Estradiol chemiluminescent enzyme immunoassay enzyme-labeled diluent and preparation method thereof

Abstract

The invention relates to the technical field of in-vitro diagnostic reagent chemiluminescence, in particular to an estradiol chemiluminescence enzyme immunoassay enzyme-labeled diluent and a preparation method thereof.The preparation method comprises the steps that 80% of purified water is taken, an MES buffer solution and sodium chloride with the corresponding mass are weighed and added into the purified water, the mixture is adjusted to the preset pH value, and a mixed solution is obtained; adding zinc chloride, magnesium chloride, casein, glucan 500, TX405 and PC300 into the mixed solution, stirring and mixing, and fixing the volume to 100% by using purified water to obtain a fixed solution; and filtering the standing solution through a nylon filter membrane to obtain a diluent. By optimizing buffer salt components, protein, saccharides, a surfactant and the like in the enzyme-labeled diluent, the thermal stability, titer, linearity and precision of a reagent can be improved, the accelerated stability of an E2 enzyme-labeled antibody in a working solution can be improved by 95% or above by adding casein and glucan 500, and therefore the problem that an existing enzyme-labeled diluent is poor in stability is solved.

Description

Technical Field

[0001] The invention relates to the technical field of chemiluminescence of in vitro diagnostic reagents, and in particular to an estradiol chemiluminescence enzyme immunoassay enzyme-labeled diluent and a preparation method thereof. Background Art

[0002] Estradiol (E2) is a steroidal estrogen, with the highest content among sex hormones, and is the main component of human estrogen. There are two types, α and β, and the α type has a stronger physiological effect. Estradiol is mainly secreted by the follicles and corpus luteum of the female ovaries, and by the adrenal cortex of men. Estradiol not only has the physiological effect of promoting and maintaining female reproductive organs and secondary sexual characteristics, but also has a significant impact on the endocrine system, cardiovascular system, body metabolism, bone growth and development, skin and other aspects. Clinically, serum estradiol levels reflect female ovarian activity and are an important indicator for monitoring follicle maturation and ovarian hyperstimulation syndrome. It is helpful for the diagnosis of amenorrhea, menstrual dysfunction, estrogen-producing tumors, precocious puberty and other diseases. Increased estradiol is common in pregnant women, female precocious puberty, irregular menstruation, ovarian tumors, hyperthyroidism and liver cirrhosis; decreased estradiol often causes female sexual immaturity, delayed puberty, secondary amenorrhea and so on.

[0003] At present, the main methods for estradiol detection include enzyme-linked immunosorbent assay, chemiluminescent immunoassay, radioimmunoassay, immunochromatographic test strips, etc. Among them, chemiluminescent immunoassay is based on the specific reaction of antigen and antibody, and releases chemiluminescent signals through sandwich or competitive reaction to detect the concentration of estradiol in the sample. At the same time, this method has high sensitivity, strong specificity, wide linear range and good repeatability, and can use a fully automatic chemiluminometer to achieve fully automatic high-throughput detection.

[0004] Among the raw materials used in in vitro diagnostic reagents, proteins such as antibodies, antigens, and enzymes are the most important key raw materials. Their specific binding and catalytic reaction functions are very sensitive to potential harmful effects during pretreatment, storage, use and processing. In order to ensure the validity and accuracy of diagnostic tests, these raw proteins must remain stable and effective. Therefore, appropriate protein protectants must be added to diagnostic reagents. Usually, adding protective proteins, sugars, and small molecule compounds to buffers can improve their stability. Sugar is the most common and widely used type of protective agent. It is a non-specific stabilizer for proteins. The main principle of its protection is to form a molecular layer on the surface of the protein, including but not limited to glucose, trehalose, mannitol, sucrose, dextran, etc.

[0005] Usually, alkaline phosphatase-labeled estradiol antibodies need to be diluted thousands of times when used. Manual operation is prone to errors, resulting in batch differences, and need to be stored at -20°C or lower. Repeated freezing and thawing can easily reduce its stability and immune activity. When stored in a solution state, the potential factors that make the antibody unstable will increase. Therefore, it is very necessary to develop a diluent that can stably use and store estradiol enzyme-labeled antibodies in working solution. Summary of the invention

[0006] The purpose of the present invention is to provide an enzyme-labeled diluent for chemiluminescent enzyme immunoassay of estradiol and a preparation method thereof, aiming to solve the problem of poor stability of the existing enzyme-labeled diluent.

[0007] To achieve the above object, in a first aspect, the present invention provides a method for preparing an enzyme-labeled dilution solution for estradiol chemiluminescent enzyme immunoassay, comprising the following steps:

[0008] Take 80% of the prepared amount of purified water, weigh and add corresponding amounts of MES buffer and sodium chloride to the purified water, adjust to a preset pH value, and obtain a mixed solution;

[0009] Add zinc chloride, magnesium chloride, casein, dextran 500, TX405, and PC300 to the mixed solution, stir and mix, and dilute to 100% with purified water to obtain a fixed solution;

[0010] The fixed solution after standing is filtered through a nylon filter membrane to obtain a diluted solution.

[0011] Among them, in “taking 80% of the prepared amount of purified water, weighing and adding corresponding masses of MES buffer and sodium chloride to the purified water, adjusting to a preset pH value, and obtaining a mixed solution”, the preparation temperature of the purified water is 25±3°C; the preset pH value is 6.8.

[0012] Among them, in the step of “add zinc chloride, magnesium chloride, casein, dextran 500, TX405 and PC300 to the mixed solution, stir and mix, and dilute to 100% with purified water to obtain a fixed solution”, the stirring and mixing time is 30 minutes.

[0013] Among them, in “filtering the fixed solution after standing through a nylon filter membrane to obtain a diluted solution”, the nylon filter membrane is a 0.2 μm nylon filter membrane, and the standing time is 30 minutes.

[0014] In a second aspect, an estradiol chemiluminescent enzyme immunoassay enzyme-labeled diluent is provided, using the preparation method of the estradiol chemiluminescent enzyme immunoassay enzyme-labeled diluent described in the first aspect.

[0015] It includes 100 mM MES, 0.9% sodium chloride, 0.01 mM zinc chloride, 5 mM magnesium chloride, 0.2% casein, 0.5% dextran 500, 0.05% TX405, 0.05% PC300, and the pH of the buffer is 6.8.

[0016] The invention discloses a method for preparing an enzyme-labeled diluent for estradiol chemiluminescent enzyme immunoassay, comprising the following steps: taking 80% of the prepared amount of purified water, weighing and adding corresponding masses of MES buffer and sodium chloride to the purified water, adjusting to a preset pH value, and obtaining a mixed solution; adding zinc chloride, magnesium chloride, casein, dextran 500, TX405, and PC300 to the mixed solution, stirring and mixing, and settling to 100% with purified water to obtain a fixed solution; filtering the fixed solution after standing through a nylon filter membrane to obtain a diluent. By optimizing the buffer salt components, proteins, sugars, surfactants, etc. in the enzyme-labeled diluent, the thermal stability, potency, linearity, and precision of the reagent can be improved, and the addition of casein and dextran 500 can improve the accelerated stability of the E2 enzyme-labeled antibody in the working solution by more than 95%. Compared with commercial enzyme diluents, the method has better performance, reduces the production cost of enterprises, and thus solves the problem of poor stability of existing enzyme-labeled diluents. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The present invention provides a flow chart of a method for preparing an enzyme-labeled dilution solution for chemiluminescent enzyme immunoassay of estradiol. DETAILED DESCRIPTION

[0019] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0020] See also Figure 1 In a first aspect, the present invention provides a method for preparing an enzyme-labeled dilution solution for estradiol chemiluminescent enzyme immunoassay, comprising the following steps:

[0021] S1 takes 80% of the prepared amount of purified water, weighs and adds corresponding amounts of MES buffer and sodium chloride to the purified water, adjusts the pH value to a preset value, and obtains a mixed solution;

[0022] The configuration temperature of the purified water is 25±3° C.; the preset pH value is 6.8.

[0023] Specifically, at room temperature of 25±3°C, take 80% of the prepared amount of purified water; add MES and sodium chloride to the purified water; after complete dissolution, adjust the pH to 6.8 with 3M hydrochloric acid;

[0024] S2: adding zinc chloride, magnesium chloride, casein, dextran 500, TX405 and PC300 to the mixed solution, stirring and mixing, and diluting the volume to 100% with purified water to obtain a fixed solution;

[0025] The stirring and mixing time is 30 minutes.

[0026] Specifically, the remaining components of zinc chloride, magnesium chloride, casein, dextran 500, TX405, and PC300 are added in sequence, and stirred at a constant speed for more than 30 minutes to fully dissolve them; after complete dissolution, purified water is added to 100%;

[0027] S3: filtering the fixed solution after standing through a nylon filter membrane to obtain a diluted solution.

[0028] The nylon filter membrane is a 0.2 μm nylon filter membrane, and the standing time is 30 minutes.

[0029] Specifically, the solution was allowed to stand for 30 minutes and then filtered using a 0.2 μm nylon filter membrane to obtain an enzyme dilution solution, which was then stored at 2° C. to 8° C. for later use.

[0030] Example 1

[0031] This example mainly optimizes the types of buffer salts in the diluent. The buffers for comparison include: 5 buffer salts of 100 mM, namely MES (2-(N-morpholino)ethanesulfonic acid), HEPES (4-hydroxyethylpiperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), Tris (tris(hydroxymethyl)aminomethane) and PBS (phosphate). Different diluents are prepared according to the following table:

[0032]

[0033] The above five different dilutions were prepared into enzyme label working solutions, S1 is the photon quantity when the calibrator concentration is 0, S2 is the low concentration point, S3 is the medium concentration point, and S4 is the high concentration point. According to the steps of the Uni-Estradiol Assay Kit (Magnetic Particle Chemiluminescence Method), the photon quantity and signal-to-noise ratio S / N of different concentrations of E2 calibrators were detected on the Uni-Estradiol Series Fully Automatic Chemiluminescence Immunoassay IA-260, and the results are shown in Table 1.

[0034]

[0035]

[0036] Table 1 Effects of different buffer salts on photon quantity and signal-to-noise ratio

[0037] From the data in Table 1, we can see the changes in the 0-value photon amount and signal-to-noise ratio of different buffer salts. The higher the 0-value photon amount, the greater the signal-to-noise ratio. Among them, the 0-value photon amount and signal-to-noise ratio of the dilution formula 1-1 are significantly higher than those of the other groups, that is, the best effect is achieved when MES buffer salt is used to prepare enzyme dilution solution.

[0038] Experiment 2 Protein Optimization

[0039] Example 2

[0040]

[0041] This embodiment mainly optimizes the protein and its concentration in the diluent to prevent the protein from being adsorbed on the container surface and stabilize the function of the labeled antibody. The proteins for comparison include 4 proteins at 2-5 g / L, namely BSA (bovine serum albumin), casein, gelatin and fish skin gelatin. Different diluents are prepared according to the following table:

[0042] The above 8 different dilutions were prepared into enzyme label working solutions, which were packaged and placed in a 2-8°C refrigerator and a 37°C oven for 7 days. The test method was the same as in Example 1, and the photon quantity, signal-to-noise ratio S / N and accelerated stability of E2 calibrators of different concentrations were detected. The results are shown in Table 2.

[0043] Table 2 Effects of different proteins and their concentrations on photon quantity, signal-to-noise ratio and acceleration stability

[0044]

[0045]

[0046] The data in Table 2 show the changes in signal-to-noise and accelerated stability at different protein concentrations. Among them, after the diluent of formula 2-3 with 2 g / L casein was accelerated at 37°C for 7 days, the photon signal retention rate was within 90-100%, which was higher than that of other groups. That is, when 2 g / L casein was added to the diluent, the stability of the reagent was the best.

[0047] Experiment 3 Sugar Optimization

[0048] Example 3

[0049] This example mainly optimizes the sugars in the diluent. Sugars can inhibit protein denaturation to improve the stability of the reagent. The sugars used for comparison include 5 g / L of four kinds of sugars, namely sucrose, trehalose, pullulan and dextran 500. Different diluents are prepared according to the following table:

[0050]

[0051]

[0052] The above four different dilutions were prepared into enzyme label working solutions, which were packaged and placed in a 2-8°C refrigerator and a 37°C oven for 7 days. The test method was the same as in Example 1, and the photon quantity, signal-to-noise ratio S / N and accelerated stability of E2 calibrators of different concentrations were detected. The results are shown in Table 3.

[0053] Table 3 Effects of different sugars on accelerated stability

[0054]

[0055] From the data in Table 3, it can be seen that different sugars have no significant effect on the signal-to-noise ratio, and the signal retention rates of the calibrants at various concentrations of formulations 3-1 and 3-4 are all above 90%. When 5 g / L of dextran 500 is added to formulation 3-4, the signal retention rate is within 95-100%, further improving the stability of the reagent, that is, when 5 g / L of dextran 500 is added to the diluent, the stability of the reagent is the best.

[0056] Experiment 4 Surfactant Optimization

[0057] Example 4

[0058] Surfactants have good wetting, solubilizing and dispersing properties, and can improve the contact between the sample and the reagent, and improve the reaction efficiency and reagent precision. This embodiment mainly optimizes the surfactant in the diluent. The surfactants compared include Tween-20, Triton X-100, Triton X-405, Brij35, and PEG400. Different diluents are prepared according to the following table:

[0059]

[0060] The above five different dilutions were prepared into enzyme label working solutions, and four different concentrations of calibrators were tested 10 times each according to the method of Example 1, and the coefficient of variation CV was calculated. The results are shown in Table 4.

[0061] Table 4 Effect of different surfactants on precision

[0062]

[0063] According to the data in Table 4, it can be seen that the CV values ​​of the calibrants of formula 4-3 at all concentrations are significantly lower than those of other surfactants, that is, adding 0.05% Triton X-405 to the diluent can significantly improve the detection precision of the detection reagent.

[0064] Comparison of Example 5 with commercial enzyme label diluent

[0065] In this example, the enzyme-labeled diluent 4-3 optimized by the present invention and the commercial enzyme-labeled diluent were used to prepare two enzyme-labeled working solutions for estradiol chemiluminescent enzyme immunoassay, and the signal-to-noise ratio, linearity, precision and stability of each group at different sample concentrations were compared. The results are shown in Table 5:

[0066] Table 5 Comparison of optimized enzyme dilution 4-3 and commercial enzyme dilution

[0067]

[0068] Experimental conclusion: According to the comparative results analysis in Table 5, the optimized dilution formula 4-3 has the best comprehensive performance for the E2 chemiluminescent enzyme immunoassay reagent, with the best signal-to-noise ratio, linearity, precision and accelerated stability. It has stable performance and can effectively reduce costs.

[0069] The above disclosure is only a preferred embodiment of an estradiol chemiluminescent enzyme immunoassay enzyme-labeled dilution solution and a preparation method thereof of the present invention. Of course, this cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for preparing an enzyme-labeled diluent for estradiol chemiluminescent enzyme immunoassay, characterized in that: The following steps are involved: Take 80% of the prepared amount of purified water, weigh and add corresponding amounts of MES buffer and sodium chloride to the purified water, adjust to a preset pH value, and obtain a mixed solution; Add zinc chloride, magnesium chloride, casein, dextran 500, TX405, and PC300 to the mixed solution, stir and mix, and dilute to 100% with purified water to obtain a fixed solution; The fixed solution after standing is filtered through a nylon filter membrane to obtain a diluted solution.

2. The method for preparing an estradiol chemiluminescent enzyme immunoassay enzyme-labeled dilution according to claim 1, wherein: In "taking 80% of the prepared amount of purified water, weighing and adding corresponding masses of MES buffer and sodium chloride to the purified water, adjusting to a preset pH value, and obtaining a mixed solution", the preparation temperature of the purified water is 25±3° C.; and the preset pH value is 6.

8.

3. The method for preparing an estradiol chemiluminescent enzyme immunoassay enzyme-labeled dilution solution as claimed in claim 1, characterized in that: In the step “add zinc chloride, magnesium chloride, casein, dextran 500, TX405 and PC300 to the mixed solution, stir and mix, and dilute to 100% with purified water to obtain a fixed solution”, the stirring and mixing time is 30 min.

4. The method for preparing an estradiol chemiluminescent enzyme immunoassay enzyme-labeled dilution solution as claimed in claim 1, characterized in that: In “filtering the fixed solution after standing through a nylon filter membrane to obtain a diluted solution”, the nylon filter membrane is a 0.2 μm nylon filter membrane, and the standing time is 30 minutes.

5. An estradiol chemiluminescent enzyme immunoassay enzyme-labeled diluent, using the method for preparing the estradiol chemiluminescent enzyme immunoassay enzyme-labeled diluent according to any one of claims 1 to 4, characterized in that: It includes 100 mM MES, 0.9% sodium chloride, 0.01 mM zinc chloride, 5 mM magnesium chloride, 0.2% casein, 0.5% dextran 500, 0.05% TX405, 0.05% PC300, and the pH of the buffer is 6.8.

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