Quantitative detection and analysis method of aptamer coupled medicine

Abstract

The invention establishes an enzyme-labeled solid-phase immunoassay method based on molecular complementary hybridization, and the enzyme-labeled solid-phase immunoassay method can be used for measuring the concentration of complete aptamer-coupled drug molecules (ApDC) in plasma. The determination method comprises the following steps: directionally coating streptavidin in micropores of an elisa plate at high density, taking a reverse complementary sequence of a nucleic acid aptamer in to-be-detected ApDC as a capture sequence, and marking the other end of the capture sequence with a biotin label; complementary pairing is carried out on the capture sequence and an aptamer in the to-be-detected ApDC to form a hybrid molecule; the method comprises the following steps of: adding a cross molecule into a pore, capturing biotin marked on a capture sequence by streptavidin on a pore plate, adding a drug antibody marked by horseradish peroxidase, and carrying out chromogenic reaction on a substrate under the conditions of antigen-antibody combination and enzyme catalysis, so that the aim of detecting the drug content is fulfilled. The method is simple, convenient, rapid and sensitive, is suitable for plasma pharmacokinetic research of aptamer-coupled drugs in different biological samples, and has a very high application value.

Description

Technical Field

[0001] The present application relates to the technical field of enzyme-linked immunosorbent assay (ELISA), and in particular to a quantitative detection and analysis method for nucleic acid aptamer-coupled drugs. Technical Background

[0002] Aptamers are single-stranded DNA or RNA that can bind to targets with high specificity and selectivity, selected by the systematic evolution of ligands by exponential enrichment (SELEX) technology. Aptamers have unique advantages such as easy synthesis, easy chemical modification, strong affinity, and low cost. They show great application potential in the precise preparation and targeted delivery of drugs, so aptamers are also called "chemical antibodies". At the same time, because aptamers have the characteristics of high specificity and selective recognition of tumors, they become "excellent candidates" for targeted delivery of drugs, that is, aptamers are coupled to small molecule drugs through linkers to synthesize aptamer drug conjugates (ApDC), which have become a class of innovative molecular targeted drugs with great potential for clinical transformation.

[0003] Pharmacokinetics (PK) mainly studies the law of drug concentration changes over time in the body, including absorption, distribution, metabolism, excretion, etc., namely ADME. ADME determines the bioavailability of drugs in the body, the duration of action and the required dosage. Pharmacokinetic parameters are effective tools for measuring the effects of drugs. Therefore, it is of great significance to conduct pharmacokinetic analysis, drug metabolism and tissue distribution evaluation of nucleic acid aptamer-coupled drugs, that is, to determine the optimal dosage according to its half-life in the body, and to minimize the number and dosage of administration; at the same time, it provides an important basis for designing and optimizing the dosing regimen for clinical research, and ensures the safety and rationality of clinical research drugs.

[0004] At present, the quantitative analysis methods of nucleic acids in nucleic acid drugs include radionuclide labeling, capillary electrophoresis, hybridization technology enzyme-linked immunosorbent assay, electrochemiluminescence immunoassay, high performance liquid chromatography, liquid chromatography-mass spectrometry, RT-PCR, etc. Among them, liquid chromatography-mass spectrometry has high specificity and a wide linear range, but usually requires complex sample pretreatment and has low throughput; at the same time, nucleic acid aptamer-coupled drugs have complex metabolism in the biological environment, and the above methods can only detect certain metabolites of drugs, but it is difficult to detect complete drug molecules.

[0005] Enzyme linked immunosorbent assay (ELISA) is an immunoassay method that combines antigens or antibodies on the surface of a solid phase carrier, uses the specific binding of antigens and the enzyme labeled on the antibody or antigen to catalyze a color reaction on a specific substrate, and thus detects the target. The detection level can reach the pmol level. Figure 1 ), which has the characteristics of high sensitivity and high throughput, is currently widely used for the quantitative analysis of oligonucleotides in biological samples and supports preclinical and clinical pharmacokinetic studies of oligonucleotide drugs; however, no one has yet used HELISA for the study of ApDC pharmacokinetics. Summary of the invention

[0006] In order to solve the problems existing in the above detection methods, the present invention provides a method for quantitative detection of complete nucleic acid aptamer-coupled drug molecules. The present application is an enzyme-labeled solid phase immunoassay method based on complementary hybridization. Specifically, the complementary chain of the nucleic acid aptamer is used as the capture sequence, and the captured substance is adsorbed on the solid phase carrier through complementary hybridization, and then an anti-small molecule toxin antibody labeled with horseradish peroxidase (HRP) is added to make it bind to the nucleic acid aptamer-coupled drug to be detected adsorbed on the solid phase carrier. After the substrate solution is added, the substrate can be converted from the original colorless reduced form to a colored oxidized form under the action of the enzyme, and then the concentration of the ApDC to be detected in the sample can be accurately detected by qualitatively or quantitatively detecting the amount of the colored product ( Figure 1 ). It should be noted that the above sample addition order cannot be adjusted, that is, the capture sequence needs to capture the ApDC molecules first, and then use the antibody to recognize the drug. This detection method is simple and fast, and has high specificity and sensitivity.

[0007] On the one hand, the present invention provides an enzyme-linked immunosorbent assay kit for detecting the concentration of nucleic acid aptamer-coupled drugs, the kit comprising a capture sequence and an antibody that can specifically recognize the drug to be detected, the capture sequence comprising a reverse complementary sequence of the nucleic acid aptamer coupled to the drug.

[0008] It should be understood that the key to the enzyme-linked immunosorbent assay kit is the capture sequence and antibody. By changing these two components according to the detection object and the purpose of the detection, the detection of any ApDC can be achieved. Because the sequence of nucleic acid aptamers and the types of drugs are inexhaustible, and the combination of the two is increased in multiples, the present invention only selects representative nucleic acid aptamers (SEQ ID NO: 1, SEQ ID NO: 7-8) and drugs (Auristatin E (MMAE), Derutec (Dxd), Exitecan (EXA)) to explore the feasibility and accuracy of HELISA to determine the concentration of the entire ApDC molecule. The results show that the types of nucleic acid aptamers and drugs do not affect the accuracy of the HELISA test results, which proves the universality of HELISA to detect the concentration of complete ApDC molecules.

[0009] In some specific embodiments, the capture sequence is a reverse complementary sequence of the full-length sequence of the nucleic acid aptamer.

[0010] Furthermore, the nucleic acid aptamer targets any one or more of the following antigens: CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, HLD-DR, CEA, TAG-72, EpCAM, MUC1, MUC15, folate-binding protein, A33, G250, PSMA, ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2receptor, tenascin, a metalloproteinase, endosialin, vascuLar endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, EGFRvIII, Her-2 / neu, MAGE A3, p53 nonmutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a Sarcomatranslocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, MSLN, PSCA, MAGE Al, MAGE-A3, sLe, CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TESL Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3. Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, C-MET, HER3, EPCAM, CA6, NAPI2B, TROP2, CLDN18.2. FAP, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PD-L1, CD204, CD71, CD206, CD301, VTCN1, VISTA. .

[0011] Specifically, the nucleic acid aptamer targets PTK7, C-MET, and CD71 antigens; more specifically, Sgc8c-Aptamer (SEQID NO: 1) targets PTK7 antigen, SL1-Aptamer (SEQ ID NO: 7) targets C-MET antigen, and HG1-9-Aptamer (SEQID NO: 8) targets CD71 antigen.

[0012] In other specific embodiments, the three nucleic acid aptamers are respectively coupled with auristatin E; when detecting the concentrations of the above-mentioned nucleic acid aptamer-coupled drugs (Sgc8c-MMAE, SL1-MMAE and HG1-9-MMAE), the detection results of the HELISA method are consistent with the theoretical values; further, the detection result of the Sgc8c-MMAE drug is the most accurate.

[0013] Furthermore, the drug includes but is not limited to any one or more of auristatin E, Dxd, exotecan, or derivatives having the same or similar core structure as auristatin E, Dxd or exotecan.

[0014] It should be understood that according to the HELISA theory, the types of drugs that can be detected by the method provided by the present invention depend on whether there is an antibody labeled with HRP that can specifically recognize the drug. Obviously, when the accuracy of HELISA detection is slightly low, in addition to the capture sequence, the antibody can also be optimized, such as the object of antibody production, the structure of the antibody, etc.

[0015] In some specific embodiments, auristatin E, Dxd and exitecan are respectively coupled with the sequence (nucleic acid aptamer) shown in SEQ ID NO: 1; when detecting the concentration of the above-mentioned nucleic acid aptamer-coupled drugs (Sgc8c-MMAE, Sgc8c-Dxd and Sgc8c-EXA), the detection results of the HELISA method are consistent with the theoretical values; further, the detection result of the Sgc8c-MMAE drug is the most accurate.

[0016] Furthermore, the capture sequence has a length of 15 to 200 bp.

[0017] It should be understood that the capture effect is best when the capture sequence is the reverse complementary sequence of the full length of the nucleic acid aptamer; the general length of the nucleic acid aptamer is between 15 and 60 bp, but when the capture sequence is too short, the efficiency of capturing the drug will be low, so the preferred length of the capture sequence is 30 to 100 bp.

[0018] In some preferred embodiments, the capture sequence is 41 bp in length.

[0019] Further, the capture sequence comprises the sequences shown in SEQ ID NO: 2 to 6 in the sequence listing, preferably SEQ ID NO 4. Further, the concentration of the capture sequence is 2 nM to 200 nM, preferably, the concentration of the capture sequence is 4 nM.

[0020] Furthermore, the kit also comprises a drug standard, an enzyme-labeled microplate, a color developing solution and a reaction termination solution.

[0021] On the other hand, the present invention provides a method for quantitative detection of nucleic acid aptamer-drug conjugates, wherein the method uses the above-mentioned kit to detect the concentration of nucleic acid aptamer-drug conjugates.

[0022] Furthermore, the method comprises the following steps:

[0023] A. Separately mix the capture sequence with the aptamer-coupled drug to be tested to form a hybrid complex under certain conditions;

[0024] B. Add the hybridization complex described in step A to the enzyme-labeled microplate and incubate;

[0025] C. Add the antibody that specifically recognizes the drug to the enzyme-labeled microplate in step B and incubate;

[0026] D. Add color developing solution to the enzyme-labeled microplate in step C, perform color development reaction, and then add reaction stop solution;

[0027] E. Determine the absorbance value of the sample to be tested, substitute it into the standard curve, and calculate the concentration of the drug in the sample.

[0028] Furthermore, the color development reaction time is 0 to 30 minutes, preferably 5 minutes.

[0029] On the other hand, the present invention provides a use of a nucleotide sequence for preparing a preparation for detecting the concentration of a complete nucleic acid aptamer-drug molecule, wherein the nucleotide sequence comprises a reverse complementary sequence of the nucleic acid aptamer and has a length of 15 to 200 bp, preferably 30 to 100 bp.

[0030] The beneficial effects of the present invention include:

[0031] 1. Generally speaking, only intact drug molecules can play a role. Therefore, understanding the in vivo distribution of intact molecules can most accurately understand the pharmacokinetic and pharmacodynamic laws of drug molecules and provide more accurate guidance for clinical drug administration. However, there is currently no method that can measure intact ApDC molecules. The molecular weight of ApDC exceeds 10kDa and is not suitable for mass spectrometry. Otherwise, ApDC in plasma must be purified, and the purification steps are cumbersome, time-consuming and labor-intensive. The present invention uses nucleic acid aptamer complementary chains as capture sequences and establishes an enzyme-linked immunosorbent assay method based on complementary hybridization, which provides a rapid and efficient detection method for the detection of intact nucleic acid aptamer-coupled drug molecules. This method has great application prospects.

[0032] 2. The ability to accurately measure the concentration of intact aptamer-drug conjugate molecules in biological samples can help study the pharmacokinetics of aptamer-drug conjugates in complex environments such as plasma, which is very important for interpreting the results of preclinical and clinical studies;

[0033] 3. The ELISA method is a classic biological sample detection method with universal applicability and easy implementation. The method is simple, rapid, and sensitive, with a detection sensitivity of nM level; and has good stability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 Schematic diagram of the HELISA principle;

[0036] Figure 2 Schematic diagram of the chemical structure of Sgc8c-MMAE;

[0037] Figure 3 is the standard curve of Sgc8c-MMAE in DPBS buffer;

[0038] Figure 4 is the standard curve of Sgc8c-MMAE in mouse plasma;

[0039] Figure 5 is the standard curve of Sgc8c-MMAE in rat plasma;

[0040] Figure 6 is the standard curve of Sgc8c-MMAE in cynomolgus monkey plasma;

[0041] Figure 7 The in vitro stability test curve of Sgc8c-MMAE at different concentrations in mouse plasma;

[0042] Figure 8 The in vitro stability test curve of Sgc8c-MMAE at different concentrations in rat plasma;

[0043] Fig. 9 The in vitro stability test curve of Sgc8c-MMAE at different concentrations in cynomolgus monkey plasma;

[0044] Fig.10 The stability of Sgc8c-MMAE in male and female SD rats is measured; Male and Female in the figure legend represent gender, and -1, -2, and -3 represent three biological replicates in the same group;

[0045] Fig.11 The effect of different color development time on the solution color;

[0046] Fig.12 The impact of different lengths of capture sequences on the detection results;

[0047] Fig.13 The effect of different concentrations of capture sequences on the detection results;

[0048] Fig.14 The effect of different plasma ratios on the test results;

[0049] Fig.15 Schematic diagram of the chemical structure of Sgc8c-Dxd;

[0050] Fig.16 is the standard curve of Sgc8c-Dxd in DPBS buffer;

[0051] Fig.17 Schematic diagram of the chemical structure of Sgc8c-EXA;

[0052] Fig.18 Schematic diagram of the chemical structure of SL1-MMAE;

[0053] Fig.19 is the standard curve of SL1-MMAE in DPBS buffer;

[0054] Fig. 20 Schematic diagram of the chemical structure of HG1-9-MMAE. DETAILED DESCRIPTION

[0055] The present invention is further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments of the specification. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0056] When the ELISA experiment is used in the present invention to detect nucleic acid aptamer-coupled drugs, three mechanical repetitions are set; the test methods used in the following embodiments are conventional methods unless otherwise specified; the in vitro experiments involved in the present invention are all carried out in rat plasma unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0057] Example 1: A method for detecting nucleic acid aptamer-coupled drugs

[0058] This embodiment provides a standard operating procedure for a quantitative detection method of a nucleic acid aptamer-drug conjugate. The specific operation can be optimized according to the characteristics of the nucleic acid aptamer and the drug. The steps of the method are as follows:

[0059] (1) Add 100 μL of PBST buffer (pH=7.4) (Sangon Biotechnology (Shanghai) Co., Ltd.) containing 2% BSA (Sangon Biotechnology (Shanghai) Co., Ltd.) to a polystyrene enzyme-labeled microplate (SA Matrix microplate) (Suzhou Beaver Biomedical Engineering Co., Ltd.) coated with streptavidin (SA) for blocking. Place the plate on a horizontal shaker and incubate at room temperature for 1 h, then wash with PBST buffer 3 to 5 times and pat dry with absorbent paper.

[0060] (2) The capture sequence modified with biotin is complementary hybridized with the nucleic acid aptamer-coupled drug standard of different concentrations to form hybrid molecules. The hybridization conditions are 75°C for 5 min, and then cooled to 4°C at a rate of 0.5°C / s;

[0061] (3) adding the hybrid molecule obtained in step (2) to the SA Matrix microplate to specifically bind thereto, reacting at 37° C. for 2 h, then washing 3 to 5 times with PBST buffer and patting dry with absorbent paper;

[0062] (4) adding 100 μL of enzyme-labeled anti-drug antibody to step (3) and reacting for 1 h, then washing 3 to 5 times with PBST buffer and patting dry with absorbent paper;

[0063] (5) Add 100 μL of TMB / H2O2 colorimetric solution to step (4) for colorimetric reaction for 5 min, and add 50 μL of HCl solution (stop solution) to terminate the reaction. The concentration of the HCl stop solution is 1 to 4 M, and 2 M is preferred in this embodiment;

[0064] (6) using a Tecan spark microplate reader to measure the absorbance of the solution obtained in step (5) at a wavelength of 450 nm, and to draw a standard curve showing the change in absorbance versus the logarithmic value of the concentration of the nucleic acid aptamer-conjugated drug standard;

[0065] (7) According to the above steps (1) to (6), the absorbance corresponding to the aptamer-drug conjugate sample to be tested is tested, and the content of the complete aptamer-drug conjugate molecules in the aptamer-drug conjugate sample to be tested can be determined by combining the linear equation of the standard curve.

[0066] The above polystyrene ELISA microplate is SA Matrix microplate. The product adopts beaver bio-nano surface technology (S-TEC) to make streptavidin (SA) high-density and directional coating in the microwells of the ELISA plate. The coating density on the plate surface is as high as 9.3×10 13 molecules / cm 2 The maximum binding amount of Biotin in SA Matrix microplate reaches 5pmol per well, and the detection limit of Biotin-IgG (Rabbit) is as low as 0.5ng.

[0067] The color developing solution and the stop solution used in the present invention are the color developing solution A, the color developing solution B and the stop solution in the AFP / alpha-fetoprotein enzyme-linked immunosorbent assay kit purchased from SinoBiological Company, with the item number: KIT12177 and the batch number: CW17AP1001. The color developing solution refers to a liquid in which the color developing solution A and the color developing solution B are mixed in equal volumes.

[0068] Example 2: Drawing of Sgc8c-MMAE standard curves in different biological samples

[0069] It is understandable that the standard operating procedure for detecting the content of nucleic acid aptamer-coupled drugs provided in Example 1 is applicable to all nucleic acid aptamer-coupled drugs. In this example, Sgc8c-MMAE (structural formula: Figure 2, the synthesis method is shown in patent: 202211647557.5) as an example, the standard process is further specified, the Sgc8c-MMAE drug refers to MMAE (manufacturer: MedChemExpress) with a nucleic acid aptamer (SEQ ID NO: 1, 41 bp in total) connected to one end, and the nucleic acid aptamer can specifically target the PTK7 antigen. The experimental steps are as follows:

[0070] (1) Blocking: Add 100 μL of PBST buffer (pH = 7.4) containing 2% BSA to the well plate for blocking, place the well plate on a horizontal shaker and incubate at room temperature for 1 h, then wash 3 to 5 times with PBST buffer and pat dry with absorbent paper;

[0071] (2) Preparation of standard samples: The highest concentration of Sgc8c-MMAE was 500 nM, which was diluted 1.5 times to 500 nM, 333.3 nM, 222.2 nM, 148.1 nM, 98.8 nM, 65.8 nM, 43.9 nM and 29.3 nM, respectively. Then, 2 μL of the above samples with known concentrations were added to 20 μL DPBS buffer (pH = 7.4, Beijing Lanjieke Technology Co., Ltd.), mouse plasma, rat plasma and cynomolgus monkey plasma, respectively.

[0072] (3) The standard sample was hybridized with a capture sequence (Capture 3, SEQ ID NO: 4) at a concentration of 4 nM in DPBS buffer, and the PCR instrument (BIO-RAD) was set to the following program: 75°C for 5 min, then cooled to 4°C at a rate of 0.5°C / s; the hybridization mixture was added to the plate at 100 μL / well; blank wells were set (no sample was added to the blank wells, and the remaining steps were the same), and incubated at 37°C for 2 h;

[0073] (4) Discard the liquid in the well plate, shake dry, add 200 μL PBST buffer to each well, let stand for 1 minute and then discard. Repeat this 3-5 times and pat dry with absorbent paper;

[0074] (5) Dilute the horseradish peroxidase (HRP)-labeled anti-MMAE antibody (HRP conjugate MMAE, purchased from ACROBiosystems Biotechnology Co., Ltd., Lot. No. PLS104-22CVF1-18A) to 1 μg / mL with DPBS, add 100 μL of the diluted antibody solution to each well, incubate at 37°C for 1 h, then wash 3 to 5 times with PBST buffer and pat dry with absorbent paper;

[0075] (6) Add 100 μL of color development solution (equal volumes of solution A and solution B) to each well, develop the color for 5 min in the dark, and add 50 μL of stop solution to terminate the reaction;

[0076] (7) Use an enzyme-labeled instrument to measure the absorbance of the resulting solution at a wavelength of 450 nm.

[0077] (8) Draw the standard curve ( Figure 3 to Figure 6 ).

[0078] Depend on Figure 3 to Figure 6 It can be seen that the R of the standard curves of the four biological samples 2 All of them are greater than 0.9, indicating that the standard curve is of very good quality, the linear relationship is very significant, and it has a high degree of credibility. The content range of Sgc8c-MMAE drug detected in rat and mouse plasma is 270.5pM-1,370pM, while that in crab-eating monkey plasma is 183.1pM-3,125pM.

[0079] Example 3: In vitro stability determination of Sgc8c-MMAE in different biological samples

[0080] 1. Sample Incubation

[0081] Sgc8c-MMAE samples of three different concentrations (2 μM, 10 μM and 20 μM) were incubated in mouse plasma, rat plasma and crab-eating macaque plasma (the specific components of the samples are shown in Table 1), and samples were taken at different time points (0 min, 5 min, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h). The sampling volume at each time point was 30 μL, and the samples were temporarily stored in a -80°C refrigerator.

[0082] Table 1 Components of Sgc8c-MMAE samples at different concentrations

[0083] Sgc8c-MMAE final concentration Sample composition 2μM 250 μL plasma + 10 μL 52 μM Sgc8c-MMAE 10μM 250μL plasma + 10μL 260μM Sgc8c-MMAE 20μM 250μL plasma + 10μL 520μM Sgc8c-MMAE

[0084] 2. ELISA method to determine sample concentration

[0085] (1) Determine the dilution factor at different time points, dilute the sample to an appropriate concentration with DPBS buffer (preferably 320 times in this embodiment), and then hybridize with 4 nM capture sequence (Capture 3, SEQ ID NO: 4). Hybridize at 75°C for 5 min in a PCR instrument and then decrease the temperature to 4°C at a rate of 0.5°C / s.

[0086] The remaining steps are the same as those described in Example 2.

[0087] 3. Data processing

[0088] Substitute the absorbance value measured in the above experiment into the standard curve of the corresponding plasma ( Figures 4 to 6) and multiply it by the corresponding dilution factor to get the corresponding concentration, and draw the time-concentration curve ( Figure 7 to Figure 9 ).

[0089] on the whole( Figure 7 to Figure 9 ), the detection limit of HELISA method is nM level; the concentration change trend of Sgc8c-MMAE samples between different biological samples detected by HELISA method is consistent, that is, with the increase of time, the complete Sgc8c-MMAE drug molecules are gradually degraded, wherein the content of Sgc8c-MMAE drug in different animal plasma after incubation for 12h, its content tends to 0, which means that the drug can only exist in plasma for 12h at most, and the experimental results are consistent with the in vitro metabolic trend of Sgc8c-MMAE studied by the inventors in the early stage, further indicating the repeatability and accuracy of HELISA detection results; in addition, the experimental results also prove that the metabolic trend of Sgc8c-MMAE drug molecules in different in vitro biological environments is relatively stable. From the results between groups, the drug metabolism trend in rat and cynomolgus monkey plasma determined by HELISA method is the most obvious, and the determination effect is the best, so HELISA method is preferred to determine the pharmacokinetics in the above two biological backgrounds; rat plasma is further preferred.

[0090] Example 4: Determination of Sgc8c-MMAE stability in rats

[0091] 1. Drug administration

[0092] The drug Sgc8c-MMAE (batch number: SM03220323) was intravenously administered to 6-8 week old male and female SD rats (n=3) (Suzhou Fangda Pharmaceutical Development Co., Ltd.), the dosage was 18 mg / kg, the administration volume was 5 mL / kg, the solvent was normal saline, and the drug was administered once every 4 days for a total of 3 times.

[0093] 2. Sampling

[0094] Blood was collected 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after administration to obtain corresponding plasma samples, which were from Suzhou Fangda Pharmaceutical Development Co., Ltd.

[0095] 3. ELISA method to determine sample concentration

[0096] The specific experimental steps are as described in Example 3.

[0097] 4. Data Processing

[0098] Substitute the absorbance value measured in the above experiment into the standard curve of rat plasma ( Figure 5 ) and multiply it by the corresponding dilution factor to get the corresponding concentration.

[0099] In this example, male and female SD rats were intravenously administered (Sgc8c-MMAE), and the pharmacokinetics of the intact drug molecule in the rats was determined using the HELISA method one day after administration. The results are as follows: Fig.10 The results showed that the metabolism of Sgc8c-MMAE in rats was independent of the sex of the rats, and that it could stably exist in rats for 4 to 8 hours, which was consistent with the results of the inventor's previous experiments, further proving the reliability and accuracy of the HELISA test results.

[0100] Example 5: Optimization of color development time

[0101] Reaction time is a factor that affects color development. Theoretically, within a certain period of time, the negative wells remain colorless, while the color of the positive wells intensifies as time goes by. Therefore, for the repeatability and reliability of the experimental results, in the quantitative determination, the reaction time after adding the substrate should be as accurate as possible as required. This example uses a Sgc8c-MMAE molecule with a final concentration of 100nM and a 4nM Capture 3 capture sequence (SEQ ID NO:4) to explore the degree of color development of the solution at different times, and ultimately determines the optimal color development time. The specific operation is the same as described in Examples 2 to 3, except that the light-protected color development time after adding the color developing solution is different. This example is light-protected for 0min, 5min, 10min, 15min, 20min and 30min, respectively. The results are as shown in the figure. Fig.11 As the color development time increases, the color of the liquid in the well gradually deepens, and black precipitation appears in the well at 10 minutes and after 10 minutes of color development, affecting the detection of absorbance. Therefore, the optimal color development time is 5 minutes to 10 minutes, preferably 5 minutes.

[0102] Example 6: Optimization of capture sequence length

[0103] According to the sequence of the nucleic acid aptamer on the drug to be tested, this embodiment designed 5 capture sequences of different lengths (see Table 2 for the specific sequences, Capture 3 is the full-length sequence, and Capture 4 to 5 are composed of the sequence of Capture 3 repeated for different times), and these 5 capture sequences were used to capture Sgc8c-MMAE, respectively. The final concentrations of the capture sequence and Sgc8c-MMAE were 4 nM and 100 nM, respectively. Finally, the absorbance at 450 nm was measured and substituted into the standard curve of Sgc8c-MMAE in rat plasma ( Figure 5 ), the measured concentration is calculated, the percentage error = (theoretical concentration - measured concentration) / theoretical concentration × 100%, the specific detection steps are the same as those described in Examples 2 to 3, and the results are as follows Fig.12 shown.

[0104] The results show (Table 2) that when the capture sequence is <50bp, the longer the capture sequence is, the higher the corresponding absorbance value is, and the calculated measured concentration is closer to the theoretical concentration. This may be because when the capture sequence is short, the capture is incomplete. Only when the capture sequence is long enough, the interchain complementary force can better capture ApDC. Therefore, the capture effect is best when the full-length reverse complementary sequence is used; when the capture sequence length is increased, the measured concentration is higher. This may be because when the sequence is too long, the non-complementary fragments in the capture sequence form a partial mismatch with ApDC, resulting in a false positive phenomenon. In summary, the drug concentration obtained by using Capture 3 (SEQ ID NO: 4) is 100.25nM, which is closest to the theoretical 100nM. Therefore, Capture 3 has the best drug capture effect, so the preferred capture sequence length is 30 to 100bp.

[0105] Table 2 Base sequence and length of captured sequence

[0106]

[0107] Example 7: Determination of capture sequence saturation concentration

[0108] The loading capacity of polystyrene enzyme-labeled microplate is limited, and the maximum binding amount of Biotin in SA Matrix microplate reaches 5pmol per well. This embodiment uses Capture 3 as the capture sequence, and sets a series of concentrations (50nM, 25nM, 10nM, 6nM, 4nM and 2nM) to explore the saturation concentration of the capture sequence. The concentration of the nucleic acid aptamer-coupled drug Sgc8c-MMAE to be tested is 100nM. In order to ensure that Biotin can completely capture the hybridization product, the hybridization product needs to be incubated in two wells for 2h respectively (i.e., after the first incubation, the supernatant is transferred to another new well for continued incubation). The remaining steps are the same as those described in Examples 2 to 3. This embodiment measures the absorbance of the hybridization mixture incubated once and the supernatant solution after incubation twice at 450nm.

[0109] The results are as follows Fig.13 As shown in the figure, as the concentration of the capture sequence decreases, the absorbance of the supernatant after incubation also decreases successively, and finally there is almost no change at around 4nM. This is because the lower the concentration of the capture sequence, the less it remains after binding with the SA coated on the well plate. Therefore, the absorbance of the supernatant solution after two incubations finally reaches a platform at the saturation concentration. Therefore, 4nM is determined to be the saturation concentration of the capture sequence bound by the SA Matrix microplate.

[0110] Example 8: Effect of plasma ratio on measurement results

[0111] In this example, the absorbance of Sgc8c-MMAE molecules of the same concentration in systems with different rat plasma ratios was measured to explore the effect of plasma ratio on the detection results. The total volume of the samples was 320 μL, of which the plasma volumes were 0 μL, 2 μL, 20 μL, 160 μL, and 320 μL, respectively. The solution volume was then replenished to 320 μL with DPBS buffer; then 2 μL, 100 nM Sgc8c-MMAE was added and hybridized with 4 nM capture sequence (Capture 3, SEQ ID NO: 4), and the remaining steps were the same as described in Examples 2 to 3.

[0112] The results are as follows Fig.14 As shown, the absorbance at 450nm wavelength of the systems with different plasma ratios hardly changed, and there was no significant difference in the measured concentrations between the groups, indicating that the dilution ratio of the sample hardly affected the measurement results.

[0113] Example 9: HELISA to detect the effect of the same nucleic acid aptamer coupled with different drugs

[0114] In this example, the same aptamer (SEQ ID NO: 1) was coupled to different drugs (MMAE, Dxd (manufacturer: MedChemExpress) and exotecan (manufacturer: MedChemExpress) to explore whether the HELISA method is suitable for the quantitative detection of different drugs. The specific research ideas are as follows: First, the drug is diluted into different gradient concentrations to prepare a concentration standard curve of the drug; then the drug of known concentration (50nM and 100nM) is detected, and the absorbance value detected at 450nm is substituted into the standard curve to obtain the measured concentration of the drug; the theoretical concentration is compared with the measured concentration, and the percentage error is calculated to evaluate the detection effect of HELISA. The solvent used in this example is DPBS buffer; the concentration of the capture sequence (Capture 3, SEQ ID NO: 4) is 4nM; and the remaining detection methods are as described in Examples 2 to 3.

[0115] The drug structure of Sgc8c-MMAE is shown in Figure 2 , standard curve see Figure 3 The test results are shown in Table 3.

[0116] Table 3 Drug determination of Sgc8c-MMAE at different concentrations

[0117] Theoretical concentration Measured concentration Percent Error 50nM 50.1246nM 0.25％ 100nM 101.4672nM 1.46％

[0118] The Sgc8c-Dxd drug (structural formula see Fig.15The highest concentration is 200 nM, and the concentration is diluted 1.5 times to 133.3 nM, 88.9 nM, 59.2 nM, 39.5 nM, 26.3 nM and 17.6 nM; the drug antibody is Anti-Dxd antibody-HPR (purchased from Shanghai Youke Biotechnology Co., Ltd., Lot. No. YKMP18020H). The results of the Sgc8c-Dxd drug standard curve are shown in the figure. Fig.16 As shown; Substitute the absorbance value obtained by the test into the standard curve to obtain the detection concentration ( Fig.16 ), the test results are shown in Table 4.

[0119] Table 4 Drug determination of Sgc8c-Dxd at different concentrations

[0120] Theoretical concentration Measured concentration Percent Error 50nM 50.2443nM 0.49％ 100nM 102.5517nM 2.55％

[0121] The drug structure of Sgc8c-EXA is shown in Fig.17 The synthesis method is shown in Patent 202411555655.5, and the test results are shown in Table 5.

[0122] Table 5 Drug determination of Sgc8c-EXA at ​​different concentrations

[0123] Theoretical concentration Measured concentration Percent Error 50nM 50.5349nM 1.06％ 100nM 103.6731nM 3.67％

[0124] In general (Tables 3 to 5), the concentrations of the above three drugs detected by the HELISA method are roughly the same as the known concentrations, indicating that the type of drug coupled to the nucleic acid aptamer does not affect the effect of quantitative detection, thereby proving that this method can be used to determine different nucleic acid aptamer-coupled drug concentrations; at the same time, it is shown that the HELISA method provided by the present invention is suitable for detecting the concentration range of ApDC molecules of 50 to 100 nM; further, by comparing the errors of detecting different drug concentrations, it is found that the detection of Sgc8c-MMAE is the smallest, followed by Sgc8c-Dxd, which means that the result of detecting the concentration of Sgc8c-MMAE is the most accurate, which may be because the antibody used to bind the MMAE drug has the highest sensitivity.

[0125] Example 10: HELISA to detect the effect of different nucleic acid aptamers coupled to the same drug

[0126] The sequence of the aptamer determines the cells, tissues or organs it targets. In this example, the same drug (MMAE) is coupled to different aptamers (Sgc8c (SEQ ID NO: 1), SL1 (SEQ ID NO: 7) and HG1-9 (SEQ ID NO: 8)) to explore the effect of the aptamer sequence on the quantitative detection effect. The research idea of ​​this example is the same as that described in Example 9; the capture sequence of this example is the reverse complementary sequence of the full-length sequence of the aptamer, and the concentration of the capture sequence is 4nM; the HELISA method is used to determine the Aptamer-MMAE drug of known concentration (500pM, 2,000pM and 5,000pM), and the solvent is DPBS buffer; the remaining detection methods are as described in Examples 2 to 3.

[0127] The drug structure of Sgc8c-MMAE is shown in Figure 2 , standard curve see Figure 3 The test results are shown in Table 6.

[0128] Table 6 Drug determination of Sgc8c-MMAE at different concentrations

[0129]

[0130]

[0131] The SL1-MMAE drug (structural formula see Fig.18 The highest concentration is 12.5 nM, which is diluted 2-fold to 6.25 nM, 3.125 nM, 1.5625 nM, 781 pM, 390.6 pM. The results of the SL1-MMAE drug standard curve are as follows: Fig.19 Substitute the detected absorbance value at 450nm into the standard curve to calculate the detection concentration ( Fig.19 ), the test results are shown in Table 7.

[0132] Table 7 SL1-MMAE drug determination at different concentrations

[0133] Theoretical concentration Measured concentration Percent Error 5,000pM 5167.73pM 3.36％ 2,000pM 2043.49pM 2.17％ 500pM 506.31pM 1.26％

[0134] The HG1-9-MMAE drug structural formula is shown in Fig. 20 The synthesis method refers to the coupling step of thiol-modified nucleic acid aptamer and vcMMAE described in patent 202211647557.5, and the detection results are shown in Table 8.

[0135] Table 8 HG1-9-MMAE drug determination at different concentrations

[0136] Theoretical concentration Measured concentration Percent Error 5,000pM 5201.13pM 4.02％ 2,000pM 2055.99pM 2.80％ 500pM 507.46pM 1.50％

[0137] In general (Tables 6 to 8), the concentrations of the above three drugs detected by the HELISA method are relatively consistent with the known concentrations, that is, the type of nucleic acid aptamer does not affect the effect of quantitative detection, thereby proving that this method can be used to determine different nucleic acid aptamer-coupled drug concentrations; and it is shown that the HELISA method provided by the present invention is suitable for detecting ApDC molecules in the concentration range of 500 to 5,000 pM; further, by comparing the errors in detecting different drug concentrations, it is found that the detection of Sgc8c-MMAE is the smallest, followed by SL1-MMAE, which means that the result of detecting the concentration of Sgc8c-MMAE is the most accurate, which may be because the length of the nucleic acid aptamer affects the accuracy of the detection result.

[0138] It should be understood that although the nucleic acid aptamers in the specific embodiments provided by the present invention are all connected to the 3' end of the drug, in fact, no matter which end of the drug the nucleic acid aptamer is connected to, the method provided by the present invention can be used to detect the concentration of intact ApDC molecules.

[0139] Example 11: Effect of capture order on HELISA test results

[0140] This example uses Sgc8c-MMAE as the research object to explore the effect of the capture sequence on the HELISA test results, that is, whether to first capture the drug with the capture sequence and then bind with the antibody, or first use the antibody to recognize the drug and then use the capture sequence to capture the drug-antibody complex. The research idea of ​​this example is the same as that described in Examples 9-10, and the concentration of the Sgc8c-MMAE drug detected is 50nM and 100nM; during the operation, in order to capture completely, the reagent (capture sequence or antibody) added first must be excessive, the specific experimental steps are the same as described above, and the results are shown in Table 9.

[0141] Table 9 Effect of capture order on detection of Sgc8c-MMAE drug concentration results As can be seen from Table 9, in order to ensure the accuracy of the HELISA detection results of the complete ApDC molecule concentration, the drug must be captured by the capture sequence first, and then the fixed drug must be recognized by the antibody. This may be because high temperature conditions are required for the complementary pairing of the capture sequence and the nucleic acid aptamer, and the antibody is denatured and inactivated under these conditions, resulting in a decrease in the horseradish peroxidase content in the reaction system, and the absorbance value obtained by the detection is significantly low, resulting in the detection value being significantly lower than the theoretical value, and then the accurate concentration of the drug to be detected cannot be obtained.

[0142] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

Claims

1. An enzyme-linked immunosorbent assay kit for detecting the concentration of nucleic acid aptamer-coupled drugs, characterized in that: The kit comprises a capture sequence and an antibody capable of specifically recognizing a drug to be detected, wherein the capture sequence comprises a reverse complementary sequence of a nucleic acid aptamer coupled to the drug.

2. The kit according to claim 1, characterized in that The specific types of cell lines in the range are CD5, CD19, CD20, CD25, CD37, CD30, CD33, and CD45. CAMPATH-1, HLD-DR, CEA, TAG-72, EpCAM, MUC1, MUC15, folate-binding protein, A33, G250, PSMA, ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-9, epidermal growth factor、p185HER2、IL-2receptor、tenascin、metalloproteinase、endosialin、vascuLar endothelial growth factor、avB3、WT1、LMP2、HPV E6、HPV E7、EGFRvIII、Her-2 / neu、MAGE A3、p53 nonmutant、NY-ESO-1、MelanA / MART1、Ras mutant、gp100、p53 mutant、PR1、bcr-abl、tyrosinase、survivin、PSA、hTERT、aSarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, MSLN, PSCA, MAGE Al, MAGE-A3, sLe, CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX,PAX5, OY-TESL Sperm protein 17. LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3 Page4 VEGFR2 MAD-CT-1 PDGFR-B MAD-CT-2 ROR2 C-MET HER3 EPCAM CA6 NAPI2B TROP2 CLDN18.2、FAP、RON、LY6E、FRA、DLL3、PTK7、LIV1、ROR1、Fos-related antigen1、VEGFR、endoglin、PD-L1、CD204、CD71、CD206、CD301、VTCN1、VISTA。.

3. The kit according to claim 2, characterized in that The drug includes but is not limited to any one or more of auristatin E, Dxd, exotecan, or derivatives having the same or similar core structure as auristatin E, Dxd or exotecan.

4. The kit according to claim 4, characterized in that The length of the capture sequence is 15 to 200 bp.

5. The kit according to claim 5, characterized in that The concentration of the capture sequence is 2nM to 200nM.

6. The kit according to claim 6, characterized in that The kit also comprises a drug standard, an enzyme-labeled microplate, a color developing solution and a reaction termination solution.

7. A quantitative detection method for aptamer-drug conjugates, characterized in that: The method uses the kit as described in any one of claims 1 to 6 to detect the concentration of the aptamer-coupled drug.

8. The method according to claim 7, characterized in that The method steps are as follows: A. Separately mix the capture sequence with the aptamer-coupled drug to be tested to form a hybrid complex under certain conditions; B. Add the hybridization complex described in step A to the enzyme-labeled microplate and incubate; C. Add the antibody that specifically recognizes the drug to the enzyme-labeled microplate in step B and incubate; D. Add color developing solution to the enzyme-labeled microplate in step C, perform color development reaction, and then add reaction stop solution; E. Determine the absorbance value of the sample to be tested, substitute it into the standard curve, and calculate the concentration of the drug in the sample.

9. The method according to claim 8, characterized in that The color development reaction time is 0 to 30 minutes.

10. A use of a nucleotide sequence for preparing a preparation for detecting the concentration of a complete nucleic acid aptamer-drug conjugate molecule, characterized in that: The nucleotide sequence comprises the reverse complementary sequence of the nucleic acid aptamer and has a length of 15 to 200 bp.

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