Method for improving unsealing stability of folic acid or vitamin B12 detection kit

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to a method for improving the unsealing stability of a folic acid or vitamin B12 detection kit. The method provided by the invention comprises the following steps: adding mineral oil with specific kinematic viscosity into alkaline sample treatment liquid in the folic acid or vitamin B12 detection kit. The folic acid or vitamin B12 detection kit prepared by the method has the advantages that the stability is obviously improved, the service life of the unsealed kit can be prolonged, and the precision is good.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and particularly to a method for improving the opening stability of a folic acid or vitamin B12 detection kit and a kit. Background Art

[0002] Folic acid (FA) is a general term for a group of compounds with similar chemical structures and biochemical characteristics. It belongs to one of the B vitamins and is one of the essential vitamins in the human body. Vitamin B12 (Vitamin B12, abbreviated as B12), also known as cobalamin, is a general term for a group of red corrinoid compounds in which cobalt binds to a corrin ring. It is a vitamin containing a metal element, and its unique structure plays an important role in maintaining the hematopoietic function and nervous system function of the human body. Folic acid supports the operation of cell functions, regulates gene activities, promotes the production of red blood cells and white blood cells, and the renewal of the skin and intestinal wall. It is a necessity for the synthesis of purines and pyrimidines. Lack of folic acid will lead to impaired cell replication and the accumulation of harmful metabolites. The role of B12 is to promote the turnover and utilization of folic acid for the synthesis of thymidine deoxynucleotide and DNA. If B12 is lacking, folic acid is difficult to be reused by the body. Therefore, anemia caused by B12 deficiency, like that caused by lack of folic acid, will also lead to megaloblastic changes in red blood cells (giant red blood cells, giant oval red blood cells). Deficiency of folic acid and B12 can severely damage the development and function of the nervous system, mostly manifested as growth retardation and nervous system defects. Clinically, if diagnosed in time and given supplementary treatment early, the progression of the disease can be stopped and even the condition can be reversed.

[0003] Paraffin oil is a mixture of alkanes extracted from mineral sources, especially petroleum fractions. Paraffin oil is transparent and colorless, mainly composed of alkanes and cyclic paraffins (related to petrolatum), and its density is about 0.8 g / cm 3 ³. Paraffin oil has stable properties, is not easy to volatilize, and is resistant to light, heat, acids and alkalis. It is widely used in traditional industrial fields such as textiles, metallurgy, rubber, plastics, building materials and batteries.

[0004] White oil is a colorless, transparent, oily liquid without odor. It is stable to acids, light and heat, insoluble in ethanol, soluble in ether, benzene, petroleum ether, etc., and can be miscible with most fatty oils. It is a mixture of liquid hydrocarbons, mainly composed of a mixture of normal and isomeric alkanes with C16 - C31. It is obtained by dewaxing, carbonization, neutralization, activated clay refining and other treatments from the high-boiling fraction (i.e., lubricating oil fraction) of petroleum fractionation. White oil is a widely used oil-soluble raw material in cosmetics and can be used to prepare almost all cosmetics such as bath oils, various skin care creams, lotions, hair care products, lipsticks, etc.

[0005] In vitro diagnostic reagents are reagents used for in vitro testing. The stability after opening refers to the stability of the reagent after the bottle is opened, and it examines the influence of factors such as air, humidity, temperature, and light on the performance indicators of in vitro diagnostic reagents under normal conditions.

[0006] Currently, the development trend of in vitro diagnostic reagents is from manual operation to full automation, and the reagents are paired with corresponding fully automatic instruments for testing. The external conditions after the reagent is opened and loaded onto the machine are somewhat different from the original storage conditions.

[0007] The detection methods for folic acid and vitamin B12 mainly include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, etc. The principles of these methods are basically similar, and they all use the competitive method principle for detection. Since most of the folic acid or vitamin B12 in serum binds to some endogenous proteins, and the proportion of free folic acid or vitamin B12 is less than half, the reagent cannot directly detect the bound folic acid or vitamin B12. Therefore, before detection, it is usually necessary to pretreat the serum sample to release all the folic acid or vitamin B12 and then perform the detection.

[0008] The methods for sample pretreatment include boiling method, organic solvent extraction method, acid-base treatment method, etc.

[0009] The boiling method and the organic solvent extraction method require manual operation, with cumbersome steps, difficult to achieve automation, and low dissociation efficiency and poor accuracy.

[0010] Folic acid or vitamin B12 is unstable under acidic conditions. When using the acid treatment method, it is necessary to add a high concentration of ascorbic acid to protect it. This method does not completely destroy the structure of endogenous proteins in serum, and the dissociation efficiency is not easy to control. At the same time, potential interfering factors in the sample, such as rheumatoid factor (RF), heterophilic antibody, complement, etc., may still interfere with the detection. Therefore, the method commonly used in current commercial kits is dissociation under alkaline conditions.

[0011] The principle is that strong alkali can denature proteins, and at the same time, a specific reducing agent is added to break the disulfide bonds of proteins, so that folic acid or vitamin B12 is released. The reason for using strong alkali is to expose the deeply buried disulfide bonds. This treatment method is very thorough, which can change the protein that wraps folic acid or vitamin B12 into a linear structure, so that folic acid or vitamin B12 is fully released. At the same time, it can also destroy other proteins in the sample, such as protein interference factors such as RF, heterophilic antibody, complement, etc., making them lose their effects, thereby further ensuring the accuracy of the detection results.

[0012] Currently, the commercial kits for the detection of folic acid and vitamin B12 usually have a shorter expiration date after opening than similar detection kits, or have harsh usage conditions, time-consuming and laborious. Summary of the Invention

[0013] In view of this, the present invention provides a method for improving the opening stability of a folic acid or vitamin B12 detection kit, solving the technical problems that the folic acid or vitamin B12 detection kit has harsh use conditions or insufficient reagent stability among the same type of detection kits.

[0014] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0015] The present invention provides the application of mineral oil in any of the following items:

[0016] i) Improving the opening stability of the detection product;

[0017] ii) Improving the precision of the detection product;

[0018] The detection product is a product for detecting folic acid or vitamin B12.

[0019] In some specific embodiments of the present invention, the product of the above application is a reagent, a kit or a device.

[0020] In some specific embodiments of the present invention, the product of the above application is a product for detecting folic acid, which may be a kit, including a magnetic particle suspension, an enzyme conjugate, a sample treatment solution and / or a calibrator;

[0021] The sample treatment solution includes an alkaline sample treatment solution.

[0022] In some specific embodiments of the present invention, the product of the above application is a product for detecting folic acid, which may be a kit, including a magnetic particle suspension, an enzyme conjugate, an intrinsic factor protein solution, a sample treatment solution and / or a calibrator;

[0023] The sample treatment solution includes an alkaline sample treatment solution.

[0024] The present invention also provides a preparation method of a detection product, including the step of mixing mineral oil and an alkaline sample treatment solution, and the detection product is a product for detecting folic acid or vitamin B12.

[0025] The present invention also provides a product for detecting folic acid or vitamin B12, which contains an alkaline sample treatment solution, and the alkaline sample treatment solution contains mineral oil.

[0026] In some specific embodiments of the present invention, the kinematic viscosity of the mineral oil in the above application, preparation method or product is 7.5 - 29 m 2 / s, 7.5 - 15 m 2 / s, 15 - 29 m 2 / s, 9.69 - 33.03 m 2 / s, 16.09 - 26.79m2 / s, 9.69 - 26.79 m 2 / s, 9.69 - 16.09 m 2 / s, 16.09 - 33.03 m 2 / s, 26.79 - 33.03 m 2 / s or 10 - 30 m 2 / s;

[0027] When the mineral oil is paraffin oil, its kinematic viscosity at 40°C is 9.69 - 33.03 m 2 / s, 16.09 - 26.79 m 2 / s, 9.69 - 26.79 m 2 / s, 9.69 - 16.09 m 2 / s, 16.09 - 33.03 m 2 / s, 26.79 - 33.03 m 2 / s or 10 - 30 m 2 / s;

[0028] When the mineral oil is white oil, its kinematic viscosity at 40°C is 7.5 - 29 m 2 / s, 7.5 - 15 m 2 / s or 15 - 29 m 2 / s.

[0029] In some specific embodiments of the present invention, the mineral oil in the above applications, preparation methods or products can be a mineral oil containing low - density paraffinized hydrocarbons or a low - density inert organic substance;

[0030] The low - density inert organic substance can be naphthenic oil or castor oil.

[0031] In some specific embodiments of the present invention, the mineral oil in the above applications, preparation methods or products can be paraffinic mineral oil, naphthenic mineral oil, aromatic mineral oil or mixed - hydrocarbon mineral oil.

[0032] In some specific embodiments of the present invention, the alkaline sample treatment solution in the above applications, preparation methods or products contains sodium hydroxide or potassium hydroxide. In some specific examples, the alkaline sample treatment solution can be 0.6 mol / L NaOH.

[0033] The present invention has the following beneficial effects.

[0034] Experiments show that for the folic acid detection reagent without added mineral oil, after being continuously loaded on the instrument for 28 days, the deviation of most samples exceeded ±10%. While for the folic acid detection reagent with added mineral oil, after being continuously loaded on the instrument for 28 days, the deviation of all samples was within 10%. For the vitamin B12 detection reagent without added mineral oil, after being continuously loaded on the instrument for 42 days, the deviation of most samples exceeded ±10%. While for the vitamin B12 detection reagent with added mineral oil, after being continuously loaded on the instrument for 42 days, the deviation of all samples was within 10%. It can be seen that mineral oil can extend the stability of the reagent loaded on the instrument and the service life after opening.

[0035] The present invention also reveals the influence of kinematic viscosity on the precision of the detection reagent. Experiments show that when the kinematic viscosity (at 40 °C) of the mineral oil is 5.95 m 2 / s, the precisions of the folic acid detection kit and the vitamin B12 detection kit are 7.33% and 6.16% respectively, with relatively poor precision; when the kinematic viscosity (at 40 °C) of the mineral oil > 50.22 m 2 / s, the precisions of both the folic acid detection kit and the vitamin B12 detection kit are > 10%, with low measured values and relatively poor precision; when the kinematic viscosity (at 40 °C) of the mineral oil is in the range of 9.69 - 33.03 m 2 / s, the precisions of both the folic acid detection kit and the vitamin B12 detection kit are within 5.0%, with relatively good precision. It can be seen that mineral oil with a specific kinematic viscosity can improve the precision of the detection reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0037] Figure 1 It shows the detection reagent after adding mineral oil, where the arrow indicates the mineral oil that forms a protective film above the detection reagent. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The present invention discloses a method for improving the opening stability of a folic acid or vitamin B12 detection kit. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technical solution of the present invention.

[0039] The folic acid detection kit involved in the present invention can be any folic acid kit containing the above-mentioned alkaline sample treatment agent. For example, the reagent mentioned in the public literature (Xie Qian. Establishment and effect evaluation of chemiluminescence immunoassay for serum folic acid using magnetic microparticles [D]. Xinxiang Medical University, 2023.), or the detection reagent mentioned in the patent application for the method for determining the content of folic acid in human red blood cells with the patent application number 200710016175.1.

[0040] The vitamin B12 detection kit involved in the present invention can be any vitamin B12 kit containing the above-mentioned alkaline sample treatment agent. For example, the detection reagent mentioned in the patent application for the vitamin B12 detection kit and method and its sample pretreatment method with the patent application number 201610165967.4.

[0041] It should be understood that the expression “one or more of...” individually includes each of the objects recited after said expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression “and / or” in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0042] The terms “comprising,” “having,” or “including,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-limiting, e.g., not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0043] It should be understood that the order of steps or the order of performing certain actions is not important as long as the present application remains operable. In addition, two or more steps or actions can be performed simultaneously.

[0044] The use of any and all examples or exemplary language such as “for example” or “including” herein is merely intended to better illustrate the present application and does not limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present application.

[0045] In addition, the numerical ranges and parameters used to define the present application are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by “about.” Here, “about” generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0046] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all ordinary commercially available products and can be purchased from the market.

[0047] The present invention will be further described below in conjunction with embodiments.

[0048] Example 1

[0049] Take mineral oil (in this example, paraffin oil with a kinematic viscosity of 16.09 m 2 / s and white oil with a kinematic viscosity of 15 m 2 / s) and use it in combination with the folic acid detection kit (magnetic particle chemiluminescence method) produced by Zhengzhou Antu Bioengineering Co., Ltd. (registration number: Henan Medical Device Approval No. 20192400299) to verify the opening (loading machine) stability of the kit. The folic acid detection kit includes a magnetic particle suspension, an enzyme conjugate, sample treatment solution A, sample treatment solution B, sample treatment solution C, and a calibrator. According to the description in its product manual, the reagent can be used on the loading machine for 14 days after opening.

[0050] Verification method: Take mineral oil and use it in combination with the folic acid detection kit, and use the folic acid detection kit alone for verification at the same time. Specifically, after opening each reagent of the folic acid detection kit, add 1.0 mL of mineral oil to the sample treatment solution A (0.5 mol / L NaOH), and then perform detection on the machine according to the steps in the manual.

[0051] Use 10 samples evenly distributed in the linear range of 1-20 ng / mL to detect 1 time respectively on the loading machine continuously for 14 days or 28 days after opening and the reagent not opened on the loading machine (loading machine day 0), and calculate the deviation between the results of 14 days or 28 days on the loading machine and the results of loading machine day 0. Deviation = (results of 14 or 28 days on the loading machine - results of loading machine day 0) / results of loading machine day 0. The deviation should be within ±10%. The results are shown in Table 1.

[0052] Table 1

[0053]

[0054] As can be seen from Table 1, compared with the reagent not loaded on the machine (loading machine day 0), the deviation of the reagent without added mineral oil after continuous loading on the machine for 14 days is within 10%, but most of the samples deviate beyond ±10% when compared with loading machine day 0 after 28 days of loading. For the reagent added with mineral oil, when compared with the reagent not loaded on the machine (loading machine day 0) after continuous loading on the machine for 14 days or 28 days, the deviation is within 10%. It can be seen that mineral oil can extend the stability of the reagent on the loading machine (extend the service life after opening).

[0055] Example 2

[0056] Take mineral oil (in this example, paraffin oil with a kinematic viscosity of 16.09 m2 Paraffin oil with a kinematic viscosity of 15 m 2 / s and white oil with a kinematic viscosity of 15 m / s) are used in combination with the Vitamin B12 Detection Kit (Magnetic Particle Chemiluminescence Method) produced by Zhengzhou Antu Bio-Engineering Co., Ltd. (Registration Certificate Number: Yu Medical Device Approval 20192400237) to verify the stability of the kit after opening (loading on the instrument). The Vitamin B12 Detection Kit includes a magnetic particle suspension, an enzyme conjugate, an intrinsic factor protein solution, sample treatment solution A, sample treatment solution B, sample treatment solution C, and a calibrator. According to the description in its product manual, the reagents can be used on the instrument for 28 days after opening.

[0057] Verification method: Use mineral oil in combination with the Vitamin B12 Detection Kit and use the Vitamin B12 Detection Kit alone for verification simultaneously. Specifically, after opening each reagent of the Vitamin B12 Detection Kit, add 1.0 mL of mineral oil to the sample treatment solution A (0.6 mol / L NaOH), and then perform the detection on the instrument according to the steps in the manual.

[0058] Use 10 samples evenly distributed in the linear range of 100 - 2000 pg / mL to perform 1 detection respectively on the instrument continuously for 28 days or 42 days after opening and the unopened reagent (loaded on the instrument on day 0). Calculate the deviation between the results of 28 days or 42 days of loading on the instrument and the results of loading on the instrument on day 0. Deviation = (Results of 28 days or 42 days of loading on the instrument - Results of loading on the instrument on day 0) / Results of loading on the instrument on day 0. The deviation should be within ±10%. The results are shown in Table 2.

[0059] Table 2

[0060]

[0061] As can be seen from Table 2, for the reagent without added mineral oil, when continuously loaded on the instrument for 28 days compared with the unloaded reagent (loaded on the instrument on day 0), the deviation is within 10%. However, when loaded on the instrument for 42 days compared with the instrument on day 0, most of the samples have a deviation exceeding ±10%. For the reagent with added mineral oil, when continuously loaded on the instrument for 28 days or 42 days compared with the unloaded reagent (loaded on the instrument on day 0), the deviation is within 10%. It can be seen that mineral oil can extend the stability of the reagent loaded on the instrument (extend the service life after opening).

[0062] Example 3

[0063] Different types of mineral oils are mainly distinguished by their kinematic viscosity. Therefore, in this example, it is explored whether different kinematic viscosities will result in different detection performances of the kit.

[0064] Use mineral oils with kinematic viscosities (at 40°C) of 5.95, 9.69, 16.09, 26.79, 33.03, 50.22 m 2Paraffin oil with a kinematic viscosity (at 40 °C) of 7.5, 15, 29, 51, 70, 100 m 2 / s and white oil were used in combination with the folic acid kit and vitamin B12 kit for precision verification.

[0065] Folic acid (FA) kit: A quality control product with a concentration of approximately 13 ng / mL was continuously detected in 10 wells at different kinematic viscosities, and the average value ( ) and standard deviation (SD) of the 10 measured concentration values were calculated. The repeatability (CV%) = (SD / ) × 100%. The results are shown in Table 3.

[0066] Table 3

[0067]

[0068] Vitamin B12 kit: A quality control product with a concentration of approximately 380 pg / mL was continuously detected in 10 wells at different kinematic viscosities, and the average value ( ) and standard deviation (SD) of the 10 measured concentration values were calculated. The repeatability (CV%) = (SD / ) × 100%. The results are shown in Table 4.

[0069] Table 4

[0070]

[0071] Conclusion: From the above results, it can be seen that when the kinematic viscosity (at 40 °C) of paraffin oil is in the range of 9.69 - 33.03 m 2 / s, the precision of both the folic acid detection kit and the vitamin B12 detection kit is within 5%, and the precision is good. When the kinematic viscosity (at 40 °C) of paraffin oil is 5.95 m 2 / s, there will be phenomena of jumping up or down, resulting in unstable detection. The precisions of the folic acid detection kit and the vitamin B12 detection kit are 7.33% and 6.16% respectively, and the precision is poor. When the kinematic viscosity (at 40 °C) > 50.22 m 2 / s, regardless of whether paraffin oil or white oil is used, the precision of both the folic acid detection kit and the vitamin B12 detection kit is > 10%, and the highest can reach 25.27%. There is an obvious situation of low measured values, resulting in poor precision. In summary, the preferred kinematic viscosity (at 40 °C) is 10 - 30 m 2 / s.

[0072] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of mineral oil in any of the following: i) Improve the unsealing stability of the tested products; ii) Improve the precision of testing products; The detection product is a product for detecting folic acid or vitamin B12.

2. The use according to claim 1, characterized in that The kinematic viscosity of the mineral oil at 40°C is 7.5-33.03 m 2 / s.

3. The use according to claim 1 or 2, characterized in that: The mineral oil is paraffin oil or white oil.

4. The use according to any one of claims 1 to 3, characterized in that The product is a reagent, a kit or a device.

5. A method for preparing a detection product, wherein the detection product is a product for detecting folic acid or vitamin B12, characterized in that: The method comprises the steps of mixing mineral oil with an alkaline sample treatment solution.

6. The preparation method according to claim 5, characterized in that: The kinematic viscosity of the mineral oil at 40°C is 7.5-33.03 m 2 / s.

7. The preparation method according to claim 5 or 6, characterized in that: The mineral oil is paraffin oil or white oil.

8. A product for detecting folic acid or vitamin B12, characterized in that: The method comprises an alkaline sample processing liquid, wherein the alkaline sample processing liquid comprises mineral oil.

9. The product according to claim 8, characterized in that The kinematic viscosity of the mineral oil at 40°C is 7.5-33.03 m 2 / s.

10. The product according to claim 8 or 9, characterized in that The mineral oil is paraffin oil or white oil.

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