A dual antibody-linked probe for detecting lactated PGK1, its preparation method and application

Abstract

This invention relates to the field of biology and discloses a dual-antibody-linked probe for detecting lactated PGK1, its preparation method, and its application. The dual-antibody-linked probe includes antibody A and antibody B, and complementary oligonucleotides conjugated to antibody A and antibody B, respectively. Antibody A is a polypeptide that recognizes the K139-lactation modification at the K139la site of PGK1. Antibody B is a polypeptide with a length of 12-15 amino acids that is ≥50 amino acids away from the K139 site in PGK1. The dual-antibody-linked probe obtained by this invention achieves ultrasensitive detection of the target protein.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, specifically to a dual antibody-linked probe for detecting lactated PGK1, its preparation method, and its application. Background Technology

[0002] In the field of tumor detection technology, existing detection methods are limited in their application in scenarios such as early diagnosis and monitoring of minimal residual disease due to their inherent limitations. Existing detection methods, including traditional immunoassays such as enzyme-linked immunosorbent assays (ELISA), suffer from problems such as insufficient sensitivity, difficulty in guaranteeing specificity, and poor clinical applicability.

[0003] For example, sensitivity is insufficient. ELISA, for instance, exhibits linear signal amplification and a high detection limit. When dealing with protein biomarkers present in extremely low concentrations (pg / mL or lower) in bodily fluids like blood, the effective signal is often drowned out by background noise, making reliable detection impossible. Specificity is difficult to guarantee. Most existing technologies rely on a "single recognition" mechanism, where a single antibody captures and identifies the target. In complex environments like plasma containing thousands of proteins, even high-quality antibodies risk cross-reactivity with structural analogs or non-specific adsorption, leading to false positives and affecting accuracy. Clinical applicability is poor. For example, while protein spectrometry (MS) can accurately identify protein modifications, its expensive equipment, cumbersome operation, and long detection cycle make it impractical for large-scale, rapid clinical screening. Other highly sensitive techniques, such as immunoPCR, are difficult to standardize and automate in clinical settings due to their complexity and susceptibility to contamination.

[0004] Due to tumor metabolic reprogramming, lactate levels are significantly elevated, accompanied by widespread lysine lactylation modifications. Among these, PGK1 (phosphoglycerate kinase 1), a key enzyme in the glycolysis pathway, not only exhibits significantly upregulated expression, but also shows a sharp increase in lactation modification levels at specific sites. Therefore, lactated PGK1 is a crucial molecular event in the development and progression of ccRCC and possesses great potential as a highly specific diagnostic biomarker. However, due to the technological limitations of existing methods, the standardized quantification of trace levels of lactated PGK1 places extremely high demands on the sensitivity and specificity of detection methods. Current technologies cannot meet the detection needs for novel, low-abundance, post-translational modification biomarkers such as lactated PGK1. Summary of the Invention

[0005] This invention addresses the problems existing in the prior art by providing a dual antibody-linked probe for detecting lactated PGK1, its preparation method, and its application.

[0006] The technical solution adopted in this invention is: a dual-antibody-linked probe for detecting lactated PGK1.

[0007] Including antibody A and antibody B, and complementary oligonucleotides conjugated to antibody A and antibody B, respectively;

[0008] The antibody A is a polypeptide A that recognizes the K139-lactation modification of the PGK1 K139la site; the sequence of polypeptide A is SEQ NO.1;

[0009] The antibody B is a polypeptide B with a length of 12 to 15 amino acids, which is used to recognize PGK1 at a distance of ≥50 amino acids from the K139 site; the sequence of polypeptide B is SEQ NO.2.

[0010] Furthermore, the complementary oligonucleotide sequence conjugated with antibody A is SEQ NO.3; and the complementary oligonucleotide sequence conjugated with antibody B is SEQ NO.4.

[0011] A method for preparing a dual-antibody-linked probe for detecting lactated PGK1 includes the following steps:

[0012] Step 1: Couple polypeptide A and polypeptide B to a carrier protein to obtain initial antigens A' and B', wherein the carrier protein includes hemocyanin KLH;

[0013] Step 2: Inject the initial antigens A' and B' into the live culture sample for immune culture. After the predetermined cycle, collect serum.

[0014] Step 3: The serum from the live samples injected with initial antigen A' and initial antigen B' in Step 2 were screened for titer and specificity using the indirect enzyme-linked immunosorbent assay (ELISA) to obtain samples A'' and B'' that meet the requirements.

[0015] Step 4: Perform booster immunization on samples A'' and B'' obtained from the initial screening in Step 3, and obtain the desired antibody A and antibody B through B cell sorting, antibody gene amplification and cloning, and recombinant antibody expression and screening, respectively.

[0016] Step 5: Connect antibody A and antibody B to complementary oligonucleotides using click chemistry to obtain the desired dual-antibody linkage probe.

[0017] Furthermore, in step 3, antibody A in the specific initial screening uses polypeptide A as the positive sieve plate and K139 ectopically modified polypeptide A1 and K139 unmodified polypeptide A2 as the negative sieve plates, selecting individuals with high antigen-specific titers and low cross-reactivity to the negative sieve plates.

[0018] Furthermore, the polypeptide sequence of A1 is SEQ NO.5, and the polypeptide sequence of A2 is SEQ NO.6.

[0019] An application of a dual antibody-linked probe for detecting lactated PGK1, the probe being used for the detection of a target protein.

[0020] Furthermore, the detection method for the target protein is as follows:

[0021] S1: Provide a sample containing the target protein, bind the target protein and the linking probe to obtain linear tag DNA;

[0022] S2: Using the DNA molecule obtained in S1 as a template, DNA polymerase is used to amplify the product through rolling circle amplification to obtain a long single-stranded repeat product containing qPCR primer binding sites.

[0023] S2: The product from step 2 can be detected by qPCR to complete the detection of the target protein.

[0024] Furthermore, the DNA molecule template sequence in S2 is SEQ NO.7.

[0025] Furthermore, the target protein is a protein that lactates PGK1.

[0026] The beneficial effects of this invention are:

[0027] This invention employs site-specific antibody A and protein-specific antibody B, which are coupled with complementary oligonucleotides to form a dual antibody probe. This dual antibody probe can be used for in situ detection and spatial distribution analysis of PGK1 K139la-K139 lactation modification in tumor tissue and cell samples, achieving ultrasensitive detection of the biomarker PGK1 in complex blood samples.

[0028] When two antibodies simultaneously bind to the same target protein molecule, the accompanying DNA probes, due to their spatial proximity, are linked into a closed circular DNA molecule by the action of the ligation probe and ligase. This "dual recognition" ensures that signal generation is strictly dependent on the presence of the "target protein in a specific modified state," guaranteeing extremely high detection specificity from the source. Using this circular DNA molecule as a template, rolling circle amplification (RCA) is initiated under the action of DNA polymerase; the single recognition time is exponentially amplified into a long-chain DNA product containing thousands of repeating sequences; the fluorescently labeled detection probe hybridizes with the RCA product, generating a strong fluorescent signal that can be quantified by conventional instruments.

[0029] This invention transforms single-molecule recognition events into macroscopic signals, achieving extremely high sensitivity and meeting the needs of early diagnosis and monitoring of trace residual lesions. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the site screening of antibody B in the probe obtained in Example 1 of the present invention. a is the KD value, b is the epitope, c is the surface, d is the disorder degree, e is Bepipred, and f is the maximum charge.

[0031] Figure 2 This is a Western blot of the polypeptide in the probe obtained in Example 1 of the present invention.

[0032] Figure 3 This is a schematic diagram of the qPCR detection results of the probe at different dilution gradients of cell positive lysates obtained in Example 1 of the present invention.

[0033] Figure 4 This is a schematic diagram of the qPCR detection results of the probe obtained in Example 1 of the present invention and the control group. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0035] A dual-antibody-linked probe for detecting lactated PGK1.

[0036] Including antibody A and antibody B, and complementary oligonucleotides conjugated to antibody A and antibody B, respectively;

[0037] The antibody A is a polypeptide A that recognizes the K139-lactation modified PGK1 K139la site;

[0038] The antibody B is a polypeptide B with a length of 12-15 amino acids, which is used to recognize PGK1 at a distance of ≥50 amino acids from the K139 site. The sequence of polypeptide A is SEQ NO.1, and the sequence of polypeptide B is SEQ NO.2. The complementary oligonucleotide sequence conjugated with antibody A is SEQ NO.3; the complementary oligonucleotide sequence conjugated with antibody B is SEQ NO.4.

[0039] A method for preparing a dual-antibody-linked probe for detecting lactated PGK1 includes the following steps:

[0040] Step 1: Couple polypeptide A and polypeptide B to a carrier protein to obtain initial antigens A' and B', wherein the carrier protein includes hemocyanin KLH;

[0041] Step 2: Inject the initial antigens A' and B' into the live culture sample for immune culture. After the predetermined cycle, collect serum.

[0042] Step 3: Serum samples from the live samples injected with initial antigen A' and initial antigen B' in Step 2 were initially screened for titer and specificity using an indirect enzyme-linked immunosorbent assay (ELISA) to obtain qualified samples A'' and B''. For the specificity screening, antibody A was screened using peptide A as the positive screening plate, and K139 ectopically modified peptide A1 and K139 unmodified peptide A2 as negative screening plates. Individuals with high antigen-specific titers and low cross-reactivity to the negative screening plates were selected. The peptide sequence of A1 is SEQ NO. 5, and the peptide sequence of A2 is SEQ NO. 6.

[0043] Step 4: Perform booster immunization on samples A'' and B'' obtained from the initial screening in Step 3, and obtain the desired antibody A and antibody B through B cell sorting, antibody gene amplification and cloning, and recombinant antibody expression and screening, respectively.

[0044] Step 5: Connect antibody A and antibody B to complementary oligonucleotides using click chemistry to obtain the desired dual-antibody linkage probe.

[0045] The probe is used for the detection of targeted proteins, and the detection method is as follows:

[0046] S1: Provide a sample containing the target protein, bind the target protein and the linking probe to obtain linear tag DNA;

[0047] S2: Using the DNA molecule obtained in S1 as a template, a long single-stranded repeat product containing qPCR primer binding sites is obtained by rolling circle amplification under the action of DNA polymerase; the DNA molecule template sequence is SEQ NO.7.

[0048] S2: The product of S2 can be detected by qPCR to complete the detection.

[0049] Example 1

[0050] A method for preparing a dual-antibody-linked probe for detecting lactated PGK1 includes the following steps:

[0051] Step 1: Couple polypeptide A and polypeptide B to a carrier protein to obtain initial antigens A' and B', wherein the carrier protein includes hemocyanin KLH.

[0052] Four healthy New Zealand rabbits, all female, weighing 2.2–2.8 kg, were selected. Two were used for the acquisition site-specific rabbit monoclonal antibody A, and the other two were used for the acquisition protein-specific rabbit monoclonal antibody B.

[0053] Antigen design, targeting site-specific antibody A (specifically recognizes the 139-lactation modification of PGK1 K139la), the polypeptide of which was determined based on the PGK1 amino acid sequence is:

[0054] Polypeptide A: KDASGN-(Lactyl)K-VKAEPA (133-145), sequence as shown in SEQ NO.1;

[0055] Design negative sieve plates A1 and A2

[0056] A1 (PGK1 K141la modified control peptide): KDASGNKV-(lactyl)K-AEPA (133-145), sequence as shown in SEQ NO.5;

[0057] A2 (PGK1 K139 unmodified control peptide): KDASGNKVKAEPA (same site unmodified), sequence as shown in SEQ NO.6.

[0058] Polypeptide B: DKFDENAKTGQA (290–301), sequence as shown in SEQ NO.2.

[0059] The screening process for site-specific antibody B (which recognizes another linear epitope of PGK1 far from K139) is as follows: Figure 1 As shown, by analyzing the hydrophobicity of proteins, such as... Figure 1 In the case of a, the linear epitope tends to be as follows: Figure 1 In the middle b, the surface is exposed as Figure 1 In the middle c, disorder / flexibility tendencies are as follows Figure 1 In the comparison of the overall epitope potential of different segments, such as d Figure 1 In the middle e, the maximum continuous charged length of K / R / D / E within the local window is as follows: Figure 1 The f-segment was used to identify fragments that might cause nonspecific adsorption, sample loading background, and solubility issues. N-glycosylation motifs NX-[S / T] (X≠P) were scanned and labeled to avoid the risk of natural glycan masking and sugar-related cross-reactions. Segments with hydrophobic background (KD below approximately 0.8), epitope and surface exposure tendencies higher than the empirical line (approximately 1.10 and 1.05), disorder / flexibility signals higher than the baseline (approximately 1.00), and a comprehensive score exceeding the reference threshold (approximately 0.50) were selected. Fragments crossing N-glycosylation motifs were excluded, and the maximum charged chain length was controlled to within 3. Linear immunopeptides meeting the criteria of being "easily recognized by B cells" (high epitope tendency), "accessible in the native conformation" (high surface exposure, low hydrophobicity), and "processable and preparable" (moderate disorder, short charged chain length, no glycosylation motifs), and free of transmembrane segments and strong hydrophobicity were selected. An amino acid sequence with a length between 12 and 15 amino acids that is ≥50 amino acids away from the K139 site.

[0060] The above peptides, A and B, were conjugated to the carrier protein KLH as initial antigens. Then, the initial antigens A' and B', A1 and A2 were conjugated to bovine serum albumin (BSA) for subsequent ELISA screening and affinity chromatography purification.

[0061] Step 2: The initial antigens A' and B' conjugated with KLH and BSA in Step 1, and A1 and A2 conjugated with BSA, were injected into different live culture samples for immune culture. After a predetermined cycle, serum was collected.

[0062] The immunization procedure for each antigen is as follows:

[0063] Each rabbit was immunized six times according to a fixed schedule: on days 1, 7, 14, 21, 28, and 40. The first immunization used complete Freund's adjuvant (CFA), and subsequent booster immunizations used incomplete Freund's adjuvant (IFA). For each immunization, 100 µg of the corresponding KLH-conjugated polypeptide immunogen was diluted with sterile saline and thoroughly emulsified with an equal volume of Freund's adjuvant. The emulsified immunogen (total volume 1 mL) was injected at four points: 0.25 mL subcutaneously in both shoulders and 0.25 mL intramuscularly in both hind legs.

[0064] On day 28, 2 mL of peripheral blood was collected from each rabbit for the first time. The blood was centrifuged at 4000 rpm for 10 minutes, and the supernatant serum was collected.

[0065] Step 3: The serum samples obtained in Step 2 were initially screened for titer and specificity using the indirect enzyme-linked immunosorbent assay (ELISA).

[0066] Screening of antibody A: The polypeptide with sequence SEQ NO.1 was used as the positive screening plate, and the K139 ectopically modified polypeptide A1 and the K139 unmodified polypeptide A2 were used as the negative screening plates;

[0067] The screening of antibody B uses a peptide with a sequence such as SEQ NO.2 as a positive screening plate.

[0068] Subsequently, 30 mL of serum was collected on days 35, 47, and 52, and the above screening test was repeated. Individuals with high antigen-specific titers and low cross-reactivity to the negative screening plate were selected to proceed to step 4.

[0069] Step 4: Using the samples obtained from the initial screening in Step 3 (samples containing antibody A and antibody B respectively), perform booster immunization. After B cell sorting, antibody gene amplification and cloning, recombinant antibody expression and screening, the desired antibody A and antibody B are obtained.

[0070] The samples containing antibody A and antibody B were processed as follows:

[0071] Three days prior to scheduled euthanasia, selected immunopositive rabbits (i.e., the samples obtained from the initial screening) (one rabbit each of antibodies A and B) underwent a final pulse immunization via the marginal ear vein, injecting 50 µg of the corresponding adjuvant-free polypeptide antigen to enrich antigen-specific plasma cells. The rabbits were euthanized between days 54 and 56, and their spleens were removed under aseptic conditions.

[0072] Tissues were gently homogenized in RPMI-1640 medium and passed through a 70 µm cell sieve to prepare a single-cell suspension. Mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation, stained with trypan blue, and counted to ensure cell viability was above 90%.

[0073] Then, fluorescence staining and flow cytometry sorting (FACS) were performed.

[0074] Cells were resuspended in PBS buffer (sorting buffer) containing 2% FBS. Active dye (to remove dead cells) and rabbit B cell surface marker antibody (anti-Rabbit IgG, used to enrich memory B cells / plasma cells that have undergone class switching) were added.

[0075] Using a flow cytometer, cells were sorted according to the following gating strategy: "live cells (viability dye negative) → IgG positive cells". Each sorted single cell was directly sorted into a 96-well PCR plate pre-filled with cell lysis buffer and reverse transcription primers.

[0076] Antibody gene amplification and cloning:

[0077] Single-cell lysates were directly reverse transcribed (RT-PCR) to synthesize cDNA. Nested PCR was performed using specific primers for the variable regions of the rabbit antibody heavy chain (HV) and light chain (VL) to efficiently amplify the antibody variable region genes. The amplified VH and VL gene fragments were cloned into mammalian cell expression vectors containing the constant region of rabbit IgG via Gibson assembly to construct paired heavy and light chain expression plasmids.

[0078] Recombinant antibody expression and screening:

[0079] Paired heavy and light chain expression plasmids were co-transfected into CHO and other suspension cells for transient expression. After 3–5 days of culture, the cell culture supernatant was collected. High-throughput screening was performed using the same ELISA method as serum screening to verify the specific binding ability of recombinant antibodies to the corresponding antigens. Plasmids of positive clones (OD value of positive sieve plate > 1.0 and significantly higher than that of negative sieve plate) were sequenced for confirmation.

[0080] Construction of stable cell lines and large-scale antibody production:

[0081] The selected positive antibody expression plasmids were transfected into the CHO-K1 cell line, and cell lines that stably and highly express antibodies were obtained through drug screening. The stable cell lines were then gradually adapted and transferred to serum-free medium for scale-up culture. When the cell density reached or exceeded 2 × 10⁻⁶ cells / year... 6 When the cell count reaches 1000 cells / mL, the culture supernatant is collected, and cells and debris are removed by centrifugation and filtration through a 0.22 µm filter membrane in preparation for purification.

[0082] Antibody purification and validation:

[0083] Purification: The clarified cell culture supernatant was loaded onto a Protein A affinity chromatography column. After loading, the column was washed with PBS buffer (pH 7.4) until the baseline stabilized. Subsequently, the bound antibody was eluted with 0.1 M glycine-hydrochloric acid buffer (pH 2.7), and the collected eluent was immediately neutralized to pH 7.2–7.4 with 1 M Tris-HCl (pH 9.0).

[0084] Western blotting was used to examine whether the preliminarily purified antibody specifically bound to the target antigen without cross-reacting with related target molecules. The process is as follows:

[0085] In different lanes of the SDS-PAGE gel, equal amounts of the target antigen and negative control were loaded respectively.

[0086] After electrophoresis, the proteins are transferred to PVDF or NC membranes and blocked with skim milk powder or BSA solution to block nonspecific sites.

[0087] The membrane was incubated with preliminarily purified antibody A (as primary antibody), washed, and then incubated with HRP-labeled secondary antibody under polarity. After washing again, ECL chemiluminescent substrate was added, and the signal was detected using an imaging system. The results are as follows: Figure 2 As shown in the figure, peptide A appears as a wash band, A1 is the control for modification site shift in the same region, and A2 is the unmodified control in the same region. Both A1 and A2 are detected. Antibody B shows a clear band, while the BSA negative control is not detected. It can be seen that both antibodies can stably recognize the target signal at the target molecular weight position, and the nonspecific background is low.

[0088] Step 5: Connect antibody A and antibody B to complementary oligonucleotides using click chemistry to obtain the desired dual-antibody linkage probe (the probe consists of two parts: a conjugate obtained by connecting antibody A to complementary oligonucleotides and a conjugate obtained by connecting antibody B to complementary oligonucleotides; the probe consists of two separate parts that exhibit complementary effects when detecting the target protein).

[0089] PLA probes with complementary extension regions were designed for antibodies A and B, respectively. Two complementary oligonucleotides, PLUS and MINUS (each approximately 50–70 nt in length), were designed, containing universal amplification primer sites and ligation / amplification docking sites to avoid self-aggregation and hairpin structures, and Tm matching.

[0090] PLUS (conjugated to antibody A, 5' azide modification for click conjugation): 5'-Azide-AGCTGATCGTTACGATCCGTAG TTTTTTTTTTCTGACTGACTGA-3', sequence as shown in SEQ NO.3.

[0091] MINUS (conjugated to antibody B, 5' azide modification for click conjugation): 5'-Azide-TGCTACGGATCGTAACGATCAGCTTTTTTTTTTTTCAGTCAGTCAG-3', sequence as shown in SEQ NO.4.

[0092] A-PLUS and B-MINUS were prepared using click chemistry (DBCO–Azide, SPAAC) methods. Antibodies A and B were respectively lightly modified with NHS-DBCO (protein:DBCO = 1:3, PBS pH 7.4, room temperature 45 min). After removing free DBCO by ultrafiltration, they were reacted with the corresponding 5'-Azide-modified PLUS or MINUS at a 1:3 molar ratio in PBS pH 7.2 at room temperature for 2 h to obtain conjugates (i.e., biantibody-linked probes). Free oligonucleotides were removed by 100 kDa ultrafiltration.

[0093] The methods for detecting targeted proteins are as follows:

[0094] The circular RCA template used in the following method (i.e., the DNA molecule template mentioned above) (after phosphorylation of the 5' end of the linear template, it is closed by T4 ligase): 5'-P-CTGACTGACTGACTGAACTTAGTGATCTCGTTAACCGTAGTTCAGTCAGTCAGTCAGTCAGT-3', the sequence of which is shown in SEQ NO.7.

[0095] Bridging oligonucleotide chain: 5'-P-TCAGTCAGTCAG-3', sequence as shown in SEQ NO.8.

[0096] qPCR primers

[0097] Primer-F: 5'-AGCTGATCGTTACGATCCGTA-3', sequence as shown in SEQ NO.9.

[0098] Primer-R: 5'-TACGGTTAACGAGATCACTA-3', sequence as shown in SEQ NO.10.

[0099] S1: Provide a sample containing the target protein, bind the target protein and the linking probe to obtain linear tag DNA;

[0100] Cell lysis buffer was used as the test sample, and an equivalent of 2×10⁶ cells was collected. 5 Cells were lysed with PBS + 0.1% NP-40 + protease inhibitor, incubated on ice for 10 min, then incubated at 12,000×g, 4 °C for 10 min, and the supernatant was collected. 5 μg of total protein was loaded per reaction.

[0101] Add the following to a 20 μL system: cell lysis buffer (containing 5 μg protein), 1 nM A-PLUS, 1 nM B-MINUS, 0.5 nM bridging oligonucleotide, 1× T4 DNA ligase buffer (containing ATP), and 5 U T4 DNA ligase. Incubate at 37°C for 30 min, allowing PLUS and MINUS to form a short double-stranded alignment domain with the Bridge at their 3' end 12 nt complementary arm, which is in close proximity to the target protein. T4 then seals the Bridge's 5'-P and MINUS's 3'-OH at the double-strand gap, as well as the Bridge's 3'-OH and the adjacent end of PLUS, sequentially generating a continuous "linear tag DNA" with an extendable 3'-OH.

[0102] S2: Using the DNA molecule obtained in S1 as a template, DNA polymerase is used to amplify the product through rolling circle amplification to obtain a long single-stranded repeat product containing qPCR primer binding sites.

[0103] After inactivating T4 DNA ligase at 65 ℃ for 10 min and cooling to room temperature, add 5 nM of pre-circularized RCA template (SEQ NO.7), 5 U of phi29 DNA polymerase, 250 μM dNTP, 8 mM MgCl2, 0.1 mg / mL BSA, and Tris-KCl-(NH4)2SO4 buffer (40 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM (NH4)2SO4) to a final volume of 25 μL. Perform rolling circle amplification at 37 ℃ for 60 min, and inactivate the polymerase at 65 ℃ for 10 min. Anneal the 3' end of the "linear tag DNA" obtained in the previous step in the complementary region (≥18 nt) of the circular template. phi29 replicates along the circular template starting from this 3'-OH, generating a long single-stranded repeat product containing qPCR primer sites.

[0104] S3: Perform qPCR detection on the product of S2 to complete the detection.

[0105] Use 2 μL of RCA product as a template to prepare 20 μL of qPCR reaction: 10 μL of 2×SYBR Green Master Mix, 0.3 μM primer-F, 0.3 μM primer-R, and make up to the required amount of nucleic acid-free water. Program: 95℃ for 2 min; followed by 40 cycles: 95℃ for 10 s, 60℃ for 30 s.

[0106] Record Ct values ​​for quantitative analysis, and the results are as follows: Figure 3 and Figure 4 As shown, to illustrate the effectiveness of the probe of this invention, Ct values ​​were measured after serial dilution of 786-O cell positive lysates, using the detection method described in Example 1. The results are as follows... Figure 3 As shown, the blank area represents the absence of positive cell lysates and serves as a control.

[0107] Figure 4 The control group used cell lysates from healthy individuals, while the case group used cell lysates from patients. All measurements were performed using the method described in Example 1. The Mann-Whitney U test was used for statistical analysis.

[0108] This invention provides a method based on the binding of a dual-antibody proximity ligation probe (PLA) to rolling circle amplification (RCA). A pair of specific antibodies are selected: antibody A specifically recognizes a PTM at a specific site on the protein, while antibody B recognizes a universal epitope of the target protein. Each antibody is conjugated to a complementary oligonucleotide. When both antibodies simultaneously bind to the same target protein molecule, the carried DNA probes, due to their spatial proximity, are ligated into a closed circular DNA molecule under the action of the ligation probe and ligase. This "dual recognition" mechanism ensures that signal generation is strictly dependent on the presence of the "target protein in a specific modified state," guaranteeing extremely high detection specificity from the source. This circular DNA molecule serves as a template, and under the action of a highly efficient strand displacement DNA polymerase (such as Phi29 polymerase), rolling circle amplification (RCA) is initiated. A single recognition event is exponentially amplified into a long-chain DNA product containing thousands of repetitive sequences. Finally, a fluorescently labeled detection probe hybridizes with the RCA product, generating a strong fluorescent signal that can be quantified by conventional instruments.

[0109] This invention transforms single-molecule recognition events into macroscopic signals, achieving extremely high sensitivity and meeting the needs of early diagnosis and monitoring of trace residual lesions. By combining biomarkers with PLA-RCA, this invention solves a key bottleneck in ccRCC blood monitoring. It achieves highly specific in-situ detection of PGK1 K139la-K139 lactation-modified PLA, possessing spatial localization, methodological specificity, and experimental reproducibility, making it suitable for histological research and potential pathological auxiliary detection applications.

Claims

1. A dual-antibody-linked probe for detecting lactated PGK1, characterized in that, Including antibody A and antibody B, and complementary oligonucleotides conjugated to antibody A and antibody B, respectively; The antibody A is a polypeptide A that recognizes the K139-lactation modification of the PGK1 K139la site; the sequence of polypeptide A is SEQ NO.1; The antibody B is a polypeptide B with a length of 12 to 15 amino acids, which is used to recognize PGK1 at a distance of ≥50 amino acids from the K139 site; the sequence of polypeptide B is SEQ NO.

2.

2. The dual-antibody-linked probe for detecting lactated PGK1 according to claim 1, characterized in that, The complementary oligonucleotide sequence conjugated with antibody A is SEQ NO.3; the complementary oligonucleotide sequence conjugated with antibody B is SEQ NO.

4.

3. The method for preparing a dual antibody-linked probe for detecting lactated PGK1 as described in any one of claims 1 to 2, characterized in that, Includes the following steps: Step 1: Couple polypeptide A and polypeptide B to a carrier protein to obtain initial antigens A' and B', wherein the carrier protein includes hemocyanin KLH; Step 2: Inject the initial antigens A' and B' into the live culture sample for immune culture. After the predetermined cycle, collect serum. Step 3: The serum from the live samples injected with initial antigen A' and initial antigen B' in Step 2 were screened for titer and specificity using the indirect enzyme-linked immunosorbent assay (ELISA) to obtain samples A'' and B'' that meet the requirements. Step 4: Perform booster immunization on samples A'' and B'' obtained from the initial screening in Step 3, and obtain the desired antibody A and antibody B through B cell sorting, antibody gene amplification and cloning, and recombinant antibody expression and screening, respectively. Step 5: Connect antibody A and antibody B to complementary oligonucleotides using click chemistry to obtain the desired dual-antibody linkage probe.

4. The method for preparing a dual antibody-linked probe for detecting lactated PGK1 according to claim 3, characterized in that, In step 3, antibody A in the specific initial screening uses polypeptide A as the positive sieve plate and K139 ectopically modified polypeptide A1 and K139 unmodified polypeptide A2 as the negative sieve plates to select individuals with high antigen-specific titers and low cross-reactivity to the negative sieve plates; the polypeptide sequence of A1 is SEQ NO.5 and the polypeptide sequence of A2 is SEQ NO.

6.

5. The application of the dual antibody-linked probe for detecting lactated PGK1 as described in any one of claims 1 to 2, characterized in that, The probe is used to target proteins for non-diagnostic purposes.

6. The application of the dual antibody-linked probe for detecting lactated PGK1 according to claim 5, characterized in that, The detection method for the target protein is as follows: S1: Provide a sample containing the target protein, bind the target protein and the linking probe to obtain linear tag DNA; S2: Using the DNA molecule obtained in S1 as a template, DNA polymerase is used to amplify the product through rolling circle amplification to obtain a long single-stranded repeat product containing qPCR primer binding sites. S2: The product from step 2 can be detected by qPCR to complete the detection of the target protein.

7. The application of the dual antibody-linked probe for detecting lactated PGK1 according to claim 6, characterized in that, The DNA template sequence in S2 is SEQ NO.

7.

8. The application of the dual antibody-linked probe for detecting lactated PGK1 according to claim 6, characterized in that, The target protein is a protein that lactates PGK1.

Images