the hairpin loop structure sequence in the partially double-stranded DNA coupled to the first DNA antibody coupled probe is reverse complementary to the root sequence of the single-stranded DNA coupled to the second DNA antibody coupled probe, and the hairpin loop structure sequence of the single-stranded DNA coupled to the second DNA antibody coupled probe is reverse complementary to the exposed single-stranded sequence in the partially double-stranded DNA coupled to the first DNA antibody coupled probe.

[0028] The basic approach offers the following benefits: 1. Due to the reverse-complementary design of the hairpin loop sequence, when the two antibody-coupled probes bind to the antigen to form a sandwich structure, precise hybridization between the DNA strands occurs. This hybridization not only enhances the stability of the sandwich structure but also improves detection specificity. The reverse-complementary sequence design allows only probes that specifically bind to the antigen to hybridize, thereby reducing the possibility of nonspecific binding. This helps reduce background signal and improve detection accuracy.

[0029] 2. The hairpin loop structure and reverse complementary sequence design enable the direct generation of a circular DNA template after sandwich formation, eliminating the need for additional DNA template preparation steps. When the two antibody-coupled probes bind to the antigen, the DNA strands between them hybridize to form a circular DNA template. The hairpin loop structure and reverse complementary sequence design make the formation of this circular DNA template more efficient and stable.

[0030] Furthermore, the length of the hairpin loop structure DNA single strand in the partially double-stranded DNA coupled to the first DNA antibody-coupled probe and the DNA single strand coupled to the second DNA antibody-coupled probe is greater than or equal to 25 bp.

[0031] The benefits of the basic approach are as follows: 1. DNA probe length is a key factor influencing hybridization efficiency. Probes greater than or equal to 25 base pairs hybridize more effectively to target DNA sequences. Probes of this length provide sufficient base pairs for stable complementary pairing, thereby improving hybridization efficiency and accuracy.

[0032] 2. Longer probe lengths help reduce the possibility of nonspecific hybridization. Shorter probes may be more likely to hybridize with incompletely matched sequences, resulting in increased background signal. Probes greater than or equal to 25 bp in length can more strictly and selectively hybridize to the target sequence, thereby reducing interference from nonspecific hybridization.

[0033] 3. During the hybridization reaction, if the probe structure is unstable or easily disturbed, it may cause problems such as nonspecific hybridization or probe degradation, resulting in false positive results. In particular, the branched structure endonuclease may cut off the double-stranded DNA end of the first DNA antibody-coupled probe. Probes with a length greater than or equal to 25 bp can avoid these problems and improve the accuracy and reliability of detection.

[0034] Furthermore, the hairpin loop structure sequence in the partially double-stranded DNA coupled to the first DNA antibody-coupled probe and the hairpin loop structure sequence in the single-stranded DNA coupled to the second DNA antibody-coupled probe are both 8-15 bp in length.

[0035] The benefits of the basic scheme are as follows: 1. When the hairpin loop sequence length is within the range of 8-15 bp, it provides sufficient base pairs for stable complementary pairing while maintaining structural flexibility, facilitating hybridization reactions. Hairpin loops of this length can more effectively bind to the target DNA sequence, improving hybridization efficiency.

[0036] 2. A moderate length of hairpin loop sequence helps reduce the possibility of nonspecific hybridization. Shorter hairpin loop sequences may be more likely to hybridize with imperfectly matching sequences, resulting in increased background signal. A length of 8-15 bp, on the other hand, allows for more stringent and selective hybridization with the target sequence, thereby reducing interference from nonspecific hybridization.

[0037] Furthermore, the circular DNA template for rolling circle amplification consists of the hairpin loop structure sequence and the 5' end sequence on the first DNA antibody-coupled probe and the second DNA antibody-coupled probe.

[0038] The basic protocol offers the following benefits: 1. The circular DNA template, consisting of the hairpin loop sequence and its 5' end sequence, is structurally stable and easy to form. This circular template binds more effectively to the primers and enzymes used in rolling circle amplification, thereby improving the efficiency and specificity of the amplification reaction. Due to the specific and complex structure of the circular DNA template, it reduces nonspecific binding and amplification of non-target sequences, thereby reducing background signal and the likelihood of false-positive results.

[0039] 2. Traditional rolling circle amplification methods require separate preparation of a circular DNA template. However, the circular DNA template in this basic protocol is directly composed of the hairpin loop structure sequence and its 5' end sequence on the antibody-coupled probe, eliminating the need for additional template preparation steps and thus simplifying the operation. Due to the reduced template preparation steps, the detection time of this method is also shortened. This helps improve work efficiency, especially in application scenarios requiring rapid detection.

[0040] Furthermore, the circular DNA template sequence includes a primer binding sequence or a fluorescent probe binding sequence for qPCR detection, and also includes a fluorescent labeling sequence for in situ fluorescence imaging detection.

[0041] The benefits of the basic protocol are as follows: 1. Because the circular DNA template sequence contains sequences for both qPCR and in situ fluorescence imaging, the protocol enables multiplexed detection. This means that in a single experiment, two different detection methods can be used simultaneously for qualitative and quantitative analysis of target DNA, providing more comprehensive and accurate test results.

[0042] 2. qPCR and in situ fluorescence imaging each have unique advantages and application scenarios. qPCR, with its high sensitivity and specificity, holds a prominent position in molecular biology research, while in situ fluorescence imaging, with its intuitive and visual nature, is favored in cell biology and histology research. This basic protocol is designed to allow these two detection methods to complement each other and adapt to different research needs and application scenarios.

[0043] 3. The primer binding sequences and fluorescent probe binding sequences within the circular DNA template sequence are carefully designed to bind to the target DNA sequence with high specificity. This specific binding helps reduce interference from nonspecific amplification and background signals, thereby improving detection accuracy and sensitivity.

[0044] 4. In situ fluorescence imaging directly observes the distribution and expression of target DNA in cells or tissues through fluorescently labeled sequences. This intuitiveness helps to more accurately determine the presence and quantity of target DNA, while also providing information on its spatial distribution and cellular localization.

[0045] Furthermore, the oligonucleotide DNA and the antibody of the first DNA-antibody-coupled probe and the second DNA-antibody-coupled probe are coupled via click chemistry.

[0046] The benefits of the basic protocol are as follows: 1. Click chemistry is an efficient and rapid coupling method that enables efficient coupling of oligonucleotide DNA to antibodies under mild conditions. This method not only offers rapid reaction speeds and high yields, but also helps ensure the stability and reliability of the coupled probes.

[0047] 2. The chemical bonds formed by click chemistry are generally highly stable, capable of maintaining the structural integrity of coupled probes in complex biological environments. This helps ensure the stability and persistence of probes in vivo. Probes coupled by click chemistry exhibit good biocompatibility and low toxicity to biological cells. This helps ensure the safety and efficacy of probes in vivo, avoiding adverse effects on experimental or therapeutic outcomes.

[0048] 3. Compared with traditional coupling methods, click chemistry coupling has simpler and faster procedures. This helps simplify the probe preparation process and improves work efficiency. Due to the mild reaction conditions and high yield of click chemistry coupling, the production cost of the probe can be reduced. This will help promote the widespread application and commercialization of the probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the structure of the DNA antibody-coupled probe in an embodiment of the present invention.

[0050] Figure 2 Schematic diagram of the method of traditional PLA probe detection and ultrasensitive immunoassay of the present invention in an embodiment of the present invention.

[0051] The reference numerals in the drawings of the specification include: 1. first DNA antibody-coupled probe; 2. second DNA antibody-coupled probe; 3. antibody; 4. root; 5. hairpin stem; 6. hairpin ring. DETAILED DESCRIPTION

[0052] The following is further described in detail through specific implementation methods:

[0053] Example 1

[0054] Basically as attached Figure 1 、 Figure 2 An ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification comprises the following steps:

[0055] Step 1: immune binding: adding the first DNA antibody-coupled probe 1 and the second DNA antibody-coupled probe 2 to the sample to bind to the antigen to form an immune complex;

[0056] Step 2: Enzymatic cleavage and release: Use a branched endonuclease to cut the 5' end branch of the DNA hairpin structure of the first DNA antibody coupled probe 1 and the second DNA antibody coupled probe 2, releasing the hairpin loop 6 DNA single strand complementary to the root 4 of the oligonucleotide DNA chain of the other coupled probe;

[0057] Step 3: Complementation and enzyme ligation. Under the influence of the proximity effect formed by the immune binding of antibody 3, the oligonucleotide DNA chains of the first DNA antibody-coupled probe 1 and the second DNA antibody-coupled probe 2 are complementary to each other, forming a circular DNA single strand with two gaps that are complementary to the oligonucleotide DNA chains of the two coupled probes. The ends of the single strands at the gaps are adjacent, and T4 ligase is added to circularize the circular DNA single strand.

[0058] Step 4: Rolling circle amplification, adding phi29 polymerase to perform rolling circle amplification of circular DNA single strands;

[0059] Step 5: Detection: Detect the content of antibody 3 by qPCR to analyze the repeated specific sequence on the rolling circle amplification product, and detect the repeated specific sequence on the rolling circle amplification product by in situ oligonucleotide fluorescence labeling detection method to perform in situ fluorescence imaging in tissue cells.

[0060] The specific implementation process is as follows: qPCR quantitative detection, using human serum as the test sample, rabbit or mouse monoclonal antibodies, and goat or rabbit polyclonal antibodies as primary antibodies.

[0061] 1. Briefly centrifuge human plasma samples at 10,000 × g for 10 min or filter through a 1.2 µm filter plate to remove cellular debris. Add 10-20 µL of the treated sample to a 100 µL reaction system containing 10 nM monoclonal antibody-DNA antibody-coupled probe or 20 nM polyclonal antibody-DNA antibody-coupled probe. Incubate at room temperature for 1 hour with gentle shaking at 300 rpm to form immune complexes. The reaction system buffer composition is 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20, 1% BSA.

[0062] 2. After the immune complex is formed, add 90 µL of cleavage buffer and 5 U / µL of branched endonuclease (FEN1) to a final concentration of 0.5 U / µL. Incubate at 37°C for 10 minutes to cleave the 5' branch of hairpin loop 6 and release the complementary sequence of hairpin loop 6. The cleavage buffer composition is 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT.

[0063] 4. Add stop buffer to terminate the enzyme digestion reaction, remove the enzyme and digestion buffer, add 100 µL of enzyme ligation buffer and 0.25 mg / mL BSA, and add T4 DNA ligase at a final concentration of 0.02 U / µL. Incubate at 37°C for 30 minutes to connect the circular DNA single strands.

[0064] 5. Terminate the enzyme ligation reaction by adding stop buffer. Circularize the DNA template by rolling circle amplification using 0.1 U / µL phi29 DNA polymerase and 100 µL RCA buffer at 30°C for 60 min, followed by inactivation at 65°C for 10 min to obtain rolling circle amplification products. The RCA buffer composition is 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, and 0.2 mM dNTPs.

[0065] 6. Collect the final eluate and perform quantitative PCR detection. The primers and fluorescent probe complementary sequences of qPCR are designed for the repetitive sequence of RCA product. A linear relationship between Ct value and concentration is established by using a gradient dilution of known antigen concentration (0.1 fg / mL to 1 ng / mL).

[0066] Test results: The limit of detection (LOD) of this method is 0.1 fg / mL (using PSA antigen as the model target), which is 10% higher than that of traditional ELISA (~10 pg / mL). 5 The linear range was 0.1 fg / mL to 1 ng / mL (R² > 0.99), covering six orders of magnitude. Cross-reactivity testing revealed no significant signal at 10 ng / mL for similar proteins (e.g., CEA, AFP, IgG). Dual-antibody validation experiments demonstrated that RCA signal was only generated when both the primary DNA antibody-conjugated probe 1 and the secondary DNA antibody-conjugated probe 2 were present; no amplification was observed in the single-probe control group.

[0067] In simulated samples containing 10% human serum, 5% hemoglobin, or 2% lipids, the recovery rate remained at 90-110%, as shown in Table 1 below.

[0068] Table 1. Recoveries in Complex Samples

[0069]

[0070] In situ fluorescence imaging detection was performed using human immortalized keratinocytes HaCat and tissue sections as detection samples.

[0071] 1. HaCat cells were seeded at a density of approximately 120,000 cells / cm² in 8-well Lab-Tek II chamber slides and cultured for 1-2 days until 70-80% confluence. BJ hTert cells were seeded at a density of approximately 47,000 cells / cm² in chamber slides and serum-starved overnight in DMEM. Subsequently, BJ hTert cells were incubated with 50 ng / ml PDGF-BB in DMEM on ice for 1 hour, washed twice with PBS, and stored at −20°C until use.

[0072] HCT116 cells were seeded in 96-well plates at approximately 15,000 cells per well and cultured for 48 hours. Subsequently, the cells were serum-starved in DMEM for 5 hours. HCT116 cells were incubated with 100 ng / ml EGF at 37°C for 3 minutes, washed twice with PBS, and used directly.

[0073] 2. Process tissue sections according to standard methods. Deparaffinize slides in 100% xylene (three consecutive incubations of 5 minutes, 5 minutes, and 1 minute). Next, rehydrate the tissue to reduce the ethanol concentration, starting with two 3-minute incubations in 99% ethanol, followed by two 5-minute and 3-minute incubations in 95% ethanol, and finally a 3-minute incubation in 70% ethanol. Wash the slides three times in water and perform antigen retrieval. In a pressure cooker, boil the slides in 1× Dako Target retrieval solution at 2 atm and 125°C for 4 minutes, followed by boiling at 90°C for 3 minutes. Allow the tissue sections to cool to room temperature and wash with water. Store the samples in PBS until use.

[0074] Thawed cells were rehydrated in PBS at room temperature for 5 minutes and permeabilized with 0.2% TritonX100 in PBS for 5 minutes at room temperature, followed by washing in PBS. For EGFR and Shp2 assays, cells were permeabilized with 0.1% TritonX100 in PBS for 5 minutes at room temperature, followed by incubation with 1% SDS in PBS for 5 minutes, and then washed extensively with PBS to identify phosphorylation sites.

[0075] Cells and tissue sections were incubated with 50% Odyssey blocking buffer and 1× Tris-buffered saline (TBS) at 37°C for 30 minutes. Paired combinations of rabbit and mouse, as well as goat and rabbit antibodies, were incubated with samples overnight at 4°C in a humidified chamber. Following incubation, slides were washed three times for 3 minutes each in TBS containing 0.05% Tween 20 (TBS-T) before adding the second conjugated probe.

[0076] 3. Dilute the conventional in situ PLA probe and the first and second DNA antibody-conjugated probes 1 and 2 of the present invention to 20, 66, 200, 600, and 1800 ng / ml, add them to the TBS-buffered sample solution, and incubate for 60 min. After incubation, wash the sample three times with TBS-T for 3 min each.

[0077] 4. Digest both probes by adding enzyme digestion buffer (20 mM Tris-HCl (pH 7.6), 30 mM NaCl, 1 mM EDTA, 100 mM KCl, and 1 nM dithiothreitol (DTT)) supplemented with 0.25 mg / ml BSA and 0.5 U / µL FEN1 and incubate at 37°C for 45 minutes. Wash the slides twice in TBS-T for 3 minutes each. Ligate the circularized oligonucleotides designed on the two proximity probes by adding 0.02 U / µL T4 DNA ligase in T4 DNA ligase buffer supplemented with 0.25 mg / ml BSA at 37°C for 30 minutes. Wash the slides twice in TBS-T for 3 minutes each.

[0078] 5. Amplify isothermally at 37°C for 60 min by adding phi29 polymerase buffer and adding 0.5 U / μl phi29 polymerase, supplemented with 0.25 mM dNTPs and 0.25 mg / ml BSA. Then wash the slides twice in TBS-T for 3 min.

[0079] 6. Incubate the slides in PBS supplemented with 0.025 μM fluorescently labeled detection oligonucleotide and 40 μg / ml Hoechst 33342, 2.5 μg / ml salmon sperm DNA, and 0.25 mg / ml BSA at 37°C for 30 minutes to visualize RCA products and cell nuclei. Before mounting with mounting medium, wash the slides twice in 1x TBS for 10 minutes each and once in 0.2x TBS for 15 minutes each.

[0080] 7. Observe and image the slides using a fluorescence microscope with filters appropriate for the fluorescence wavelengths of DAPI, FITC, and Cy3. During image acquisition, the exposure time for DAPI is 40 ms, the exposure time for FITC is 510 ms, and the exposure time for Cy3 is 1.5 s.

[0081] Detection Results: To achieve the optimal signal-to-noise ratio, a dilution series of the DNA antibody-conjugated probe was used in the assay. The DNA antibody-conjugated probe for conventional in situ PLA was tested at concentrations of 50, 100, 200, 500, 1200, and 1800 ng / ml. Analysis revealed that the DNA antibody-conjugated probe at 500 ng / ml was suitable for studying receptor signaling in cells, while that at 1800 ng / ml was suitable for fluorescence imaging in tissue sections. For the DNA antibody-conjugated probe of the present invention, concentrations of 20, 50, 100, 200, 400, and 600 ng / ml were tested, with the optimal signal-to-noise ratio being achieved at 50 ng / ml. However, at higher DNA antibody-conjugated probe concentrations, the RCA product became so dense that the amount of nonspecific detection signal in the results increased significantly. This phenomenon is similar to that observed with existing DNA antibody-conjugated probes for in situ PLA. When one primary antibody is omitted, the amount of signal from cross-reactivity and nonspecific binding is low. Omitting any enzyme will cause the disappearance of all signals in cell fluorescence imaging, which means that the hairpin loop 6 structure can completely avoid the complementarity between nonspecific circular DNA templates and probes.

[0082] The efficiency of detecting phosphorylated proteins using conventional PLA probes and the probes of the present invention was compared. Immortalized fibroblast BJ hTert cells were starved or stimulated with platelet-derived growth factor-BB (PDGF-BB). Two DNA antibody-conjugated probes, both using anti-PDGFR and anti-pan-phospho-pY100 primary antibodies, detected increased PDGFR-β phosphorylation in stimulated cells. At both probe concentrations (50 and 500 ng / ml), the probes of the present invention were able to reveal more phosphorylation events than conventional in situ PLA. Furthermore, specific phosphorylation of epidermal growth factor receptor (EGFR) at tyrosine 1068 (pY1068) was quantified in HCT116 colorectal cancer cells following EGF stimulation. Consistent with previous results, the probes of the present invention, at a concentration of 50 ng / ml, increased the fluorescence signal intensity fivefold compared to conventional PLA probes under EGF-stimulated conditions.

[0083] Example 2

[0084] The difference from the above embodiment is that, as shown in the attached Figure 1 、 Figure 2As shown: the antibody 3 protein on the first DNA antibody-coupled probe 1 and the second DNA antibody-coupled probe 2 can bind to the antigen to form a sandwich immune complex, and the oligonucleotide DNA and antibody 3 of the first DNA antibody-coupled probe 1 and the second DNA antibody-coupled probe 2 are coupled by click chemistry; the first DNA antibody-coupled probe 1 is coupled with a portion of double-stranded DNA, in which one DNA single strand has a hairpin loop 6 structure and a 3'-5' end sequence; the second DNA antibody-coupled probe 2 is coupled with a DNA single strand, which has a hairpin loop 6 structure and a 3'-5' end sequence. 5' end sequence; the hairpin loop 6 structure sequence in the partially double-stranded DNA coupled to the first DNA antibody coupled probe 1 is reverse complementary to the root 4 sequence of the DNA single strand coupled to the second DNA antibody coupled probe 2, and the hairpin loop 6 structure sequence of the DNA single strand coupled to the second DNA antibody coupled probe 2 is reverse complementary to the exposed single strand sequence in the partially double-stranded DNA coupled to the first DNA antibody coupled probe 1; the length of the hairpin loop 6 structure DNA single strand in the partially double-stranded DNA coupled to the first DNA antibody coupled probe 1 and the DNA single strand coupled to the second DNA antibody coupled probe 2 is greater than or equal to 25 bp; the hairpin loop 6 structure sequence in the partially double-stranded DNA coupled to the first DNA antibody-coupled probe 1 and the hairpin loop 6 structure sequence in the single-stranded DNA coupled to the second DNA antibody-coupled probe 2 are both 8-15 bp in length; the circular DNA template for rolling circle amplification consists of the hairpin loop 6 structure sequence on the first DNA antibody-coupled probe 1 and the second DNA antibody-coupled probe 2 and their 5' end sequences, and the circular DNA template sequence contains a primer binding sequence or a fluorescent probe binding sequence for qPCR detection, and also contains a fluorescent label sequence for in situ fluorescence imaging detection.

[0085] The specific implementation process is as follows: 1. Design two corresponding DNA sequences, including the root 4, hairpin stem 5 and hairpin loop 6 sequences, where the sequences of the hairpin loop 6 and the root 4 of the two DNA sequences are complementary to each other, and amplify them through chemical synthesis and use of primers modified with 3' end azide, and then purify and recover them through gel electrophoresis to increase the concentration.

[0086] 2. In a copper-free cyclooctyne-catalyzed modification method, N-hydroxysuccinimide ester (NHS ester) was used to modify the amino group of antibody 3 to add a linker group to antibody 3. The recovered DNA chain was then used for a click chemistry reaction. The buffer was PBS with a pH of 7.4. The molar ratio of antibody 3 to DNA chain was 1:5. The reaction was shaken overnight at 4°C in the dark. The purified DNA-antibody-coupled probe was frozen at ultra-low temperature for future use.

[0087] Example 3

[0088] The difference from the above embodiment is that, as shown in the attached Figure 1 、 Figure 2Traditional PLA probes have been shown to improve the sensitivity of protein detection in binding assays, in which an antibody is used to capture the target from the sample, followed by rolling circle amplification using two DNA antibody-conjugated probes. Proximity extension reactions (PEA) rely on small reaction distances and dilute background reduction, but do not require washing before ligation or polymerization.

[0089] Compared to traditional in situ PLA probes, the probes of the present invention demonstrated enhanced signal generation in cells and tissues. The two approaches were compared by detecting proteins captured in solution on a solid support. The probes were immobilized in microtiter wells to capture IL-6. Conventional ELISA assays were then performed using enzyme-conjugated antibody reagents. The captured antigens were detected using either traditional in situ PLA or the probes of the present invention. Results demonstrated that the probes of the present invention achieved new limits of detection (LODs) compared to traditional in situ PLA designs, demonstrating superior performance compared to ELISA. Antigen dilutions using 10% or 100% cell lysate also demonstrated superior performance compared to traditional in situ PLA probes and ELISA-based assays.

[0090] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0091] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. An ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification, characterized by: The following steps are involved: Step 1: immune binding, adding a first DNA antibody-coupled probe (1) and a second DNA antibody-coupled probe (2) to the sample to bind to the antigen to form a sandwich immune complex; Step 2: Enzymatic cleavage and release: using a branched structure endonuclease to cut the 5' end branches of the DNA hairpin structure of the first DNA antibody coupled probe (1) and the second DNA antibody coupled probe (2), releasing the hairpin loop (6) DNA single strand complementary to the root of the oligonucleotide DNA chain (4) of the other coupled probe; Step 3: complementation and enzyme ligation. Under the influence of the proximity effect formed by antibody immune binding, the oligonucleotide DNA chains of the first DNA antibody-coupled probe (1) and the second DNA antibody-coupled probe (2) are mutually complementary, forming a circular DNA single strand with two gaps that is complementary to the oligonucleotide DNA chains of the two coupled probes, and the ends of the single strands at the gaps are adjacent. T4 ligase is added to circularize the circular DNA single strand. Step 4: Rolling circle amplification, adding phi29 polymerase to perform rolling circle amplification of circular DNA single strands; Step 5: Detection: Detect the content of the antibody (3) by qPCR to analyze the repeated specific sequence on the rolling circle amplification product, and perform in situ fluorescence imaging in tissue cells by in situ oligonucleotide fluorescence labeling detection method to detect the repeated specific sequence on the rolling circle amplification product; Wherein, the second DNA antibody coupled probe (2) is coupled with a DNA single strand, the DNA single strand is a hairpin loop (6) structure, and is a 3' end-5' end sequence; The first DNA antibody-coupled probe (1) is coupled to a portion of double-stranded DNA, wherein one DNA single strand is a hairpin loop (6) structure and has a 3'-5' end sequence; The hairpin loop (6) structural sequence in the partially double-stranded DNA coupled to the first DNA antibody coupled probe (1) is reverse complementary to the DNA single-stranded root (4) sequence coupled to the second DNA antibody coupled probe (2), and the DNA single-stranded hairpin loop (6) structural sequence coupled to the second DNA antibody coupled probe (2) is reverse complementary to the exposed single-stranded sequence in the partially double-stranded DNA coupled to the first DNA antibody coupled probe (1); The lengths of the hairpin loop (6) structure sequence in the partially double-stranded DNA coupled to the first DNA antibody coupled probe (1) and the hairpin loop (6) structure sequence in the single-stranded DNA coupled to the second DNA antibody coupled probe (2) are both 8-15 bp.

2. The ultrasensitive immunoassay method according to claim 1, wherein: The circular DNA template for rolling circle amplification consists of the hairpin loop (6) structure sequence and its 5' end sequence on the first DNA antibody-coupled probe (1) and the second DNA antibody-coupled probe (2).

3. The ultrasensitive immunoassay method according to claim 2, wherein: The circular DNA template sequence includes a primer binding sequence or a fluorescent probe binding sequence for qPCR detection, and also includes a fluorescent labeling sequence for in situ fluorescence imaging detection.

4. The ultrasensitive immunoassay method according to claim 1, wherein: The oligonucleotide DNA and the antibody (3) of the first DNA-antibody-coupled probe (1) and the second DNA-antibody-coupled probe (2) are coupled via click chemistry.

Images