IL-6 electrochemiluminescence immunoassay method and detection kit

Abstract

The invention relates to an IL-6 electrochemiluminescence immunodetection method and a detection kit, and solves the technical problems of narrow detection range, low sensitivity, tedious operation steps and radioactive contamination of the existing IL-6 detection method. An electrochemiluminescence immunoassay platform is applied, the surface of an electrochemiluminescence plate is directly modified with a captured antibody, then a double-antibody sandwich structure is formed among the captured antibody, IL-6 and a ruthenium-labeled detection antibody, and an IL-6 direct electrochemiluminescence immunoassay system is established.

Description

Electrochemiluminescence immunoassay method and reagent kit for IL-6 Technical Field

[0001] This invention relates to the field of interleukin-6 (IL-6) detection technology, and more specifically, to an electrochemiluminescence immunoassay method and detection kit for IL-6. Background Technology

[0002] Interleukin-6 (IL-6) is a type of interleukin, a multifunctional cytokine that plays an important role in human metabolism, differentiation of autoimmune cells, and disease treatment.

[0003] Currently, commonly used clinical methods for IL-6 detection include traditional ELISA and radioimmunoassay. Traditional ELISA is often cumbersome and time-consuming (several hours), requires manual washing, and has relatively limited sensitivity and a narrow detection range. Radioimmunoassay has disadvantages such as short shelf life and radioactive contamination. Therefore, there is an urgent need to develop a simple, rapid, wide-range, and highly sensitive IL-6 detection method. Summary of the Invention

[0004] This application aims to address the technical problems of existing IL-6 detection methods, such as narrow detection range, low sensitivity, cumbersome operation procedures, and radioactive contamination, by providing an IL-6 electrochemiluminescence immunoassay method and detection kit.

[0005] 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.

[0006] This invention establishes a direct electrochemiluminescence immunoassay for IL-6 by directly modifying the surface of an electrochemiluminescence plate with a capture antibody, followed by the formation of a double-antibody sandwich structure between the capture antibody, IL-6, and ruthenium-labeled detection antibody.

[0007] A first aspect of the present invention provides an electrochemiluminescence immunoassay method for detecting IL-6, comprising the following steps: First, adding IL-6 capture antibody coating solution to an electrochemiluminescence plate and incubating; Second, adding washing buffer to each well of the electrochemiluminescence plate for washing; Third, preparing samples: preparing IL-6 samples by diluting human IL-6 with sample diluent; preparing labeled detection reagents by diluting labeled detection antibodies with sample diluent; Fourth, adding samples: adding IL-6 samples to the electrochemiluminescence plate, incubating, and washing the plate with washing buffer after incubation; then adding the labeled detection reagent, incubating, and washing the plate with washing buffer after incubation; Fifth, adding electrochemiluminescence substrate solution; Sixth, reading the values.

[0008] Preferably, the labeled detection reagent is a ruthenium-labeled detection reagent, which is prepared by mixing the ruthenium-labeled detection antibody with a sample diluent; the electrochemiluminescence substrate solution is tri-n-propylamine as a co-reactant.

[0009] Preferably, the washing buffer is PBST buffer.

[0010] Preferably, the concentration of the IL-6 capture antibody coating solution is 1.0–8.0 μg / mL; and the concentration of the ruthenium-labeled detection reagent is 1.0–6.0 μg / mL.

[0011] Preferably, the concentration of the IL-6 capture antibody coating solution is 5.0 μg / mL, and the concentration of the ruthenium-labeled detection reagent is 1.0 μg / mL.

[0012] A second aspect of the present invention provides a detection kit comprising a monoclonal antibody, a washing buffer, a labeled detection reagent, an electrochemiluminescence plate, and an electrochemiluminescence substrate solution.

[0013] Preferably, the monoclonal antibody pair is a mouse anti-human IL-6 monoclonal antibody (Cap) and a mouse anti-human IL-6 monoclonal antibody (Det); the light chain amino acid sequence of the mouse anti-human IL-6 monoclonal antibody (Cap) is shown in SEQ ID NO.1; the heavy chain amino acid sequence of the mouse anti-human IL-6 monoclonal antibody (Cap) is shown in SEQ ID NO.2; the light chain amino acid sequence of the mouse anti-human IL-6 monoclonal antibody (Det) is shown in SEQ ID NO.3; and the heavy chain amino acid sequence of the mouse anti-human IL-6 monoclonal antibody (Det) is shown in SEQ ID NO.4.

[0014] Preferably, the labeling detection reagent is a ruthenium-labeled detection reagent.

[0015] The beneficial effect of this disclosure is that a double-antibody sandwich structure is constructed on an electrochemiluminescence microplate, and the electrochemiluminescence signal of ruthenium is directly detected by introducing a ruthenium-labeled detection antibody into the double-antibody sandwich structure. This method has advantages such as simple operation, wide detection range, high specificity, high sensitivity, good selectivity, and good stability.

[0016] 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

[0017] Figure 1 shows the fitted standard curve; Figure 2 compares the detection results under different concentrations of capture antibody and detection antibody; Figure 3 shows the electrochemiluminescence signal values ​​of different concentrations of IL-6; Figure 4 shows the precision and accuracy results of 6 tests; Figure 5 shows the results of the minimum dilution experiment; Figure 6 shows the dilution linearity and hook effect results; Figure 7 shows the parallelism verification results; Figure 8 shows the selectivity experiment results; Figure 9 shows the specificity verification data; Figure 10 shows the stability assessment results. Detailed Implementation

[0018] The application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] 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.

[0020] The preferred labeling reagent is the ruthenium labeling reagent from Wenzhou Kangrui Biotechnology Co., Ltd.

[0021] 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%.

[0022] Commercially available monoclonal antibodies can be selected, and mouse anti-human IL-6 monoclonal antibody (Cap) and mouse anti-human IL-6 monoclonal antibody (Det) from Wenzhou Kangrui Biotechnology Co., Ltd. are preferred monoclonal antibody pairs.

[0023] The light chain amino acid sequence of the mouse anti-human IL-6 monoclonal antibody (Cap) is shown in SEQ ID NO.1: TQGSNLRPSCAETPTVEYSVEGSNLVGLYAEYKQASKNTSQPAFGAASQSSTVQPHGTAVTPLIYTKVLSGRPPEDGSFNSNTVASACGPYDKILQGYATCSVALNTSYFTVLTAGNNAVWTEQYGQVKAEHTSGIVTRASVQTCYFPSLGTVAIETKKPVTLTLLPCQS VSSA. The heavy chain amino acid sequence of the mouse anti-human IL-6 monoclonal antibody (Cap) is shown in SEQ ID NO.1: TQGSNLRPSCAETPTVEYSVEGSNLVGLYAEYKQASKNTSQPAFGAASQSSTVQPHGTAVTPLIYTKVLSGRPPEDGSFNSNTVASACGPYDKILQGYATCSVALNTSYFTVLTAGNNAVWTEQYGQVKAEHTSGIVTRASVQTCYFPSLGTVAIETKKPVTLTLLPCQS VSSA. The light chain amino acid sequence of mouse anti-human IL-6 monoclonal antibody (Det) shown in NO.2 is as follows: LQTGSVSNKITDYSPVLSLVYWKSGPLKTSGSSSYPRDTSVWQTGVHQDTQYTKVSLTVAVYPSDLVYNLSTGPGSPTSTSGTFATPDKGPTLQVLTSKQTSGYYVGIKGDPRHWSSTVKLDSKGSQEVAAPFSNVTVQVNCPYAHGVESLQSSQVLPGQYSLVFWKNSHLVFSYDTRTQVPSKKGSTSLYQAVSDIYTTGHPVLAIQTPEGVGQLNVWSSRTVGDTLHLVAYKQQGSPS is as shown in SEQ ID. The heavy chain amino acid sequence of the mouse anti-human IL-6 monoclonal antibody (Det) shown in NO.3 is as shown in SEQ ID NO.As shown in Figure 4: The IL-6 electrochemiluminescence immunoassay method includes the following steps: First, add 100.0 μL of a certain concentration of IL-6 capture antibody coating solution to a 96-well electrochemiluminescence plate and freeze at 4°C. o C. Leave overnight.

[0024] The concentration of the IL-6 capture antibody coating solution can be 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL or 8.0 μg / mL.

[0025] 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.

[0026] The third step is sample preparation: Human IL-6 is prepared into an IL-6 sample with a concentration of 10 pg / mL using sample diluent; similarly, ruthenium-labeled detection antibodies are prepared into ruthenium-labeled detection reagents using sample diluent at a specific ratio. The concentration of the ruthenium-labeled detection reagents can be 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, or 6.0 μg / mL.

[0027] Preparation of Ruthenium-labeled detection antibody: The IL-6 detection antibody was labeled using the CRIVVIN Ruthenium labeling kit. The ruthenium labeling of the IL-6 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 100.0 mg / mL were prepared. Simultaneously, commercially available ruthenium (Ru) labeled lyophilized powder was reconstituted to 20.0 mg / mL. Then, the solutions were mixed at a volume ratio of Ru solution:NHS solution:EDC solution = 80:15:15 to prepare an activation premix. The mixture was 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 antibody. After activation, the above mixture was mixed with the IL-6 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.

[0028] Step 4, sample addition: Add 100.0 μL of IL-6 sample at a concentration of 10 pg / mL to the electrochemiluminescence plate, 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.

[0029] Step 5, add Read buffer: Add 100.0 μL of tri-n-propylamine Read buffer to each well.

[0030] Step 6, Reading: Read the electrochemiluminescence value at a wavelength of 620 nm.

[0031] The data results are shown in Figure 2. The S / N values ​​are highest at 5.0 μg / mL capture antibody and 1.0 μg / mL detection antibody. Therefore, the optimal concentrations of capture antibody and detection antibody are 5.0 μg / mL and 1.0 μg / mL, respectively.

[0032] 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: Step (1), add 100.0 μL of IL-6 capture antibody coating solution with a concentration of 5.0 μg / mL to the 96-well electrochemiluminescence plate and freeze at 4°C. oIncubate overnight at C. Add 300 μL of PBST buffer to each well of the electrochemiluminescence plate, blot dry, and wash 3 times.

[0033] Step (2), Sample preparation: Use sample diluent to prepare human IL-6 into standard curve points of different concentrations; similarly, use sample diluent to prepare ruthenium-labeled detection antibody into 1.0 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0034] Step (3), sample addition: Add 100.0 μL of IL-6 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 1.0 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.

[0035] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0036] Step (5), reading: read the electrochemiluminescence value at a wavelength of 620 nm.

[0037] A standard curve for IL-6 was established and fitted using a four-parameter 4-PL model. The goodness of fit R² ≥ 0.990, and the standard curve fitting plot is shown in Figure 1. Experimental results are shown in Figure 3. The coefficient of variation (%CV) and relative error (%RE) were both within 20%. The standard curve exhibited good linearity in the range of 2.0–1,000.0 pg / mL, with a lower limit of quantification (LLOQ) of 2.0 pg / mL.

[0038] 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 1000.0 pg / mL, the high quality control (HQC) is 750.0 pg / mL, the moderate quality control (MQC) is 60.0 pg / mL, the low quality control (LQC) is 6.0 pg / mL, and the LLOQ is 2.0 pg / mL. Each sample is replicated in two wells. The specific steps are as follows: Step (1): Add 100.0 μL of IL-6 capture antibody coating solution (5.0 μg / mL) to the 96-well electrochemiluminescence plate and freeze at 4°C. o Incubate overnight at C. Add 300 μL of PBST buffer to each well, blot dry, and wash 3 times.

[0039] Step (2): Sample preparation: Use sample diluent to prepare human IL-6 into standard curve points and quality control points of different concentrations; similarly, use sample diluent to prepare ruthenium-labeled detection antibody into 1.0 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0040] Step (3): Sample addition: Add 100.0 μL of IL-6 samples 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 1.0 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.

[0041] Step (4): Add Read buffer: Add 100.0 μL of tri-n-propylamine Read buffer to the co-reactant.

[0042] Step (5): Reading value: Read the electrochemiluminescence value at a wavelength of 620 nm.

[0043] The experimental results are shown in Figure 4. The coefficient of variation (%CV) of the standard curve and the quality control point is ≤20.0%, and the relative error (|%RE|) is ≤0.0%. Among them, ULOQ and LLOQ are %CV≤25.0% and |%RE|≤25.0%, respectively, indicating that the method has good precision and accuracy.

[0044] In the development and validation of bioanalytical methods, determining the minimum required dilution (MRD) is a crucial step to ensure data accuracy and reliability. The specific steps are as follows: Step (1): Add 100.0 μL of 5.0 μg / mL IL-6 capture antibody coating solution 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.

[0045] Step (2): Sample preparation: Select mixed blood and #9 blood to spike human IL-6 to 1000.0 pg / mL, and use sample diluent to perform a series of gradient dilutions (1:1, 1:5, 1:10, 1:50, 1:100); similarly, use sample diluent to prepare ruthenium-labeled detection antibody to 1.0 μg / mL ruthenium-labeled detection reagent.

[0046] Step (3): Sample addition: Add IL-6 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 1.0 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.

[0047] Step (4): Add Read buffer: Add 100.0 μL of tri-n-propylamine Read buffer to the co-reactant.

[0048] Step (5): Reading: Read the electrochemiluminescence value at a wavelength of 620 nm. The experimental results are shown in Figure 5. 1:10 was selected as the optimal MRD.

[0049] Dilution linearity is crucial for verifying the accuracy of bioanalytical methods (especially immunoassays) in quantifying high-concentration samples. It requires that the measured concentration after dilution be strictly proportional to the dilution factor; otherwise, the accuracy of the results cannot be guaranteed. This verification 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 in high-concentration regions. Therefore, complete dilution linearity verification goes beyond conventional linearity testing; it is a necessary quality control step to identify potential method defects and ensure correct response even in extremely high-concentration samples. The specific steps are as follows: Step (1), add 100.0 μL of 5.0 μg / mL IL-6 capture antibody coating solution 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, blot dry, and wash 3 times.

[0050] Step (2), Sample preparation: Prepare IL-6 to 10,000.0 pg / mL, and use sample diluent to serially dilute it 2-fold to 5,000.0 pg / mL and 2,500.0 pg / mL, and then serially dilute it to 2.0 pg / mL; Similarly, use sample diluent to prepare ruthenium-labeled detection antibody to prepare ruthenium-labeled detection reagent at a certain ratio.

[0051] Step (3), sample addition: Add 100.0 μL of IL-6 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 1.0 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.

[0052] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0053] Step (5): Read the electrochemiluminescence value at a wavelength of 620 nm. The experimental results are shown in Figure 6. All prepared hook-shaped samples were analyzed in one experimental batch. The electrochemiluminescence values ​​of all samples with concentrations higher than ULOQ were higher than the ULOQ signal value, thus indicating that no hook effect was generated. The samples exceeding the upper limit of quantitation were serially diluted to the quantitation range. The %CV of the quality control samples was ≤20.0%, and |%RE| was ≤20.0%, indicating that the method has good dilution linearity.

[0054] In bioanalysis, parallelism validation is a crucial experiment for assessing the applicability of analytical methods to actual samples. The specific steps are as follows: Step (1), add 100.0 μL of 5.0 μg / mL IL-6 capture antibody coating solution 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.

[0055] Step (2), Sample preparation: Select three individual serum samples and prepare IL-6 to 1,000.0 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 set to 10. Similarly, use sample diluent to prepare ruthenium-labeled detection antibody to 1.0 μg / mL ruthenium-labeled detection reagent.

[0056] Step (3), sample addition: Add 100.0 μL of IL-6 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 1.0 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.

[0057] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0058] Step (5), reading: read the electrochemiluminescence value at a wavelength of 620 nm. The experimental results are shown in Figure 7. The precision is ≤30.0%, indicating that the method has good parallelism.

[0059] 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: Step (1), add 100.0 μL of 5.0 μg / mL IL-6 capture antibody coating solution 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, blot dry, and wash 3 times.

[0060] Step (2), Sample preparation: Select ten individual serum samples and add IL-6 at HQC and LQC quality control levels respectively; similarly, use sample diluent to prepare ruthenium-labeled detection antibody into 1.0 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0061] Step (3), sample addition: Add 100.0 μL of IL-6 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 1.0 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.

[0062] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0063] Step (5), reading: read the electrochemiluminescence value at a wavelength of 620 nm. The experimental results are shown in Figure 8. By analyzing at least 10 blank biological matrix samples from different sources, and adding target analytes at HQC and LQC 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.

[0064] 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: Step (1), add 100.0 μL of 5.0 μg / mL IL-6 capture antibody coating solution 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, blot dry, and wash 3 times.

[0065] Step (2), Sample preparation: Add 10,000.0 pg / mL, 5,000.0 pg / mL and 2,500.0 pg / mL of IL-9 to the blank matrix, and add IL-6 at the HQC and LQC quality control levels respectively; similarly, use sample dilution buffer to prepare ruthenium-labeled detection antibody into 1.0 μg / mL ruthenium-labeled detection reagent at a certain ratio.

[0066] Step (3), sample addition: Add 100.0 μL of IL-6 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 1.0 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.

[0067] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0068] Step (5), reading: read the electrochemiluminescence value at a wavelength of 620 nm. The experimental results are shown in Figure 9. IL-9 was added to the blank matrix at concentrations of 10,000.0 pg / mL, 5,000.0 pg / mL, and 2,500.0 pg / mL to examine the accuracy of IL-6 at HQC and LQC quality control levels. At least 80% of the samples should have %CV ≤ 20.0% and |%RE| ≤ 20.0%. The results show that the method has good specificity, and all test samples were not affected by the addition of IL-9. The method can tolerate the concentration of the interfering IL-9 at least 10,000.0 pg / mL.

[0069] Stability assessment can be performed to ensure that each step in the sample preparation, processing, and analysis process, as well as storage conditions, does not affect the concentration of the analyte. Stability verification should include 24 h room temperature stability, 3-day and 7-day stability at 2-8 °C, and 5 freeze-thaw cycles. Stability verification samples include 3 sets of samples (each set contains 1 HQC sample and 1 LQC sample, all in duplicate). The specific steps are as follows: Step (1), add 100.0 μL of 5.0 μg / mL IL-6 capture antibody coating solution to the 96-well electrochemiluminescence plate and freeze at 4 °C. o Incubate overnight at C. Add 300 μL of PBST buffer (1X) to each well, blot dry, and wash 3 times.

[0070] Step (2), sample preparation: Under the conditions of 24 h room temperature stability, 3 days and 7 days stability at 2-8 ℃ and 5 freeze-thaw cycles, IL-6 at HQC and LQC quality control levels were added respectively; similarly, the ruthenium-labeled detection antibody was prepared into 1.0 μg / mL ruthenium-labeled detection reagent by using sample dilution buffer.

[0071] Step (3), sample addition: Add 100.0 μL of IL-6 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 1.0 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.

[0072] Step (4), add Read buffer: Add co-reactant tri-n-propylamine Read buffer, 100.0 μL / well.

[0073] Step (5), reading: read the electrochemiluminescence value at a wavelength of 620 nm. The experimental results are shown in Figure 10. All stability samples were analyzed in one experimental batch, and the results all met the requirement of |%Bias|≤30.0%. The results show that the samples met the standard after being placed at room temperature for 24 h, at 2~8 ℃ for 3 days and 7 days, and after 5 freeze-thaw cycles.

Claims

1. An electrochemiluminescence immunoassay method for detecting IL-6, characterized in that, Includes the following steps: First, add IL-6 capture antibody coating solution to the electrochemiluminescence plate and incubate; second, add washing buffer to each well of the electrochemiluminescence plate for washing. The third step is sample preparation: human IL-6 is prepared into an IL-6 sample using sample dilution buffer; the marker-labeled detection antibody is prepared into a labeled detection reagent using sample dilution buffer; the fourth step is sample addition: IL-6 sample is added to the electrochemiluminescence plate, incubated, and the plate is washed with washing buffer after incubation; the labeled detection reagent is then added, incubated, and the plate is washed with washing buffer after incubation. Step 5: Add the electrochemiluminescence substrate solution; Step 6: Read the value.

2. The IL-6 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 a ruthenium-labeled detection antibody with a sample diluent; the electrochemiluminescence substrate solution is tri-n-propylamine as a co-reactant.

3. The electrochemiluminescence immunoassay method for IL-6 according to claim 2, characterized in that, The washing buffer is PBST buffer.

4. The electrochemiluminescence immunoassay method for IL-6 according to claim 2 or 3, characterized in that, The concentration of the IL-6 capture antibody coating solution is 1.0–8.0 μg / mL; the concentration of the ruthenium-labeled detection reagent is 1.0–6.0 μg / mL.

5. The electrochemiluminescence immunoassay method for IL-6 according to claim 4, characterized in that, The concentration of the IL-6 capture antibody coating solution is 5.0 μg / mL, and the concentration of the ruthenium-labeled detection reagent is 1.0 μg / mL.

6. The electrochemiluminescence immunoassay method for IL-6 according to claim 4, characterized in that, The monoclonal antibody pair is mouse anti-human IL-6 monoclonal antibody (Cap) and mouse anti-human IL-6 monoclonal antibody (Det); the light chain amino acid sequence of mouse anti-human IL-6 monoclonal antibody (Cap) is shown in SEQ ID NO.1; the heavy chain amino acid sequence of mouse anti-human IL-6 monoclonal antibody (Cap) is shown in SEQ ID NO.2; the light chain amino acid sequence of mouse anti-human IL-6 monoclonal antibody (Det) is shown in SEQ ID NO.3; and the heavy chain amino acid sequence of mouse anti-human IL-6 monoclonal antibody (Det) is shown in SEQ ID NO.

4.

7. A test kit, characterized in that, It includes monoclonal antibodies, washing buffer, labeled detection reagents, electrochemiluminescence plates, and electrochemiluminescence substrate solutions.

8. The detection kit according to claim 7, characterized in that, The labeling detection reagent is a ruthenium-labeled detection reagent.

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