Preparation process of myoglobin for chemical luminescence immunoassay quality control product

Abstract

The application discloses a kind of myoglobin preparation process for chemiluminescence immunoassay quality control product, it is related to myoglobin preparation technical field, comprising the following steps: raw material pretreatment: after removing impurities of fresh pig heart muscle tissue, it is placed in automatic cleaning cutting equipment and is cut and cleaned, myoglobin extraction: muscle tissue after pretreatment is placed in grinding mechanism, and extraction fluid is added to grind, centrifugal separation: after extraction, mixed liquid is placed in high-speed centrifugal separation equipment and is centrifuged, to obtain supernatant, purification treatment: supernatant is purified by multistage filtration separation equipment, stabilization treatment: after purification, myoglobin solution is added with stabilizer, placed in gradient temperature control stirring system and stirred and mixed, low-temperature preservation, solve the problem that muscle tissue is cut into meat foam by manual shearing mode, grinding is not enough, so that separation effect is poor, leading to the problem that myoglobin purity is not high and activity is easily affected.

Description

Technical Field

[0001] This invention relates to the field of myoglobin preparation technology, specifically a process for preparing myoglobin for chemiluminescent immunoassay quality control. Background Technology

[0002] Chemiluminescence immunoassay technology is widely used in clinical diagnosis due to its advantages such as high sensitivity and high specificity. As an important component of this technology, the quality of quality control materials directly affects the reliability of test results. Myoglobin is a common component of quality control materials, and the optimization of its preparation process is crucial.

[0003] Chinese Patent Publication No. CN112851801A discloses a method for preparing antigens for chemiluminescent immunoassay calibrators or quality control products, comprising the following steps: homogenization of porcine heart tissue, ammonium sulfate precipitation, ion exchange chromatography, and hydrophobic chromatography. This invention uses porcine myoglobin instead of human myoglobin, solving the raw material source problem. Furthermore, the preparation method is simple, yields high myoglobin production, and has a high effective activity concentration, meeting the requirements for large-scale production. Simultaneously, it meets the requirements for chemiluminescent immunoassay calibrators or quality control products, exhibiting good linear correlation and more stable performance than recombinant myoglobin, with a 7-day accelerated degradation rate of ≤5% at 37°C. However, when extracting myoglobin from porcine heart, the method involves manually cutting the muscle tissue into a minced form, resulting in insufficient grinding and poor separation, leading to low myoglobin purity and easily affected activity. Summary of the Invention

[0004] The purpose of this invention is to provide a process for preparing myoglobin for chemiluminescent immunoassay quality control products, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a process for preparing myoglobin for chemiluminescent immunoassay quality control, comprising the following steps:

[0006] Raw material pretreatment: After removing impurities from fresh pig heart muscle tissue, it is placed into an automatic cleaning and cutting equipment for cutting and cleaning;

[0007] Myoglobin extraction: The pretreated muscle tissue is placed in a grinding mechanism, and the extraction solution is added for grinding;

[0008] Centrifugal separation: The extracted mixture is placed in a high-speed centrifuge for centrifugal separation to obtain the supernatant;

[0009] Purification process: The supernatant is purified by passing it through a multi-stage filtration and separation device;

[0010] Stabilization treatment: Add stabilizer to the purified myoglobin solution and mix it in a gradient temperature-controlled stirring system;

[0011] Low-temperature preservation: The stabilized myoglobin solution is aliquoted and stored in a low-temperature preservation device.

[0012] Preferably, the automatic cleaning and cutting equipment includes equally spaced cutting blades and a cleaning spray mechanism. The cleaning spray mechanism uses high-pressure nozzles arranged in a ring to spray rinsing fluid onto the surface of muscle tissue.

[0013] Preferably, the grinding mechanism includes a grinding jar, a grinding pestle, and a driving device. The inner wall of the grinding jar is provided with a replaceable wear-resistant grinding lining, and the surface of the lining is distributed with protrusions of different sizes and densities.

[0014] Preferably, the rinsing solution is 0.9% physiological saline.

[0015] Preferably, the extraction solution is a buffer solution containing 0.05 mol / L Tris-HCl, 0.1 mol / L NaCl, and a pH of 7.2-7.4.

[0016] Preferably, the stabilizer is a mixture of bovine serum albumin and glycerol, with bovine serum albumin concentration of 0.1%-0.5% and glycerol concentration of 5%-10%, and a stirring speed of 100-200 r / min.

[0017] Preferably, the multi-stage filtration and separation device consists of multiple filtration units connected in series. Each filtration unit uses a filter membrane with a different pore size. The filtration units are connected by adjustable connecting pipes and are equipped with an internal backwashing system.

[0018] Preferably, the filtration units of the multi-stage filtration and separation device use filter membranes with pore sizes of 1μm, 0.45μm, and 0.22μm in sequence, and the filtration speed is 100ml / min.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention, by incorporating an automatic cleaning and cutting device and a grinding mechanism, produces more uniform meat pieces compared to manual cutting, and is more efficient. The cut muscle tissue fragments are thoroughly ground to improve subsequent separation. The grinding size can be adjusted as needed, solving the problem that manual cutting of muscle tissue into minced meat results in insufficient grinding, poor separation, low purity of myoglobin, and compromised activity.

[0021] This invention features a multi-stage filtration and separation device, consisting of multiple filtration units connected in series. Each filtration unit uses a filter membrane with a different pore size, and the filtration units are connected by adjustable connecting pipes. An internal backwashing system is included to progressively improve the purity of myoglobin and effectively remove impurities. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the overall process flow of the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Please see Figure 1 This invention provides a technical solution: a process for preparing myoglobin for chemiluminescent immunoassay quality control, comprising the following steps:

[0025] Raw material pretreatment: After removing impurities from fresh pig heart muscle tissue, it is placed into an automatic cleaning and cutting equipment for cutting and cleaning;

[0026] Myoglobin extraction: The pretreated muscle tissue is placed in a grinding mechanism, and the extraction solution is added for grinding;

[0027] Centrifugal separation: The extracted mixture is placed in a high-speed centrifuge for centrifugal separation to obtain the supernatant;

[0028] Purification process: The supernatant is purified by passing it through a multi-stage filtration and separation device;

[0029] Stabilization treatment: Add stabilizer to the purified myoglobin solution and mix it in a gradient temperature-controlled stirring system;

[0030] Low-temperature preservation: The stabilized myoglobin solution is aliquoted and stored in a low-temperature preservation device.

[0031] Furthermore, the automatic cleaning and cutting equipment includes equally spaced cutting blades and a cleaning spray mechanism. The cleaning spray mechanism uses high-pressure nozzles arranged in a ring to spray rinsing liquid onto the surface of muscle tissue. Compared with manual cutting, the resulting meat pieces are more uniform in size and the work efficiency is higher.

[0032] Furthermore, the grinding mechanism includes a grinding jar, a grinding pestle, and a driving device. The inner wall of the grinding jar is provided with a replaceable wear-resistant grinding lining. The surface of the lining is distributed with protrusions of different sizes and densities to fully grind the cut muscle tissue fragments, thereby improving the subsequent separation effect. The grinding size can be adjusted as needed.

[0033] Furthermore, the rinsing solution is 0.9% physiological saline, which can effectively remove impurities without damaging the structure of myoglobin.

[0034] Furthermore, the extraction solution is a buffer solution containing 0.05 mol / L Tris-HCl and 0.1 mol / L NaCl, with a pH of 7.2-7.4, which facilitates the separation of myoglobin.

[0035] Furthermore, the stabilizer is a mixture of bovine serum albumin and glycerol, with bovine serum albumin concentration of 0.1%-0.5% and glycerol concentration of 5%-10%. The stirring speed is 100-200 r / min to ensure that the stabilizer is fully dissolved and binds to myoglobin, which can significantly improve the stability of myoglobin. When stored at -20℃ for more than 6 months, the activity of myoglobin can still be maintained at more than 90%.

[0036] Furthermore, the multi-stage filtration and separation device consists of multiple filtration units connected in series. Each filtration unit uses a filter membrane with a different pore size. The filtration units are connected by adjustable connecting pipes. An internal backwashing system is provided to progressively improve the purity of myoglobin and effectively remove impurities.

[0037] Furthermore, the filtration units of the multi-stage filtration and separation device sequentially use filter membranes with pore sizes of 1μm, 0.45μm, and 0.22μm, with a filtration speed of 100ml / min, to gradually remove impurities and improve the efficiency and effectiveness of filtration and separation.

[0038] Working principle: Fresh pig heart muscle tissue is selected, placed in an automatic cleaning and cutting device for cutting and cleaning, drained at 4℃, and then placed in a grinding mechanism with extraction solution. The grinding size is adjusted as needed to meet the requirements of subsequent processing. Then, centrifugation is performed to obtain supernatant, which is then filtered and purified step by step through a multi-stage filtration separation device to remove impurities and improve the purity of myoglobin. Stabilizer is then added and stirred to fully dissolve the stabilizer and bind it to myoglobin, which can significantly improve the stability of myoglobin. Finally, it is packaged and stored at low temperature to obtain myoglobin for chemiluminescent immunoassay quality control.

[0039] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0040] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A process for preparing myoglobin for chemiluminescent immunoassay quality control, characterized in that: Includes the following steps: Raw material pretreatment: After removing impurities from fresh pig heart muscle tissue, it is placed into an automatic cleaning and cutting equipment for cutting and cleaning; Myoglobin extraction: The pretreated muscle tissue is placed in a grinding mechanism, and the extraction solution is added for grinding; Centrifugal separation: The extracted mixture is placed in a high-speed centrifuge for centrifugal separation to obtain the supernatant; Purification process: The supernatant is purified by passing it through a multi-stage filtration and separation device; Stabilization treatment: Add stabilizer to the purified myoglobin solution and mix it in a gradient temperature-controlled stirring system; Low-temperature preservation: The stabilized myoglobin solution is aliquoted and stored in a low-temperature preservation device.

2. The process for preparing myoglobin for chemiluminescent immunoassay quality control according to claim 1, characterized in that: The automatic cleaning and cutting equipment includes equally spaced cutting blades and a cleaning spray mechanism. The cleaning spray mechanism uses high-pressure nozzles arranged in a ring to spray rinsing fluid onto the surface of muscle tissue.

3. The process for preparing myoglobin for chemiluminescent immunoassay quality control according to claim 1, characterized in that: The grinding mechanism includes a grinding jar, a grinding pestle, and a driving device. The inner wall of the grinding jar is provided with a replaceable wear-resistant grinding lining, and the surface of the lining is distributed with protrusions of different sizes and densities.

4. The process for preparing myoglobin for chemiluminescent immunoassay quality control according to claim 2, characterized in that: The flushing solution is 0.9% physiological saline.

5. The process for preparing myoglobin for chemiluminescent immunoassay quality control according to claim 1, characterized in that: The extraction solution is a buffer solution containing 0.05 mol / L Tris-HCl and 0.1 mol / L NaCl, with a pH of 7.2-7.

4.

6. The process for preparing myoglobin for chemiluminescent immunoassay quality control according to claim 1, characterized in that: The stabilizer is a mixture of bovine serum albumin and glycerol, with bovine serum albumin concentration of 0.1%-0.5% and glycerol concentration of 5%-10%, and a stirring speed of 100-200 r / min.

7. The process for preparing myoglobin for chemiluminescent immunoassay quality control according to claim 1, characterized in that: The multi-stage filtration and separation equipment consists of multiple filtration units connected in series. Each filtration unit uses a filter membrane with a different pore size. The filtration units are connected by adjustable connecting pipes and are equipped with an internal backwashing system.

8. The process for preparing myoglobin for chemiluminescent immunoassay quality control according to claim 1, characterized in that: The multi-stage filtration and separation device uses filter membranes with pore sizes of 1μm, 0.45μm, and 0.22μm in sequence, with a filtration rate of 100ml / min.

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