Protein detection method and kit based on enzyme-mediated proximity extension assay

Abstract

The present application relates to a proximity extension assay kit based on enzyme site-specific coupling and a protein detection method, and belongs to the technical field of biomedical detection, comprising at least one pair of amino-modified oligonucleotide-antibody conjugate probes; wherein the oligonucleotide-antibody conjugate is prepared by deglycosylation treatment of glycopeptidase F and then using microbial transglutaminase (mTGase) to catalyze the covalent connection of oligonucleotide to the specific glutamine residue of the antibody Fc region. The proximity extension assay kit based on enzyme site-specific coupling and the protein detection method adopt PNGase F and mTGase mediated site-specific coupling, first use PNGase F to deglycosylate the N297 site of the IgG antibody Fc region, then use mTGase to specifically recognize the Q295 site of the deglycosylated IgG antibody Fc region, and connect it as a modified site with the amino-modified double-arm single-stranded DNA probe, and the connection site is away from the Fab region, completely retaining the antigen binding activity of the antibody.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a proximity extension analysis kit and protein detection method based on enzyme-based site-specific coupling. More specifically, this invention utilizes microbial transglutaminase to achieve site-specific coupling of antibodies with a single-stranded DNA probe containing two arms, and applies the resulting conjugate to construct a high-performance PEA multiplex protein detection system, suitable for the simultaneous and accurate quantification of dozens to hundreds of low-abundance protein biomarkers in scientific research, translational medicine, and in vitro diagnostics. Background Technology

[0002] Proximity extension analysis (PEA) is an advanced variant of the highly sensitive protein detection technique, specifically neighbor-linked assay (PLA). Its core principle is as follows: a pair of antibodies targeting different epitopes of the same target protein are each coupled with a partially complementary single-stranded DNA probe. When both antibodies bind to the target protein simultaneously, the DNA probes hybridize due to their spatial proximity, forming a transient double-stranded region. Subsequently, under the action of DNA polymerase, an extension reaction occurs using the other antibody as a template, generating a complete, amplifiable double-stranded DNA product. This product is quantified by qPCR or NGS, and the signal intensity is correlated with the target protein concentration. Compared to traditional PLA, PEA offers advantages such as lower background noise, higher specificity, and easier achievement of high multiplicity, making it an important tool for high-throughput proteomics research.

[0003] Despite the promising prospects of PEAs, their performance is highly dependent on the quality of the antibody-DNA probe conjugate. Currently, both academia and industry widely employ chemical conjugation methods to prepare probes, which have the following inherent drawbacks: First, random coupling leads to probe heterogeneity—modification occurs on multiple lysine residues on the antibody surface, resulting in partial loss of antibody antigen-binding activity. The number and location of DNA coupled to each antibody molecule are not uniform, resulting in poor batch-to-batch reproducibility and difficulty in standardization.

[0004] Secondly, the steric hindrance effect is significant—randomly linked DNA may interfere with the binding of antibodies to antigens or hinder effective hybridization and extension between DNA probes.

[0005] Third, while enzymatic site-specific and ratio-based coupling can partially address the issue of site randomness, glycosylation of N297 affects the efficiency of site-specific coupling at nearby Q295, and ratio-based coupling limits the amount of DNA bound to each antibody, thus limiting further amplification of nucleic acid signals. Furthermore, current enzymatic techniques have neither revealed nor taught the synergistic effects that can be achieved by combining PNGaseF and mTGase-mediated Fc-terminal site-specific coupling with the DNA hybridization-extension mechanism of PEA.

[0006] Therefore, the present invention aims to provide a proximity extension analysis kit and protein detection method based on enzyme-based site-specific coupling, which solves the problem of random coupling and achieves synergistic amplification of nucleic acid signals, thereby breaking through the sensitivity and repeatability bottlenecks of existing PEA technology. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a proximity extension analysis kit and protein detection method based on enzyme-based site-specific coupling. It has advantages such as uniform DAR values, high sensitivity, dual-arm probe signal amplification, excellent signal-to-noise ratio, and high throughput, solving the problems of uneven DAR, poor repeatability, and steric interference in traditional chemical coupling.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A proximity extension assay kit based on enzyme-based site-conjugated coupling, the kit comprising at least one pair of oligonucleotide-antibody conjugate probes; The oligonucleotide-antibody conjugate is prepared by deglycosylation modification with glycosylpeptidase F (PNGaseF) and catalysis by microbial transglutaminase (mTGase) to covalently link the oligonucleotide to a specific glutamine residue in the Fc region of the antibody. Furthermore, the two-arm oligonucleotide sequences on the pair of probes contain partially complementary regions, which can hybridize when spatially adjacent and serve as extension templates for each other; The kit also includes DNA polymerase, a mixture of dNTPs, PEA reaction buffer, and reagents for qPCR or NGS library construction.

[0009] Furthermore, the specific glutamine residue is the Q295 site of the Fc segment of a human IgG1, IgG2, or IgG4 antibody; wherein the N297 site of the antibody Fc terminus is deglycosylated by glycosylpeptidase F (PNGase F) before coupling.

[0010] Furthermore, in the oligonucleotide-antibody conjugate, each antibody molecule is conjugated with 1.8–2.2 oligonucleotide molecules.

[0011] Furthermore, the oligonucleotide also contains a universal primer binding region and a specific tag sequence for multiplex detection and differentiation using qPCR or high-throughput sequencing.

[0012] Furthermore, the PEA reaction buffer contains 10–50 mM Tris-HCl, 20–100 mM KCl, 1–5 mM MgCl2, 0.1%–1% BSA, and a stabilizer.

[0013] This invention also provides a protein detection method based on an enzyme-based site-joint proximity extension assay kit, comprising the following steps: S1. Incubate the sample to be tested with at least one pair of oligonucleotide-antibody conjugate probes to allow the probes to bind to the target protein; S2. Add DNA polymerase and dNTPs, and perform proximity hybridization and extension reactions in PEA reaction buffer to generate double-stranded DNA products. S3. Quantify the DNA product using a qPCR instrument or a high-throughput sequencing platform.

[0014] Furthermore, in S1, the deglycosylation modification reaction conditions before antibody conjugation are: a molar ratio of antibody to PNGase F of 1:0.04, a reaction temperature of 30°C, and a reaction time of 1 hour; and the oligonucleotide-antibody conjugation reaction conditions are: a molar ratio of antibody to probe to mTGase of 1:5:0.05, a reaction temperature of 30°C, and a reaction time of 2 hours.

[0015] Furthermore, in S2, the extension reaction conditions are: in PEA reaction buffer, polymerase is activated at 96°C for 1 minute, extended at 60°C for 5 minutes, and inactivated at 95°C for 5 minutes.

[0016] Furthermore, the method can simultaneously detect 10 to 384 different target proteins.

[0017] Compared with existing technologies, this invention provides a proximity extension analysis kit and protein detection method based on enzyme-based site-specific coupling, which has the following advantages: 1. This enzyme-based site-specific conjugation assay kit and protein detection method utilizes PNGase F and mTGase-mediated site-specific conjugation. First, PNGase F is used to deglycosylate the N297 site of the Fc region of the IgG antibody. Then, mTGase specifically recognizes the Q295 site of the deglycosylated IgG antibody Fc region and uses it as a modification site to precisely ligate to a probe of a double-armed single-stranded DNA. The ligation site is far from the Fab region, completely preserving the antibody's antigen-binding activity (retention rate >97%). Each antibody molecule is precisely conjugated to two DNA probes, with uniform DAR values ​​(4.0±0.2) and batch-to-batch coefficient of variation <8%, fundamentally solving the industry pain points of wide DAR value distribution and poor batch-to-batch reproducibility in traditional chemical conjugation methods.

[0018] 2. This enzyme-based site-coupled proximity extension assay kit and protein detection method utilizes a dual-arm oligonucleotide probe design—each antibody is coupled with two DNA probe arms. When a pair of probes hybridizes adjacently under the mediation of the target protein, it is equivalent to two nucleic acid signals participating in the extension reaction simultaneously, achieving synergistic amplification of nucleic acid signals. Compared with traditional single-arm probes, dual-arm probes significantly improve the amplification efficiency of DNA products, increase detection sensitivity by more than an order of magnitude, and achieve a detection limit of 0.1–1 fg / mL, meeting the detection requirements of ultra-low abundance targets such as exosomes and rare cytokines.

[0019] 3. This enzyme-based site-specific coupling proximity extension analysis kit and protein detection method utilizes oligonucleotide probes whose 3' extension complementary regions contain only short complementary sequences of 5-8 bases. Under conditions of spatial proximity without target antigen mediation, the thermodynamic stability of this sequence is insufficient to maintain double-strand hybridization in its free state, fundamentally eliminating non-specific background extension. Site-specific coupling avoids DNA aggregation and non-specific adsorption caused by random coupling, further reducing background signal. The signal-to-noise ratio is more than 10 times higher than that of the traditional chemically coupled PEA system.

[0020] 4. This enzyme-based site-coupled proximity extension assay kit and protein detection method is compatible with users' own qPCR or NGS platforms, breaking down the closed system barriers of existing PEA technology and exhibiting good openness and versatility. Through the design of unique tag sequences in the probes, it can simultaneously detect 10 to 384 different target proteins, making it suitable for large-scale proteomics research scenarios such as inflammatory factor profiling, tumor immune microenvironment characterization, and screening for biomarkers of neurodegenerative diseases. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the working principle of antibody-site-directed conjugation of nucleic acid according to the present invention.

[0022] Figure 2 This is a schematic diagram illustrating the working principle of the PEA of the present invention.

[0023] Figure 3 This is a comparison chart of the PEA standard curve of the present invention.

[0024] Figure 4 This is a non-reducing electrophoresis image of SDS-PAGE according to the present invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Please see Figures 1 to 4 .

[0027] Example 1: Preparation of anti-IL-6 PEA probe pairs (identical sequences on both arms) S1: Design of two-armed oligonucleotide probes To target the IL-6 protein, a pair of PEA probes (A1 and B1) were designed, with identical single-stranded DNA sequences on both amino-modified arms:

[0028] [IL6-A1] and [IL6-B1] are partially complementary sequences (~15 nt), and the rest are universal primer regions.

[0029] PNGase F-mediated antibody deglycosylation modification: Antibody: 1 mg / mL anti-IL-6 monoclonal antibody (IgG1), dissolved in PBS buffer.

[0030] Deglycosylation modification reaction ratio: antibody PNGase F = 1:0.04 (molar ratio).

[0031] Reaction conditions: Incubation in a water bath at 30°C for 1 hour, which allows the glycosyl group of asparagine at the N297 site of the Fc segment of the PNGaseF-catalyzed antibody to be cleaved to form aspartic acid (D297), thus eliminating its interference with Q295.

[0032] S2: mTGase site-directed coupling Antibody: 1 mg / mL anti-IL-6 monoclonal antibody (IgG1), dissolved in PBS buffer.

[0033] Coupling reaction ratio: antibody: probe: mTGase = 1:5:0.05 (molar ratio).

[0034] Reaction conditions: Incubation in a water bath at 30°C for 2 hours to allow the 5' amino group of the mTGase catalytic probe to form a covalent bond with the glutamine residue at the Q295 site of the antibody Fc segment.

[0035] S3: Probe Purification and Validation Purification: Uncoupled probes and mTGase enzymes were removed using a PD-10 desalting column.

[0036] DAR value determination: MALDI-TOF mass spectrometry confirmed DAR = 4.0 ± 0.2 (each antibody is conjugated with two double-stranded probes, i.e., 4 single-stranded DNAs).

[0037] Activity validation: The antigen-binding activity of the conjugated antibody was detected by ELISA, and the results showed that the antigen-binding activity retention rate was >97%.

[0038] Example 2: Preparation of anti-IL-6 PEA probe pairs (different sequences for each arm) S1: Design of two-armed oligonucleotide probes To target the IL-6 protein, another pair of PEA probes (A2 and B2) were designed, with different single-stranded DNA sequences on the amino-modified arms:

[0039] The 3' ends of probes A2 and B2 contain two complementary sequences (approximately 15 nt each) to ensure adjacent hybridization and extension. The single-stranded DNA sequences on the two arms are different, and each antibody molecule is coupled with two different single-stranded DNA arms.

[0040] S2 and S3: PNGase F antibody modification, mTGase conjugation, purification, and validation were performed as in Example 1. DAR = 4.0 ± 0.2, antigen binding activity retention rate > 96%.

[0041] Example 3: PEA reagent kit preparation and detection process Kit components: 96 pairs of mTGase-conjugated antibody-DNA probes (lyophilized powder); PEA Master Mix: Contains hot-start DNA polymerase, dNTPs, Mg²⁺, BSA, and trehalose; PEA Buffer: 20mM Tris-HCl (pH 8.0), 50mM KCl, 2mM DTT; qPCR Mix: SYBR Green premix.

[0042] Operating steps: Step a: Mix sample (1µL serum) with probe solution (4µL) and incubate at 4°C for 16 hours to allow the probe to bind to the target protein; Step b: Add PEA Master Mix (5µL), heat at 96℃ for 1 minute (activate polymerase), heat at 60℃ for 5 minutes (near-neighbor hybridization and extension reaction), and heat at 95℃ for 5 minutes (inactivate polymerase). Step c: Take 3 µL of the extension product for qPCR quantitative analysis.

[0043] Quantitative calculation: The concentration of IL-6 in the sample was calculated by comparing the Ct value obtained by qPCR with the standard curve.

[0044] Example 4: Background Suppression Design Verification Experimental design: The background signals of the short complementary sequence probe (3' complementary region 5-8 nt) and the long complementary sequence probe (3' complementary region 15-20 nt) of the present invention were compared under target protein-free conditions. The verification results are shown in Table 1.

[0045] Table 1 Results: The short complementary sequence probe of this invention has extremely low background signal (Ct≈35, close to the detection limit) in the absence of target protein, while the signal is strong and the signal-to-noise ratio is high when target protein is present, proving that the short complementary sequence design effectively suppresses nonspecific background extension.

[0046] Example 5: Construction of a multiplex protein detection system S1: Multi-target probe pair design Ten pairs of PEA probes were designed for each of the ten target proteins in the inflammatory cytokine spectrum (IL-6, TNF-α, IFN-γ, IL-1β, IL-10, IL-8, IL-12p70, GM-CSF, MCP-1, and IP-10). Each probe pair contains an antibody targeting the specific target and a two-armed oligonucleotide probe. The oligonucleotide sequence of each probe pair contains a unique tag sequence (barcode) for NGS sequencing to distinguish different targets.

[0047] PNGase F-mediated antibody deglycosylation modification: Using the same method as in Example 1, 10 pairs of deglycosylated modified antibodies were prepared.

[0048] S2: mTGase site-directed coupling Using the same method as in Example 1, antibody-oligonucleotide conjugates with 10 pairs of probes were prepared respectively.

[0049] S3: Multiple Detection Mix 10 pairs of antibody-oligonucleotide conjugate probes and incubate them together with the sample to be tested (1µL); After neighbor-to-neighbor hybridization and polymerase extension, an NGS library was constructed. The read counts of each tag sequence were obtained by high-throughput sequencing, and the concentrations of each target protein were calculated by comparing them with a standard curve.

[0050] Simultaneously, 10 inflammatory factors were detected, with the detection range covering fg / mL to ng / mL.

[0051] Comparative Example 1: Comparison of Chemical Coupling Methods The same antibody and probe sequences were used, but the traditional NHS-ester chemical conjugation method (non-site random conjugation) was employed, and the remaining steps were the same as in Example 3.

[0052] result: The DAR value has a wide distribution (1.5–4.5), with an inter-batch CV of 18%. SDS-PAGE shows a diffuse trailing stripe; The antigen binding activity retention rate was only 72%. IL-6 detection limit (LOD): 0.89 fg / mL in this invention vs. 25 fg / mL in the comparative example.

[0053] Comparative Example 2: Single-arm probe comparison A single-arm probe (each antibody conjugated to one single-stranded DNA, DAR≈2) was used instead of the double-arm probe of the present invention. The conjugation method was the same as in Example 1 (mTGase site-specific conjugation), and the other detection conditions were the same.

[0054] result: The IL-6 LOD of the single-arm probe system was 8.5 fg / mL; The IL-6 LOD of the two-arm probe system (the present invention) is 0.89 fg / mL; The signal-to-noise ratio of the dual-arm probe system is about 3 times higher than that of the single-arm system.

[0055] Comparative conclusion: The dual-arm probe significantly improves detection sensitivity through the synergistic amplification of dual nucleic acid signals.

[0056] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0057] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A proximity extension analysis kit based on enzyme-mediated site-specific coupling, characterized in that, The kit contains at least one pair of oligonucleotide-antibody conjugate probes; The oligonucleotide-antibody conjugate is prepared by deglycosylation of glycosylpeptidase F (PNGase F) and then covalently linking the oligonucleotide to a specific glutamine residue in the Fc region of the antibody using microbial transglutaminase (mTGase). Furthermore, the two-arm oligonucleotide sequences on the pair of probes contain partially complementary regions, which can hybridize when spatially adjacent and serve as extension templates for each other; The kit also includes DNA polymerase, a mixture of dNTPs, PEA reaction buffer, and reagents for qPCR or NGS library construction.

2. The proximity extension analysis kit based on enzyme-mediated site coupling according to claim 1, characterized in that, The specific glutamine residue is the Q295 site of the Fc segment of human IgG1, IgG2 or IgG4 antibody; The antibody Fc-terminal N297 site is deglycosylated by glycosylpeptidase F (PNGase F) before coupling.

3. The proximity extension analysis kit based on enzyme-mediated site coupling according to claim 1, characterized in that, In the oligonucleotide-antibody conjugate, each antibody molecule is conjugated with 1.8–2.2 oligonucleotide molecules.

4. The proximity extension analysis kit based on enzyme-mediated site coupling according to claim 1, characterized in that, The oligonucleotide also contains a universal primer binding region and a specific tag sequence for multiplex detection and differentiation using qPCR or high-throughput sequencing.

5. The proximity extension analysis kit based on enzyme-mediated site coupling according to claim 1, characterized in that, The PEA reaction buffer contains 10–50 mM Tris-HCl, 20–100 mM KCl, 1–5 mM MgCl2, 0.1%–1% BSA, and a stabilizer.

6. A protein detection method using the proximity extension assay kit based on enzyme-based site-specific coupling as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Probe Preparation: Design one or more pairs of oligonucleotide-antibody conjugate probes. Each pair of probes contains a first oligonucleotide probe conjugated to a first antibody and a second oligonucleotide probe conjugated to a second antibody. The first antibody and the second antibody specifically recognize different epitopes of the target protein, respectively. Each of the first oligonucleotide probe and the second oligonucleotide probe contains two amino-modified single-stranded DNA arms, and the 3' ends of the two probes contain partially complementary sequences. The N297 of the Fc end of the antibody is deglycosylated with PNGase F. Then, using microbial transglutaminase (mTGase) as a catalyst, the oligonucleotide probes are covalently linked to the conserved glutamine residue (Q295) of the Fc segment of the antibody, and the DAR value is confirmed after purification. S2, Proximity Extension Reaction: The sample to be tested is incubated with the probe pair to allow the probe pair to bind to the target protein, and the complementary sequences at the 3' ends hybridize under spatial proximity conditions; DNA polymerase and dNTPs are added to carry out the extension reaction to generate double-stranded DNA product; S3. Quantitative detection: The DNA product is quantitatively detected by qPCR or high-throughput sequencing to determine the product amount and calculate the target protein concentration based on the standard curve.

7. The protein detection method of the proximity extension analysis kit based on enzyme site coupling according to claim 6, characterized in that, In S1, the antibody deglycosylation modification reaction conditions are: a molar ratio of antibody to PNGase F of 1:0.04, a reaction temperature of 30°C, and a reaction time of 1 hour; the oligonucleotide-antibody conjugation reaction conditions are: a molar ratio of antibody to probe to mTGase of 1:5:0.05, a reaction temperature of 30°C, and a reaction time of 2 hours.

8. The protein detection method of the proximity extension analysis kit based on enzyme site coupling according to claim 6, characterized in that, In S2, the extension reaction conditions are: in PEA reaction buffer, activate polymerase at 96°C for 1 minute, extend at 60°C for 5 minutes, and inactivate at 95°C for 5 minutes.

9. The protein detection method of the proximity extension analysis kit based on enzyme site coupling according to claim 6, characterized in that, The method can simultaneously detect 10 to 384 different target proteins.

10. The application of the enzyme-based site-coupled proximity extension analysis kit according to claim 1 in inflammatory factor profiling, tumor immune microenvironment analysis, or screening for biomarkers of neurodegenerative diseases.

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