CN118256595A - Probe and marker combination and method for determining ASO by using same - Google Patents

Abstract

The invention discloses a probe and marker combination and a method for determining ASO by using the same. The probe and label combination includes a capture probe, a detection probe, and a label; the 3 'end of the capture probe is provided with modification which can be combined with a solid phase material, and the 5' end is combined with a detection probe; the detection probes are hybridizable to the oligonucleotides and have modifications that can be detected by the labels. The method uses the probe and label combination for detection quantification. The probe and the marker have high combined sensitivity and flux, the method has good reproducibility and usability (non-enzymatic heterogeneous system), the flow is simple, and the time consumption is short (3-4 h).

Description

Probe and marker combination and method for determining ASO by using same

Technical Field

The invention belongs to the field of biological detection, and relates to a probe and marker combination and a method for determining ASO by using the same.

Background

Oligonucleotide drugs refer to short DNA or RNA sequences and analogues thereof designed and synthesized artificially to have disease treatment function. The medicine can be paired with DNA, mRNA or pre-mRNA through a base complementary pairing principle, and can regulate gene expression through a series of mechanisms such as gene silencing, non-coding RNA inhibition, gene activation and the like, so that the pathogenic protein expression is down-regulated or normal protein is recovered to be expressed, and the medicine plays a role in treating diseases at the gene level. Oligonucleotide drugs are mainly of the following types: ASO (antisense oligonucleotide), siRNA (small interfering RNA), miRNA (microrna), aptamer (Aptamer). Wherein antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the main forms of oligonucleotide drugs developed clinically. Currently, 17 nucleic acid drugs are marketed globally, of which 10 are ASO and 5 are siRNA.

Compared with traditional small molecule drugs and antibody drugs, oligonucleotide drugs have multiple technical advantages, including: (1) The specificity is high, and the expression of pathogenic genes is regulated from the root; (2) long-acting, can reduce the frequency of administration; (3) The research and development period is short, and the success rate of clinical transformation and research and development is relatively high; (4) the targets are rich, and the indications are wide; and (5) the programmable design and the synthesis are simpler.

In the past, oligonucleotide drugs have not been successful in clinical applications because of lack of effective delivery systems and susceptibility to off-target effects and immune responses. In recent years, the development of drug delivery systems and chemical modification techniques has led to significant improvements in tissue targeting specificity, cellular uptake and potency of oligonucleotides, which have had a dramatic impact on efficacy and thus reduced effective doses. Quantitative analysis of oligonucleotides in biological samples is of great importance in Pharmacokinetic (PK), pharmacodynamics (PD) and toxicological safety evaluation, not only provides important information such as PK and toxicological generation for oligonucleotide drugs, but also plays a guiding role in structural modification of the drugs and optimization of delivery systems, and achieves the purposes of improving the stability of the drugs and improving the delivery efficiency.

In order to adequately address the exposure-reaction relationships in complex matrices, different bioanalytical platforms need to be selected depending on the physicochemical properties of the drug, the chemical modification and the nature of the drug delivery system and conjugate. With the development and advancement of scientific instruments and molecular biology techniques, many analytical techniques are used for detection of oligonucleotide drugs in biological matrices, and are mainly classified into 4 major categories: PCR (polymerase chain reaction), LC-MS/MS (liquid chromatography-mass spectrometry platform), LC-FL (liquid phase fluorescence platform), hybridization ELISA (hybrid enzyme-linked immunosorbent assay platform), the main characteristics of these methods are as follows:

(1) PCR platforms rely on hybridization of analytes to complementary strands, requiring selection, design, and synthesis of reagents, and thus the robustness of the method will depend on the design and availability of reagents; the method is intolerant to chemical modification of oligonucleotide drugs and is mainly used for quantifying unmodified oligonucleotides; in addition, no clear regulation guidance exists at present, and only advice given by white paper is insufficient to support the development of normative bioanalytical research, so that the method is generally used for early development.

(2) The LC-MS/MS platform can separate and distinguish the metabolite and the interfering substance through the chromatographic separation, and can also separate and distinguish the metabolite and the original drug form through the molecular weight, so that the oligonucleotide drug and the metabolite thereof can be quantitatively detected simultaneously, and the specificity and the selectivity are higher; the platform does not need the design and synthesis of probes, and the development period of the method is short; but its sensitivity (5-10 ng/mL) is inferior to that of hybridization-based methods (e.g., hybridization ELISA, LC-FL) and becomes lower as the length of the oligonucleotide increases, so LC-MS/MS is suitable for analysis of oligonucleotides of shorter length (< 13 mer) and with less stringent sensitivity requirements.

(3) The LC-FL platform combines a probe complementarily paired with the oligonucleotide to be detected, performs separation through a liquid phase, performs detection through fluorescence, combines a molecular hybridization technology and a liquid chromatography, and has the advantages of being complementary to each other. The sensitivity of detection is improved (about 1 ng/mL) compared with the LC-MS method; compared with the molecular hybridization method, the method has the advantages that the selectivity and the specificity of the method are improved, the method can be selected when the specificity and the sensitivity of the method are important, but the LC-FL analysis method needs to design special primers or probes, the analysis time of single medicines is long (10-15 min) and is unfavorable for mass sample analysis, and the oligonucleotide metabolites cannot be specifically identified.

(4) The Hybridization ELISA platform is in base complementary pairing with a target sequence through the specificity of the probe, and signal detection is carried out by utilizing the reaction of the labeled enzyme in the enzyme-linked immunosorbent assay and the substrate. The sensitivity of the method is obviously increased along with the increase of the length of the nucleic acid to be detected, the method is suitable for analyzing oligonucleotide medicines with the nucleotide quantity more than 24, the overall sensitivity (which can reach the level of 0.1-1 ng/mL) is better than that of LC-MS, and the method has strong inclusion for chemical modification. For Hybridization ELISA methods, there are various options such as enzyme-labeled instrument, MSD, etc., wherein MSD platform is based on 96-well plate, and uses SULFO-TAG (ruthenium terpyridyl) as luminescent substrate, and its molecular weight is about 1kDa (about 1/40 of HRP), so that steric hindrance is small, easy to label antibody, and not easy to hinder its binding with the object to be tested or other antibodies. SULFO-TAG loses electrons on the surface of the anode, is oxidized, and can generate high-efficiency, stable and continuous electrochemical luminescence (ECL) signals under catalysis of tripropylamine cation radicals and excitation of triangle pulse voltage. Detection is performed using an electrochemiluminescence signal. The MSD platform has significant advantages over ELISA in that the generation and collection of detection signals is significantly improved: MSD sensitivity can reach pg/ml level; the effective linear range reaches 6log; on the sample dosage, no matter single index or multiple indexes, only less than or equal to 25ul of samples are needed to finish detection, and more data are provided for the study of rare samples. Although this method cannot be used for qualitative and quantitative analysis of metabolites, it has not only a higher sensitivity but also little need for sample processing, and can be selected according to the analysis requirements, as compared with other analysis platforms.

In summary, the inherent polyanionic nature of oligonucleotides makes the sensitivity of bioassay assays largely dependent on the length and chemical modification of the oligonucleotide molecule. In general, increasing the length/size of the oligonucleotide will cause a decrease in the detection sensitivity of the LC-MS/MS, but will increase the sensitivity of hybridization-based methods (e.g., hybridization ELISA, LC-FL). For absolute quantitative bioassays of oligonucleotides, hybridization ELISA and LC-FL assays are more sensitive, while LC-MS/MS assays are more specific. Currently, LC-MS/MS is generally more suitable for quantitative analysis of oligonucleotides containing 13 or fewer nucleotide units, whereas Hybridization ELISA and LC-FL based assays are more suitable for quantitative analysis of oligonucleotides containing 24 or more nucleotide units.

In order to study the pharmacokinetics and biodistribution of ASO-type oligonucleotides, the present invention selects ASO drug ISIS2302 detected in the method of non-patent document 1 (Anal biochem.2002May 1;304 (1): 19-25.), which is a thio-oligonucleotide having antisense activity against human intercellular adhesion molecule-1 mRNA, for use in the treatment of various inflammatory diseases including organ transplant rejection, inflammatory bowel disease, arthritis, psoriasis, and the like. And provides a probe and marker combination and a method for determining ASO by using the same, wherein the principle (figure 1) is as follows: (1) Coating a biotin-labeled capture probe on a streptavidin-pre-coated MSD 96-well plate; (2) Mixing ASO substrate to be detected with complementary upstream probes and downstream probes on a PCR instrument for hybridization, adding the hybridization mixture into a 96-well plate, and carrying out secondary hybridization with capture probes on the plate; (3) And then adding a Sulfo-Tag labeled anti-digoxin antibody, adding a Read Buffer for electrochemiluminescence signal detection, fitting relevant parameters of a standard curve by a four-parameter regression model through biological analysis software, and calculating the concentration of the sample.

In order to develop a quantitative oligonucleotide method meeting high requirements in drug development, the inventor develops an oligonucleotide bioanalytical method with high sensitivity and flux, good reproducibility and usability (non-enzymatic reaction), simple flow and short time consumption (3-4 h) based on the existing Hybridization ELISA platform, thereby completing the invention.

Disclosure of Invention

The invention aims to solve the technical problems that the oligonucleotide detection and quantification method is complex, long in time consumption, small in flux and poor in reproducibility in the prior art, and provides a non-enzymatic heterogeneous Hybridization ELISA method which has no participation of catalytic enzyme, and a secondary hybridization step is added, so that compared with the conventional Hybridization ELISA method, the sensitivity is ensured, and the reproducibility, the usability and the specificity are better.

The invention solves the technical problems through the following technical proposal.

In a first aspect the invention provides a probe and label combination comprising a capture probe, a detection probe and a label.

In the present invention, the 3 'end of the capture probe has a modification that can bind to a solid phase material, and the 5' end of the capture probe binds to a detection probe.

In some embodiments of the invention, the 3' end of the capture probe has a biotin modification.

In the present invention, the detection probe is hybridizable to the oligonucleotide and has a modification that can be detected by the label.

In the present invention, the detection probe comprises a downstream probe and an upstream probe, wherein the upstream probe is bound to the capture probe and hybridized with the oligonucleotide together with the downstream probe, and the 3' -end of the downstream probe has a non-biotin modification that can be detected by a label.

In some embodiments of the invention, the non-biotin modification at the 3' end of the downstream probe is a digoxin modification.

In the invention, the marker is anti-digoxin antibody identification and marking with SULFO-TAG; and/or, the oligonucleotide is an antisense oligonucleotide (ASO).

In some embodiments of the invention, the ASO is from a serum sample.

In a second aspect the invention provides a kit comprising a probe and label combination as described in the first aspect.

In the present invention, the kit further comprises one or more of a solid phase material, an anti-digoxin antibody and a hybridization buffer.

In some embodiments of the invention, the solid phase material is a streptavidin pre-coated solid phase material; the anti-digoxin antibody is SULFO-TAG anti-digoxin antibody; the hybridization buffer included 60mM Na ₂ HPO4, 1M NaCl, 5mM EDTA, and 0.02% Tween 20; the percentages are by volume.

In some embodiments of the invention, the solid phase material may be conventional in the art, preferably a 96-well plate.

In a third aspect the present invention provides a method of detecting and quantifying an oligonucleotide using a probe and label combination as described in the first aspect or a kit as described in the second aspect, comprising the steps of:

(1) Coating the capture probes of the first aspect on a solid phase material; preferably, the solid phase material is a 96-well plate pre-coated with streptavidin;

(2) Hybridizing the sample to be detected with the detection probe according to the first aspect to generate a hybridization mixture; the hybridization reaction is carried out, for example, in a PCR instrument, incubator, metal bath;

(3) Adding the hybridization mixture into the solid phase material coated with the capture probes for hybridization;

(4) Detecting the detection probe of the first aspect with the label of the first aspect; preferably, the detection probes comprise an upstream probe and a downstream probe; more preferably, the 3' end of the downstream probe is provided with digoxin modification, and the marker is an anti-digoxin antibody;

(5) Detecting and quantifying the electrochemical signal; preferably, the label carries SULFO-TAG, which can be luminescent and quantified by an electrochemical reaction.

In some embodiments of the invention, the reaction conditions for coating the capture probes are: incubating for 1 h+ -5min at 25+ -1deg.C under shaking at 450rpm and in the dark; and/or the reaction conditions for hybridization of the detection probe and the sample to be detected are as follows: hybridization was performed at 90℃for 5min and at 35℃for 30min.

In some embodiments of the invention, the reaction conditions for the second hybridization are: at 37+/-1 ℃, standing and incubating for 1 h+/-5 min in dark; and/or, the reaction conditions for labeling digoxin on the downstream probe with SULFO-TAG anti-digoxin antibody are: incubating for 1 h+ -5 min at 25+ -1deg.C under shaking at 450rpm and in the dark; preferably, the 96-well plate is washed before coating, before the second hybridization, and after the second hybridization.

In a fourth aspect the present invention provides the use of a probe and label combination as described in the first aspect for the preparation of a diagnostic agent for detecting an oligonucleotide.

In the present invention, the oligonucleotide may be conventional in the art, preferably an antisense oligonucleotide.

In some embodiments of the invention, the oligonucleotide is from a blood sample, such as serum.

The invention has the positive progress effects that:

(1) The non-enzyme heterogeneous reaction system established by the invention has high sensitivity (up to 0.400 ng/mL), good reproducibility and easy usability.

(2) The oligonucleotide bioanalytical method has high flux, simple flow and short time consumption (3-4 h).

Drawings

FIG. 1 is a schematic view of the operation mode

FIG. 2 shows RUN1 standard curve and parameters thereof in SD rat serum

FIG. 3 shows RUN2 standard curves and parameters thereof in SD rat serum

FIG. 4 Standard Curve and parameters thereof in liver tissue homogenate supernatant of Mixed SD rat

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1: configuration of reagents

Preparation of mixed SD rat liver tissue homogenate supernatant:

5 SD rat liver tissues from different sources were taken, each sample was weighed, and about 1000mg each was cut into fine pieces and placed into a 50mL centrifuge tube. The lysate (Clarity OTX Lysis-Loading Buffer v 2.0) was added in a proportion of 100. Mu.l of lysate per 10mg of tissue. Samples were placed on ice and homogenized with a manual homogenizer. Centrifuge at 4000rpm for 5min at 4deg.C (set temperature) and collect the supernatant. Thus obtaining the prepared mixed SD rat liver tissue homogenate supernatant.

Preparation of hybridization buffer:

The hybridization buffer comprises the following components: 60mM Na ₂HPO₄, 1M NaCl, 5mM EDTA, 0.02% Tween 20.

The specific preparation method comprises the following steps: 1.7035g of anhydrous disodium hydrogen phosphate is weighed, added into 100mL of ultrapure water, 40mL of 5M NaCl solution, 2mL of 0.5M EDTA solution and 40 mu L of Tween20 reagent are weighed into the ultrapure water, and the mixture is uniformly mixed until the mixture is completely dissolved, and then the ultrapure water is subjected to constant volume to 200mL. The refrigerator (2-8 ℃) is preserved, and the effective period is 1 month.

Preparation of 1 XKPL wash:

KPL Wash Solution Concentrate (20×) mother liquor was diluted 20 times with ultrapure water and mixed well for use.

Preparation of ISIS2302 (ASO substrate)/capture probe/upstream probe and downstream probe complex solution:

The substrate complex solution or probe complex solution was diluted with DEPC water to give a concentration of 100. Mu.M.

Preparation of SULFO-TAG-anti-Digoxigenin antibody:

Anti-Digoxigenin antibody was SULFO-TAG labeled using MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack 1 kit, and the labeled reagent was designated SULFO-TAG-Anti-Digoxigeninantibody.

Preparing a capture probe working solution:

the capture probe multiplex solution was diluted to 50nM with PBS.

Preparing a mixed working solution of an upstream probe and a downstream probe:

The upstream probe and downstream probe complex solutions were diluted with hybridization buffer to a final concentration of 50nM, respectively. Preparation of SULFO-TAG-anti-Digoxigenin antibody working solution:

SULFO-TAG-anti-Digoxigenin antibody was diluted 500-fold with hybridization buffer.

Preparation of 2×read Buffer:

MSD Read Buffer T (4×) was diluted with ultrapure water to a 2×read Buffer.

TABLE 1 reagents and materials information

Example 2: experimental procedure

(1) Washing the plate: plates were washed by adding 1 XKPL wash to MSD 96-well plates after equilibration to room temperature.

(2) Coating: adding a capture probe working solution into each well of an MSD 96 well plate, sealing a plate, placing in a biochemical incubator at 25+/-1 ℃, oscillating at 450rpm, and carrying out light-proof incubation for 1 h+/-5 min.

(3) Sample pretreatment: ISIS2302 (ASO substrate) was diluted according to the concentration gradient in tables 1 and 2 and then diluted 20-fold with hybridization buffer.

(4) Off-plate hybridization: adding the mixed working solution of the upstream probe and the downstream probe into a PCR plate, adding the diluted and pretreated standard curve quality control and sample into the PCR plate, uniformly mixing by vibration, and then placing the PCR plate into a PCR amplification instrument for off-plate hybridization under the following hybridization conditions: denaturation at 90℃for 5min, hybridization at 35℃for 30min, and final holding at 12 ℃.

(5) In-plate hybridization: according to the experimental layout, adding the off-board hybridization compound into the corresponding hole, sealing the plate by a sealing plate membrane, and standing and incubating for 1 h+/-5 min at 37+/-1 ℃ in a biochemical incubator.

(6) Washing the plate: plates were washed by adding 1 XKPL wash to MSD 96-well plates after equilibration to room temperature.

(7) Adding SULFO-TAG-anti-Digoxigenin antibody working solution, sealing the plate membrane, sealing the plate, shaking at 450rpm in a biochemical incubator at 25+ -1deg.C, and incubating for 1 h+ -5 min under dark conditions.

(8) Washing the plate: plates were washed by adding 1 XKPL wash to MSD 96-well plates after equilibration to room temperature.

(9) Add Read Buffer: 2 XRead Buffer was added to MSD 96 well plates and Read in 1-10 min.

(10) Detecting and reading: usingSector S600 electrochemiluminescence was read from MSD 96 well plates.

(11) Data analysis: data analysis was performed using SoftMax Pro software.

Example 3: preparation of standard curve and quality control sample

The related preparation listed in the method is only a clear preparation method, and the specific preparation volume can be adjusted proportionally according to actual conditions.

Table 2 standard curve sample formulation procedure

In the table: STD is an abbreviation for Standard.

TABLE 3 preparation of precision and accuracy samples

In the table: ULOQ is the upper limit of quantitation (Upper Limit of Quantitation), HQC is the high concentration quality control (High Concentration Quality Control), MQC is the medium concentration quality control (Medium Concentration Quality Control), LQC is the low concentration quality control (Low Concentration Quality Control), and LLOQ is the lower limit of quantitation (Lower Limit of Quantitation).

Example 4: standard curve regression method

And taking the concentration of the sample to be detected as an abscissa, taking the Signal value difference between the complex Kong Junzhi of the object to be detected and the blank complex Kong Junzhi as an ordinate, fitting a standard curve through a four-parameter regression model, and calculating the actual measurement concentration of the analyte in the sample by using the obtained regression equation.

The measured concentration of analyte in the sample is calculated from the following regression equation:

Y＝(A-D)/[1+(x/C)^B]+D

Wherein A: estimating the asymptote under the curve

D: asymptote estimation on a curve

B: slope of curve

C: corresponding concentration at half maximum binding

Weight factor: 1/y ²

TABLE 4 summary of standard curve results

From the data in the table, the standard curve for ASO detection in blood according to the present invention is stable in the concentration range of 0.4 ng/mL-40 ng/mL, and the standard curve is shown in FIGS. 2 and 3.

Table 5 summary of quality control sample precision and accuracy results

In the table: CV% is the coefficient of variation (Coefficient of variation), RE% is the Relative deviation (Relative Error), TE% is the Total Error of the method (Total Error).

According to the table, the comparison method is used for measuring ASO, the result value and the theoretical value are lower in CV%, RE% and TE%, and the accuracy and the sensitivity of measuring ASO in serum are high, and the sensitivity can reach 0.400ng/mL.

Example 5: results of the hook effect

The hook effect procedure is detailed in example 2.

TABLE 6 hook effect sample formulation table

In the table: 5 parts of DL 1-DL 2 hook effect samples are prepared in parallel, and the samples are named as DL1-1, DL1-2, DL1-3, DL1-4 and DL1-5 respectively; DL2-1, DL2-2, DL2-3, DL2-4, DL2-5.

TABLE 7 hook effect results

According to the above table, ASO samples with theoretical concentrations of 6000ng/mL and 600ng/mL were tested for the hook effect, and found that the signal values were higher than the signal values corresponding to the concentration points of ULOQ, indicating no hook effect.

Example 6: liver tissue sample results

The procedure for liver tissue samples is detailed in example 2.

Table 8 standard curve sample formulation procedure

TABLE 9 preparation of precision and accuracy samples

Table 10 summary of Standard Curve results

As can be seen from the data in the table, the standard curve for ASO detection in mixed SD rat liver tissue homogenate supernatant according to the present invention is stable in the concentration range of 0.2 ng/mL-40 ng/mL, and the standard curve is shown in FIG. 4.

Table 11 summary of quality control sample precision and accuracy results

According to the table, the comparison method shows that the result value and the theoretical value of ASO are lower in CV%, RE% and TE%, and the accuracy and the sensitivity of ASO in liver tissue homogenate supernatant of a mixed SD rat are high, and the sensitivity can reach 0.200ng/mL.

The above-described embodiments are merely preferred embodiments of the present invention, and it should be noted that modifications or substitutions can be made by those skilled in the art without departing from the principles of the present invention, which should also be considered as the scope of the present invention.

Claims (10)

1. A probe and label combination, wherein the probe and label combination comprises a capture probe, a detection probe, and a label; the 3 'end of the capture probe is provided with modification which can be combined with a solid phase material, and the 5' end is combined with a detection probe; the detection probes are hybridizable to the oligonucleotides and have modifications that can be detected by the labels.

2. The probe and label combination of claim 1, wherein the capture probe has a biotin modification at the 3' end.

3. The probe and label combination of claim 1, wherein the detection probe comprises a downstream probe and an upstream probe, wherein the upstream probe is conjugated to the capture probe and hybridized to the oligonucleotide in conjunction with the downstream probe, and wherein the 3' end of the downstream probe has a non-biotin modification, such as digoxin modification, that can be detected by the label.

4. The probe and label combination of claim 3, wherein the label is an anti-digoxin antibody recognition and label bearing SULFO-TAG; and/or the oligonucleotide is an antisense oligonucleotide (ASO), e.g., from a serum or tissue sample.

5. A kit comprising the probe and label combination of any one of claims 1-4.

6. The kit of claim 5, further comprising one or more of a solid phase material, an anti-digoxin antibody, and a hybridization buffer;

preferably, the solid phase material is a streptavidin pre-coated solid phase material such as a 96-well plate, the anti-digoxin antibody is SULFO-TAG anti-digoxin antibody, and the hybridization buffer comprises 60mM Na ₂HPO₄, 1M NaCl, 5mM EDTA, and 0.02% Tween20; the percentages are by volume.

7. A method for detecting and quantifying an oligonucleotide, wherein the method uses the probe and label combination of any one of claims 1-4, or the kit of claim 5 or 6, for detection quantification, comprising:

(1) Coating the capture probes on a solid phase material; preferably, the solid phase material is a 96-well plate pre-coated with streptavidin;

(2) Hybridizing a sample to be detected with a detection probe to generate a hybridization mixture; the hybridization reaction is carried out, for example, in a PCR instrument, incubator, metal bath;

(3) Adding the hybridization mixture into the solid phase material coated with the capture probes for hybridization;

(4) Detecting the detection probe with a label; preferably, the detection probes comprise an upstream probe and a downstream probe; more preferably, the 3' end of the downstream probe is provided with digoxin modification, and the marker is an anti-digoxin antibody;

(5) Detecting and quantifying the electrochemical signal; preferably, the label carries SULFO-TAG, which can be luminescent and quantified by an electrochemical reaction.

8. The method of claim 7, wherein the reaction conditions for coating the capture probes are: incubating for 1 h+ -5 min at 25+ -1deg.C under shaking at 450rpm and in the dark; and/or the reaction conditions for hybridization of the detection probe and the sample to be detected are as follows: hybridization was performed at 90℃for 5min and at 35℃for 30min.

9. The method of claim 7, wherein the reaction conditions for the second hybridization are: at 37+/-1 ℃, standing and incubating for 1 h+/-5 min in dark; and/or, the reaction conditions for labeling digoxin on the downstream probe with SULFO-TAG anti-digoxin antibody are: incubating for 1 h+ -5 min at 25+ -1deg.C under shaking at 450rpm and in the dark; preferably, the 96-well plate is washed before coating, before the second hybridization, and after the second hybridization.

10. Use of a probe and label combination according to any one of claims 1-4 for the preparation of a diagnostic agent for detecting oligonucleotides; preferably, the oligonucleotide is an antisense oligonucleotide; and/or the oligonucleotides are from a blood or tissue sample, such as serum.