Ferritin determination kit based on homogeneous chemiluminescence method and determination method thereof
By employing homogeneous chemiluminescence, using antibodies conjugated with luminescent donor and acceptor molecules, and optimized buffer solutions, the complexity and sensitivity issues of existing ferritin assay methods have been resolved, achieving efficient and accurate ferritin detection suitable for various sample types.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-29
- Publication Date
- 2026-04-03
AI Technical Summary
Existing methods for ferritin assays are complex to operate, have low sensitivity and poor accuracy, making it difficult to meet the detection needs of samples with different concentrations, and they are also insufficient in handling interfering samples such as those with hemolysis or high lipid content.
A homogeneous chemiluminescence method was adopted, using ferritin-specific antibodies coupled with luminescent donor molecules and ferritin-specific antibodies coupled with luminescent acceptor molecules. Signal detection was performed based on the principle of fluorescence resonance energy transfer. Combined with optimized reaction buffer and pretreatment solution, a rapid and simple ferritin assay was achieved.
It achieves highly sensitive ferritin detection with a detection limit as low as 0.08 ng/mL at low concentrations, a wide linear range, and is suitable for detecting samples of different concentrations. It is easy to operate, adaptable to complex samples, and has high detection efficiency and accuracy, making it suitable for various clinical scenarios.
Abstract
Description
Technical Field
[0001] This invention relates to the field of photo-induced chemiluminescence technology, and in particular to a ferritin assay kit and method based on homogeneous chemiluminescence. Background Technology
[0002] Ferritin is an important iron storage protein widely distributed in various tissues and cells of the human body. Its serum concentration level is closely related to the diagnosis and monitoring of many diseases, especially in clinical applications such as tumor marker detection, anemia diagnosis, and assessment of iron metabolism abnormalities. Therefore, accurately measuring the ferritin content in biological samples is of great clinical significance for the diagnosis and treatment of cancer.
[0003] Currently, commonly used methods for ferritin assays include electrochemiluminescence immunoassay and enzyme-linked immunosorbent assay (ELISA). However, existing ferritin detection methods still have some shortcomings: First, traditional heterogeneous immunoassay methods require solid-phase carriers and multiple washing steps, making the operation complex and time-consuming; second, some detection methods have limited sensitivity and linear range, making it difficult to meet the detection requirements of samples with different concentrations; third, they lack the ability to handle interfering samples such as hemolyzed or hyperlipidemic samples, affecting the accuracy of the test results.
[0004] Therefore, developing a simple, accurate, and rapid method for ferritin determination is of great practical significance. Summary of the Invention
[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a ferritin assay kit based on homogeneous chemiluminescence to solve the problems of complex operation, low sensitivity, and poor accuracy of existing ferritin assay methods. At the same time, this invention will also provide a method for ferritin assay based on this kit.
[0006] To achieve the above and other related objectives, the present invention provides the following technical solutions: A first aspect of the present invention provides a ferritin assay kit based on homogeneous chemiluminescence, comprising reagent 1 and reagent 2, wherein: The reagent 1 includes ferritin-specific antibody 1 coupled with a luminescent donor molecule and a reaction buffer; The reagent 2 includes ferritin-specific antibody 2 coupled with a luminescent receptor molecule and a reaction buffer.
[0007] In recent years, homogeneous chemiluminescence immunoassay has emerged as a novel detection technique and has received widespread attention and application in the field of biomarker detection. Based on the principle of fluorescence resonance energy transfer (FRET), this technique achieves signal detection through energy transfer between luminescent donor and acceptor molecules, offering advantages such as no need for separation and washing, ease of operation, and rapid detection speed. Specifically, ferritin-specific antibodies 1 and ferritin-specific antibodies 2 bind to ferritin in the sample to form a sandwich immune complex, which triggers a chemiluminescent reaction, generating a detectable light signal.
[0008] Furthermore, the luminescent donor molecule is selected from europium (Eu). 3+ ) chelates, Tb 3+ Chelates, coumarin derivatives, or naphthalimide fluorescent molecules, preferably Eu-TTA, Eu-β-NTA, 7-hydroxycoumarin, coumarin-3-carboxylic acid, etc.
[0009] Furthermore, the luminescent acceptor molecule is selected from at least one of anthocyanin dyes, fluorescein derivatives, quantum dots, porphyrin compounds, and gold nanoparticles, preferably at least one of Cy3.5, Cy5, rhodamine B, tetraphenylporphyrin sulfonate (TPPS), quantum dot CdSe, and quantum dot ZnS.
[0010] Furthermore, the luminescent donor molecule and the luminescent acceptor molecule are chemically modified.
[0011] Furthermore, the luminescent donor molecule is modified by introducing a carboxyl group and activating it into an NHS ester, or by introducing an amino group or a maleimide group; the luminescent acceptor molecule is modified by activating a carboxyl group, introducing a maleimide group, or a PEG linker.
[0012] Furthermore, the reaction buffer comprises 40–60 mmol / L HEPES buffer (pH 7.2–7.5), 0.1–0.2% BSA, and 0.02–0.10% Triton X-100. BSA is used to stabilize the reaction and reduce background noise; Triton X-100 reduces nonspecific adsorption.
[0013] Furthermore, the reaction buffer also includes at least one of the following: 10-100 mmol / L NaCl, 1-5 mmol / L MgCl2, and 0.01-0.05% sodium azide. NaCl increases ionic strength to promote antibody-ferritin binding; MgCl2 promotes the immune response of antibody-ferritin binding; and sodium azide inhibits bacterial and fungal growth.
[0014] Furthermore, the reaction buffer also includes at least one of the following: 0.5-3.0% PEG 6000, 0.1-1.0% glycine, or 0.01-0.05% PMSF. PEG 6000 and glycine are used to eliminate the influence of interfering substances in complex samples such as those with hemolysis and high lipid content; PMSF (phenylmethyl sulfonyl fluoride) inhibits the activity of proteases in the sample, preventing antibody degradation.
[0015] A second aspect of the present invention provides a method for determining ferritin based on the above-described kit, comprising the following steps: (1) Mix the sample to be tested with reagents 1 and 2 in the kit and incubate to obtain the test mixture; (2) Excite the above-mentioned mixture to be tested with excitation light to induce a chemiluminescence reaction, and measure the luminescence intensity at different detection wavelengths, and calculate the ratio of the two as the signal value; (3) Establish a standard curve of “sample concentration-signal value” based on the known concentration of the sample, and calculate the ferritin content in the sample to be tested by referring to the above standard curve.
[0016] Furthermore, samples with severe hemolysis (hemoglobin ≥10g / L) or high lipid content (triglycerides ≥10mmol / L) require pretreatment with a pretreatment solution, which is a 50mmol / L HEPES buffer containing 1% Triton X-100 and 0.05% EDTA.
[0017] Furthermore, the volume ratio of sample to pretreatment solution is 3 to 5:1, preferably 4:1.
[0018] Furthermore, in step (1), the volume ratio of the sample to be tested, reagent 1, and reagent 2 is 0.1~0.3:1:1.
[0019] Furthermore, in step (1), the incubation temperature is 37°C and the incubation time is 10~30 min.
[0020] Furthermore, in step (2), the excitation wavelength is 320 nm; the detection wavelengths are 665 nm and 620 nm.
[0021] Furthermore, in step (2), the emission wavelength is determined using a fully automated chemiluminescence analyzer.
[0022] As described above, the ferritin assay kit and method based on homogeneous chemiluminescence of the present invention have the following beneficial effects: 1. High sensitivity: Compared with existing ferritin assays such as electrochemiluminescence and enzyme-linked immunosorbent assay, this invention uses homogeneous chemiluminescence technology, combined with optimized luminescent donor-acceptor combination and reaction conditions, to accurately detect low concentrations of ferritin, with a detection limit as low as 0.08 ng / mL, meeting the needs of early tumor screening.
[0023] 2. Wide linear range: Covering the concentration range of 0.1ng / mL to 1000ng / mL, it can be adapted to the detection of samples with different concentration levels, such as healthy people and cancer patients, without the need for multiple dilutions.
[0024] 3. Simple operation: No complicated pretreatment and cleaning / separation steps are required. The sample and reagent are mixed and incubated before detection. Results can be obtained in as little as 25 minutes. The detection efficiency is high and it is suitable for batch samples and emergency scenarios.
[0025] 4. High accuracy: Through the design of dual antibody specific recognition, dual wavelength signal ratio detection, and the addition of interference inhibitors, non-specific reactions and environmental interference are reduced, with a relative deviation of ≤±8% and excellent inter-batch and intra-batch precision.
[0026] 5. Wide range of applications: By adjusting the reaction buffer, it can be adapted to routine samples such as serum and plasma, as well as complex samples such as hemolyzed and high-lipid samples, to meet the testing needs of different clinical scenarios. Detailed Implementation
[0027] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.
[0028] Example 1 This embodiment provides a ferritin assay kit based on homogeneous chemiluminescence, suitable for routine clinical testing, including: Reaction buffer: 50 mmol / L HEPES buffer, pH=7.4; 0.1% BSA; 0.05% Triton X-100; 100 mmol / L NaCl; 1 mmol / L MgCl2; 0.03% sodium azide.
[0029] Reagent 1: Select Eu 3+ -β-NTA chelate was used as a luminescent donor. A carboxyl group was introduced through succinic anhydride modification. After activation by NHS and DCC, it was incubated with ferritin-specific antibody 1 at a molar ratio of 8:1 in 50 mmol / L HEPES buffer at pH 7.4 at 4°C for 2 h. After coupling, it was diluted with the above reaction buffer to a final concentration of antibody 1 of 1.0 mg / mL.
[0030] Reagent 2: Cy3.5 (maleimide modified) and Cy5 (maleimide modified) were selected as luminescent acceptors. Ferritin-specific antibody 2 was reduced with DTT to obtain thiol groups, and then incubated with the two acceptors at a molar ratio of 1:5 in 50 mmol / L HEPES buffer at pH 7.2 at room temperature for 1 h. The two conjugates were mixed at a volume ratio of 1:1 and diluted with the above reaction buffer to a final concentration of antibody 2 of 1.0 mg / mL.
[0031] This embodiment also provides a method for determining ferritin based on the above kit, including the following steps: (1) Mix 20 μL of the sample to be tested with 80 μL of reagent 1 and 80 μL of reagent 2, and incubate at 37°C for 15 min to obtain the mixture to be tested; (2) Excite the above-mentioned mixture to undergo a chemiluminescence reaction using 320nm excitation light, and measure the luminescence intensity at wavelengths of 620nm and 665nm, and calculate the ratio R=I. 665 / I 620 ; (3) The concentration gradient of ferritin standard is 0.5 ng / mL, 5 ng / mL, 50 ng / mL, 200 ng / mL, 500 ng / mL and 1000 ng / mL. A "sample concentration-R" standard curve is established based on the standard concentration. The ferritin content in the sample to be tested is calculated by referring to the above standard curve.
[0032] Detection results: Limit of detection 0.3 ng / mL; Linear range 0.5 ng / mL~1000 ng / mL, R 2 ≥0.995; relative deviation ≤±5%; intra-batch CV ≤3.5%, inter-batch CV ≤5.0%.
[0033] Example 2 This embodiment provides a ferritin assay kit based on homogeneous chemiluminescence, suitable for high-sensitivity scenarios, including: Reaction buffer: 50 mmol / L HEPES buffer, pH=7.4; 0.2% BSA; 0.05% Triton X-100; 100 mmol / L NaCl; 1 mmol / L MgCl2.
[0034] Reagent 1: Select Tb 3+ -TTA chelate was used as a luminescent donor. After introducing the aminocaproic acid linker, it was activated into NHS ester. It was then incubated with ferritin-specific antibody at a molar ratio of 10:1 in 50 mmol / L HEPES buffer at pH 7.5 for 3 h at 4 °C. After purification by Sephadex G-25 column, the antibody was diluted with the above reaction buffer to a final concentration of 1.5 mg / mL.
[0035] Reagent 2: Sulfonated tetraphenylporphyrin (TPPS, NHS ester modified) and rhodamine B (activated after PEG linker modification) were selected as luminescent acceptors and conjugated with ferritin-specific antibody 2 at a molar ratio of 6:1. The two conjugates were mixed at a volume ratio of 1:1 and diluted with the above reaction buffer to a final antibody concentration of 1.5 mg / mL.
[0036] This embodiment also provides a method for determining ferritin based on the above kit, including the following steps: (1) Mix 20 μL of the sample to be tested with 100 μL of reagent 1 and 100 μL of reagent 2, and incubate at 37 °C for 20 min to obtain the mixture to be tested; (2) Excite the above-mentioned mixture to undergo a chemiluminescence reaction using 320nm excitation light, and measure the luminescence intensity at wavelengths of 620nm and 665nm, and calculate the ratio R=I. 665 / I 620 ; (3) The concentration gradient of ferritin standard is 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL and 800 ng / mL. A "sample concentration-R" standard curve is established based on the standard concentration. The ferritin content in the sample to be tested is calculated by referring to the above standard curve.
[0037] Detection results: Limit of detection 0.08 ng / mL; Linear range 0.1 ng / mL~800 ng / mL, R 2 ≥0.996; detection deviation ≤±8% when hemoglobin ≤5g / L and bilirubin ≤200μmol / L; performance degradation ≤10% after 30 days of storage at 2~8℃.
[0038] Example 3 This embodiment provides a ferritin assay kit based on homogeneous chemiluminescence, suitable for rapid emergency scenarios, including: Reaction buffer: 50 mmol / L HEPES buffer, pH=7.4; 0.15% BSA; 0.05% Triton X-100.
[0039] Reagent 1: 7-hydroxycoumarin was selected as the luminescent donor. After being modified with chloroacetic acid to introduce a carboxyl group and activated, it was coupled with ferritin-specific antibody 1 at a molar ratio of 6:1 and incubated at room temperature for 1 h. The antibody was then diluted with the above reaction buffer to a final concentration of 0.8 mg / mL and stored at 2-8 °C.
[0040] Reagent 2: Cy5.5 (NHS ester modified) and rhodamine 6G modified as luminescent acceptors were selected and coupled with ferritin-specific antibody 2 using a rapid conjugation process (30 min). After mixing, the mixture was diluted with the above reaction buffer to a final antibody concentration of 0.8 mg / mL and stored at 2~8℃.
[0041] This embodiment also provides a method for determining ferritin based on the above kit, including the following steps: (1) Mix 20 μL of the sample to be tested with 80 μL of reagent 1 and 80 μL of reagent 2, and incubate at 37 °C for 10 min to obtain the mixture to be tested; (2) Excite the above-mentioned mixture to undergo a chemiluminescence reaction using 320nm excitation light, and measure the luminescence intensity at wavelengths of 620nm and 665nm, and calculate the ratio R=I. 665 / I 620 ; (3) The concentration gradient of ferritin standard is 1 ng / mL, 10 ng / mL, 50 ng / mL, 300 ng / mL and 600 ng / mL. A "sample concentration-R" standard curve is established based on the standard concentration. The ferritin content in the sample to be tested is calculated by referring to the above standard curve.
[0042] Test results: Total detection time ≤ 25 min; Limit of detection 0.5 ng / mL; Linear range 1 ng / mL ~ 600 ng / mL, R 2 ≥0.990; compatible with fully automated chemiluminescence analyzers.
[0043] Example 4 This embodiment provides a ferritin assay kit based on homogeneous chemiluminescence, suitable for complex samples, including: Reaction buffer: 50 mmol / L HEPES buffer, pH=7.4; 0.2% BSA; 0.1% Triton X-100; 100 mmol / L NaCl; 1 mmol / L MgCl2; 0.03% sodium azide; 2% PEG 6000; 0.5% glycine; 0.01% PMSF.
[0044] Reagent 1: Select Eu 3+ -β-NTA chelate was used as a luminescent donor. A carboxyl group was introduced through succinic anhydride modification. After activation by NHS and DCC, it was incubated with ferritin-specific antibody 1 at a molar ratio of 8:1 in 50 mmol / L HEPES buffer at pH 7.4 at 4°C for 2 h. After coupling, it was diluted with the above reaction buffer to a final concentration of antibody 1 of 1.0 mg / mL.
[0045] Reagent 2: Cy3.5 (maleimide modified) and Cy5 (maleimide modified) were selected as luminescent acceptors. Ferritin-specific antibody 2 was reduced with DTT to obtain thiol groups, and then incubated with the two acceptors at a molar ratio of 1:5 in 50 mmol / L HEPES buffer at pH 7.2 at room temperature for 1 h. The two conjugates were mixed at a volume ratio of 1:1 and diluted with the above reaction buffer to a final concentration of antibody 2 of 1.0 mg / mL.
[0046] Pretreatment solution: 50 mmol / L HEPES buffer containing 1% Triton X-100 and 0.05% EDTA. This embodiment also provides a method for determining ferritin based on the above kit, including the following steps: (1) Mix the samples with severe hemolysis (hemoglobin ≥10g / L) or high lipid (triglycerides ≥10mmol / L) with the pretreatment solution at a volume ratio of 4:1 and incubate at room temperature for 5min to obtain the test sample; (2) Mix 20 μL of the sample to be tested with 80 μL of reagent 1 and 80 μL of reagent 2, and incubate at 37°C for 10 min to obtain the mixture to be tested; (3) Excite the above-mentioned mixture to undergo a chemiluminescence reaction using 320nm excitation light, and measure the luminescence intensity at wavelengths of 620nm and 665nm, and calculate the ratio R=I. 665 / I 620 ; (4) The concentration gradient of ferritin standard is 0.5 ng / mL, 5 ng / mL, 50 ng / mL, 200 ng / mL, 500 ng / mL and 1000 ng / mL. A "sample concentration-R" standard curve is established based on the standard concentration. The ferritin content in the sample to be tested is calculated by referring to the above standard curve.
[0047] Test results: When hemoglobin ≤15g / L, triglycerides ≤15mmol / L, and bilirubin ≤300μmol / L, the detection deviation is ≤±6%; the consistency with the test results of the interference-free samples is ≥95%.
[0048] In summary, this invention employs homogeneous chemiluminescence technology, combined with optimized luminescent donor-acceptor combinations and reaction conditions, to accurately detect low concentrations of ferritin, achieving a detection limit as low as 0.08 ng / mL with a wide linear range. The operation is simple, requiring no complex pretreatment or washing / separation steps, significantly improving detection efficiency. Through designs such as dual-antibody specific recognition, dual-wavelength signal ratio detection, and the addition of interference inhibitors, non-specific reactions and environmental interference are reduced, resulting in high accuracy. It is adaptable to routine samples such as serum and plasma, as well as complex samples such as those with hemolysis and hyperlipidemia, meeting the needs of various clinical scenarios. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and possesses high industrial application value.
[0049] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.
Claims
1. A ferritin assay kit based on homogeneous chemiluminescence, characterized in that, Including reagent 1 and reagent 2, wherein: The reagent 1 includes ferritin-specific antibody 1 coupled with a luminescent donor molecule and a reaction buffer; The reagent 2 includes ferritin-specific antibody 2 coupled with a luminescent receptor molecule and a reaction buffer.
2. The reagent kit according to claim 1, characterized in that, The luminescent donor molecule is selected from Eu 3+ Chelates, Tb 3+ The luminescent acceptor molecule is selected from at least one of chelates, coumarin derivatives, or naphthalimide fluorescent molecules; the luminescent acceptor molecule is selected from at least one of anthocyanin dyes, fluorescein derivatives, quantum dots, porphyrin compounds, and gold nanoparticles.
3. The reagent kit according to claim 2, characterized in that, The luminescent donor molecule is Eu-TTA, Eu-β-NTA, 7-hydroxycoumarin, or coumarin-3-carboxylic acid; the luminescent acceptor molecule is at least one of Cy3.5, Cy5, rhodamine B, sulfonated tetraphenylporphyrin, quantum dot CdSe, and quantum dot ZnS.
4. The reagent kit according to any one of claims 1 to 3, characterized in that, The luminescent donor molecule and the luminescent acceptor molecule are chemically modified. The luminescent donor molecule is modified by introducing a carboxyl group and activating it to form an NHS ester, or by introducing an amino group or a maleimide group. The luminescent acceptor molecule is modified by activating a carboxyl group, introducing a maleimide group, or introducing a PEG linker arm.
5. The reagent kit according to claim 1, characterized in that, The reaction buffer comprises 40-60 mmol / L HEPES buffer, 0.1-0.2% BSA, and 0.02-0.10% Triton X-100.
6. The reagent kit according to claim 5, characterized in that, The reaction buffer also includes at least one of the following: 10-100 mmol / L NaCl, 1-5 mmol / L MgCl2, and 0.01-0.05% sodium azide.
7. The reagent kit according to claim 5, characterized in that, The reaction buffer also includes at least one of the following: 0.5-3.0% PEG6000, 0.1-1.0% glycine, or 0.01-0.05% PMSF.
8. A method for determining ferritin based on the kit according to any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Mix the sample to be tested with reagents 1 and 2 in the kit and incubate to obtain the test mixture; (2) Excite the above-mentioned mixture to be tested with excitation light to induce a chemiluminescence reaction, and measure the luminescence intensity at different detection wavelengths, and calculate the ratio of the two as the signal value; (3) Establish a standard curve of "sample concentration-signal value" based on the known concentration of the sample, and calculate the ferritin content in the sample to be tested by referring to the above standard curve.
9. The method according to claim 8, characterized in that, Severely hemolyzed or hyperlipidemic samples require pretreatment with a pretreatment solution, which is a 50 mmol / L HEPES buffer containing 1% Triton X-100 and 0.05% EDTA.
10. The method according to claim 8, characterized in that, In step (2), the excitation wavelength is 320 nm; the detection wavelengths are 665 nm and 620 nm.