Method for unmarked quantitative distinguishing of antibody nucleic acid coupling probe stoichiometric ratio

Abstract

The invention discloses a method for unmarked quantitative distinguishing of the stoichiometric ratio of an antibody nucleic acid coupling probe. The method comprises the following steps: 1) dissolving the antibody nucleic acid coupling probe in a CZE separation buffer solution to prepare a sample solution; 2) setting proper capillary zone electrophoresis conditions, and separating the sample by using a capillary zone electrophoresis apparatus; 3) monitoring the electrophoretic separation process in real time through an ultraviolet detector or a mass spectrum detector; and (4) realizing the stoichiometric ratio of the antibody nucleic acid coupling probe for label-free quantitative analysis according to the peak position and peak area in the electrophoretogram. According to the invention, based on the capillary zone electrophoresis technology, accurate and convenient analysis of oligonucleotide-antibody stoichiometric ratios obtained by different coupling methods is realized, and even coupling products with the difference of only one oligonucleotide can be distinguished. The method provides an important tool for quality control and optimization of the AOCs, and is expected to promote further application of the AOCs in the field of biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of antibody-nucleic acid conjugate probes, and more particularly to a method for quantitatively and discriminatively analyzing the stoichiometric ratio of antibody-nucleic acid conjugate probes without labeling. Background Art

[0002] Antibody-nucleic acid conjugate probes (AOCs) have shown great potential in the fields of molecular imaging, diagnosis and treatment of major diseases, etc. However, the high efficiency and specificity of AOCs in recognizing target molecules highly depend on their homogeneity, especially the oligonucleotide-to-antibody ratio (OAR). Currently, traditional OAR analysis methods (such as gel electrophoresis, ultraviolet-visible spectroscopy, etc.) have limitations such as low resolution, complex operation or the need for labeling, and it is difficult to meet the quality control requirements of AOCs.

[0003] Therefore, it is of great significance to develop an accurate, convenient and label-free OAR analysis method. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for quantitatively and discriminatively analyzing the stoichiometric ratio of antibody-nucleic acid conjugate probes without labeling, so as to solve the problems that traditional OAR analysis methods have limitations such as low resolution, complex operation or the need for labeling, and it is difficult to meet the quality control requirements of AOCs.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] According to the present invention, there is provided a method for quantitatively and discriminatively analyzing the stoichiometric ratio of antibody-nucleic acid conjugate probes without labeling, comprising the following steps: 1) dissolving the antibody-nucleic acid conjugate probe in a CZE separation buffer to prepare a sample solution; 2) setting appropriate capillary zone electrophoresis conditions and separating the sample using a capillary zone electrophoresis instrument; 3) monitoring the electrophoresis separation process in real time through an ultraviolet detector or a mass spectrometry detector; 4) realizing label-free quantitative analysis of the stoichiometric ratio of the antibody-nucleic acid conjugate probe according to the peak positions and peak areas in the electrophoresis pattern.

[0007] According to the present invention, the antibody-nucleic acid conjugate probe is a complex formed by covalently or non-covalently connecting an antibody and an oligonucleotide through a chemical or biological method.

[0008] According to the present invention, the antibody-nucleic acid conjugate probe is a sample of an antibody-nucleic acid conjugate probe prepared using a click reaction, a cross-linking agent or a biotin-based non-covalent reaction.

[0009] According to the present invention, the method can distinguish antibody-nucleic acid conjugate probes that differ by only one oligonucleotide.

[0010] In step 1), the AOCs sample is dissolved in a CZE separation buffer suitable for CZE analysis to prepare a sample to be tested. The CZE separation buffer can be a phosphate buffer (PBS) or other buffer systems suitable for electrophoresis. The sample concentration is usually 0.1 - 1.0 mg / mL to ensure detection sensitivity.

[0011] According to the present invention, the selection conditions for the CZE separation buffer solution are: 1) having a strong buffering capacity within the selected pH range; 2) having a low UV absorption at the detection wavelength; 3) having a small mobility (i.e., large volume, low charge ions) to reduce the generated current.

[0012] Common buffer systems include: 1. Sodium phosphate system: wide buffering range; 2. Sodium borate system: high pH range; 3. Tris-HCl system: low pH range; 4. Acetic acid - ammonium acetate system: a commonly used system for CE / MS.

[0013] According to the research findings of the present invention, most preferably, the CZE separation buffer is a buffer containing 50 mM borax, 25 mM HP-β-CD, and 10 mM SDS, with a pH of approximately 9.0. Preparation method: Prepare 200 mM borax, 100 mM HP-β-CD (dissolved in 20% acetonitrile), and 100 mM SDS stock solutions respectively. Then, take 2.5 mL, 2.5 mL, and 1 mL of the above solutions in sequence, add water to a total volume of 10 mL, and mix evenly.

[0014] According to a preferred embodiment of the present invention, after dissolving the AOCs sample in the CZE separation buffer, a protein concentrator tube (100k) with a centrifugal filtration function is used to desalt the AOCs sample to exclude interference. High charge ions may generate a relatively large current in the capillary, affecting the separation of the sample. High charge ions may also cause heating, affecting the separation effect. Therefore, after this desalting treatment, unnecessary interference can be effectively excluded.

[0015] Preferably, in step 2), the capillary zone electrophoresis conditions used are: adopting an SCIEX PA 800Plus pharmaceutical analysis system, matching a diode array detector, full wavelength scanning, acquisition frequency: 2 Hz, window slit: 100×200 μm; fused silica capillary, 50 μm inner diameter, 50 / 60.2 cm (effective length / total length); injection conditions: 0.5 psi, 5 s for the sample, 1 psi, 5 s for the separation buffer; separation voltage: 10 kV, 40 min; capillary temperature: 25 °C, sample chamber temperature: 15 °C.

[0016] Preferably, in step 2), the method for flushing between capillary needles is as follows: flushing with 0.1 mol / L sodium hydroxide solution at a pressure of 50 psi for 3 min, then flushing with deionized water at a pressure of 50 psi for 2 min, and finally flushing with CZE separation buffer at a pressure of 50 psi for 3 min.

[0017] Preferably, in step 4), according to the peak positions and peak areas in the electrophoresis pattern, different components in the AOCs products are quantitatively analyzed, including free antibodies, free oligonucleotides, and AOCs products with different OARs. The coupling efficiency and OAR distribution are calculated.

[0018] According to a method for quantitatively resolving the stoichiometry ratio of antibody-nucleic acid conjugate probes without labeling by capillary zone electrophoresis provided by the present invention, accurate and convenient analysis of the oligonucleotide-antibody stoichiometry ratio (OAR) obtained by different coupling methods is achieved. Based on this strategy, we quantitatively resolved different components and their ratios in the AOC S products, including free antibodies and oligonucleotides, as well as AOC S products with different OARs. In particular, coupling products that differ by only one oligonucleotide (21 nt) can also be distinguished. This resolution ability is superior to traditional gel electrophoresis and has been verified by high performance liquid chromatography-mass spectrometry. We analyzed the AOC S products obtained by three covalent or non-covalent coupling methods using this strategy. This study provides a new tool for the quality control of AOC S .

[0019] It should be known that the basic principle of capillary zone electrophoresis analysis technology is as follows: in an electrolyte solution, due to the dissociation of the solute (analyte) itself or its interaction with certain components in the electrolyte solution, the solute particles carry a certain charge. When a direct current electric field is applied across the capillary, these charged particles will migrate directionally under the action of the electric field. Different charged particles have different migration speeds in the electric field due to differences in the number of charges carried, particle size, and shape, etc., thus achieving separation. So far, in the field of biochemistry, capillary zone electrophoresis analysis technology is usually used for the separation and analysis of biological macromolecules such as proteins, polypeptides, and nucleic acids, such as the purity identification of proteins, the sequence analysis of polypeptides, and the fragment separation of nucleic acids.

[0020] However, if the existing capillary zone electrophoresis technology is directly applied to the analysis of antibody-nucleic acid conjugate probes, there are still many technical difficulties, such as: 1) Molecular size and charge complexity: The large complex (>150 kDa) formed after the conjugation of an antibody (~150 kDa) and a nucleic acid (such as a 20-mer oligonucleotide ~6.8 kDa) has its migration rate in CZE affected by both hydrodynamic volume and charge, resulting in peak overlap and insufficient resolution; the heterogeneity caused by differences in conjugation sites and nucleic acid chain lengths exacerbates the separation difficulty; Capillary adsorption problem: The adsorption of proteins (antibodies) by uncoated capillaries can lead to peak tailing or sample loss, and dynamic coatings (such as polyethylene oxide) may interfere with nucleic acid detection signals; Detection sensitivity limitation: Low-concentration conjugates require high-sensitivity detection (such as LIF or mass spectrometry coupling), while conventional CZE-UV detectors may not meet the requirements. Therefore, up to now, researchers usually prefer to use mature technologies such as size exclusion chromatography (SEC), ion exchange chromatography (IEX), or microfluidic chip electrophoresis, because these methods have a higher degree of standardization for the separation of macromolecules.

[0021] The present invention first conducted capillary electrophoresis experiments in different modes (SDS-CGE / CZE) respectively, and determined that capillary zone electrophoresis technology is more suitable. Then, by optimizing experimental conditions such as buffer solutions, it was experimentally confirmed that CZE can provide high-resolution separation effects. The isoelectric point of the antibody is 8.0 - 9.2, and buffer solutions with different components near pH 9.0 were selected for separation. According to the research findings of the present invention, under these conditions, the antibody is positively charged, the oligonucleotide and AOCs are all negatively charged. After applying a positive electric field, under the combined action of electroosmotic flow, the particles all move from the anode to the cathode. Among them, the positively charged particles have the fastest migration speed, and the negatively charged particles have a slower migration speed. Moreover, the larger the mass-to-charge ratio of the negatively charged molecule, the slower its migration speed. Therefore, the positively charged antibody peaks first. The mass-to-charge ratio is mainly determined by the number of charges and the molecular weight. Free IgG has the largest molecular weight and a relatively small number of negative charges, so its mass-to-charge ratio is small and it will peak first. Free oligonucleotides have the smallest molecular weight and the highest number of charges, so their mass-to-charge ratio is the largest and they will peak last. For AOCs, their molecular weight does not change much compared with that of the antibody, while the number of charges increases with the conjugation of oligonucleotides. Therefore, their migration speed will be slower than that of the antibody, and the more oligonucleotides are conjugated, the more the charge increases and the slower the migration speed.

[0022] The positive progressive effects of the present invention compared with the prior art are as follows:

[0023] 1) Optimize the quality control process of AOCs through CZE and develop high-throughput automated analysis technology to meet the requirements of industrial production;

[0024] 2) Explore more efficient and stable conjugation strategies to improve the consistency and drug performance of OAR;

[0025] 3) Expand the application potential of AOCs in the fields of gene editing, targeted therapy, and molecular imaging;

[0026] 4) Combine multi-dimensional characterization techniques such as mass spectrometry imaging and single-molecule fluorescence microscopy to more comprehensively analyze the structure and function of AOCs;

[0027] 5) Deeply study the influence of coupling strategies on the stability and pharmacokinetics of AOCs, clarify its mechanism in cancer immunotherapy and drug delivery at the molecular level, and it is expected to be applied in the fields of targeted drug delivery, molecular imaging, and cancer immunotherapy.

[0028] In summary, the present invention provides a method for quantitatively resolving the stoichiometric ratio of antibody-nucleic acid conjugate probes without labeling, which is used to accurately determine the OAR of AOCs and evaluate their coupling efficiency. This method has the advantages of high resolution, label-free, and simple operation. It can distinguish AOC products that only differ by one oligonucleotide (such as 21 nt) and quantify the efficiency of different coupling methods. This strategy can not only quantitatively compare the results of different coupling methods, but also provides an important tool for the quality control and optimization of AOCs, and is expected to promote the further application of AOCs in the biomedical field. The breakthrough application of the CZE method in the quantitative determination of AOC coupling efficiency and component separation has laid an important foundation for the quality evaluation of antibody-nucleic acid conjugate probes and the development of new treatment methods, and has broad application prospects. Brief Description of the Drawings

[0029] Figure 1 It is a schematic diagram of AOCs. The blue represents the antibody part, the red represents the oligonucleotide part, and the black represents the linker;

[0030] Figure 2 It is the SDS-CGE non-reducing (a) and reducing (b) electrophoresis diagrams of ODN;

[0031] Figure 3 It is the non-reducing SDS-CGE-NR electrophoresis result of (a) RTX, the non-reducing SDS-CGE-R electrophoresis result of (b) AOCs, the reducing SDS-CGE electrophoresis result of (c) RTX, and the reducing SDS-CGE electrophoresis result of (d) AOCs;

[0032] Figure 4 It is the CZE chromatograms of RTX (a) and AOCs (b) at pH = 3;

[0033] Figure 5 It is the CZE chromatograms of RTX (a) and AOCs (b) at pH = 5.5;

[0034] Figure 6CZE chromatograms of RTX (a) and AOCs (b) at pH = 7.5;

[0035] Figure 7 are the CZE electrophoresis results of (a) blank sample at pH = 9.0, (b) ODN, (c) RTX, and (d) AOCs;

[0036] Figure 8 Reaction formulas of three methods for coupling RTX with oligonucleotides to prepare AOCs;

[0037] Figure 9 is the CEZ spectrum of AOC linked by crosslinker in Example 2;

[0038] Figure 10 is the OAR analysis chart of AOC linked by crosslinker in Example 2;

[0039] Figure 11 is the CZE chromatogram of AOC sample linked by click reaction in Example 3;

[0040] Figure 12 is the OAR analysis chart of AOC linked by click reaction in Example 3;

[0041] Figure 13 is the CZE chromatogram of AOC sample linked by biotin-based non-covalent bond in Example 4;

[0042] Figure 14 is the OAR analysis chart of AOC linked by crosslinker in Example 4. Detailed implementation manners

[0043] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions in the following embodiments, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0044] The AOCs used in the following embodiments of the present invention are AOCs drugs in which a monoclonal antibody is conjugated with an oligonucleotide, and the antibody is Rituximab (RTX). Its structure is as Figure 1 shown. The oligonucleotide is a 21-nt single-stranded nucleic acid with different modifications, and the sequence is GTGGTAGTTAGGAGTGAATGC. The theoretical molecular weight of IgG is 147244 Da, and the theoretical molecular weight of oligonucleotide (oligodeoxynucleotide, ODN) is 6581 Da. Before being linked to IgG, ODN needs to be modified first. The 5'-end phosphate backbone of ODN reacts with HS-C6 structure to form HS-C6 modified ODN (molecular weight 6777 Da).

[0045] Example 1: Screening of Capillary Electrophoresis Conditions

[0046] First, we employed SDS capillary gel electrophoresis (SDS-CGE), which is based on a separation principle similar to SDS-PAGE. In this method, polyacrylamide gel is used as the screening matrix in the capillary, and an electric field is applied for separation according to molecular size and shape. Depending on the presence of a reducing agent, SDS-CGE can be divided into non-reducing (SDS-CGE-NR) and reducing (SDS-CGE-R) modes, both of which are used to characterize antibodies and AOCs. Free ODNs elute at approximately 12.7 minutes in SDS-CGE ( Figure 2 ).

[0047] Under non-reducing conditions, the disulfide bonds of antibody molecules remain intact, and the rituximab sample shows a single antibody peak ( Figure 3 a). In the AOCs sample, since free oligonucleotides (ODNs) are significantly smaller in molecular weight and size compared to AOCs, they elute first ( Figure 3 b, peak 1). However, the retention times of antibody molecules ( Figure 3 a, peak 1) and AOCs-ODN conjugates ( Figure 3 b, peak 2) are similar (about 28.1 minutes), and the broadening of the AOC peak indicates the presence of a heterogeneous mixture of AOCs molecules conjugated with ODNs. However, effective separation between these two substances could not be achieved. In contrast, under reducing conditions, the disulfide bonds are broken, and AOCs decompose into two distinct peaks corresponding to their light and heavy chains, eluting at 15.7 minutes ( Figure 3 c, peak 1) and 19.8 minutes ( Figure 3 c, peak 2) respectively. In the AOCs sample, in addition to the peaks corresponding to free ODNs ( Figure 3 d, peak 1) and the free light and heavy chains of AOCS ( Figure 3 d, peak 2 and peak 4), additional peaks appear at slightly longer retention times ( Figure 3 d, peak 3 and peak 5). These peaks may be the light and heavy chains of AOCs conjugated with different numbers of ODNs. However, the resolution between these peaks is still not ideal. Overall, although SDS-CGE can distinguish AOCs from free ODNs in both reducing and non-reducing modes, it fails to achieve sufficient resolution to distinguish AOCs with different numbers of ODN conjugations. Therefore, this method is not sufficient to accurately quantify the conjugation efficiency or the oligonucleotide-antibody ratio (OAR). This limitation may stem from the fact that the changes in molecular weight and size after ODN conjugation are not sufficient to achieve effective separation in the SDS-CGE system.

[0048] Although the separation method based on molecular weight differences failed to effectively distinguish AOCs products with different ODN conjugation ratios (OARs) due to the limited mass difference between RTX and ODN, the significant negative charge of ODN led to charge heterogeneity among AOC variants. Capillary zone electrophoresis (CZE) separates analytes based on the charge-to-mass ratio, which gives it a particular advantage in separating substances with similar molecular weights but different charges, suggesting its potential in AOC separation.

[0049] To further optimize the separation, this example also tested buffer systems with a pH range between 3.5 and 9.0 (pH 3, 5.5, 7.5, and 9.0, see Figures 4 - 7 ).

[0050] First, we tried buffer conditions with pH 3 - 9.0 (pH 3, 5.5, 7.5, 9.0). At pH 3, IgG and AOC could be separated, but there was no resolution between AOCs with different numbers of DNA attached; at pH 5.5, proteins adsorbed on the inner wall of the capillary and no peaks appeared; at pH 7.5, IgG and AOC could be separated, and isomers of AOC with different numbers of DNA attached could also be separated; at pH 9.0, not only could IgG and AOC be separated, isomers of AOC with different coupling numbers could be separated, and free DNA could also be detected. The separation of AOC from impurities and the separation of isomers of AOC with different DNA coupling numbers were achieved under one separation condition. Therefore, pH 9.0 was finally selected as the optimal separation condition.

[0051] When using the boric acid / HP-β-CD / SDS buffer system with pH 9.0, all components of the AOCs mixture were effectively separated ( Figure 7 ). As shown in the CEZ electrophoresis diagram, except for the system peak ( Figure 7 , peak 1), the migration times of ODN and RTX were 21.0 minutes (peak 2, Figure 7 b) and 6.25 minutes (peak 2, Figure 7 c). In the AOCs sample ( Figure 7In d), peak 2 aligns with free RTX, while peaks 3 - 5 (7.0 - 8.5 minutes) correspond to RTX conjugated with increasingly more ODN molecules. Evidence shows that their migration times are between those of RTX and free ODN. Specifically, peak 3 represents the least conjugated AOC (with the fewest ODN molecules), while peak 5 reflects the maximum ODN loading. Peak 8 matches free ODN. Peaks 6 - 7 migrate slightly faster than free ODN, but their UV spectral characteristics are the same as those of the ODN standard. Considering the 5'-S-S-C6 modification of the ODN standard, we attribute peak 6 to a disulfide-linked ODN dimer, and peak 7 to an ODN with a truncated 5'-C6 chain, both of which are generated during the synthesis of AOCs.

[0052] From the comparison results, it can be seen that at pH = 9.0, a resolution effect can be achieved to distinguish AOC products that differ by only one oligonucleotide.

[0053] The preparation methods of AOCs in Examples 2 - 4 of the present invention are all prior arts. Three different methods were respectively used to couple RTX with 21-base oligodeoxyribonucleotides (ODN) to prepare AOCs, and the reaction formulas are as Figure 8 shown. Since this preparation method is not the technical key point of the present invention, it will only be briefly described herein.

[0054] Example 2: Analysis of Crosslinker-Linked Covalent Conjugated AOCS

[0055] Sample preparation: In this example, the heterobifunctional crosslinker Sulfo-SMCC was used to couple the amino group on the lysine residue of RTX with the modified thiol group on ODN to prepare a crosslinker-linked covalent conjugated AOCs. As Figure 8 shown in a. Then, the AOC sample prepared based on the SMCC reaction was dissolved in PBS buffer at a concentration of 1.0 mg / mL.

[0056] CZE conditions: The SCIEX PA 800Plus pharmaceutical analysis system was used, equipped with a diode array detector for full-wavelength scanning, acquisition frequency: 2 Hz, window slit: 100×200 μm; fused silica capillary, 50 μm inner diameter, 50 / 60.2 cm (effective length / total length); injection conditions: 0.5 psi, 5 s for the sample, 1 psi, 5 s for the CZE separation buffer (a buffer of 50 mM borax, 25 mM HP-β-CD, and 10 mM SDS, pH 9.0); separation voltage: 10 kV, 40 min; capillary temperature: 25 °C, sample chamber temperature: 15 °C.

[0057] Spectrum analysis: Figure 9The middle peak 1 is the antibody monomer peak, and peaks 2, 3, and 4 are the peaks of IgG conjugated with different numbers of ODN molecules. The number of ODN molecules conjugated to peak 2 is the least, and the number of ODN molecules conjugated to peak 5 is the most. Calculate Figure 9 the peak areas of each peak in Figure 10 .

[0058] It can be seen from the calculation that in this example, mainly single-conjugated products (IgG-1ODN, 40.5%) were formed, and the proportions of multi-conjugated products (such as IgG-2ODN, 23.2%, IgG-3ODN, 3.8%) were relatively low.

[0059] CZE quantification (RTX: 32.5%, RTX-1ODN: 40.5%; RTX-2ODN: 23.2%; RTX-3ODN: 3.8%) To verify the consistency with the actual situation, a comparison was made with the conjugation stoichiometry of HPLC-MS (RTX: 34.2%, RTX-1ODN: 47.4%; RTX-2ODN: 15.8%; RTX-3ODN: 2.6%). A strong consistency was shown.

[0060] Example 3: Analysis of covalently conjugated AOCs connected by click reaction

[0061] Sample preparation: The preparation method of covalently conjugated AOCs in this example is as follows: A copper-free click reaction occurs between the N3 group added to the Fc end of the antibody under the catalysis of β-1,4-galactosyltransferase and the DBCO group modified on the ODN. As Figure 8 shown in b. Then, the AOCS sample prepared based on the click reaction was dissolved in PBS buffer at a concentration of 0.5 mg / mL.

[0062] CZE conditions: An SCIEX PA 800Plus pharmaceutical analysis system was used, equipped with a diode array detector, full-wavelength scanning, acquisition frequency: 2 Hz, window slit: 100×200 μm; fused silica capillary, 50 μm inner diameter, 50 / 60.2 cm (effective length / total length); injection conditions: 0.5 psi, 5 s sample, 1 psi, 5 s CZE separation buffer; separation voltage: 10 kV, 40 min; capillary temperature: 25 °C, sample chamber temperature: 15 °C.

[0063] Spectrum analysis: Figure 11 The middle peak 1 is the antibody monomer peak, and peaks 2, 3, 4, and 5 are the peaks of IgG conjugated with different numbers of ODN molecules. The number of ODN molecules conjugated to peak 2 is the least, and the number of ODN molecules conjugated to peak 5 is the most. Calculate Figure 11 the peak areas of each peak in Figure 12 .

[0064] Click-chemistry-based conjugation achieved a high efficiency of 98.5% and presented a wide distribution of ODN conjugation ratios (OAR) (OAR 1–4; OAR = 1 at 12.5%, OAR = 2 at 39.4%, OAR = 3 at 26.0%, OAR = 4 at 20.6%).

[0065] Example 4: Analysis of Biotin-based Non-covalent Coupled AOCs

[0066] Sample preparation: In this example, the preparation method of covalently coupled AOCs was as follows: Biotinylated RTX and ODN were non-covalently coupled with avidin respectively, as shown in Figure 8 c. Then, the AOC sample prepared by biotin-based non-covalent coupling reaction was dissolved in PBS buffer at a concentration of 0.5 mg / mL.

[0067] CZE conditions: The SCIEX PA 800Plus pharmaceutical analysis system was used, equipped with a diode array detector for full-wavelength scanning. Acquisition frequency: 2 Hz, window slit: 100×200 μm; fused silica capillary, 50 μm inner diameter, 50 / 60.2 cm (effective length / total length); injection conditions: 0.5 psi, 5 s for the sample, 1 psi, 5 s for the CZE separation buffer; separation voltage: 10 kV, 40 min; capillary temperature: 25 °C, sample chamber temperature: 15 °C.

[0068] Spectrum analysis: Figure 13 In, peak 1 was the antibody monomer peak, and peaks 2, 3, and 4 were the peaks of IgG conjugated with different numbers of ODN molecules. Peak 2 had the fewest ODN molecules conjugated, and peak 4 had the most ODN molecules conjugated. Calculate Figure 13 the peak areas of each peak in, and obtain the OAR of each component as shown in Figure 14 . Non-covalent linkage mainly formed multi-conjugated products, mainly double-conjugated (IgG-2ODN, 64.6%), followed by single-conjugated (IgG-1ODN, 18.5%) and triple-conjugated (IgG-3ODN, 2.4%). The proportion of IgG was moderate (14.5%), and no tetra-conjugated products were formed.

[0069] The above are only the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. The above embodiments of the present invention can also be subject to various changes. That is, all simple, equivalent changes and modifications made in accordance with the claims and the content of the specification of the present invention application fall within the scope of protection of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for quantitatively and discriminatively determining the stoichiometric ratio of an antibody-nucleic acid conjugate probe without labels, characterized in that, It includes the following steps: 1) Dissolve the antibody-nucleic acid conjugate probe in the CZE separation buffer to prepare a sample solution; 2) Set appropriate capillary zone electrophoresis conditions and use a capillary zone electrophoresis instrument to separate the sample; 3) Monitor the electrophoresis separation process in real time through an ultraviolet detector or a mass spectrometry detector; 4) Based on the peak positions and peak areas in the electrophoresis pattern, perform label-free quantitative analysis of the stoichiometric ratio of the antibody-nucleic acid conjugate probe.

2. The method according to claim 1, wherein The antibody-nucleic acid conjugate probe is a complex formed by covalently or non-covalently linking an antibody and an oligonucleotide through chemical or biological methods.

3. The method according to claim 2, wherein The antibody-nucleic acid conjugate probe is a sample of an antibody-nucleic acid conjugate probe prepared using click reaction, cross-linking agent or biotin-based non-covalent reaction.

4. The method according to claim 1, wherein The method can distinguish antibody-nucleic acid conjugate probes that differ by only one oligonucleotide.

5. The method according to claim 1, characterized in that, In step 1), the CZE separation buffer includes: sodium phosphate system, sodium borate system, Tris-HCl system, acetic acid-ammonium acetate system.

6. The method according to claim 5, wherein In step 1), the composition of the CZE separation buffer is: 50 mM borax, 25 mM HP-β-CD and 10 mM SDS, pH 9.

0.

7. The method according to claim 1, characterized in that In step 1), the sample concentration in the prepared sample solution is 0.1 - 1.0 mg / mL.

8. The method according to claim 1, characterized in that In step 2), the capillary zone electrophoresis conditions adopted are: SCIEX PA 800Plus drug analysis system, equipped with a diode array detector, full wavelength scanning, acquisition frequency: 2 Hz, window slit: 100×200 μm; fused silica capillary, 50 μm inner diameter, effective length 50 cm, total length 60.2 cm; injection conditions: 0.5 psi, 5 s sample, 1 psi, 5 s CZE separation buffer; separation voltage: 10 kV, 40 min; capillary temperature: 25 °C, sample chamber temperature: 15 °C.

9. The method according to claim 1, wherein In step 2), the method for rinsing between capillary needles adopted is: rinse with 0.1 mol / L sodium hydroxide solution at 50 psi for 3 min, then rinse with deionized water at 50 psi for 2 min, and finally rinse with CZE separation buffer at 50 psi for 3 min.

10. The method according to claim 1, characterized in that, In step 4), based on the peak positions and peak areas in the electrophoresis pattern, quantitatively analyze different components in the antibody-nucleic acid conjugate probe: including free antibody, free oligonucleotide and antibody-nucleic acid conjugate probes with different stoichiometric ratios, and calculate the coupling efficiency and stoichiometric ratio distribution.

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