Electrochemiluminescence immunoassay method for detecting il-9 and detection kit

Abstract

The application relates to the technical field of interleukin 9 (IL-9) detection, and provides an IL-9 electrochemiluminescence immunoassay method and a detection kit, which solve the technical problems of narrow detection range, low sensitivity, complicated operation steps and radioactive pollution of the existing IL-9 detection method; the electrochemiluminescence immunoassay platform is applied, the capture antibody is directly modified to the surface of an electrochemiluminescence plate, then a double-antibody sandwich structure is formed among the capture antibody, IL-9 and a ruthenium-labeled detection antibody, and an IL-9 direct electrochemiluminescence immunoassay system is established.

Description

Technical Field

[0001] This invention relates to the field of interleukin-9 (IL-9) detection technology, and more specifically, to an IL-9 electrochemiluminescence immunoassay method and detection kit. Background Technology

[0002] Interleukin 9 (IL-9) is a type of interleukin, a multifunctional cytokine that plays an important role in the formation of immune memory, regulation of Th2 immune responses, and tumor immunotherapy. Its value in the diagnosis and prognostic assessment of autoimmune diseases, allergic diseases, and tumors is increasingly prominent. Therefore, developing a highly sensitive, wide-range, and easy-to-use IL-9 detection method is of significant clinical importance.

[0003] Currently, the commonly used clinical methods for IL-9 detection mainly include traditional ELISA and radioimmunoassay. However, these traditional techniques have significant limitations: traditional ELISA is often cumbersome and time-consuming (requiring several hours) and relies on manual washing; its sensitivity is relatively limited, and its detection range is narrow, making it difficult to meet the detection needs of low-concentration samples or complex matrix samples. Radioimmunoassay has disadvantages such as short reagent shelf life, risk of radioactive contamination, and difficult waste disposal, which limits its widespread application.

[0004] To overcome the aforementioned technical bottlenecks, there is an urgent need in this field for a new IL-9 detection scheme that is simple, fast, has a wide detection range, and high sensitivity. Summary of the Invention

[0005] This application aims to solve the technical problems of existing IL-9 detection methods, such as narrow detection range, low sensitivity, cumbersome operation steps, and radioactive contamination, and provides an IL-9 electrochemiluminescence immunoassay method and detection kit.

[0006] Electrochemiluminescence immunoassay analyzers are quantitative analysis platforms that combine highly sensitive electrochemiluminescence technology with highly specific immunoassay technology. This detection platform has ultra-high sensitivity and a wide detection range. At the same time, the platform has strong resistance to matrix interference and is especially suitable for direct detection of complex samples such as serum, plasma, and cell culture supernatant, with stable and reliable results.

[0007] This invention employs pre-coated plate technology. By pre-immobilizing the capture antibody onto the surface of an electrochemiluminescence plate, an IL-9 pre-coated plate is prepared. This eliminates the need for cumbersome coating and blocking steps during use, shortening the experimental procedure and overall detection time, and reducing the complexity of experimental operations and batch-to-batch variability. The prepared IL-9 pre-coated plate forms a double-antibody sandwich structure with IL-9 standards and ruthenium-labeled detection antibodies, establishing a direct electrochemiluminescence immunoassay for IL-9.

[0008] Based on the above technical solutions, this invention further optimizes the key reagents in the immune reaction system and deeply improves the core reagents. By optimizing the coating diluent, a modified antibody coating buffer system is used to replace the traditional phosphate-buffered saline (PBS) or carbonate buffer (CB), significantly improving the fixation efficiency and activity of antibodies on the solid-phase carrier, ensuring the spatial conformation of the captured antibody, which is more conducive to antigen binding, thereby improving the initial efficiency of the reaction. Simultaneously, to address matrix interference in complex serum, this invention specifically optimizes the sample diluent formulation, introducing fructose and glycine into the conventional diluent to reduce matrix interference and enhance the antigen-antibody binding reaction efficiency, ensuring that the target antigen is fully released and remains active in the sample, thereby further improving the detection signal intensity and sensitivity.

[0009] A first aspect of this application provides an electrochemiluminescence immunoassay method for detecting IL-9, comprising the following steps:

[0010] The first step is to add IL-9 capture antibody coating solution to the electrochemiluminescence plate and incubate it; the IL-9 capture antibody coating solution contains HEPES, sodium chloride, glycine, and the remainder is deionized water;

[0011] The second step is to add washing buffer to each well of the electrochemiluminescence plate for cleaning;

[0012] Step 3: Prepare the sample.

[0013] Human IL-9 was prepared into an IL-9 sample using sample dilution buffer; the marker-labeled detection antibody was prepared into a labeled detection reagent using sample dilution buffer.

[0014] Step 4, adding samples:

[0015] Add IL-9 sample to the electrochemiluminescence plate, incubate, and wash the plate with washing buffer after incubation; then add labeled detection reagent, incubate, and wash the plate with washing buffer after incubation.

[0016] Step 5: Add the electrochemiluminescence substrate solution;

[0017] Step 6: Read the value.

[0018] Preferably, in the IL-9 capture antibody coating solution, the mass concentration of HEPES is 1.7–2.9 g / L, the mass concentration of sodium chloride is 6–11 g / L, and the mass concentration of glycine is 0.5–0.9 g / L. More preferably, in the IL-9 capture antibody coating solution, the mass concentration of HEPES is 2.38 g / L, the mass concentration of sodium chloride is 8.77 g / L, and the mass concentration of glycine is 0.75 g / L.

[0019] Preferably, the labeled detection reagent is a ruthenium-labeled detection reagent, which is prepared by mixing ruthenium-labeled detection antibodies with a sample diluent;

[0020] The electrochemiluminescence substrate solution is tri-n-propylamine, a co-reactant.

[0021] Preferably, the concentration of the IL-9 capture antibody coating solution is 1.0 μg / mL, and the concentration of the labeled detection reagent is 0.5 μg / mL.

[0022] Preferably, the sample diluent is a casein blocking solution with added fructose and glycine, wherein the fructose concentration is 10–35 g / L and the glycine concentration is 0.75–2.5 g / L. More preferably, the sample diluent contains 15 g / L fructose and 1.5 g / L glycine.

[0023] A second aspect of this application provides a detection kit comprising an electrochemiluminescence plate, an IL-9 capture antibody coating solution, an IL-9 standard, a washing buffer, a labeled detection reagent, a sample diluent, and an electrochemiluminescence substrate solution; wherein the IL-9 capture antibody coating solution comprises HEPES, sodium chloride, and glycine, with the remainder being deionized water; and the sample diluent comprises fructose and glycine.

[0024] Preferably, the labeling detection reagent is a ruthenium-labeled detection reagent.

[0025] The beneficial effects of this disclosure are that a stable double-antibody sandwich structure is constructed on an electrochemiluminescence microplate, and the direct detection of electrochemiluminescence signals is achieved by introducing ruthenium-labeled detection antibodies; at the same time, by optimizing the antibody coating buffer system and sample dilution composition, the antibody immobilization efficiency and antigen-antibody binding efficiency are significantly improved, and non-specific adsorption and matrix interference are reduced, thus giving this method the advantages of simple operation, wide detection range, high specificity, high sensitivity, good selectivity and stability.

[0026] Further features and aspects of this disclosure will be clearly described in the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0027] Figure 1 It is the fitted standard curve;

[0028] Figure 2 It compares the detection results under different concentrations of capture antibody and detection antibody;

[0029] Figure 3 This is a comparison of the detection results for phosphate buffer, carbonate buffer, and modified buffer.

[0030] Figure 4This is a comparison of the detection results of sample diluents, high-performance blocking buffer, casein blocking buffer, and modified sample diluent;

[0031] Figure 5 These are the results of different concentrations of IL-9 and their corresponding electrochemiluminescence signal values;

[0032] Figure 6 These are the results of six precision and accuracy tests;

[0033] Figure 7 These are the results from the minimum dilution experiment;

[0034] Figure 8 This is the result of dilution linearity and hook effect;

[0035] Figure 9 This is the result of the parallelism verification;

[0036] Figure 10 These are results from selective experiments;

[0037] Figure 11 This is specificity verification data;

[0038] Figure 12 This is the result of the stability assessment. Detailed Implementation

[0039] The application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] The specific embodiments described below are merely preferred embodiments of this application, and the scope of protection of this application is not limited thereto. Those skilled in the art can make modifications or variations based on the principles, concepts, and spirit of this application, and the resulting technical solutions should all be covered within the scope of protection of this application.

[0041] The preferred labeling reagent is the ruthenium labeling reagent from Wenzhou Kangrui Biotechnology Co., Ltd.

[0042] The electrochemiluminescence substrate solution is preferably an electrochemiluminescence solution containing tri-n-propylamine, more preferably with a tri-n-propylamine content of 3% to 8%, and even more preferably with a tri-n-propylamine content of 5%.

[0043] The preferred monoclonal antibody pairs are the rabbit anti-human IL-9 monoclonal antibody (Cap) and the humanized IL-9 monoclonal antibody (Det) from Wenzhou Kangrui Biotechnology Co., Ltd.

[0044] The light chain amino acid sequence of the rabbit anti-human IL-9 monoclonal antibody (Cap) is shown in SEQ ID NO.1:

[0045] ALVMTQTPASVEAAVGGTVTIKCQANENIYSSLAWYQQKPGQRPKLLIYGASTLASGVPSRFKGSRSGTEYTLTISDLECADAATYYCLGGYSDTTVESAFGGGTEL EILCDPIAPTVLLFPPSADQVTTETVTIVCVANKYFPDVTVTWKVDGTTQTTGIENSTTPQSPEDCTYNLSSTLSLTKAQYNSHSEYTCEVTQGTGSIVQSFNRGDC

[0046] The heavy chain amino acid sequence of the rabbit anti-human IL-9 monoclonal antibody (Cap) is shown in SEQ ID NO.2:

[0047] QSLEESGGGLVKPGASLTLTCTASGFSISSYYGMCWVRQAPGKGLEWIGSIYAGGASAYYANWAKGRFTISKTSSTTVDLKMTSLTAADTATYFCARGYIGDGYAAGAFDP WGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTC SKPTCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKT ISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0048] The light chain amino acid sequence of the humanized IL-9 monoclonal antibody (Det) is shown in SEQ ID NO.3:

[0049] DIQMTQSPSSSLSASVGDRVTITCRASQHVIAHTTWYQQKPGQAPKLLIYGVSNAFSGVPSRFSGSGSGTDFTLSISSLQPEDFATYYCAQYYSVPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0050] The heavy chain amino acid sequence of the humanized IL-9 monoclonal antibody (Det) is shown in SEQ ID NO.4:

[0051] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGVGIEWVRQAPGQGFEWMGEINPGCGPNKKALKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARADYYGQDNVKGDVWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0052] The IL-9 electrochemiluminescence immunoassay method includes the following steps:

[0053] First, add 100.0 μL of a certain concentration of IL-9 capture antibody coating solution to a 96-well electrochemiluminescence plate and freeze at 4°C. o C. Leave overnight.

[0054] The concentration of the IL-9 capture antibody coating solution can be 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL or 4.0 μg / mL.

[0055] The second step is to add 300 μL of PBST buffer to each well of the electrochemiluminescence plate, blot dry, and wash three times. Ensure that there is no residual liquid in the wells after the last blot, otherwise it will affect the readings and results.

[0056] Step 3: Prepare the sample.

[0057] Human IL-9 was prepared into an IL-9 sample with a concentration of 20 pg / mL using sample diluent; similarly, ruthenium-labeled detection antibodies were prepared into ruthenium-labeled detection reagents using sample diluent at a specific ratio. The concentration of the ruthenium-labeled detection reagents could be 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, or 4.0 μg / mL.

[0058] Preparation process of ruthenium-labeled detection antibody: using KanryECL TM The ruthenium labeling kit labels the IL-9 detection antibody. The ruthenium labeling of the IL-9 detection antibody is based on the carbodiimide (EDC / NHS) coupling chemistry principle. The specific procedure is as follows: First, using 2-morpholine ethanesulfonic acid buffer (MES) as the solvent, solutions of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide sodium salt (NHS) with a concentration of 50.0 mg / mL are prepared. Simultaneously, commercially available ruthenium (Ru) labeled lyophilized powder is reconstituted to 20.0 mg / mL. Then, the solutions are mixed at a volume ratio of Ru solution:NHS solution:EDC solution = 100:15:15 to prepare an activation premix. The mixture is then incubated at 25°C in the dark with shaking for 25 minutes to ensure sufficient activation of the carboxyl groups of the Ru labeled compound. After activation, the above mixture was mixed with the IL-9 detection antibody to be labeled at a 1:1 molar ratio and incubated for another 2 hours under the same conditions (25°C, protected from light, and shaking) to complete covalent coupling. Finally, the free small molecule reagents in the reaction system were removed using an ultrafiltration purification device to obtain purified ruthenium-labeled antibody.

[0059] Step 4, adding samples:

[0060] Add IL-9 sample at a concentration of 20 pg / mL to the electrochemiluminescence plate, 100.0 μL per well, and incubate with shaking at 25°C for 1 h. After incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of ruthenium-labeled detection reagent, and incubate with shaking at 25°C for 1 h. After incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0061] Step 5, add Read buffer: Add 100.0 μL of tri-n-propylamine Read buffer to each well.

[0062] Step 6, read the value:

[0063] Electrochemiluminescence values ​​were read at a wavelength of 620 nm.

[0064] Data results as follows Figure 2 As shown, the S / N values ​​are highest at 1.0 μg / mL capture antibody and 0.5 μg / mL detection antibody. Therefore, the optimal concentrations of capture antibody and detection antibody are 1.0 μg / mL and 0.5 μg / mL, respectively.

[0065] To obtain a coating buffer system suitable for the preparation of IL-9 pre-coated plates, this invention optimized the selection of different coating conditions. Specifically, IL-9 capture antibody coating solutions of the same concentration were prepared using phosphate-buffered saline (PBS), carbonate buffer (CB), and a modified coating buffer, respectively.

[0066] Step (1): Prepare different coating buffer systems (including phosphate buffer (PBS), carbonate buffer (CB) and modified coating buffer).

[0067] PBS preparation: Dissolve sodium chloride (NaCl, 8.0 g / L), potassium chloride (KCl, 0.2 g / L), disodium hydrogen phosphate (Na2HPO4, 1.44 g / L), and potassium dihydrogen phosphate (KH2PO4, 0.24 g / L) in deionized water and stir until homogeneous; adjust the pH of the solution to 7.4; and bring the volume to 1 L.

[0068] Preparation of CB: Dissolve sodium carbonate (Na2CO3, 1.59 g / L) and sodium bicarbonate (NaHCO3, 2.94 g / L) in deionized water and stir until homogeneous; adjust the pH of the solution to 9.6; and bring the volume to 1 L.

[0069] Preparation of modified coating buffer: Dissolve HEPES (2.38 g / L), sodium chloride (NaCl, 8.77 g / L), and glycine (0.75 g / L) in deionized water and stir until homogeneous; adjust the pH of the solution to 7.8; and bring the volume to 1 L.

[0070] Prepare an IL-9 capture antibody coating solution with a concentration of 1.0 μg / mL using the above coating buffer, add 100.0 μL to a 96-well electrochemiluminescence plate, and incubate at 4°C. o Incubate overnight at C to prepare IL-9 pre-coated plates with different coating conditions. Add 300 μL of PBST buffer to each well of the electrochemiluminescence plate, pat dry, and wash 3 times.

[0071] Step (2), Sample preparation: Prepare human IL-9 samples at concentrations of 2,000 pg / mL, 200 pg / mL, and 20 pg / mL using sample diluent; similarly, prepare ruthenium-labeled detection antibody at a certain ratio using sample diluent to prepare ruthenium-labeled detection reagent at a concentration of 0.5 μg / mL.

[0072] Step (3), sample addition: Add IL-9 samples with concentrations of 2,000 pg / mL, 200 pg / mL, and 20 pg / mL to the electrochemiluminescence plate, 100.0 μL per well, and incubate with shaking at 25°C for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of ruthenium-labeled detection reagent at 0.5 μg / mL, and incubate with shaking at 25°C for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0073] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0074] Step (5), reading: read the electrochemiluminescence value at a wavelength of 620 nm.

[0075] Data results as follows Figure 3 As shown, the pre-coated plate prepared by the modified coating buffer of the present invention has the highest S / N value under the same detection conditions, indicating that it is beneficial to improve the fixation efficiency and activity retention of the capture antibody, and is suitable for constructing a stable IL-9 pre-coated plate.

[0076] To obtain a sample dilution system suitable for IL-9 detection, this invention compared conventional and modified diluents, and screened for optimized sample dilution conditions. The modified diluent is based on the conventional diluent with the addition of fructose and glycine, which can improve detection sensitivity.

[0077] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution with a concentration of 1.0 μg / mL to a 96-well electrochemiluminescence plate and freeze at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well of the electrochemiluminescence plate, blot dry, and wash 3 times.

[0078] Step (2), Preparation of improved sample dilution solution: Add fructose and glycine to the casein blocking solution. The mass concentration of fructose is 15 g / L and the mass concentration of glycine is 1.5 g / L. Stir well and adjust the pH of the solution to 7.5.

[0079] Sample preparation: Human IL-9 was prepared into IL-9 samples of 2,000 pg / mL, 200 pg / mL, and 20 pg / mL using conventional sample diluent and modified sample diluent; similarly, ruthenium-labeled detection antibody was prepared into ruthenium-labeled detection reagent of 0.5 μg / mL using conventional sample diluent and modified sample diluent.

[0080] Step (3), sample addition: Add IL-9 samples with concentrations of 2,000 pg / mL, 200 pg / mL, and 20 pg / mL to the electrochemiluminescence plate, 100.0 μL per well, and incubate with shaking at 25°C for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate with shaking at 25°C for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0081] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0082] Step (5), reading: read the electrochemiluminescence value at a wavelength of 620 nm.

[0083] Data results as follows Figure 4 As shown, by comparing the conventional diluent with the modified diluent, the modified sample diluent of the present invention can achieve sample dilution conditions with a higher S / N value.

[0084] To reflect the relationship between the concentration of the analyte and the electrochemiluminescence value, a stable and reliable standard curve was established for this analytical method. The specific steps are as follows:

[0085] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution with a concentration of 1.0 μg / mL to a 96-well electrochemiluminescence plate and freeze at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well of the electrochemiluminescence plate, blot dry, and wash 3 times.

[0086] Step (2), Sample preparation: Use sample diluent to prepare human IL-9 into standard curve points of different concentrations; similarly, use sample diluent to prepare ruthenium-labeled detection antibody into 0.5 μg / mL ruthenium-labeled detection reagent in a certain proportion.

[0087] Step (3), sample addition: Add 100.0 μL of IL-9 sample of different concentrations to each well, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0088] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0089] Step (5), reading: read the electrochemiluminescence value at a wavelength of 620 nm.

[0090] A standard curve for IL-9 was established and fitted using a four-parameter 4-PL model. The goodness of fit R² ≥ 0.990. The fitted curve is shown in the attached figure. Figure 1 As shown. The experimental results are as follows. Figure 5 As shown, the coefficient of variation (%CV) and relative error (%RE) were both within 20%, and the standard curve showed good linearity in the range of 20.6–10,000.0 pg / mL, with a lower limit of quantification (LLOQ) of 20.0 pg / mL.

[0091] In bioanalysis, the validation of precision and accuracy is the cornerstone of ensuring the reliability of analytical methods and the trustworthiness of data. Its core significance lies in the fact that precision measures the repeatability of a method (random error), ensuring stable and consistent experimental results; while accuracy measures the correctness of a method (systematic error), ensuring that the measured values ​​are infinitely close to the true values. This invention involves conducting multiple independent analytical batches on different days, on the same plate, and on different plates of electrochemical microplates. Each analytical batch includes one set of standard curves and three sets of 5-level quality control points, where the upper limit of quantitation (ULOQ) is 10,000.0 pg / mL, the high quality control (HQC) is 6,000.0 pg / mL, the moderate quality control (MQC) is 600.0 pg / mL, the low quality control (LQC) is 60.0 pg / mL, and the LLOQ is 20.0 pg / mL. Each sample is replicated in two wells. The specific steps are as follows:

[0092] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution (1.0 μg / mL) to a 96-well electrochemiluminescence plate and incubate at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well, blot dry, and wash 3 times.

[0093] Step (2): Sample preparation: Use sample diluent to prepare human IL-9 into standard curve points and quality control points of different concentrations; similarly, use sample diluent to prepare ruthenium-labeled detection antibody into 0.5 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0094] Step (3): Sample addition: Add 100.0 μL of IL-9 sample of different concentrations to each well, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0095] Step (4): Add Read buffer: Add 100.0 μL of tri-n-propylamine Read buffer to the co-reactant.

[0096] Step (5): Reading value: Read the electrochemiluminescence value at a wavelength of 620 nm.

[0097] Experimental results are as follows Figure 6 As shown, the coefficient of variation (%CV) of the standard curve and the quality control points is ≤20.0%, and the relative error (|%RE|) is ≤20.0%, with ULOQ and LLOQ being ≤25.0% and ≤25.0% respectively, indicating that the method has good precision and accuracy.

[0098] In the development and validation of bioanalytical methods, determining the minimum required dilution (MRD) is a crucial step in ensuring data accuracy and reliability. The specific steps are as follows:

[0099] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution (1.0 μg / mL) to a 96-well electrochemiluminescence plate and incubate at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well, blot dry, and wash 3 times.

[0100] Step (2): Sample preparation: Select mixed blood and #21 blood to spike human IL-9 to 2,000.0 pg / mL, and perform a series of gradient dilutions on the sample using sample diluent (1:2, 1:5, 1:10, 1:50, 1:100); similarly, use sample diluent to prepare ruthenium-labeled detection antibody into 0.5 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0101] Step (3): Sample addition: Add IL-9 samples of different dilution gradients, 100.0 μL per well, and incubate with shaking at 25°C for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate with shaking at 25°C for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0102] Step (4): Add Read buffer: Add 100.0 μL of tri-n-propylamine Read buffer to the co-reactant.

[0103] Step (5): Reading: Read the electrochemiluminescence value at a wavelength of 620 nm. Experimental results are as follows: Figure 7 As shown, 1:5 is selected as the optimal MRD.

[0104] Dilution linearity validation is crucial for verifying the accurate quantification of high-concentration samples using bioanalytical methods, especially immunoassays. It requires that the diluted assay concentration be strictly proportional to the dilution factor; otherwise, the accuracy of the results cannot be guaranteed. This validation is particularly important because it reveals the "hook effect" caused by antigen overload. This effect stems from the saturation of antibody binding sites or the formation of complexes that do not produce a detection signal, leading to an anomalous "plateau" or "negative bias" in the dose-response curve at high concentrations. Therefore, complete dilution linearity validation goes beyond conventional linearity assessment; it is a necessary quality control step to identify potential methodic defects and ensure correct response even in extremely high-concentration samples. The specific steps are as follows:

[0105] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution (1.0 μg / mL) to a 96-well electrochemiluminescence plate and incubate at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well, blot dry, and wash 3 times.

[0106] Step (2), Sample preparation: Prepare IL-9 to 200,000.0 pg / mL, and use sample diluent to serially dilute it 2-fold to 100,000.0 pg / mL and 50,000.0 pg / mL, and then serially dilute it to 20.6 pg / mL; Similarly, use sample diluent to prepare ruthenium-labeled detection antibody into 0.5 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0107] Step (3), sample addition: Add 100.0 μL of IL-9 sample of different concentrations to each well, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0108] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0109] Step (5): Read the values ​​at a wavelength of 620 nm. The experimental results are as follows... Figure 8 As shown, all prepared hook-shaped samples were analyzed in a single experimental batch. All samples with concentrations higher than ULOQ showed electrochemiluminescence values ​​higher than the ULOQ signal value, thus indicating no hook effect. Samples exceeding the upper limit of quantitation were serially diluted to the quantitation range; the quality control samples showed %CV ≤ 20.0% and |%RE| ≤ 20.0%, indicating good dilution linearity of the method.

[0110] In bioanalysis, parallelism validation is a crucial experiment for assessing the applicability of analytical methods to real-world samples. The specific steps are as follows:

[0111] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution (1.0 μg / mL) to a 96-well electrochemiluminescence plate and incubate at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well, blot dry, and wash 3 times.

[0112] Step (2), Sample preparation: Select three individual serum samples and prepare IL-9 to 2,000.0 pg / mL using the spiked method. Then, use blank human serum to perform a series of gradient dilutions on the sample (1:1, 1:2, 1:4, 1:8, 1:16), with MRD selected as 5. Similarly, use the sample diluent to prepare ruthenium-labeled detection antibody to prepare ruthenium-labeled detection reagent to 0.5 μg / mL.

[0113] Step (3), sample addition: Add 100.0 μL of IL-9 sample of different concentrations to each well, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0114] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0115] Step (5), reading: Read the electrochemiluminescence value at a wavelength of 620 nm. Experimental results are as follows... Figure 9 As shown, the precision is ≤30.0%, indicating that the method has good parallelism.

[0116] Selectivity is a key indicator of method specificity, that is, the ability to accurately determine the target analyte in a complex sample matrix without interference from other components. Validating selectivity aims to confirm the specificity of the detection signal, which is a prerequisite for ensuring accurate and reliable data. The specific steps are as follows:

[0117] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution (1.0 μg / mL) to a 96-well electrochemiluminescence plate and incubate at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well, blot dry, and wash 3 times.

[0118] Step (2), Sample preparation: Select ten individual serum samples and add IL-9 at ULOQ and LLOQ quality control levels respectively; similarly, use sample diluent to prepare ruthenium-labeled detection antibody into 0.5 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0119] Step (3), sample addition: Add 100.0 μL of IL-9 sample of different concentrations to each well, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0120] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0121] Step (5), reading: Read the electrochemiluminescence value at a wavelength of 620 nm. Experimental results are as follows... Figure 10As shown, by analyzing at least 10 blank biological matrix samples from different sources and adding target analytes at ULOQ and LLOQ quality control levels respectively, the results showed that at least 80% of the samples had %CV ≤ 20.0% and |%RE| ≤ 20.0%, indicating that the method has good selectivity.

[0122] Specificity refers to the ability of an analytical method to accurately and specifically determine a target analyte; its core lies in distinguishing the target analyte from other components that may be present in a biological sample. The specific steps are as follows:

[0123] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution (1.0 μg / mL) to a 96-well electrochemiluminescence plate and incubate at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well, blot dry, and wash 3 times.

[0124] Step (2), Sample preparation: Add 100,000.0 pg / mL, 50,000.0 pg / mL and 25,000.0 pg / mL of IL-6 to the blank matrix, and add IL-9 at the HQC and LQC quality control levels respectively; Similarly, use sample dilution buffer to prepare ruthenium-labeled detection antibody into 0.5 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0125] Step (3), sample addition: Add 100.0 μL of IL-9 sample of different concentrations to each well, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0126] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0127] Step (5), reading: Read the electrochemiluminescence value at a wavelength of 620 nm. Experimental results are as follows... Figure 11 As shown, IL-6 was added to a blank matrix at concentrations of 100,000.0 pg / mL, 50,000.0 pg / mL, and 25,000.0 pg / mL to investigate the accuracy of IL-9 at HQC and LQC quality control levels. At least 80% of the samples should have %CV ≤ 20.0% and |%RE| ≤ 20.0%. The results indicate that the method has good specificity, and all test samples were not affected by the addition of IL-6. The method can tolerate the interfering IL-6 concentration of at least 100,000.0 pg / mL.

[0128] Stability assessments can be performed to ensure that each step in sample preparation, processing, and analysis, as well as storage conditions, does not affect the concentration of the analyte. Stability validation should include 24-hour stability at room temperature, stability at 2–8 °C and -20 °C, and stability after five freeze-thaw cycles. Stability validation samples consist of three sets (each set contains one HQC sample and one LQC sample, all in duplicate). The specific steps are as follows:

[0129] Step (1): Add 100.0 μL of IL-9 capture antibody coating solution (1.0 μg / mL) to a 96-well electrochemiluminescence plate and incubate at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well, blot dry, and wash 3 times.

[0130] Step (2), sample preparation: Under the conditions of 24 h room temperature stability, 2~8 ℃ and -20 ℃ stability and 5 freeze-thaw cycles stability, IL-9 at HQC and LQC quality control levels were added respectively; Similarly, the ruthenium-labeled detection antibody was prepared into 0.5 μg / mL ruthenium-labeled detection reagent by using sample dilution buffer.

[0131] Step (3), sample addition: Add 100.0 μL of IL-9 sample of different concentrations to each well, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times. Then add 100.0 μL of 0.5 μg / mL ruthenium-labeled detection reagent, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST buffer, 300 μL / well, 3 times.

[0132] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0133] Step (5), reading: Read the electrochemiluminescence value at a wavelength of 620 nm. Experimental results are as follows... Figure 12 As shown, all stability samples were analyzed in one test batch, and the results all met the requirement of |%Bias|≤30.0%. The results indicate that the stability of the samples after 24 h at room temperature, at 2~8 ℃ and at -20 ℃, as well as after 5 freeze-thaw cycles, all meet the standards.

Claims

1. An electrochemiluminescence immunoassay method for detecting IL-9, characterized in that, Includes the following steps: The first step is to add IL-9 capture antibody coating solution to the electrochemiluminescence plate and incubate it; the IL-9 capture antibody coating solution contains HEPES, sodium chloride, glycine, and the remainder is deionized water. The second step is to add washing buffer to each well of the electrochemiluminescence plate for cleaning; Step 3: Prepare the sample. Human IL-9 was prepared into an IL-9 sample using sample dilution buffer; the marker-labeled detection antibody was prepared into a labeled detection reagent using sample dilution buffer. Step 4, adding samples: Add IL-9 sample to the electrochemiluminescence plate, incubate, and wash the plate with washing buffer after incubation; then add labeled detection reagent, incubate, and wash the plate with washing buffer after incubation. Step 5: Add the electrochemiluminescence substrate solution; Step 6: Read the value.

2. The IL-9 electrochemiluminescence immunoassay method according to claim 1, characterized in that, The IL-9 capture antibody coating solution contained HEPES at a concentration of 1.7–2.9 g / L, sodium chloride at a concentration of 6–11 g / L, and glycine at a concentration of 0.5–0.9 g / L.

3. The IL-9 electrochemiluminescence immunoassay method according to claim 2, characterized in that, The IL-9 capture antibody coating solution contained HEPES at a concentration of 2.38 g / L, sodium chloride at a concentration of 8.77 g / L, and glycine at a concentration of 0.75 g / L.

4. The IL-9 electrochemiluminescence immunoassay method according to claim 1, characterized in that, The labeled detection reagent is a ruthenium-labeled detection reagent, which is prepared by mixing ruthenium-labeled detection antibodies with a sample diluent. The electrochemiluminescence substrate solution is tri-n-propylamine, a co-reactant.

5. The IL-9 electrochemiluminescence immunoassay method according to claim 1, characterized in that, The concentration of the IL-9 capture antibody coating solution is 1.0 μg / mL, and the concentration of the labeled detection reagent is 0.5 μg / mL.

6. The IL-9 electrochemiluminescence immunoassay method according to claim 1, characterized in that, The sample diluent was prepared by adding fructose and glycine to casein blocking solution. The fructose concentration was 10–35 g / L and the glycine concentration was 0.75–2.5 g / L.

7. The IL-9 electrochemiluminescence immunoassay method according to claim 6, characterized in that, The sample dilution contained fructose at a mass concentration of 15 g / L and glycine at a mass concentration of 1.5 g / L.

8. A test kit, characterized in that, The apparatus includes an electrochemiluminescence plate, an IL-9 capture antibody coating solution, an IL-9 standard, a washing buffer, a labeled detection reagent, a sample diluent, and an electrochemiluminescence substrate solution; the IL-9 capture antibody coating solution contains HEPES, sodium chloride, and glycine, with the remainder being deionized water; the sample diluent contains fructose and glycine.

9. The detection kit according to claim 8, characterized in that, The labeling detection reagent is a ruthenium-labeled detection reagent.

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