Method for detecting content of collagenase in biological matrix by electrochemiluminescence immunoassay

Abstract

The invention discloses a method for detecting the content of type II collagenase in a biological matrix, which comprises the following steps: Biotin incubation: adding a Bio working solution into a microwell plate, and incubating, the Bio working solution being a biotin-labeled capture antibody dilution buffer solution; sample incubation: adding the sample solution into the microwell plate, and incubating; a Ru working solution is added into the microwell plate, incubation is conducted in a dark place, and the Ru working solution is a diluted buffer solution containing the Ru labeled detection antibody; reading: adding plate reading liquid into the microwell plate, and reading a signal through an electrochemical luminescence analyzer; and calculation: collecting data and calculating the content of collagenase. The content determination method disclosed by the invention has the advantages of higher sensitivity, wider linear range and good precision and repeatability, saves the use amount of samples, and is suitable for determining the content of the type II collagenase.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis and testing, and in particular to a method for detecting the content of collagenase in a biological matrix by electrochemiluminescence immunoassay and an application thereof. Background Art

[0002] In 1993, the Shanghai Institute of Pharmaceutical Industry successfully developed collagenase (hereinafter referred to as "collagenase"), which received national Class I new drug certification. This drug is primarily used to treat intervertebral disc herniation, providing patients with a new alternative to surgical treatment. Internationally, XIAFLEX®, the first FDA-approved collagenase-containing drug, is a fixed-ratio mixture of collagenase H (ColH) and collagenase G (ColG) extracted from Clostridium histolyticum. Its formulation is a lyophilized powder injection and is clinically used to treat Dupuytren's contracture and Peyronie's disease. In July 2020, QWO® Collagenase from Clostridium histolyticum (CCH-aaes) subcutaneous injection received FDA approval for its third indication: the treatment of moderate to severe cellulite on the buttocks of adult women. This marks the product's debut in the medical aesthetics field.

[0003] In clinical use, the most common adverse reactions (incidence ≥1%) of collagenase drugs are primarily localized at the injection site, including bruising, pain, nodules, itching, erythema, discoloration, swelling, and fever. To reduce the risk of drug-induced tissue damage and improve drug safety and product stability, the development of a reliable method for measuring collagenase levels in subject serum samples is crucial for evaluating drug safety and accurately assessing clinical trial results.

[0004] Currently, conventional methods for measuring collagenase content include: colorimetry (based on the principle of gelatin degradation), fluorescence, radioisotope analysis, ELISA or antibody labeling, and SDS-PAGE electrophoresis. For example, according to the FDA's publicly available technical approval information, QWO® uses an ELISA method to detect type II Clostridium histolyticum collagenase in human serum, with a sensitivity of 25 ng / mL.

[0005] Electrochemiluminescence immunoassay (ECLIA), a highly sensitive detection technology that integrates electrochemistry, chemiluminescence, and immunoassay, has been widely used in biomedical research, clinical diagnostics, and drug analysis. The core principle of this technology is to trigger a chemiluminescence signal through an electrochemical reaction, combined with specific antigen-antibody recognition, to achieve precise quantitative detection of target substances. The advantages of ECLIA technology are mainly reflected in: high sensitivity: it can detect extremely low concentrations of target substances; wide linear range: it is suitable for accurate measurement of samples of varying concentrations; rapid and efficient: fully automated operation and a simple process improve experimental efficiency; strong stability: the luminescence signal is triggered by electrochemistry, effectively avoiding the photobleaching problem of traditional chemiluminescence.

[0006] To date, there are no reports in the published literature on the use of electrochemiluminescence immunoassay (ECLIA) to detect collagenase content in biological matrices. This technical gap provides a potential direction for innovative research on collagenase detection methods. Summary of the Invention

[0007] Technical Problems to be Solved by the Invention

[0008] As mentioned above, there are no prior reports on the use of electrochemiluminescence immunoassay (ECLIA) to detect type II collagenase in biological matrices. This present invention utilizes an ECLIA for the first time to detect type II collagenase in human serum, facilitating the assessment of type II collagenase safety and drug metabolism during clinical trials. The detection method of this invention boasts high sensitivity (2.00 ng / mL), a wide quantitative range (2.00–200.00 ng / mL), is non-radioactive, rapidly and accurately quantifies, has a wide detection range, and is simple to operate.

[0009] Technical solutions to technical problems

[0010] The present invention provides a method for determining the concentration of type II collagenase (e.g., Clostridium histolyticum type II collagenase) in human serum based on an electrochemiluminescence immunoassay (ECLIA). This method involves adding Bio working solution to a pre-sealed SA-MSD plate. A calibration standard of known concentration of type II collagenase and a quality control sample solution are then added to designated wells. Finally, Ru working solution is added and incubated. The Bio working solution contains a biotinylated capture antibody dilution buffer that binds to streptavidin on the bottom of the SA-MSD plate, coating the plate. The antibody specifically binds to type II collagenase, capturing it. Finally, Ru working solution is added as a detection reagent. The detection antibody specifically binds to type II collagenase, and Ru converts the electrical signal into a light signal. After sufficient reaction, a SA-Bio-type II collagenase-Ru complex is formed. Unbound detection reagent is washed away, and the plate is read on an MSD ultrasensitive multi-factor electrochemiluminescence analyzer. The light signal (RLU) is positively correlated with the type II collagenase concentration in the sample.

[0011] The present invention includes the following contents.

[0012] A method for detecting the content of type II collagenase in a biological matrix, comprising:

[0013] Biotin incubation: Add Bio working solution to the microplate and incubate. The Bio working solution is a biotin-labeled capture antibody dilution buffer.

[0014] Sample incubation: Add sample solution to the microplate and incubate;

[0015] Detection antibody incubation: Add Ru working solution to the microplate and incubate in the dark. The Ru working solution is a dilution buffer containing Ru-labeled detection antibody.

[0016] Reading: Add plate reader solution to the microplate and read the signal using an electrochemiluminescence analyzer;

[0017] Calculation: Collect data and calculate the collagenase content.

[0018] In the method for detecting the content of type II collagenase of the present invention, the samples include calibration standards, quality control samples, blank samples and samples to be tested.

[0019] In the method for detecting the content of type II collagenase of the present invention, the collagenase is type II Clostridium histolyticum collagenase.

[0020] In the method for detecting type II collagenase content of the present invention, the biological matrix is human serum or whole blood.

[0021] The method for detecting type II collagenase content of the present invention further includes a sample dilution step before the sample incubation step: diluting the sample with a dilution buffer, wherein the dilution buffer solution is a phosphate buffer solution containing BSA, Tween-20 and Proclin-300, and the dilution ratio is 1:2 to 1:20, preferably 1:5.

[0022] In the method for detecting type II collagenase content of the present invention, a blocking step is further included before the biotin incubation step: adding a blocking buffer to a microplate containing avidin and / or streptavidin, wherein the blocking buffer is a phosphate buffer solution containing BSA, Tween-20 and Proclin-300.

[0023] In the method for detecting type II collagenase content of the present invention, the biotin incubation time is preferably 30 minutes to 1 hour and 30 minutes, the sample incubation time is preferably 1 hour to 2 hours, and the detection antibody incubation time is preferably 30 minutes to 1 hour and 30 minutes.

[0024] In the method for detecting type II collagenase content of the present invention, a washing step is further included before the Biotin incubation step: washing the microplate with a washing solution, wherein the washing solution is a phosphate buffer solution containing Tween-20.

[0025] In the method for detecting type II collagenase content of the present invention, a washing step is further included before the sample incubation step: washing the microplate with a washing solution, wherein the washing solution is a phosphate buffer solution containing Tween-20.

[0026] In the method for detecting type II collagenase content of the present invention, a washing step is further included before the detection antibody incubation step: washing the microplate with a washing solution, wherein the washing solution is a phosphate buffer solution containing Tween-20.

[0027] In the method for detecting type II collagenase content of the present invention, the microplate contains streptavidin and / or avidin.

[0028] In the method for detecting type II collagenase content of the present invention, the concentration of the Biotin working solution is 0.20 μg / mL to 0.30 μg / mL.

[0029] In the method for detecting type II collagenase content of the present invention, the concentration of the Ru working solution is 0.40 μg / mL to 0.60 μg / mL.

[0030] In the method for detecting type II collagenase content of the present invention, the plate reading buffer is MSD ReadBuffer T containing a surfactant.

[0031] In the method for detecting type II collagenase content of the present invention, the biotin incubation, sample incubation and / or detection antibody incubation are performed under shaking conditions.

[0032] In the method for detecting type II collagenase content of the present invention, the biotin incubation, sample incubation and / or detection antibody incubation are performed at room temperature.

[0033] Beneficial technical effects

[0034] The present invention successfully detects type II collagenase in human serum using electrochemiluminescence immunoassay (ECLIA) for the first time. Compared with the existing enzyme-linked immunosorbent assay (ELISA) analysis method, the electrochemiluminescence immunoassay (ECLIA) of the present invention has the following advantages:

[0035] 1) Higher sensitivity, capable of detecting lower concentrations of collagenase in samples;

[0036] 2) A wider linear range, allowing for the detection of both high and low abundance collagenases;

[0037] 3) Save sample volume. The sample volume for ELISA usually requires 50-100 μL, while the sample volume for ECLIA is ≤25 μL. This has obvious advantages for extremely precious test samples.

[0038] 4) Higher precision and repeatability, improving the accuracy of inspection;

[0039] 5) High sample compatibility, small matrix effect, can exclude many non-specific signals, and is compatible with different samples;

[0040] 6) The ECLIA experimental process is simple and fast, which liberates labor and improves experimental efficiency;

[0041] 7) The ECLIA signal is stable and is not affected by the color development order. The experimental process does not require light protection and is not affected by personnel differences. The data stability is extremely high. DETAILED DESCRIPTION

[0042] Features, characteristics, compounds, and methods described in conjunction with an embodiment or example of the present invention are to be understood as applicable to any other embodiment or example described herein, unless incompatible therewith. All features disclosed in this specification (including claims and abstract) and / or all steps of any method or process disclosed therein may be combined in any manner, except where at least some of these features and / or steps are mutually exclusive. The present invention is not limited to the details of any embodiment. The present invention extends to any new feature or any new combination of features disclosed in this specification (including claims and abstract), or to any new step or any new combination of steps of any method or process disclosed therein.

[0043] definition

[0044] In the examples of the present invention, unless otherwise specified, all instruments, materials, chemical reagents, pharmaceuticals, compounds, and biological samples are commercially available products familiar to those skilled in the art. Unless otherwise specified, all technical means used in the examples of the present invention are conventional means known to those skilled in the art. The progress of the reactions of the present invention can be monitored using conventional methods in the art (e.g., TLC, HPLC, LCMS, or NMR), with the disappearance of the reaction substrate generally considered the endpoint of the reaction.

[0045] Experimental methods in which specific conditions are not specified in the examples of the specification are generally carried out in accordance with conventional conditions in the art or the conditions recommended by the manufacturer. In the present invention, unless otherwise specified, "above", "below", and "within" are intended to include the number itself. When "comprising" or "including" are used to describe an embodiment herein, other similar embodiments described as "consisting of..." and / or "consisting essentially of..." are also provided. When "consisting essentially of..." is used to describe an embodiment herein, other similar embodiments described as "consisting of..." are also provided. The term "and / or" as used in phrases such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0046] In this disclosure, "±" is used to indicate the error range, upper and lower limits, measurement accuracy, or statistical range of a value. For example, "10 ± 2" means the value is between 8 and 12, or between 8 and 12 after rounding. "25 ± 5°C" means between 20°C and 30°C, or between these two ranges after rounding. When referring to measurements, "±" is used to indicate the accuracy of the measuring instrument. In statistics or experimental data, "±" is used to indicate the confidence interval or standard deviation of the data.

[0047] 1. Overview of detection methods

[0048]

[0049] 2. Analysis Steps

[0050] Blocking: Take an MSD microplate and add 200 μL of dilution buffer solution (DBS, 1% BSA, 0.05% Tween-20, 0.05% Proclin-300 in 1× PBS) to each well. After applying the sealing film, place it on a microplate shaker at room temperature, set the speed to 500 rpm, and incubate for at least 1 hour.

[0051] Washing: After blocking the MSD microplate, wash each well three times with 300 μL of washing buffer (WBS, 1× PBS containing 0.05% Tween-20) and pat dry on absorbent paper.

[0052] Biotin incubation: Add 100 μL of Bio working solution (0.25 μg / mL Biotin-labeled capture antibody in dilution buffer) to each well of the MSD microplate. After sealing the plate with film, place the plate on a microplate shaker at 500 rpm and incubate at room temperature for 1 hour ± 10 minutes.

[0053] Wash the plate: Wash each well of the MSD plate three times with 300 μL of washing buffer and pat dry on absorbent paper.

[0054] Sample MRD (minimum dilution factor) dilution: dilute the recombinant collagenase calibration standard, quality control sample, blank sample and other samples to be tested 1:5 in a dilution plate (take 25μL sample + 100μL dilution buffer on the dilution plate), mix well and set aside.

[0055] Sample incubation: Add 100 μL of diluted MRD solution to each well of the MSD plate, apply the sealing film, and incubate on a microplate shaker at 500 rpm for 1.5 h ± 10 min.

[0056] Wash the plate: Wash each well of the MSD microplate three times with 300 μL of washing buffer and pat dry on absorbent paper.

[0057] Detection antibody incubation: Add 100 μL of Ru working solution (containing 0.50 μg / mL Ru-labeled detection antibody in dilution buffer) to each well of the MSD microplate. After sealing the plate, incubate on a microplate shaker at 500 rpm in the dark for 1 hour ± 10 minutes.

[0058] Wash the plate: Wash each well of the MSD microplate three times with 300 μL of washing buffer and pat dry on absorbent paper.

[0059] Reading: Add 150 μL of 2×MSD Read Buffer with Surfactant to each well of the MSD microplate and read the values on the MSD.

[0060] 3. Contents of Analytical Method Verification

[0061] 1. Standard curve and quantitative range

[0062] The quantitative range of the standard curve is determined by the LLOQ of the lowest concentration of the calibration standard and the ULOQ of the highest concentration of the calibration standard. Only analytical batches that meet the acceptance criteria for the standard curve and the quantitative range are acceptable validation batches.

[0063] Validation results showed that all reported analytical batch standard curves met the expected standards, with a quantitative range of 2.00-200.00 ng / mL.

[0064] 2. Precision and accuracy

[0065] Six analytical runs were analyzed by at least two analysts over at least two days for precision and accuracy assessment. Each precision and accuracy run included a standard curve, a matrix blank, and at least three sets of quality control samples. Each set of quality control samples included at least five concentration levels: LLOQ, LQC, MQC, HQC, and ULOQ. The results showed acceptable intra- and inter-run precision and accuracy for all six runs.

[0066]

[0067] 3. Robustness

[0068] The robustness of an analytical method is the ability to measure the method's ability to remain unaffected. During the validation process, factors such as sample incubation time, instrumentation, and analytical personnel are evaluated. Method robustness is verified through accuracy and precision assessments, demonstrating the robustness and reliability of the method.

[0069]

[0070] 4. Matrix selectivity

[0071] The matrix may contain nonspecific components that interfere with the detection of the target analyte. The selectivity of a method refers to its ability to accurately measure the analyte in the presence of nonspecific components in the matrix.

[0072] Healthy Individual Matrix: Blank serum from 10 different healthy individuals was spiked with the analytes at both the LLOQ and ULOQ levels, and the results met the acceptance criteria. Responses in the unspiked blank matrix were below the LLOQ.

[0073] Hemolytic Matrix (HE): Five individual serum matrices containing 2% blood cell lysate from different sources spiked with analytes met the acceptance criteria, and five individual matrices without analytes had responses below the LLOQ.

[0074] Lipid Matrix (HL): Five individual serum matrices containing 300 mg / dL fat emulsion from different sources were spiked with the analytes. The results met the acceptance criteria, and the responses of the five individual matrices without analytes were below the LLOQ.

[0075]

[0076] 5. Dilution Linearity / Hook Effect

[0077] Dilution linearity was verified by diluting quality control samples. A diluted quality control sample UHQC (concentration: 10,000.00 ng / mL) was prepared using pooled human serum and stored frozen in a -80°C refrigerator for at least 12 h. On the day of analysis, the sample was serially diluted with pooled human serum and named DL-1 to DL-8.

[0078] At the same time, the signal response values measured at analyte concentrations of 4000.00 mg / mL and 400.00 ng / mL were greater than the ULOQ signal response value, indicating that there was no hook effect at 4000.00 ng / mL.

[0079]

[0080] 6. Sample stability

[0081] The stability of the samples at room temperature, freeze-thaw, refrigeration and freezing conditions for a certain period of time was investigated.

[0082]

[0083] Example

[0084] All clinical subjects fully understood and voluntarily signed informed consent forms that complied with international standards and national laws and regulations and submitted them for ethical review. During the implementation phase, the present invention strictly followed the sample processing protocol to protect the privacy and safety of all clinical subjects.

[0085] Example 1: Detection of recombinant collagenase concentration in serum samples of clinical subjects

[0086] Step 1: Solution preparation

[0087] Wash Buffer (WBS, 1× PBS containing 0.05% Tween-20): Measure an appropriate volume of 20× PBS and add it to the corresponding volume of ultrapure water. Then, add an appropriate volume of Tween-20 and mix thoroughly. Fill in the reagent label and prepare the appropriate amount of Wash Buffer according to the volume shown in the table below. Store at room temperature. The shelf life is 1 month.

[0088]

[0089] Dilution Buffer (DBS, 1× PBS containing 1% BSA, 0.05% Tween-20, and 0.05% Proclin-300): Measure an appropriate volume of 20× PBS and add it to a container containing the corresponding volume of ultrapure water to obtain 1× PBS. Add the appropriate amount of BSA to the container and mix thoroughly. Then, add the appropriate volume of Proclin-300 and Tween-20. Mix thoroughly and set aside. Fill the reagent label. Prepare the appropriate amount of Dilution Buffer according to the volume shown in the table below. Store at 2–8°C. The shelf life is 1 month.

[0090]

[0091] Bio working solution: dilution buffer containing 0.25 μg / mL Biotin-labeled capture antibody, stored at room temperature, ready for use.

[0092] Ru working solution: dilution buffer containing 0.50 μg / mL Ru-labeled purified antibody. Store at room temperature and prepare immediately for use.

[0093] Ru is SULFO-TAG™ NHS Ester (CAS 482618-42-8), chemical structure: ruthenium bipyridine sulfonic acid complex + NHS ester group + sodium salt

[0094] Purified antibodies refer to anti-collagenase antibodies

[0095] Preparation process of Ru-labeled purified antibody: prepare anti-collagenase antibody, label the anti-collagenase antibody with Sulfo-Tag, and purify to obtain Ru-labeled purified antibody.

[0096] 2× Read Buffer: Measure an appropriate volume of MSD Read Buffer T (4×) With Surfactant and add it to the corresponding volume of ultrapure water. Mix well and store at room temperature. Prepare and use immediately.

[0097]

[0098] Step 2: Sample preparation

[0099] Calibration standard preparation: Prepare the sample according to the following dilution method. The stock is CU-20401 (type II collagenase) for injection (purchased from Cody Biotechnology), with a concentration of 0.30 mg / mL. The matrix is mixed human serum.

[0100]

[0101] Preparation of quality control samples: Prepare by dilution according to the table below. The stock is CU-20401 (type II collagenase) for injection (purchased from Cody Biotechnology), with a concentration of 0.30 mg / mL, and the matrix is mixed human serum.

[0102]

[0103] Preparation of serum samples to be tested: Mix the whole blood sample by inversion 5 times, let it stand at room temperature for 15-60 minutes, and centrifuge the sample (centrifugal parameters: centrifugal force 1700g, temperature 4°C, time 10 minutes). After centrifugation, transfer the upper serum sample, transfer at least 0.8ml to the test tube, and transfer the remaining sample to a backup tube. The aliquoted serum sample is transferred to -60~-90°C for storage. The sample is taken out of the refrigerator, thawed at room temperature, and then incubated.

[0104] Step 3: Analysis and testing

[0105] Blocking: Take the MSD plate, add 200 μL of dilution buffer solution to each well, apply the sealing film, and incubate on a microplate shaker at room temperature at 500 rpm for at least 1 hour.

[0106] Wash the plate: Wash each well of the MSD plate three times with 300 μL of washing buffer and pat dry on absorbent paper.

[0107] Biotin incubation: Add 100 μL of Bio working solution to each well of the MSD plate, apply the sealing film, and incubate on a microplate shaker at 500 rpm at room temperature for 1 hour ± 10 minutes.

[0108] MRD dilution: dilute the recombinant collagenase calibration standard, quality control sample, blank sample and serum sample to be tested 1:5 in a dilution plate (take 25 μL sample + 100 μL dilution buffer on the dilution plate), mix well and set aside.

[0109] Wash the plate: Wash each well of the MSD plate three times with 300 μL of washing buffer and pat dry on absorbent paper.

[0110] Sample incubation: Add 100 μL of diluted MRD solution to each well of the MSD plate, apply the sealing film, and incubate on a microplate shaker at 500 rpm for 1.5 h ± 10 min.

[0111] Wash the plate: Wash each well of the MSD plate three times with 300 μL of washing buffer and pat dry on absorbent paper.

[0112] Detection antibody incubation: Add 100 μL of Ru working solution to each well of the MSD plate, apply the sealing film, and incubate on a microplate shaker at 500 rpm in the dark for 1 hour ± 10 minutes.

[0113] Wash the plate: Wash each well of the MSD plate three times with 300 μL of washing buffer and pat dry on absorbent paper.

[0114] Reading: Add 150 μL of 2× plate reading solution to each well of the MSD plate and read the value on the MSD.

[0115] Step 4: Data collection and calculation

[0116] The data were collected using DISCOVERY WORKBENCH 4.012, processed using Watson LIMS 7.5, and summarized and calculated using Excel.

[0117] Results: An electrochemiluminescence immunoassay (ECLIA) was used to quantify type II collagenase concentrations in human serum. The accuracy and precision of the quality control samples met acceptable standards, demonstrating the reliability of the assay. Of the 20 serum samples tested, type II collagenase concentrations were below the limit of quantification (2 ng / mL) in all samples. This result indicates low systemic exposure to the drug in vivo and suggests a potentially favorable safety profile. However, further evaluation of its clinical significance requires consideration of the drug's therapeutic window, pharmacodynamic properties, and individual variability.

[0118] Quality control sample test results

[0119]

[0120] Test results of serum samples to be tested

[0121]

[0122] BLQ: Below the Limit of Quantitation.

[0123] While the present invention has been described with reference to certain preferred embodiments thereof, those skilled in the art will appreciate that the foregoing description is provided as a further detailed description of the present invention in conjunction with specific embodiments thereof, and that the present invention is not limited to these descriptions. Within the spirit and scope of the present invention, those skilled in the art may make various changes in form and detail, including simple deductions or substitutions, which also constitute a part of the present invention.

Claims

1. A method for detecting the content of type II collagenase in a biological matrix, comprising: Biotin incubation: Add Bio working solution to the microplate and incubate. The Bio working solution is a biotin-labeled capture antibody dilution buffer. Sample incubation: Add sample solution to the microplate and incubate; Detection antibody incubation: Add Ru working solution to the microplate and incubate in the dark. The Ru working solution is a dilution buffer containing Ru-labeled detection antibody. Reading: Add plate reader solution to the microplate and read the signal using an electrochemiluminescence analyzer; Calculation: Collect data and calculate the collagenase content.

2. The detection method according to claim 1, wherein the collagenase is type II Clostridium histolyticum collagenase and / or The biological matrix is human serum or whole blood.

3. The detection method according to claim 1 or 2, further comprising before the sample incubation step: Sample dilution step: diluting the sample with a dilution buffer, wherein the dilution buffer solution is a phosphate buffer solution containing BSA, Tween-20 and Proclin-300; and / or The dilution ratio is 1:2~1:20, preferably 1:

5.

4. The detection method according to claim 1, further comprising: Blocking step: Add blocking buffer to the microplate containing avidin and / or streptavidin. The blocking buffer is a phosphate buffer solution containing BSA, Tween-20 and Proclin-300.

5. The detection method according to claim 1, wherein the biotin incubation time is 30 minutes to 1 hour and 30 minutes, and / or The sample incubation time is 1 hour to 2 hours, and / or The detection antibody incubation time is 30 minutes to 1 hour and 30 minutes.

6. The detection method according to claim 4, wherein each step of biotin incubation, sample incubation, detection antibody incubation and reading further comprises: Washing step: Wash the microplate with a washing solution containing phosphate buffered saline (PBS) containing Tween-20. The detection method according to claim 1 , wherein the microplate contains streptavidin and / or avidin.

8. The detection method according to claim 1, wherein the concentration of the Biotin working solution is 0.20 μg / mL to 0.30 μg / mL; and / or The concentration of the Ru working solution is 0.40 μg / mL to 0.60 μg / mL.

9. The detection method according to claim 1, wherein the plate reading buffer is MSD Read Buffer T containing a surfactant.

10. The detection method according to claim 1, wherein the biotin incubation, sample incubation and / or detection antibody incubation are performed under shaking conditions; and / or The biotin incubation, sample incubation and / or detection antibody incubation are performed at room temperature.

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