CN109613280B

CN109613280B - Serum iron determination kit and preparation method and application thereof

Info

Publication number: CN109613280B
Application number: CN201811642261.8A
Authority: CN
Inventors: 王飞; 解苇生; 刘安娜; 张强
Original assignee: Zhongtuo Biotechnology Co ltd
Current assignee: Zhongtuo Biotechnology Co ltd
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2022-01-28
Anticipated expiration: 2038-12-29
Also published as: CN109613280A

Abstract

The invention discloses a serum iron determination kit, which comprises a reagent R1 and a reagent R2; wherein the reagent R1 consists of: buffer solution, ascorbic acid, interference removing agent, stabilizing agent, surfactant and preservative; the reagent R2 contains the following components: buffer solution and bathophenanthroline; also relates to a preparation method and application of the serum iron determination kit. The stable serum iron is a liquid double reagent, does not need to be re-dissolved for preparation, and can be directly used after opening a bottle. The stability and the reducibility of the ascorbic acid are favorably maintained by adding a stabilizer to the reagent R1. The reagent R1 is added with an interference removing agent, and thiourea is mixed with ascorbic acid solution, which can be used as a reducing agent and a masking agent to remove the interference of other ions. The reagent has higher sensitivity. The reagent is convenient to use, high in specificity and accuracy, and free of pollution to a biochemical analyzer, and is favorable for further popularization of the reagent in the market.

Description

Serum iron determination kit and preparation method and application thereof

Technical Field

The invention relates to the field of in-vitro diagnostic reagents for medical immunization, in particular to a serum iron determination kit, and also relates to a preparation method and application of the serum iron determination kit.

Background

Iron is one of the most abundant essential trace elements of the human body, is a main raw material for synthesizing hemoglobin in red blood cells, and is widely involved in the metabolic process in the human body. The average amount of iron in normal adults is about 3-4.5g, about 70% of all iron in the whole body is present in hemoglobin and in myoglobin, while iron in various enzymes and in a plasma transport state only accounts for a very small part of the iron in the whole body, and iron deficiency or excessive iron can cause various diseases. Transferrin, also known as Transferrin (TRF), is the major iron-containing protein in plasma, responsible for carrying iron absorbed by the digestive tract and released by degradation of erythrocytes, as TRF-Fe³⁺Into the bone marrow for the production of mature red blood cells. Only one third of transferrin in serum binds Iron, the remaining two thirds are unbound, the potential Iron Binding capacity of transferrin is called unsaturated Iron Binding capacity (uibc), (unreacted Iron Binding capacity), the maximum amount of Iron that can bind is called total Iron Binding capacity (tibc), (total Iron Binding capacity), which is equal to the sum of the Binding capacity of serum Iron and unsaturated Iron. The determination of serum iron is of great significance for diagnosing blood system, liver and kidney diseases and protein deficiency.

The method for measuring serum iron includes colorimetry, atomic absorption spectrophotometry, stripping voltammetry, and the like. The atomic absorption spectrophotometry and the stripping voltammetry are sensitive, rapid and accurate, but need expensive instruments and equipment, have complex operation process, are rarely used for routine analysis of serum iron, are the main methods for measuring serum iron, are recommended as reference methods by IFCC, have high specificity and simple and convenient operation, are suitable for full-automatic biochemical analyzers and are suitable for detection of large batches of samples. The clinical serum iron colorimetric method determination principle is as follows: dissociating iron ion from transferrin under acidic condition, adding reducing agent such as hydroxylamine, ascorbic acid, thioglycolic acid, hydrazine, etc. to reduce iron ion to ferrous ion, de-complexing the ferrous ion with a chromogen capable of complexing with ferrous ion, and performing colorimetric determination. The chromogens widely used in clinic are ferrocazine, furan triazine disodium salt (ferene), Bathophenanthroline (BPZ), tripyridyl Triline (TPZ) and the like, the most commonly used chromogen is the ferrochromazine (such as a kit disclosed in CN 106814063A), but the reagents generally have the defects of poor sensitivity and specificity, and the invention improves the defects of the existing serum iron determination kit so as to meet the requirements of clinical detection and chemical analysis.

Disclosure of Invention

In order to solve the problems, the invention provides a serum iron determination kit, a preparation method and application thereof, and the kit is a liquid kit with high sensitivity, strong stability and high accuracy.

The invention is realized by the following technical scheme:

a serum iron determination kit comprises a reagent R1, a reagent R2;

wherein the reagent R1 consists of:

50-80 mmol/L buffer solution

Ascorbic acid 30-50 g/L

0-100 mmol/L of interference removing agent

20-90 mmol/L stabilizer

1-10 g/L of surfactant

0.1-10 g/L of preservative

The reagent R2 contains the following components:

buffer solution of 30-50 mmol/L

2-4 g/L bathophenanthroline

Preferably, the pH of the reagent R1 is 4.0-6.0; the pH value of the reagent R2 is 2.0-6.0.

Preferably, the stabilizer in the reagent R1 is selected from one or more of hydroxylamine hydrochloride, sodium metabisulfite, sodium sulfite, sodium bisulfite, mercaptoethanol, thioglycolic acid and dithiothreitol.

Preferably, the interference eliminator in the reagent R1 is one or more selected from thiourea, thiosemicarbazide, N-dimethylthiourea and isothiourea.

Preferably, the buffer solution selected by the reagent R1 is one or more of an acetic acid buffer solution, a citric acid buffer solution, a glycine buffer solution, a phosphate-citrate buffer solution and a phosphate-hydrochloric acid buffer solution; the buffer solution selected by the reagent R2 is one or more of acetic acid buffer solution, citric acid buffer solution, glycine buffer solution, phosphate-citrate and phosphate-hydrochloric acid buffer solution;

preferably, the surfactant in the reagent R1 is selected from one or more of the group consisting of triton series, tween series, brij-35 and dilauryl glycerol sulphate.

Preferably, the preservative in the reagent R1 is one or more of sodium azide, proclin300, MIT, gentamicin sulfate and thimerosal.

Preferably, the volume ratio of the reagent R1 to the reagent R2 is 1-5: 1, and more preferably 4: 1.

The preparation method of the serum iron determination kit is characterized by comprising the following steps: adding buffer solution into the reagents R1 and R2, adjusting the pH value with hydrochloric acid or sodium hydroxide, adjusting the pH value of the reagent R1 to 4.0-6.0 and the pH value of the reagent R2 to 2.0-6.0, and adding other substances according to the proportion for dissolving to prepare the serum iron determination kit.

The application of the serum iron determination kit is used for determining the concentration of iron ions in serum for non-disease diagnosis and treatment purposes.

Advantageous effects

1) The stable serum iron is a liquid double reagent, does not need to be re-dissolved for preparation, and can be directly used after opening a bottle.

2) Ascorbic acid is unstable and easily decomposed in a solution state, and the addition of a stabilizer to the reagent R1 is advantageous for maintaining the stability and reducibility of ascorbic acid. The reagent R1 is added with an interference removing agent, and thiourea is mixed with ascorbic acid solution, which can be used as a reducing agent and a masking agent to remove the interference of other ions.

3) The reagent selects bathophenanthroline as the chromogen, the bathophenanthroline and ferrous ions form a stable red compound, and compared with the ferrous oxazine, the molar absorption coefficient of the chromogen is higher, so that the reagent has higher sensitivity.

4) The reagent is convenient to use, high in specificity and accuracy, and free of pollution to a biochemical analyzer, and is favorable for further popularization of the reagent in the market.

Drawings

FIG. 1 is a correlation curve for comparative example 1 and example 1;

figure 2 stability of example 1 compared to comparative examples 3 and 4.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

In the kit of this embodiment, when in use, the determination method is to use a micheli 800 full-automatic biochemical analyzer with double reagent functions, and perform determination by an endpoint method, where the dominant wavelength is 578nm, and the operations are as follows:

adding 20 μ l of physiological saline, sample or calibrator, adding 200 μ l of R1 reagent, pre-incubating for 5min, reading absorbance A1, adding 50 μ l of R2 reagent, reacting for 5min, reading absorbance A2, and calculating Δ A.

Sample concentration =

X standard concentration

Sample requirements: fresh serum. The specimen can be preserved for one week at 4 ℃.

Example 1

A conventional serum iron assay kit comprises a reagent R1 and a reagent R2.

Reagent R1:

glycine buffer 60mmol/L

Ascorbic acid 35 g/L

Thiourea 20mmol/L

Hydroxylamine hydrochloride of 30mmol/L

Tween-20 1 g/L

Sodium azide 1 g/L

pH 5

Reagent R2:

glycine buffer 50mmol/L

Bathophenanthroline 2 g/L

pH 3

In this embodiment, the reagents R1 and R2 are prepared by preparing a buffer solution, adjusting the pH with hydrochloric acid and sodium hydroxide, adjusting the pH to an appropriate value, and then adding other substances to dissolve the buffer solution to obtain the assay reagent.

Example 2

Glycine buffer 60mmol/L

Ascorbic acid 35 g/L

Thiosemicarbazide 30mmol/L

Hydroxylamine hydrochloride 35 mmol/L

Tween-20 1 g/L

Sodium azide 1 g/L

pH 5

Reagent R2:

glycine buffer 40mmol/L

3g/L bathophenanthroline

pH 3

The preparation method is the same as example 1.

Example 3

Acetic acid buffer 50mmol/L

Ascorbic acid 35 g/L

Thiosemicarbazide 40mmol/L

Sodium metabisulfite 35 mmol/L

Tween-20 1 g/L

Sodium azide 1 g/L

pH 5.5

Reagent R2:

glycine buffer 45mmol/L

3g/L of bathophenanthroline;

pH 2.5

the preparation method is the same as example 1.

Comparative example 1

Imported Landau iron kit

Comparative example 2

Serum iron determination kit adopted in patent CN 106814063A

The reagent R1 is composed of:

pH4.2 Tris buffer 60mmol/L

Ascorbic acid 100mmol/L

Sodium azide 10mmol/L

Surfactant TritonX-1001%

The reagent R2 is composed of:

pH4.2 Tris buffer 60mmol/L

50mmol/L of ferrocazine

Sodium azide 10mmol/L

In this example, reagent R1 and reagent R2 are prepared by adding buffer Tris, adjusting to an appropriate pH with hydrochloric acid, and then adding other substances.

Comparative example 3

The difference from the serum iron assay kit of example 1 is only that reagent 1 does not contain hydroxylamine hydrochloride, and the other steps are the same as those of example 1.

Comparative example 4

The reagent kit is different from the serum iron assay kit in example 1 only in that the reagent 1 does not contain thiourea, and the other steps are the same as those in example 1.

Performance verification

Test No.)

The example 1 and the comparative example 1 were subjected to a correlation test according to the following test scheme: example 1 and comparative example 1, 40 clinical serum samples were tested simultaneously, the test results are shown in table 2, correlation analysis was performed on the test results, and a correlation coefficient r was calculated; relative deviation (Bias%) of 40 pairs of data was calculated, respectively, using the results of the test of comparative example 1 as a target value. It is required that r is not less than 0.990 and the relative deviation is not more than. + -. 10%.

TABLE 1 correlation comparative experiment results

As can be seen from the detection data in Table 1 and FIG. 1, the correlation between the detection results of the detection kit in example 1 and the detection kit in comparative example 1 is 0.9980, and the correlation is relatively good, which indicates that the kit of the present invention has high consistency with the serum iron assay kit which is approved in the market and has excellent accuracy, so that the detection reagent in example 1 provided by the present invention has good correlation with imported reagent, and can completely replace imported reagent.

Test No. two

The serum iron determination kit is used for testing a sample with the known concentration of 17.9 [ mu ] mol/L, and the absorbance difference value is recorded. The test was carried out using the reagents of example 1 and comparative examples 1 and 2, respectively. The results are shown in Table 2.

TABLE 2 analytical sensitivity test results

Theoretical concentration	Example 1 measurement result Δ A	Comparative example 1 test result Δ a	Comparative example 2 test result Δ a
				17.9 µmol/L	0.046	0.051	0.021

According to the detection data, the absorbance difference of the detection kit in the embodiment 1 is higher than that of the detection kit in the embodiment 3, which indicates that the reagent uses bathophenanthroline as the chromogen instead, and the bathophenanthroline and ferrous ions form a stable red compound, so that the response signal is enlarged, the reaction sensitivity is enhanced, and the analysis sensitivity of the detection kit is improved.

Experiment three

The serum iron determination reagents provided in example 1 and comparative examples 2 and 3 were subjected to a stability test according to the following protocol: the reagents provided in example 1 and comparative examples 2 and 3 were placed together in a 37 ℃ water bath, a control with a target value of 19.5. + -. 1.75 μmol/L L was detected every day, and the change in the measured value of the control was monitored.

TABLE 3 verification of thermal stability of reagents

As can be seen from Table 3 and FIG. 2, the reagent of example 1 provided by the invention has basically no change in the water bath condition at 37 ℃ within 10 days, and has good stability; while the reagents of comparative examples 2 and 3 changed significantly within 10 days under 37 ℃ water bath conditions. The stability of the kit of the example 1 is superior to that of the kits of the comparative examples 2 and 3, and the stability and reducibility of ascorbic acid are favorably maintained by adding the stabilizer hydroxylamine hydrochloride into the reagent, so that the stability of the reagent is favorably maintained.

Experiment four

The serum iron determination kit provided in example 1 and comparative examples 2 and 4 was subjected to an anti-interference test according to the following protocol: dividing Landao biochemical quality control products with target values of 19.5 +/-1.75 mu mol/L into 4 equal parts, dividing each equal part into 5 equal parts, adding different interference substances to ensure that the concentration of the interference substances in serum meets the requirements of table 3, and then respectively adopting the reagents obtained in example 1 and comparative examples 2 and 4 to measure the content of iron in the quality control products, wherein the relative deviation (%) = (the measured mean value of an interference sample-the measured mean value of a control sample)/the measured mean value of the control sample is multiplied by 100%.

TABLE 4 comparison of anti-interference Performance test results

As can be seen from Table 4, the kit of the invention has no obvious interference on the detection result, and the comparative example reagent is obviously interfered when the interfering substances with the above concentrations exist, which shows that the anti-interference capability of the kit of the invention is better than that of the comparative example reagent, and that after the interference removing agent thiourea is added into the reagent, the interference of other substances is effectively avoided, and the anti-interference performance of the reagent is improved.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

1. A serum iron assay kit, characterized in that, the kit contains reagent R1, reagent R2;

wherein the reagent R1 consists of:

glycine buffer 60mmol/L

Ascorbic acid 35 g/L

Thiourea 20mmol/L

Hydroxylamine hydrochloride of 30mmol/L

Tween-20 1 g/L

Sodium azide 1 g/L

pH 5；

Reagent R2:

glycine buffer 50mmol/L

Bathophenanthroline 2 g/L

pH 3；

The volume ratio of the reagent R1 to the reagent R2 is 4: 1.

2. The method for preparing the serum iron assay kit according to claim 1, characterized by comprising the following steps: adding buffer solution into the reagents R1 and R2, adjusting the pH value with hydrochloric acid or sodium hydroxide, adjusting the pH value of the reagent R1 to 5 and the pH value of the reagent R2 to 3, adding other substances according to the proportion, and dissolving to prepare the serum iron determination kit.

3. Use of the serum iron assay kit of claim 1 for the determination of the concentration of iron ions in serum for non-disease diagnostic and therapeutic purposes.