Unsaturated iron binding capacity assay kit and use thereof

Abstract

The application discloses an unsaturated iron binding capacity determination kit and application thereof, and comprises reagent R1 and reagent R2, wherein the reagent R1 comprises a BICINE buffer, an iron salt and a first preservative; the reagent R2 comprises a KCl-HCl buffer, a reducing agent, a chromogenic agent and a second preservative; the reagent R1 adopts a N, N-dihydroxyethyl glycine (BININE) buffer, so that the stability of the reagent R1 is improved, and the kit can maintain good stability without the additional introduction of a chelating agent and the addition of a stabilizer; the reagent R2 adopts a KCl-HCl biological buffer, the buffer has weak buffering capacity, does not affect the alkaline environment after the reagent R1 and the reagent R2 are mixed, and KCl can maintain electrolyte balance of the system, so that the reagent can maintain good stability without the addition of a stabilizer.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a kit for determining the binding force of unsaturated iron and its application. Background Technology

[0002] Iron is an essential trace element for the human body and a major raw material for the synthesis of hemoglobin in red blood cells. Serum iron (Fe) is bound to transferrin (TRF). Each TRF molecule can bind two ferric ions, but only a portion of the transferrin molecule is saturated, while the other portion remains unsaturated, a phenomenon known as unsaturated iron-binding capacity (UIBC). When serum transferrin is fully saturated, the amount of iron bound is the total iron-binding capacity, which is the sum of serum iron and unsaturated iron-binding capacity. Measuring serum iron is helpful in diagnosing various diseases. For example, hemolytic anemia, aplastic anemia, megaloblastic anemia, acute hepatitis, and lead poisoning can all cause elevated serum iron levels; while iron deficiency anemia, chronic blood loss, menorrhagia, pregnancy, infectious diseases, malignant tumors, and cirrhosis can cause decreased serum iron levels. Elevated serum total iron-binding capacity is seen in iron deficiency anemia, acute hepatitis, etc., while decreased total iron-binding capacity is seen in cirrhosis, nephropathy, uremia, and hemochromatosis. Total iron-binding capacity increases in iron deficiency anemia, late pregnancy, oral contraceptive use, and viral hepatitis, and decreases in chronic infection, iron poisoning, kidney disease, nephrotic syndrome, and thalassemia.

[0003] Currently, most commercially available unsaturated iron binding capacity assay kits use a colorimetric method. This involves using excess iron ions under alkaline conditions to bind all transferrin in serum. The remaining iron ions are then reduced to ferrous ions using a reducing agent, and the colorimetric determination yields the unsaturated iron binding capacity. For example, patents CN102323430A and CN104483494A add ferric ions to reagent 1 and thiourea as an interference remover to eliminate copper ion interference. However, thiourea itself has reducing properties and can undergo a redox reaction with the ferric ions in reagent 1, reducing them to ferrous ions and affecting the reagent's stability. In patent CN104483494A, water-soluble carbonates or bicarbonates are added to reagent 1. This can cause a double hydrolysis reaction with the iron ions in the reagent, and the reagent is prone to precipitation upon heating, affecting its performance.

[0004] It is evident that since the binding force of unsaturated iron needs to be measured under alkaline conditions, iron ions in alkaline buffer solutions usually form ferric hydroxide precipitate or hydrolyze to produce ferric hydroxide colloid. During long-term storage, especially during thermal degradation, the reagent will gradually precipitate obvious reddish-brown ferric hydroxide colloid, thus affecting the stability of the reagent. Therefore, solving the problem of iron ion stability in alkaline buffer solutions is crucial. Summary of the Invention

[0005] In view of this, this application provides a reagent kit for determining the binding force of unsaturated iron and its application, which has good stability, high accuracy and high precision.

[0006] To achieve the above technical objectives, this application adopts the following technical solution:

[0007] In a first aspect, this application provides a kit for determining the binding force of unsaturated iron, comprising reagent R1 and reagent R2, wherein reagent R1 comprises BICINE buffer, iron salt, and a first preservative, and reagent R2 comprises KCl-HCl buffer, reducing agent, colorimetric agent, and a second preservative.

[0008] Preferably, the concentration of BICINE buffer is 50 mmol / L to 150 mmol / L.

[0009] Preferably, the pH value of the BICINE buffer is 8.0-9.0.

[0010] Preferably, the concentration of the iron salt is 10 μmol / L to 20 μmol / L.

[0011] Preferably, the iron salt includes one or more of ferric sulfate, potassium ferric sulfate, ammonium ferric sulfate, and ferric chloride.

[0012] Preferably, the concentration of the KCl-HCl buffer is 150 mmol / L to 300 mmol / L.

[0013] Preferably, the pH value of the KCl-HCl buffer solution is 1.5 to 2.5.

[0014] Preferably, the concentration of the reducing agent is 50 mmol / L to 200 mmol / L.

[0015] Preferably, the concentration of the colorimetric reagent is 1 mmol / L to 3 mmol / L.

[0016] Secondly, this application provides an application of an unsaturated iron binding force determination kit in detecting unsaturated iron binding force, wherein the volume ratio of reagent R1, reagent R2 and the sample to be tested is 10:2:1.

[0017] The beneficial effects of this application are as follows:

[0018] In this protocol, reagent R1 uses N,N-dihydroxyethylglycine (BININE) buffer, which improves the stability of R1 without the need for additional chelating agents or stabilizers to maintain the good stability of the kit. Reagent R2 uses KCl-HCl biological buffer, which has a low buffering capacity and will not affect the alkaline environment after mixing reagents R1 and R2. Furthermore, KCl can maintain the electrolyte balance of the system, so the reagents can maintain good stability even without the addition of stabilizers. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] As those skilled in the art know, in an alkaline buffer solution with an excess of iron ions, all the transferrin in the sample that is not bound to iron binds to iron ions. The remaining iron ions are then reduced and react with the chromogenic agent to form a red complex. The unsaturated iron binding force in the sample can be calculated by calculating the amount of iron ions reduced in the buffer solution.

[0021] The experimental principle dictates that iron ion detection must be performed in an alkaline buffer solution. However, in a conventional alkaline buffer solution, iron ions inevitably undergo hydrolysis or precipitation, which severely affects the accuracy of the detection results. Typically, a certain amount of chelating agent capable of complexing iron ions is added to the buffer solution. However, conventional chelating agents, such as tartrates and citrates, only achieve optimal complexing effects when added in appropriate amounts. If too little chelating agent is added, iron ions will still hydrolyze or precipitate. If too much chelating agent is added, it will alter the pH of the buffer solution, making it difficult to maintain a constant pH and potentially interfering with the determination of iron ions. Often, the chelation effect is not particularly ideal.

[0022] Based on the above, this invention was created.

[0023] In a first aspect, this application provides a kit for determining the binding force of unsaturated iron, comprising reagent R1 and reagent R2, wherein reagent R1 comprises BICINE buffer, iron salt, and a first preservative, and reagent R2 comprises KCl-HCl buffer, reducing agent, colorimetric agent, and a second preservative.

[0024] The high stability of the unsaturated iron binding capacity assay kit in this application is due to the fact that the BICINE buffer in reagent R1 is an amphoteric amino acid buffer, possessing the properties of both amino acids and aminoethanol. It can act as a stable biological buffer to maintain a constant pH in the system, and it can also form stable glycine-based ferric complexes with iron ions. These complexes do not affect the determination of iron ions, but they ensure the stable existence of iron ions under alkaline conditions. In other words, this buffer can simultaneously function as a buffer, chelating agent, and stabilizer, thus greatly improving the stability of the reagent. Reagent R2 uses a biological buffer, KCl-HCl buffer. This buffer has a low buffering capacity and will not affect the alkaline environment after mixing reagents R1 and R2, thus significantly improving the measured values ​​and ensuring good accuracy. Furthermore, KCl maintains the electrolyte balance of the system, ensuring good reagent stability even without the addition of a stabilizer.

[0025] The concentration of BICINE buffer is 50 mmol / L to 150 mmol / L. If the concentration is too low, it will not be enough to completely complex the ferric ions, and the stability of the reagent will not be good enough. If the concentration is too high, it will increase the cost of the reagent.

[0026] Preferably, the pH value of the BICINE buffer is 8.0-9.0. When the pH of the reagent is lower than 8.0, the buffering capacity is weak and the stability of the reagent is not good enough.

[0027] To ensure that the amount of iron ions added to the reagent is excessive, it is necessary to guarantee that all transferrin in the sample is bound by ferric ions, allowing the remaining ferric ions to participate in the next reduction reaction. However, if too many ferric ions are added, the concentration of the BICINE buffer in the reagent will also need to be increased accordingly. Therefore, the concentration of the BICINE buffer and the concentration of the ferric salt must be relatively appropriate. In this protocol, the concentration of the ferric salt is 10 μmol / L to 20 μmol / L, and the ferric salt includes one or more of ferric sulfate, potassium ferric sulfate, ammonium ferric sulfate, and ferric chloride.

[0028] Preferably, the concentration of the KCl-HCl buffer is 150 mmol / L to 300 mmol / L. This buffer has the best stability within this range. On the other hand, if the concentration is too high, the pH of the mixture of reagents R1 and R2 will not reach the optimal value, which will reduce the accuracy of the reagents.

[0029] KCl-HCl buffer solution has a relatively weak buffering capacity. When the pH is below 1.5, the buffering capacity is too weak. When the pH is above 2.5, the buffering capacity gradually decreases, resulting in poor reagent stability. The pH value of KCl-HCl buffer solution is 1.5 to 2.5.

[0030] Preferably, the concentration of the reducing agent is 50 mmol / L to 200 mmol / L, and the reducing agent includes one or more of hydroxylamine hydrochloride, sodium sulfite, sodium bisulfite, and ascorbic acid.

[0031] Preferably, the concentration of the colorimetric reagent is 1 mmol / L to 3 mmol / L, and the colorimetric reagent is furan triazine disodium salt (Ferene).

[0032] In reagent R1, the first preservative is sodium azide at a concentration of 0.1% to 1.0%. In reagent R2, the second preservative has a concentration of 0.1% to 1.0%, and the second preservative includes one or more of benzoic acid, sodium benzoate, sorbic acid, and potassium sorbate.

[0033] In this scheme, the preparation process of reagent R1 is as follows: prepare a BICINE buffer solution with a concentration of 50 mmol / L to 150 mmol / L and a pH of 8.0 to 9.0, add iron salt of 10 μmol / L to 20 μmol / L and preservative of 0.1% to 1.0% to the buffer solution, and mix well after adjusting the volume to obtain reagent R1.

[0034] The preparation process of reagent R2 is as follows: prepare a KCl-HCl buffer solution with a concentration of 150 mmol / L to 300 mmol / L and a pH of 1.5 to 2.5. Add a reducing agent of 50 mmol / L to 200 mmol / L, a colorimetric reagent of 1 mmol / L to 3 mmol / L, and a preservative of 0.1% to 1.0% to the buffer solution in sequence. After adjusting the volume, mix well to obtain reagent R2.

[0035] This application provides an application of an unsaturated iron binding force determination kit in detecting unsaturated iron binding force. The unsaturated iron binding force in the sample is detected using the unsaturated iron binding force determination kit of this application according to the endpoint method commonly used by those skilled in the art. The volume ratio of reagent R1, reagent R2 and the sample to be tested is 10:2:1.

[0036] The following describes the specific implementation methods of this solution.

[0037] Example 1

[0038] A kit for determining the binding capacity of unsaturated iron includes reagent R1 and reagent R2. Reagent R1 includes BICINE buffer, iron salt, and a first preservative. The concentration of the BICINE buffer is 100 mmol / L, the pH is 8.0, the iron salt is 10 μmol / L ferric sulfate, and the first preservative is 0.1% sodium azide. Reagent R2 includes KCl-HCl buffer, a reducing agent, a colorimetric agent, and a second preservative. The concentration of the KCl-HCl buffer is 150 mmol / L, the pH is 1.5, the reducing agent is 100 mmol / L ascorbic acid, the colorimetric agent is 2 mmol / L disodium furanyltriazine, and the second preservative is 0.1% sodium benzoate.

[0039] Example 2

[0040] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the concentration of the BICINE buffer is 50 mmol / L.

[0041] Example 3

[0042] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the concentration of the KCl-HCl buffer is 300 mmol / L.

[0043] Example 4

[0044] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the pH of the BICINE buffer is 9.0.

[0045] Example 5

[0046] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the pH of the KCl-HCl buffer is 2.5.

[0047] Example 6

[0048] A kit for determining the binding force of unsaturated iron is provided, which is otherwise identical to that in Example 1, except that the concentration of ferric sulfate is 20 μmol / L.

[0049] Example 7

[0050] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the pH of the BICINE buffer is 8.5.

[0051] Example 8

[0052] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the concentration of BICINE buffer is 150 mmol / L.

[0053] Example 9

[0054] A kit for determining the binding force of unsaturated iron is provided, which is otherwise identical to that in Example 1, except that the concentration of the KCl-HCl buffer is 200 mmol / L.

[0055] Example 10

[0056] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the pH of the KCl-HCl buffer solution is 2.

[0057] Comparative Example 1

[0058] A kit for determining unsaturated iron binding capacity is identical to that in Example 1, except that the BICINE buffer is replaced with Tris buffer and 50 mmol / L thiourea is added to reagent R1.

[0059] Comparative Example 2

[0060] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the KCl-HCl buffer is replaced with acetate buffer, and 5 mmol / L hydroxylamine hydrochloride and 10 mmol / L sodium thiosulfate are added to reagent R2 as stabilizers.

[0061] Comparative Example 3

[0062] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the BICINE buffer is replaced with Tris buffer, 50 mmol / L thiourea is added to reagent R1, the KCl-HCl buffer is replaced with acetate buffer, and 5 mmol / L hydroxylamine hydrochloride and 10 mmol / L sodium thiosulfate are added to reagent R2 as stabilizers.

[0063] Comparative Example 4

[0064] A kit for determining unsaturated iron binding capacity is identical to that in Example 1, except that the BICINE buffer is replaced with tris(hydroxymethyl)methylglycine buffer.

[0065] Comparative Example 5

[0066] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the KCl-HCl buffer is replaced with acetate buffer.

[0067] Comparative Example 6

[0068] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the pH of the BICINE buffer is 7.5.

[0069] Comparative Example 7

[0070] A kit for determining the binding force of unsaturated iron is provided, which is otherwise identical to that in Example 1, except that the concentration of BICINE buffer is 30 mmol / L.

[0071] Comparative Example 8

[0072] A kit for determining the binding force of unsaturated iron is identical to that in Example 1, except that the pH of the KCl-HCl buffer solution is 3.

[0073] Comparative Example 9

[0074] A kit for determining the binding force of unsaturated iron is provided, which is otherwise identical to that in Example 1, except that the concentration of the KCl-HCl buffer is 400 mmol / L.

[0075] Evaluation Test

[0076] The components in Examples 1-10 and Comparative Examples 6-9 are summarized in Table 1. By combining the modified components in Comparative Examples 1-5, different kits for determining the binding force of unsaturated iron are obtained.

[0077] Table 1. Components of each kit

[0078]

[0079] The unsaturated iron binding force in the sample was determined using the endpoint method. The operating parameters were as follows: temperature: 37℃, main wavelength: 600nm, secondary wavelength: 700nm, sample volume: 20μL, R1: 200μL, R2: 40μL, reaction direction: forward, reaction time: 10min, calibration method: two-point calibration. The operating steps were as follows: 20μL of the sample to be tested was mixed with 200μL of reagent R1, incubated at 37℃ for 5 minutes, the blank tube was zeroed, and the absorbance A1 of each tube was measured at 600nm. Then, 40μL of reagent R2 was added, mixed, and incubated at 37℃ for 5 minutes to measure the absorbance A2 of each tube. Calculate the sample to be tested ΔA = A2 - A1, and similarly calculate the blank sample ΔA' and the calibration sample ΔA". Based on the above values, evaluate the performance of each kit. The evaluation criteria for the kit are as follows: (1) Blank absorbance: The reagent blank absorbance at a wavelength of 600 nm should be ≤0.8000; (2) Accuracy: Calculate the deviation between the measured average value and the target value for the quality control sample. The relative deviation should be ≤10%; (3) Precision: Under repeatability conditions, test the same serum sample or control substance with the kit 10 times and compare the coefficient of variation (CV). The CV should be ≤6%; (4) Analytical sensitivity: When the kit tests 20.0 μmol / L of the analyte, the absorbance difference (ΔA) should be ≥0.0200; (5) Linearity: In the range of 1.0 μmol / L to 100 μmol / L, the linear correlation coefficient r ≥0.995.

[0080] The thermal stability and open-bottle stability of each reagent kit were evaluated, and the results are shown in Tables 2 and 3 (Table 2 shows the thermal stability performance evaluation and the performance evaluation after 30 days of opening; Table 3-1 shows the deviation results during the opening process of the examples; Table 3-2 shows the deviation results during the opening process of the comparative examples). The evaluation methods are as follows: Thermal stability: After the reagents were placed in a 37℃ constant temperature water bath for 14 days of thermal degradation and on a shaker at 42℃ for 5 days, the performance of the reagent blank absorbance, accuracy, precision, analytical sensitivity, and linearity were evaluated; Open-bottle stability: The reagents were opened at 2℃~8℃, and the quality control samples were tested. The values ​​were measured three times and the average value was taken. The tests were performed every few days, and the relative deviation was compared with that on day 0. After 30 days of opening, the performance of the reagent blank absorbance, accuracy, precision, analytical sensitivity, and linearity were evaluated.

[0081] Table 2. Results of reagent kit performance testing

[0082]

[0083]

[0084]

[0085] As shown in Table 2, the reagents in Examples 1-10 showed very little decrease in blank absorbance and analytical sensitivity after being subjected to heat damage at 37°C for 14 days, shaking at 42°C for 5 days, and opening for 30 days, compared with the unopened reagents stored at 2-8°C. The accuracy, precision, and linear correlation coefficient of the quality control samples also showed little change. This indicates that there were no significant differences in the stability of the BICINE buffer solution (concentration between 50 mmol / L and 150 mmol / L, pH between 8 and 9), the KCl-HCl buffer solution (concentration between 150 mmol / L and 300 mmol / L, pH between 8 and 9), and the ferric sulfate solution (concentration between 10 μmol / L and 20 μmol / L).

[0086] Compared with the examples, Comparative Examples 1-5 showed poor accuracy. The data after the reagents were subjected to heat damage at 37°C for 14 days, shaking at 42°C for 5 days, and after opening for 30 days showed a significant decrease in blank absorbance and analytical sensitivity compared with the unopened reagents stored at 2-8°C. The precision and linearity also deteriorated, resulting in poor stability.

[0087] Compared to Comparative Examples 1-5, Comparative Examples 6-9 showed a significant improvement in accuracy, but compared to Examples 1-10, both accuracy and precision were slightly lower. Furthermore, after 14 days of heat damage at 37°C, 5 days of shaking at 42°C, and 30 days after opening, the reagents in Comparative Examples 6-9 exhibited worse blank absorbance, analytical sensitivity, and linearity compared to unopened reagents stored at 2-8°C. Overall, their performance was superior to Comparative Examples 1-5, but worse than Examples 1-10. This indicates that when the BICINE buffer concentration is outside the range of 50 mmol / L to 150 mmol / L and the pH is outside the range of 8 to 9, and when the KCl-HCl buffer concentration is outside the range of 150 mmol / L to 300 mmol / L and the pH is outside the range of 1.5 to 2.5, the performance of the reagents, especially their thermal stability and stability after opening, is not good enough.

[0088] Table 3-1 Deviation results during the opening process of the reagent kits for each example

[0089]

[0090] Table 3-2 Deviation results during the opening process of each comparative example reagent kit

[0091]

[0092] As shown in Table 3, the open-bottle stability of Comparative Examples 1-5 was generally only stable for 14-18 days, after which the stability gradually increased to over 10%, indicating poor open-bottle stability. Comparative Examples 6-9 showed significant improvement compared to Comparative Examples 1-5, but the stability still exceeded 10% after 31 days. Examples 1-10 showed that the increase in values ​​after 31 days compared to day 0 was still controlled within 8%, demonstrating a significant advantage in open-bottle stability compared to the comparative examples, indicating good stability.

[0093] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A kit for determining the binding force of unsaturated iron, characterized in that, The reagent includes reagent R1 and reagent R2. Reagent R1 includes a BICINE buffer solution with a concentration of 50 mmol / L to 150 mmol / L and a pH of 8.0 to 9.0, an iron salt with a concentration of 10 μmol / L to 20 μmol / L, and a first preservative, and reagent R1 does not contain thiourea. Reagent R2 includes a KCl-HCl buffer solution with a concentration of 150 mmol / L to 300 mmol / L and a pH of 1.5 to 2.5, a reducing agent, a colorimetric agent, and a second preservative.

2. The unsaturated iron binding force determination kit according to claim 1, characterized in that, The iron salts include one or more of ferric sulfate, potassium ferric sulfate, ammonium ferric sulfate, and ferric chloride.

3. The unsaturated iron binding force determination kit according to claim 1, characterized in that, The concentration of the reducing agent is 50 mmol / L to 200 mmol / L.

4. The unsaturated iron binding force determination kit according to claim 1, characterized in that, The concentration of the colorimetric reagent is 1 mmol / L to 3 mmol / L.

5. The application of the unsaturated iron binding force determination kit as described in any one of claims 1-4 in the detection of unsaturated iron binding force, characterized in that, The volume ratio of reagent R1, reagent R2 and the sample to be tested is 10:2:1.

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