Iron metabolism marker detection kit with high anti-interference performance and application thereof

Abstract

The invention discloses an iron metabolism marker detection kit with high anti-interference performance and application thereof, and belongs to the technical field of in-vitro diagnostic reagents. The invention provides a composite interference neutralization reagent group. The composite interference neutralization reagent group comprises the following working concentration components: 1 to 10 mmol / L of cyclohexanediamine tetraacetic acid, 20 to 100 mmol / L of triethanolamine, 0.5 to 3.0 mmol / L of potassium ferrocyanide, 0.1 to 1.0% w / v of polyethylene glycol, 0.5 to 5.0 mmol / L of sodium nitrite, 0.05 to 0.5% w / v of poloxamer 407 and 0.1 to 2.0 mmol / L of N-ethylmaleimide. The invention also provides an iron metabolism marker detection kit containing the composite interference neutralization reagent group. According to the invention, the interference of endogenous and exogenous substances is effectively eliminated through the synergistic interaction among the compound interference neutralization reagent groups, and the accuracy and reliability of a detection result are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, specifically relating to a reagent for detecting serum iron metabolism markers (transferrin, serum iron, and unsaturated iron binding capacity). Background Technology

[0002] Serum iron and its metabolites detection refers to the determination of the content of iron and its metabolites in serum. The results can indicate the type and degree of anemia, guide treatment and evaluate the treatment efficacy, and can also indicate certain diseases. The main targets of iron metabolism marker detection include transferrin, serum iron, and unsaturated iron binding capacity: (1) Transferrin detection kit is used to quantitatively determine the content of transferrin (TF) in human serum in vitro. (2) Serum iron detection kit is used to quantitatively determine the content of iron (FE) in human serum in vitro. Under acidic conditions, serum iron is released from the transferrin bound to it, and at the same time, ferric iron is reduced to ferrous ions (ferrous ions). Ferrous ions react with ferrous ions to form a purple complex. The absorbance change is measured at 560 nm and is proportional to the iron ion concentration in the sample. (3) Unsaturated iron binding capacity detection kit is used to quantitatively determine the unsaturated iron binding capacity in human serum in vitro. In an alkaline buffer solution with excess iron ions, all transferrin in the sample that was not bound to iron binds to iron ions. The remaining iron ions react with the reducing agent ascorbic acid and the colorimetric agent disodium furan triazine to form a blue complex (600 nm). The unsaturated iron binding force of the sample can be calculated by calculating the amount of iron ions reduced in the buffer solution.

[0003] However, existing iron metabolism reagents, which use transferrin, serum iron, and unsaturated iron binding capacity as core detection indicators, are easily interfered with by various substances in clinical biochemical tests. For example, (1) hemolysis (hemoglobin-Hb): causes spectral and chemical interference. (2) jaundice (bilirubin-Bil): causes spectral interference and possible reaction competition. (3) lipemia (chylotriglycemia-TG): causes light scattering, increasing light absorption. (4) other metal ions (Cu) 2+ Zn 2+ ): Competitively binds with the colorimetric reagent. (5) Reducing substances (such as ascorbic acid-VitC): Affect the redox reaction process. In addition, existing reagent formulations mostly use single or small amounts of masking agents, which are difficult to cope with the challenges of multiple interferences coexisting in complex clinical samples, and the additives may affect each other, resulting in limited effectiveness. Summary of the Invention

[0004] To address the shortcomings of existing technologies and to solve the problem of easy interference from various substances in the detection of iron metabolism markers (transferrin, serum iron, and unsaturated iron binding capacity), the present invention aims to design and provide a novel and highly efficient composite interference neutralizing reagent set, which is integrated into the reagent formulation. Through the synergistic effect between the components, the interference of endogenous and exogenous substances can be effectively eliminated, significantly improving the accuracy and reliability of the detection results.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] On one hand, the present invention provides a composite interference neutralizing reagent kit for the detection of iron metabolism markers, comprising the following components at working concentrations: 1-10 mmol / L cyclohexanediaminetetraacetic acid, 20-100 mmol / L triethanolamine, 0.5-3.0 mmol / L potassium ferrocyanide, 0.1%-1.0% w / v polyethylene glycol, 0.5-5.0 mmol / L sodium nitrite, 0.05%-0.5% w / v poloxamer 407, and 0.1-2.0 mmol / L N-ethylmaleimide.

[0007] 1-10 mmol / L cyclohexanediaminetetraacetic acid and 20-100 mmol / L triethanolamine were used as metal ion masking synergists. Interfering with metal ions (Cu... 2+ Zn 2+ TEA possesses extremely strong chelating ability, but at high concentrations it may affect the main reaction. The addition of TEA not only has a weak masking effect itself, but also forms a synergistic effect with CDTA, enhancing the selective masking of specific metal ions, while simultaneously stabilizing CDTA to prevent it from affecting Fe. 2+ , / Fe3 + , and the reaction with chromogenic agents or transferrin.

[0008] Potassium ferrocyanide (K4[Fe(CN)6]) and polyethylene glycol-6000 (PEG-6000) are used as hemoglobin scavengers and stabilizers. Potassium ferrocyanide reacts with hemoglobin to form stable cyanogenic methemoglobin, eliminating its interference. The addition of PEG-6000 promotes the soluble aggregation of the reaction product, preventing precipitation, while also stabilizing colloids and reducing lipid light scattering, achieving two benefits at once.

[0009] Sodium nitrite (NaNO2) and poloxamer 407 serve as bilirubin oxidation and lipid emulsifiers. Under acidic buffer conditions, sodium nitrite slowly releases trace amounts of nitrous acid, effectively oxidizing bilirubin to its colorless derivatives. Poloxamer 407 is a nonionic polymeric surfactant that efficiently emulsifies lipid particles and significantly reduces light scattering interference. Its use in conjunction with sodium nitrite provides a suitable microenvironment for bilirubin oxidation.

[0010] N-Ethylmaleimide (NEM) is used as a neutralizing agent for reducing substances. NEM is a highly efficient thiol reagent that can specifically and rapidly undergo addition reactions with reducing substances (such as ascorbic acid and glutathione) to neutralize their reducing properties, thereby eliminating their interference with the reduction step without affecting the subsequent colorimetric reaction.

[0011] The above-mentioned composite interference neutralizing reagent group was used as the core anti-interference component and added to the buffer system of reagent 1 (R1) in the kit.

[0012] The composite interference neutralizing reagent kit for the detection of iron metabolism markers, wherein the volume ratio of cyclohexanediaminetetraacetic acid, triethanolamine, potassium ferrocyanide, polyethylene glycol, sodium nitrite, poloxamer 407, and N-ethylmaleimide is [missing information].

[0013] Secondly, the present invention provides an interference-resistant iron metabolism biomarker detection kit, comprising reagent 1, reagent 2 and the aforementioned composite interference neutralizing reagent group.

[0014] The aforementioned interference-resistant iron metabolism marker detection kit, when the iron metabolism marker detection kit is a transferrin assay kit, comprises:

[0015] The reagent 1 is a solution containing 3%-8% w / v polyethylene glycol, 10-50 mmol / L pH 7.2-7.6 buffer solution and 100-200 mmol / L ionic strength adjuster;

[0016] The reagent 2 is a solution containing 5%-15% transferrin antibody, 10-50 mmol / L pH 7.2-7.6 buffer, and 100-200 mmol / L ionic strength adjuster;

[0017] The standard is transferrin;

[0018] And, the aforementioned composite interference neutralizing reagent group;

[0019] The buffer solution is selected from at least one of Tris / HCl buffer, HEPES buffer, phosphate-buffered saline, and MOPS buffer.

[0020] The ionic strength modifier is sodium chloride or potassium chloride.

[0021] Preferably, the HEPES buffer solution has a concentration of 10-50 mmol / L and a pH of 7.2-7.6.

[0022] Preferably, the phosphate buffer solution is 10-50 mmol / L phosphate, contains 100-200 mmol / L NaCl, and has a pH of 7.2-7.6;

[0023] Preferably, the PEG is a PEG with an average molecular weight between 4000 and 8000, such as PEG-4000 or PEG-8000.

[0024] For the transferrin detection kit (immunoturbidimetric assay): The composite interference neutralizing reagent group of this invention can effectively eliminate background absorbance interference and non-specific turbidity, so that turbidity changes only originate from specific antigen-antibody reactions.

[0025] The aforementioned interference-resistant iron metabolism marker detection kit, when the iron metabolism marker detection kit is an iron detection kit, comprises:

[0026] Reagent 1 is a solution containing 80-120 mmol / L acetate buffer, pH 4.0-4.4, reducing agent, 0.05%-0.5% (v / v) surfactant and 0.01%-0.1% preservative;

[0027] The reagent 2 is a solution containing 3-8 mmol / L ferroazine and 0.01%-0.1% preservative;

[0028] And the aforementioned composite interference neutralizing reagent group.

[0029] The iron metabolism marker detection kit with anti-interference properties, wherein the reducing agent is at least one selected from 10-100 mmol / L ascorbic acid, 10-50 mmol / L hydroxylamine hydrochloride, 5-20 mmol / L mercaptoethanol, or 10-50 mmol / L thiourea.

[0030] The aforementioned interference-resistant iron metabolism marker detection kit, wherein the surfactant is a nonionic surfactant;

[0031] Preferably, the nonionic surfactant is Triton X-100 series, Tween-20 series, or Brij series.

[0032] The interference-resistant iron metabolism marker detection kit, wherein the preservatives in reagent 1 and reagent 2 are at least one of Proclin 300, Proclin 950, sodium azide, sodium benzoate or potassium sorbate;

[0033] Preferably, the preservative is 0.05% - 0.1% (v / v) Proclin 300, 0.05% - 0.1% (v / v) Proclin 950, 0.05% - 0.1% (w / v) sodium azide, 0.1% - 0.2% (w / v) sodium benzoate or 0.1% - 0.2% (w / v) potassium sorbate.

[0034] The aforementioned interference-resistant iron metabolism marker detection kit, when the iron metabolism marker detection kit is an unsaturated iron binding capacity detection kit, comprises:

[0035] Reagent 1 is a solution containing 0.4-0.8 mol / L Tris buffer solution with pH 8.0-9.0, 0.05-0.2 mol / L thiourea, and 10-20 μmol / L ferrous ammonium sulfate;

[0036] Reagent 2 is a solution containing 6 mmol / L furan triazine disodium salt and 30 mmol / L ascorbic acid;

[0037] The calibrator is deferrotransferrin;

[0038] And, the aforementioned composite interference neutralizing reagent group;

[0039] Preferably, the buffer solution provides a stable buffer within an alkaline range of pH > 8.0 and does not precipitate metal ions in the solution;

[0040] More preferably, the buffer solution is a Tris buffer, a glycine buffer, or a borate buffer;

[0041] Preferably, the ascorbic acid can also be replaced with hydroxylamine hydrochloride or other substances that are stable in the solution of reagent 2 and can rapidly reduce Fe. 3+ The reagent.

[0042] In this invention, the synergistic effect of furan triazine disodium salt and thiourea ensures the high sensitivity and stability of the detection method, which is the core of this invention.

[0043] For the iron detection kit (ferriazine method) and the unsaturated iron binding force detection kit (Ferene method): This composite interference neutralizing reagent group creates a clean environment at the initial stage of the reaction, ensuring the specificity of the colorimetric reaction and the accuracy of absorbance changes.

[0044] Thirdly, the present invention provides the application of the aforementioned composite interference neutralizing reagent group, or any of the aforementioned kits, in the detection of iron metabolism markers. During detection, the composite interference neutralizing reagent group is mixed with reagent 1 at a volume ratio of 1:5 to 1:20 to prepare a ready-to-use working solution.

[0045] Preferably, the volume ratio is 1:10;

[0046] The volume ratio of (reagent 1 + composite interference neutralizing reagent group) to reagent 2 is (3-4):1.

[0047] The instruments applicable to the above three iron metabolism marker detection kits (transferrin detection kit; iron assay kit; unsaturated iron binding capacity detection kit) include, but are not limited to, the following: Hitachi 7180 / 7600 fully automated biochemical analyzer, Toshiba TBA-40FR / 120FR fully automated biochemical analyzer, Abbott C16000 fully automated biochemical analyzer, Siemens ADVIA2400 fully automated biochemical analyzer, Beckman AU5800 / AU680 fully automated biochemical analyzer, Olympus AU2700 fully automated biochemical analyzer, and Mindray BS-480 fully automated biochemical analyzer.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. The composite interference neutralizing reagent group of this invention produces a synergistic effect, rather than simply adding masking agents. Cyclohexanediaminetetraacetic acid (a strong chelating agent), as the main active ingredient, preferentially forms stable chelates with metal ions, directly blocking their interaction with transferrin antibodies (transferrin detection), ferroazine (serum iron detection), and transferrin binding sites (unsaturated iron binding capacity detection). Triethanolamine, as a synergistic ingredient, enhances the chelating ability of cyclohexanediaminetetraacetic acid by adjusting the pH of the detection system; furthermore, its amino group can form auxiliary coordination bonds with metal ions, further strengthening the chelate structure and preventing high concentrations of metal ions from overcoming the masking effect of a single chelating agent. Compared to using the traditional masking agent EDTA alone, this combination improves the masking efficiency for metal ions while avoiding additional interference from pH fluctuations to the three types of detection reactions.

[0050] 2. Precise targeting: Each type of interference has a corresponding combination solution, which is highly targeted and avoids the side effects that may be caused by a single high-concentration masking agent.

[0051] 3. Versatile formulation: As a whole module, this composite system can be flexibly embedded into the existing R1 reagent formulations of immunoturbidimetric, ferrizimine, and Ferene methods, making it highly universal and easy to promote in industrial applications.

[0052] 4. Significant improvement: It can greatly improve the performance of the kit in difficult clinical samples, improve the accuracy and repeatability of the test, and reduce the retesting rate and the risk of misdiagnosis.

[0053] 5. Highly innovative: This invention is the first to propose the use of NEM as an iron metabolism reagent to resist interference from reducing substances, and the use of the NaNO2 / Poloxamer 407 combination for the dual treatment of bilirubin and lipemia. These combinations are not obvious and have outstanding substantive features. Detailed Implementation

[0054] The present invention will be further illustrated by the following examples.

[0055] Example 1: Transferrin Detection Kit

[0056] The reagent composition for preparing the transferrin detection kit:

[0057] (1) Reagent 1 (R1): 6% (w / v) polyethylene glycol, 20 mmol / L, pH 7.4 Tris / HCl buffer, 150 mmol / L sodium chloride. Reagent 1 is a colorless and clear liquid.

[0058] (2) Reagent 2 (R2): 10% transferrin antibody, 20 mmol / L, pH 7.4 Tris / HCl buffer, 150 mmol / L sodium chloride. Reagent 2 is a colorless or pale yellow clear liquid.

[0059] (3) Calibrator: transferrin (liquid).

[0060] (4) Compound interference neutralizing reagent group: 5 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 60 mmol / L triethanolamine (TEA), 2.0 mmol / L potassium ferrocyanide, 0.5% (w / v) polyethylene glycol-6000, 3 mmol / L sodium nitrite (NaNO2), 0.2% (w / v) poloxamer 407, 1.0 mmol / L N-ethylmaleimide (NEM).

[0061] Example 2:

[0062] The reagent composition for preparing the transferrin detection kit:

[0063] (1) Reagent 1 (R1): 3% (w / v) polyethylene glycol, 10 mmol / L, pH 7.2 HEPES buffer, 100 mmol / L potassium chloride. Reagent 1 is a colorless and clear liquid.

[0064] (2) Reagent 2 (R2): 5% transferrin antibody, 10 mmol / L, pH 7.6 Tris / HCl buffer, 100 mmol / L potassium chloride. Reagent 2 is a colorless or pale yellow clear liquid.

[0065] (3) Calibrator: transferrin (liquid).

[0066] (4) Compound interference neutralizing reagent group: 1 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 20 mmol / L triethanolamine (TEA), 0.5 mmol / L potassium ferrocyanide, 0.1% (w / v) polyethylene glycol-4000, 0.5 mmol / L sodium nitrite (NaNO2), 0.05% (w / v) poloxamer 407, 0.1 mmol / L N-ethylmaleimide (NEM).

[0067] Example 3:

[0068] The reagent composition for preparing the transferrin detection kit:

[0069] (1) Reagent 1 (R1): 8% (w / v) polyethylene glycol, 50 mmol / L, pH 7.4 Tris / HCl buffer, 200 mmol / L sodium chloride. Reagent 1 is a colorless and clear liquid.

[0070] (2) Reagent 2 (R2): 15% transferrin antibody, 50 mmol / L Tris / HCl buffer, pH 7.2, 200 mmol / L sodium chloride. Reagent 2 is a colorless or pale yellow clear liquid.

[0071] (3) Calibrator: transferrin (liquid).

[0072] (4) Compound interference neutralizing reagent group: 10 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 100 mmol / L triethanolamine (TEA), 3.0 mmol / L potassium ferrocyanide, 1.0% (w / v) polyethylene glycol-6000, 5.0 mmol / L sodium nitrite (NaNO2), 0.5% (w / v) poloxamer 407, 2.0 mmol / L N-ethylmaleimide (NEM).

[0073] Example 4: Performance testing of the transferrin detection kit

[0074] 1. Standard Curve

[0075] (1) Reagent preparation: Take the reagents from the kit in Example 1.

[0076] (2) Experimental procedure:

[0077] Turn on the Toshiba TBA-40FR / 120FR fully automated biochemical analyzer, preheat it, and use the endpoint method. Add a test item to the fully automated biochemical analyzer and set the test item parameters as follows: Standard: (Reagent 1 + Compound Interference Neutralizing Reagent Set): Reagent 2 = 2μL / 150μL / 50μL; where the volume ratio of Reagent 1 to Compound Interference Neutralizing Reagent Set is 5:1; main wavelength 600nm, secondary wavelength 700nm; reaction temperature 37℃; total reaction time 10min; reaction direction is ascending. Prepare standard concentrations of 0g / L, 0.25g / L, 1.26g / L, 2.27g / L, 3.28g / L, 4.29g / L, 5.30g / L, and 6.3g / L. Take 2μL of each of the above different concentration standards and mix them with 150μL of Reagent 1 + Compound Interference Neutralizing Reagent Set. Incubate at 37℃ for 5 minutes and read the absorbance value A1. Add 50 μL of reagent 2, mix well, incubate at 37℃ for 5 minutes, and then measure the absorbance A2. Calculate ΔA = A2 - A1. Plot a standard curve based on the absorbance change rate (ΔA) and the corresponding standard concentration (mg / L).

[0078] Results: Within the concentration range of 0.25 g / L to 5.30 g / L, the linear correlation coefficient r ≥ 0.9900; when the concentration is in the range of [0.25, 2.0] g / L, the absolute deviation does not exceed ±0.2 g / L; when the concentration is in the range of [2.0, 5.30] g / L, the relative deviation does not exceed ±10%. This indicates a good linear relationship.

[0079] 2. Sensitivity Detection

[0080] A serum sample with a concentration of approximately 1.3 g / L was used as the test sample. The kit prepared in Example 1 was used, and the composite interference neutralizing reagent group was added to the buffer system of the first reagent (R1) of the kit as the core anti-interference component. The test sample was tested, and the absorbance change produced by the kit was recorded. The absorbance difference (ΔA) of the 1.3 g / L sample was converted. The result ΔA was 0.1100. The ΔA result was between 0.0500 and 0.1500, which proved that the kit of the present invention has high sensitivity.

[0081] 3. Accuracy:

[0082] Using the kit prepared in Example 1, the reference substance: human serum standard (ERM-DA470K / IFCC) was measured three times. The results of the three measurements were calculated as M. The relative deviation (Bias%) was calculated according to formula (1). The results are shown in Table 1 below. The relative deviation results of the three measurements did not exceed ±10%, which proves that the kit of the present invention has high accuracy.

[0083] Bias(%)=(MT) / T×100% formula (1)

[0084] Where Bias (%) is the relative deviation, M is the test result, and T is the reference material label value.

[0085] Table 1 Test Results

[0086]

[0087] The transferrin detection kits obtained in Examples 2 and 3 were tested using the same methods described above. The results were similar to those described above; the transferrin detection kits prepared in Examples 2 and 3 also exhibited high sensitivity and accuracy.

[0088] Example 5: Iron Detection Kit

[0089] The reagent composition for preparing the iron detection kit is as follows:

[0090] (1) Reagent 1 (R1): 100 mmol / L, pH 4.20 acetate buffer, 50 mmol / L ascorbic acid, 0.2% (v / v) Tween-20, 0.05% potassium sorbate.

[0091] (2) Reagent 2 (R2): 5 mmol / L ferriazine, 0.05% potassium sorbate.

[0092] (3) Compound interference neutralizing reagent group: 5 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 60 mmol / L triethanolamine (TEA), 2.0 mmol / L potassium ferrocyanide, 0.5% (w / v) polyethylene glycol-6000, 3 mmol / L sodium nitrite (NaNO2), 0.2% (w / v) poloxamer 407, 1.0 mmol / L N-ethylmaleimide (NEM).

[0093] Example 6:

[0094] The reagent composition for preparing the iron detection kit is as follows:

[0095] (1) Reagent 1 (R1): 80 mmol / L, pH 4.40 acetate buffer, 20 mmol / L hydroxylamine hydrochloride, 0.5% (v / v) Tween-20, 0.01% sodium benzoate.

[0096] (2) Reagent 2 (R2): 3 mmol / L ferriazine, 0.01% sodium benzoate.

[0097] (3) Compound interference neutralizing reagent group: 1 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 20 mmol / L triethanolamine (TEA), 0.5 mmol / L potassium ferrocyanide, 0.1% (w / v) polyethylene glycol-4000, 0.5 mmol / L sodium nitrite (NaNO2), 0.05% (w / v) poloxamer 407, 0.1 mmol / L N-ethylmaleimide (NEM).

[0098] Example 7:

[0099] The reagent composition for preparing the iron detection kit is as follows:

[0100] (1) Reagent 1 (R1): 120 mmol / L, pH 4.40 acetate buffer, 50 mmol / L ascorbic acid, 0.5% (v / v) Tween-20, 0.1% potassium sorbate.

[0101] (2) Reagent 2 (R2): 8 mmol / L ferriazine, 0.1% potassium sorbate.

[0102] (3) Compound interference neutralizing reagent group: 10 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 100 mmol / L triethanolamine (TEA), 3.0 mmol / L potassium ferrocyanide, 1.0% (w / v) polyethylene glycol-6000, 5.0 mmol / L sodium nitrite (NaNO2), 0.5% (w / v) poloxamer 407, 2.0 mmol / L N-ethylmaleimide (NEM).

[0103] Example 8: Performance testing of the iron detection kit

[0104] 1. Standard Curve

[0105] (1) Reagent preparation: Take the reagents from the kit in Example 5.

[0106] (2) Experimental procedure:

[0107] Turn on the Hitachi 7180 fully automated biochemical analyzer, preheat it, and use the endpoint method. Add the test item to the fully automated biochemical analyzer and set the test item parameters as follows: Standard: (Reagent 1 + Complex Interference Neutralizing Reagent Group): Reagent 2 = 20μL / 200μL / 50μL; where the volume ratio of Reagent 1 to Complex Interference Neutralizing Reagent Group is 10:1; main wavelength 570nm, secondary wavelength 700nm; reaction temperature 37℃; total reaction time 10min; reaction direction is ascending reaction. Prepare standard solutions at concentrations of 0 g / L, 5 g / L, 40 g / L, 75 g / L, 110 g / L, 145 g / L, 180 g / L, and 200 g / L. Take 20 μL of each of these standard solutions and mix them with 200 μL of the reagent 1 + interference neutralizing reagent group. Incubate at 37°C for 5 minutes. Then add 50 μL of reagent 2, mix well, and incubate at 37°C for 1 minute. Record the absorbance change rate ΔA / min over the next 180 seconds. Plot a standard curve based on the absorbance change rate (ΔA / min) and the corresponding standard concentration (mg / L).

[0108] Results: Within the range of 5 μmol / L to 180 μmol / L, the linear correlation coefficient r ≥ 0.990; for concentrations in the range of [5, 25] μmol / L, the absolute deviation should not exceed ±2.5 μmol / L; for concentrations in the range of (25, 180) μmol / L, the relative deviation should not exceed ±10%. This indicates a good linear relationship.

[0109] 2. Sensitivity

[0110] Serum with a concentration of approximately 35 μmol / L was used as the test sample. The kit prepared in Example 3 was used, and the composite interference neutralizing reagent group was added to the buffer system of the first reagent (R1) of the kit as the core anti-interference component. The test sample was tested, and the absorbance change produced by the kit was recorded. The absorbance change rate (ΔA / min) of the 35 μmol / L sample was converted. The result ΔA / min was 0.0800, which proves that the kit of the present invention has high sensitivity.

[0111] 3. Accuracy

[0112] Using the kit prepared in Example 3, the reference substance: iron single element solution (GBW08616) was measured three times. The results of the three measurements were calculated as M. The relative deviation (Bias%) was calculated according to the above formula (1). The results are shown in Table 2 below. The relative deviation results of the three measurements did not exceed ±10%, which proves that the kit of the present invention has high accuracy.

[0113] Table 2 Test Results

[0114]

[0115] The iron detection kits obtained in Examples 6 and 7 were tested using the same methods described above. The results were similar to those described above; the iron detection kits prepared in Examples 6 and 7 also exhibited high sensitivity and accuracy.

[0116] Example 9: Unsaturated iron binding force test kit

[0117] The reagent composition for preparing the unsaturated iron binding force test kit is as follows:

[0118] (1) Reagent 1 (R1): 0.6 mol / L Tris buffer, 0.12 mol / L thiourea, 14 μmol / L ferrous ammonium sulfate.

[0119] (2) Reagent 2 (R2): 6 mmol / L furan triazine disodium salt, 30 mmol / L ascorbic acid.

[0120] (3) Calibrator: Deferrotransferrin (liquid).

[0121] (4) Compound interference neutralizing reagent group: 5 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 60 mmol / L triethanolamine (TEA), 2.0 mmol / L potassium ferrocyanide, 0.5% (w / v) polyethylene glycol-6000, 3 mmol / L sodium nitrite (NaNO2), 0.2% (w / v) poloxamer 407, 1.0 mmol / L N-ethylmaleimide (NEM).

[0122] Example 10:

[0123] The reagent composition for preparing the unsaturated iron binding force test kit is as follows:

[0124] (1) Reagent 1 (R1): 0.4 mol / L Tris buffer, 0.05 mol / L thiourea, 10 μmol / L ferrous ammonium sulfate.

[0125] (2) Reagent 2 (R2): 4 mmol / L furan triazine disodium salt, 20 mmol / L ascorbic acid.

[0126] (3) Calibrator: Deferrotransferrin (liquid).

[0127] (4) Compound interference neutralizing reagent group: 1 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 20 mmol / L triethanolamine (TEA), 0.5 mmol / L potassium ferrocyanide, 0.1% (w / v) polyethylene glycol-4000, 0.5 mmol / L sodium nitrite (NaNO2), 0.05% (w / v) poloxamer 407, 0.1 mmol / L N-ethylmaleimide (NEM).

[0128] Example 11:

[0129] The reagent composition for preparing the unsaturated iron binding force test kit is as follows:

[0130] (1) Reagent 1 (R1): 0.8 mol / L Tris buffer, 0.2 mol / L thiourea, 20 μmol / L ferrous ammonium sulfate.

[0131] (2) Reagent 2 (R2): 8 mmol / L furan triazine disodium salt, 40 mmol / L ascorbic acid.

[0132] (3) Calibrator: Deferrotransferrin (liquid).

[0133] (4) Compound interference neutralizing reagent group: 10 mmol / L cyclohexanediaminetetraacetic acid (CDTA), 100 mmol / L triethanolamine (TEA), 3.0 mmol / L potassium ferrocyanide, 1.0% (w / v) polyethylene glycol-6000, 5.0 mmol / L sodium nitrite (NaNO2), 0.5% (w / v) poloxamer 407, 2.0 mmol / L N-ethylmaleimide (NEM).

[0134] Example 12: Performance Testing of the Unsaturated Iron Binding Force Detection Kit

[0135] 1. Standard Curve

[0136] (1) Reagent preparation: Take the reagents from the kit in Example 9.

[0137] (2) Experimental procedure:

[0138] Turn on the Olympus AU2700 fully automated biochemical analyzer, preheat it, and use the endpoint method. Add the test item to the fully automated biochemical analyzer and set the test item parameters as follows: Standard: (Reagent 1 + Complex Interference Neutralizing Reagent Set): Reagent 2 = 15μL / 200μL / 50μL; where the volume ratio of Reagent 1 to the Complex Interference Neutralizing Reagent Set is 20:1; main wavelength 600nm, secondary wavelength 700nm; reaction temperature 37℃; total reaction time 10min; reaction direction is ascending. Prepare standard concentrations of 0g / L, 4g / L, 31g / L, 58g / L, 85g / L, 112g / L, 140g / L, and 150g / L. Take 15μL of each of the above different concentrations of standard and mix it with 200μL of Reagent 1 + Complex Interference Neutralizing Reagent Set. Incubate at 37℃ for 5 minutes and read the absorbance value A1. Add 50 μL of reagent 2, mix well, incubate at 37℃ for 5 minutes, and then measure the absorbance A2. Calculate ΔA = A2 - A1. Plot a standard curve based on the absorbance change rate (ΔA) and the corresponding standard concentration (mg / L).

[0139] Results: Within the range of 4 μmol / L to 140 μmol / L, the linear correlation coefficient r ≥ 0.9900; when the concentration is [4, 20] μmol / L, the absolute deviation does not exceed ±2 μmol / L; when the concentration is [20, 140] μmol / L, the relative deviation does not exceed ±10%. This indicates a good linear relationship.

[0140] 2. Sensitivity Detection

[0141] Serum with a concentration of approximately 60 μmol / L was used as the test sample. The kit prepared in Example 5 was used, and the composite interference neutralizing reagent group was added to the buffer system of the first reagent (R1) of the kit as the core anti-interference component. The test sample was tested, and the absorbance change produced by the kit was recorded. The absorbance difference (ΔA) of the 60 μmol / L sample was converted. The result ΔA was 0.0900. The ΔA result was between 0.0500 and 0.1500, which proved that the kit of the present invention has high sensitivity.

[0142] 3. Accuracy testing

[0143] Using the kit prepared in Example 1, the enterprise reference material was measured three times. The results of the three measurements were calculated as M, and the relative deviation (Bias%) was calculated according to the above formula (1).

[0144] Table 3 Test Results

[0145]

[0146] The results are shown in Table 3 above. The relative deviations calculated three times did not exceed ±10%. This demonstrates that the kit of the present invention has high accuracy.

[0147] The performance of the unsaturated iron binding force test kits obtained in Examples 10 and 11 was tested using the same method as described above. The results were similar to those described above; the unsaturated iron binding force test kits prepared in Examples 10 and 11 also exhibited high sensitivity and accuracy.

Claims

1. A composite interference neutralizing reagent kit for the detection of iron metabolism markers, characterized in that, The components include the following working concentrations: 1-10 mmol / L cyclohexanediaminetetraacetic acid, 20-100 mmol / L triethanolamine, 0.5-3.0 mmol / L potassium ferrocyanide, 0.1% - 1.0% w / v polyethylene glycol, 0.5-5.0 mmol / L sodium nitrite, 0.05%-0.5% w / v poloxamer 407, and 0.1-2.0 mmol / L N-ethylmaleimide.

2. A reagent kit for detecting iron metabolism biomarkers that resists interference, characterized in that, It comprises reagent 1, reagent 2, and the composite interference neutralizing reagent group as described in claim 1.

3. The iron metabolism biomarker detection kit with anti-interference properties as described in claim 2, characterized in that, When the iron metabolism marker assay kit is a transferrin assay kit, it contains: The reagent 1 is a solution containing 3%-8% w / v polyethylene glycol, 10-50 mmol / L pH 7.2-7.6 buffer solution and 100-200 mmol / L ionic strength adjuster; The reagent 2 is a solution containing 5%-15% transferrin antibody, 10-50 mmol / L pH 7.2-7.6 buffer, and 100-200 mmol / L ionic strength regulator; The standard is iron-saturated transferrin; And, the composite interference neutralizing reagent group as described in claim 1.

4. The iron metabolism biomarker detection kit with anti-interference properties as described in claim 3, characterized in that, The buffer solution is selected from at least one of Tris / HCl buffer, HEPES buffer, phosphate-buffered saline, and MOPS buffer. The ionic strength modifier is sodium chloride or potassium chloride.

5. The iron metabolism biomarker detection kit as described in claim 2, characterized in that, When the iron metabolism marker detection kit is an iron detection kit, it contains: Reagent 1 is a solution containing 80-120 mmol / L acetate buffer, pH 4.0-4.4, reducing agent, 0.05%-0.5% (v / v) surfactant and 0.01%-0.1% preservative; The reagent 2 is a solution containing 3-8 mmol / L ferroazine and 0.01%-0.1% preservative; And, the composite interference neutralizing reagent group as described in claim 1.

6. The interference-resistant iron metabolism marker detection kit as described in claim 5, characterized in that, The reducing agent is at least one of 10-100 mmol / L ascorbic acid, 10-50 mmol / L hydroxylamine hydrochloride, 5-20 mmol / L mercaptoethanol, or 10-50 mmol / L thiourea. The buffer solution is an acetate buffer, a citrate buffer, a formate buffer, or a glycine-HCl buffer.

7. The iron metabolism biomarker detection kit with anti-interference properties as described in claim 5, characterized in that, The surfactant is a nonionic surfactant.

8. The interference-resistant iron metabolism marker detection kit as described in claim 5, characterized in that, The preservatives in both reagent 1 and reagent 2 are at least one of Proclin 300, Proclin 950, sodium azide, sodium benzoate, or potassium sorbate.

9. The interference-resistant iron metabolism biomarker detection kit as described in claim 2, characterized in that, When the iron metabolism marker assay kit is an unsaturated iron binding capacity assay kit, it contains: Reagent 1 is a solution containing 0.4-0.8 mol / L, pH 8.0-9.0 buffer solution, 0.05-0.2 mol / L thiourea and 10-20 μmol / L ferrous ammonium sulfate; Reagent 2 is a solution containing 4-8 mmol / L furan triazine disodium salt and 20-40 mmol / L ascorbic acid; The calibrator is deferrotransferrin; And, the composite interference neutralizing reagent group as described in claim 1.

10. The application of the composite interference neutralizing reagent kit as described in claim 1, or the kit as described in claim 2, in the detection of iron metabolism biomarkers, characterized in that, During testing, the composite interference neutralizing reagent group is mixed with reagent 1 at a volume ratio of 1:5 to 1:20 to prepare a ready-to-use working solution.

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