CN111718404A - Device for separating high-density lipoprotein in serum, preparation method thereof and method for separating high-density lipoprotein in serum - Google Patents
Device for separating high-density lipoprotein in serum, preparation method thereof and method for separating high-density lipoprotein in serum Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
Abstract
The invention relates to a device for separating high-density lipoprotein in serum, which comprises a pipe body and a filtering component, wherein the pipe body is provided with a pipeline, two ends of the pipeline are communicated with the outside, the filtering component is arranged in the pipeline of the pipe body, the filtering component comprises a low-density lipoprotein blocking filter membrane, and the low-density lipoprotein blocking filter membrane is prepared by soaking the filter membrane in a treatment solution and then drying the filter membrane; the treatment solution comprises a low-density lipoprotein blocking reagent with the concentration of 20-30 mg/mL and 0.2-0.3M MgCl2Preservative with mass concentration of 0.016-0.024% and water. The invention has the advantages of low equipment cost, high popularization rate, easy preparation and acquisition, quick reaction and convenient operation.
Description
Technical Field
The invention relates to a device for separating high-density lipoprotein in serum, a preparation method thereof and a method for separating the high-density lipoprotein in the serum.
Background
High Density Lipoprotein (HDL) is the most abundant Lipoprotein in the plasma of most mammals, but the level of HDL in human plasma is low in Low Density Lipoprotein (LDL), and can be subdivided into several subclasses, HDL1, HDL2, HDL3, etc., according to Density differences. Apolipoprotein in plasma HDL is mainly apoAI and apoAII, which bind loosely to HDL and tightly to apoAII. HDL is spherical in shape and is a group of lipoproteins that are most dense, least particulate, and very heterogeneous in plasma. The main function of HDL is to participate in Reverse Cholesterol Transport (RCT) of the body, which can remove the cholesterol from the cells of the surrounding tissues and transport it to the liver for conversion and elimination, thus having the potential role of resisting the occurrence and development of Atherosclerosis (AS). Is mainly used for treating hyperlipidemia, coronary heart disease, atherosclerosis and other diseases clinically. For the needs of biological and medical research, researchers often need to prepare pure lipoprotein by ultracentrifugation, chemical precipitation (such as dextran sulfate precipitation), gel filtration, high pressure liquid chromatography, and the like.
So far, the density gradient ultracentrifugation separation and purification of various lipoproteins is still a good method with high purity, convenience and practicability. The ultracentrifugation method can be divided into a preparation type and an analysis type, the preparation type density gradient centrifugation method can be divided into an equal density centrifugation method and a speed-zone density gradient centrifugation method, and the selected rotating head has an angle head, a throwing head, a vertical head and a zone head. The specific method is as follows:
1. preparing a gradient solution:
solution A: 0.1g of EDTA-Na was weighed using a balance2And 11.40g NaCl, the weighed reagents are put into a 1L beaker, 500mL of pure water is measured by a measuring cylinder and added into the beaker, 1mL of NaOH with the concentration of 1M is added, and the mixture is stirred until the reagents are completely dissolved. Pure water was added to 1000 mL. Finally, 3mL of pure water was added. The NaCl concentration in the final solution was 0.195mol, and the final density of the solution was 1.006g/cm3,pH10.37。
Solution B24.98 g NaBr was weighed out using a balance and added to 100mL of solution A. The concentration of NaCl in the final solution is 0.195mol, the concentration of NaBr is 2.44mol, and the final density of the solution is 1.182g/cm3，pH 9.72。
Solution C78.32 g NaBr were weighed out using a balance and added to 100mL of solution A. The concentration of NaCl in the final solution is 0.195mol, the concentration of NaBr is 7.65mol, and the final density of the solution is 1.478g/cm3，pH 8.86。
2. Centrifuge, rotor, centrifuging tube:
1) a centrifuge: hitachi CP-MX series ultracentrifuges or other similar units, such as Hitachi CS-150GXL miniultracentrifuges.
2) Turning head and centrifuge tube: s140 AT: 140,000rpm, 1,050,000 xg, 10 × 2mL, K5, 1PC thick wall tube. S80 AT 3: 80,000rpm,415,000 Xg, 8X 8mL, 23, 6PC thick walled tube. S70 AT: 70,000rpm,505,000 xg, 8 × 40mL, K44, 40PA tube.
3. Centrifugal operation
1) Plasma-corpuscular separation: whole blood, angle rotary head or swing rotary head 3000rpm x 20min, precipitate as blood cell, supernatant as blood plasma, for use.
2) Chylomicron (CM) isolation: any rotor can be used, and the centrifuge is set at 4X 106(g X min) at 10 ℃. The centrifuge tube contained plasma in the bottom 3/4 volume and 0.15M NaCl +0.3M EDTA in the upper 1/4 volume, pH 7.4. After centrifugation, CM will float to the upper layer. At this point the centrifuge tube was tilted and the CM was slowly aspirated using a pipette gun.
3) Very Low Density Lipoprotein (VLDL) isolation: using a S140AT (or similar type) rotor, 30mL of plasma was added to the lower portion of the centrifuge tube and 15mL of solution A was added to the upper portion, and the tube was centrifuged at 140,000rpm at 16 ℃ for 50min (acceleration 5, deceleration 7). Upon completion, distinct layers were observed in the tube, with the upper 1/3 being VLDL. At this point the centrifuge tube was tilted and very low density lipoprotein was slowly aspirated using a pipette gun.
4) Low Density Lipoprotein (LDL) (intermediate density lipoprotein-containing IDL) separation: using S140AT (or the same type of rotor), 15mL of solution B was added to the surface of the remaining 30mL of blood sample in the centrifuge tube and centrifuged at 140,000rpm at 16 ℃ for 80min (acceleration 9, deceleration 7). Upon completion, the tubes were visibly separated, with LDL + IDL in the top 1/3. At this time, the centrifuge tube was tilted, and LDL + IDL was slowly aspirated off using a pipette gun.
5) High Density Lipoprotein (HDL) separation: after removing LDL + IDL from the centrifuge tube using an S140AT (or similar type) rotor, the remaining 30mL of blood was transferred to another centrifuge tube, 15mL of solution C was added to the surface, and the tube was centrifuged at 140,000rpm at 16 ℃ for 140min (acceleration 9, deceleration 7). After the separation, the centrifugal tube is obviously layered, the upper part is HDL, and the lower part is serum albumin and globulin. At this point the centrifuge tube was tilted and HDL was slowly aspirated using a pipette gun.
To this end, the separation of chylomicron, LDL and HDL in plasma was completed.
The scheme of ultracentrifugation method for separating LDL and HDL, which is commonly adopted at present, needs to use an expensive ultra-high speed centrifuge as a main separation device, and has the disadvantages of multiple experimental steps, very complex operation, preparation of various reagents, most importantly, very time consumption, and the whole separation time often exceeds 6 hours.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a device which is inexpensive and can efficiently remove LDL in plasma within 30 minutes to obtain HDL of higher purity.
Another technical problem to be solved by the present invention is to provide a method for manufacturing the device.
The third technical problem to be solved by the present invention is to provide a method for efficiently removing LDL in plasma to obtain HDL of higher purity by using the above-mentioned apparatus.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention aims to provide a device for separating high-density lipoprotein in serum, which comprises a pipe body and a filtering component, wherein the pipe body is provided with a pipeline, two ends of the pipeline are communicated with the outside, the filtering component is arranged in the pipeline of the pipe body, the filtering component comprises a low-density lipoprotein blocking filter membrane, and the low-density lipoprotein blocking filter membrane is prepared by soaking the filter membrane in a treatment solution and then drying the filter membrane; the treatment solution comprises a low-density lipoprotein blocking reagent with the concentration of 20-30 mg/mL and 0.2-0.3M MgCl2Preservative with mass concentration of 0.016-0.024% and water.
Preferably, the treatment solution comprises a low-density lipoprotein blocking reagent with the concentration of 22-28 mg/mL and 0.22-0.28M MgCl2Preservative with mass concentration of 0.018-0.022% and water.
More preferably, the treatment solution comprises a low-density lipoprotein blocking reagent with a concentration of 24-26 mg/mL and 0.24-0.26M MgCl2Preservative with mass concentration of 0.019% -0.021% and water.
Preferably, the low-density lipoprotein blocking reagent is one or more of dextran sulfate and its salt, magnesium phosphotungstate, and polyethylene glycol 20000.
Further preferably, the low density lipoprotein blocking reagent is dextran sulfate and its salt.
More preferably, the low density lipoprotein blocking reagent is dextran sulfate sodium salt.
Preferably, the preservative is Procline 300.
Preferably, the filter membrane is a glass fiber filter membrane, filter paper or gauze.
Further preferably, the filter membrane is a glass fiber membrane, including but not limited to millipore AQFA type, whatman GFB membrane, corbel PES membrane.
More preferably, the glass fiber filter membrane is a whatman GFB membrane.
The thickness of the filter membrane is 650-700 microns, and the filtering speed is 400-450 microns/second.
Further preferably, the thickness of the glass fiber filter membrane is 670-680 micrometers, and the filtering speed is 430-440 micrometers/second.
Preferably, 3-5 low-density lipoprotein blocking filter membranes are compacted and compacted in the pipeline of the pipe body to form the filter assembly.
Preferably, the tube body is a pipette tip.
The pipette tip used in the present invention may have various specifications, for example, 5mL, 10mL, and the like.
Another object of the present invention is to provide a method for preparing the device for separating high density lipoprotein in serum, comprising the following steps:
cutting the filter membrane into circular sheets;
step (2) adding the low-density lipoprotein blocking reagent and the MgCl2Dissolving the preservative in water to prepare the treatment solution;
soaking the filter membrane cut in the step (1) in the treatment solution prepared in the step (2) for 50-70 min, and then drying at 35-40 ℃ for 50-70 min to prepare the low-density lipoprotein barrier filter membrane;
and (4) plugging the low-density lipoprotein blocking filter membrane prepared in the step (3) into the pipeline of the tube body to prepare the device for removing the low-density lipoprotein in the serum.
The third purpose of the invention is to provide a method for separating high-density lipoprotein in serum, which adopts a plurality of devices to perform multiple rounds of filtration on serum to remove low-density lipoprotein in serum, wherein the filtrate is the high-density lipoprotein, and the specific method of each round of filtration is as follows: adding serum into the tube body from one end of the tube body, pressurizing the serum in the tube body by using a pipette gun to promote the serum to filter the filter assembly, and collecting filtrate.
Preferably, 2-5 rounds of the filtration are carried out.
The multi-round filtration in the invention refers to that 1 device is adopted to filter the serum and then collect the filtrate, and the 1 round filtration is adopted, and the collected filtrate is filtered by another device and then collected, and the 2 round filtration is adopted.
The principle of the invention is based on that the surfaces of LDL (low density lipoprotein), VLDL (very low density lipoprotein) and Chylomicron (CM) mainly contain apolipoprotein B (ApoB) with anions, low density lipoprotein blocking agent such as dextran sulfate and Mg2+Can make these lipoprotein components aggregate to form precipitate, so as to attain the goal of separating HDL and LDL. In this scheme, we used a glass fiber filtration membrane as the carrier, which was soaked in dextran sulfate and Mg2+The related reagent is saturated and adsorbed on the filter membrane, and the filter membrane is plugged into a pipette tip to prepare a simple filter for filtering serum. When the serum passes through the filter membrane, LDL and other related components in the serum are mixed with dextran sulfate and Mg in the filter membrane2+The HDL components can pass through the filter membrane, thereby realizing separation of the two components.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
the invention provides a simple and easy device and a method for separating high-density lipoprotein (HDL) in serum, the reagent required for preparing the device is simple, the material is cheap and easy to obtain, 90 percent of LDL in serum can be removed within 30 minutes, and more than 50 percent of high-density lipoprotein (HDL) is reserved. Can efficiently remove LDL in plasma to obtain HDL of high purity.
The invention has the advantages of low equipment cost, high popularization rate, easy preparation and acquisition, quick reaction and convenient operation.
Drawings
FIG. 1 is a schematic view of an apparatus;
FIG. 2 is a schematic view of the apparatus in use;
FIG. 3 is a graph showing the results of detection in example 1.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. All other embodiments obtained by a person skilled in the art without making any inventive step are within the scope of protection of the present invention.
The reagents used in the invention are commercially available, and the specific types are as follows:
| name of reagent | Type (molecular weight) | Use of | Origin of origin |
| Dextran sulfate sodium salt | 6,500～10,000 | Low density lipoprotein blocking reagent | Commercially available |
| Dextran sulfate sodium salt | 50,000 | Low density lipoprotein blocking reagent | Commercially available |
| Sodium phosphotungstate hydrate | - | Low density lipoprotein blocking reagent | Commercially available |
| Proclin 300 | - | Preservative | Commercially available |
| MgCl2.2H2O | - | Coenzyme | Commercially available |
Example 1: including having the pipeline just the both ends of pipeline and the body of external intercommunication, setting be in the pipeline of body in the filter assembly, filter assembly include low density lipoprotein separation filter membrane:
(1) cutting the GFB glass fiber filter membrane into a wafer with the diameter of 13mm for later use;
(2) preparing 15mL of treatment solution, wherein: dextran Sulfate (DS) sodium salt (6,500-10,000): 25mg/ml MgCl20.25M, Procline300, 0.02 percent by mass;
(3) soaking the round filter membrane prepared in the first step in a treatment solution for 1 hour, and drying the round filter membrane in a blast oven for 1 hour at 37 ℃ to prepare a low-density lipoprotein blocking filter membrane 1 for later use;
(4) plugging 4 prepared round low-density lipoprotein blocking filter membranes 1 into a 5mL pipette tip 2, and compacting and tamping the low-density lipoprotein blocking filter membranes 1 in the pipette tip 2 by using a glass rod to prepare a simple LDL filter head, namely the device;
(5) serum 5 is fed into the LDL filter head from the end with the larger opening of the LDL filter head by means of a pipette 3, and the serum in the filter head is pressurized by means of the pipette at the end with the larger opening, thereby facilitating filtration through the low-density lipoprotein-blocking filter 1. The filtered serum is collected with an EP tube or a small beaker 4 at the tip of the LDL filter head;
(6) 3 rounds of filtration were performed on 1 serum using 3 LDL filters, and HDL-c and LDL-c were detected using Hitachi 7180 full-automatic biochemical analyzer before and after each filtration, and the results are shown in FIG. 3. It can be seen that after 3 rounds of filtration, nearly 60% of the original HDL still remains in the serum (HDL concentration is reduced from 1.377mmol/L to 0.819mmol/L), while LDL concentration is reduced from 4.333mmol/L to 0.423mmol/L, and the reduction range is 90%.
Example 2: including having the pipeline just the both ends of pipeline and the body of external intercommunication, setting be in the pipeline of body in the filter assembly, filter assembly include low density lipoprotein separation filter membrane:
(1) cutting the Millipore glass fiber filter membrane into a circular sheet with the diameter of 13mm for later use;
(2) preparing 15mL of treatment solution, wherein: sodium phosphotungstate: 10mg/ml MgCl20.25M, Procline300, 0.02 percent by mass;
(3) soaking the round filter membrane prepared in the first step in a treatment solution for 1 hour, and drying the round filter membrane in a blast oven for 1 hour at 37 ℃ to prepare a low-density lipoprotein blocking filter membrane 1 for later use;
(4) plugging 4 prepared round low-density lipoprotein blocking filter membranes 1 into a 5mL pipette tip 2, and compacting and tamping the low-density lipoprotein blocking filter membranes 1 in the pipette tip 2 by using a glass rod to prepare a simple LDL filter head, namely the device;
(5) serum 5 is fed into the LDL filter head from the end with the larger opening of the LDL filter head by means of a pipette 3, and the serum in the filter head is pressurized by means of the pipette at the end with the larger opening, thereby facilitating filtration through the low-density lipoprotein-blocking filter 1. The filtered serum is collected with an EP tube or a small beaker 4 at the tip of the LDL filter head;
(6) 3 rounds of filtration were performed on 1 serum using 3 LDL filters, and HDL-c and LDL-c were detected using Hitachi 7180 full-automatic biochemical analyzer before and after each filtration, and the results are shown in FIG. 3. It can be seen that after 3 rounds of filtration, nearly 50% of the original HDL still remains in the serum (HDL concentration is reduced from 1.377mmol/L to 0.652mmol/L), while LDL concentration is reduced from 4.333mmol/L to 0.816mmol/L, and the reduction reaches 80%.
The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.
Claims (14)
1. A device for separating high-density lipoprotein in serum is characterized in that: the device comprises a pipe body and a filtering component, wherein the pipe body is provided with a pipeline, two ends of the pipeline are communicated with the outside, the filtering component is arranged in the pipeline of the pipe body, the filtering component comprises a low-density lipoprotein blocking filter membrane, and the low-density lipoprotein blocking filter membrane is prepared by soaking the filter membrane in a treatment fluid and then drying the filter membrane; the treatment solution comprises a low-density lipoprotein blocking reagent with the concentration of 20-30 mg/mL and 0.2-0.3M MgCl2Preservative with mass concentration of 0.016-0.024% and water.
2. The device for separating high-density lipoprotein in serum according to claim 1, wherein: the treatment solution comprises a low-density lipoprotein blocking reagent with the concentration of 22-28 mg/mL and 0.22-0.28MgCl of M2Preservative with mass concentration of 0.018-0.022% and water.
3. The device for separating high-density lipoprotein in serum according to claim 2, wherein: the treatment solution comprises a low-density lipoprotein blocking reagent with the concentration of 24-26 mg/mL and 0.24-0.26M MgCl2Preservative with mass concentration of 0.019% -0.021% and water.
4. The device for separating high-density lipoprotein in serum according to any one of claims 1 to 3, characterized in that: the low-density lipoprotein blocking reagent is one or more of dextran sulfate and salt thereof, magnesium phosphotungstate and polyethylene glycol 20000.
5. The device for separating high-density lipoprotein in serum according to claim 4, wherein: the low-density lipoprotein blocking reagent is dextran sulfate and salt thereof.
6. The device for separating high-density lipoprotein in serum according to any one of claims 1 to 3, characterized in that: the preservative is Procline 300.
7. The device for separating high-density lipoprotein in serum according to any one of claims 1 to 3, characterized in that: the filter membrane is a glass fiber filter membrane, filter paper or gauze, the thickness of the filter membrane is 650-700 microns, and the filtering speed is 400-450 microns/second.
8. The device for separating high-density lipoprotein in serum according to claim 7, wherein: the filter membrane is a glass fiber membrane, and the glass fiber filter membrane is a millipore AQFA type, a whatman GFB membrane or a Cobaltt PES membrane.
9. The device for separating high-density lipoprotein in serum according to claim 8, wherein: the glass fiber filter membrane is a whatman GFB membrane.
10. The device for separating high-density lipoprotein in serum according to any one of claims 1 to 3, characterized in that: 3-5 low-density lipoprotein obstructing filter membranes are compacted and compacted in the pipeline of the pipe body to form the filter assembly.
11. The device for separating high-density lipoprotein in serum according to any one of claims 1 to 3, characterized in that: the tube body is a liquid-transfering gun head.
12. A method for preparing the device for separating high-density lipoprotein in serum according to any one of claims 1 to 11, wherein: the method comprises the following steps:
cutting the filter membrane into circular sheets;
step (2) adding the low-density lipoprotein blocking reagent and the MgCl2Dissolving the preservative in water to prepare the treatment solution;
soaking the filter membrane cut in the step (1) in the treatment solution prepared in the step (2) for 50-70 min, and then drying at 35-40 ℃ for 50-70 min to prepare the low-density lipoprotein barrier filter membrane;
and (4) plugging the low-density lipoprotein blocking filter membrane prepared in the step (3) into the pipeline of the tube body to prepare the device for removing the low-density lipoprotein in the serum.
13. A method for separating high-density lipoprotein in serum, which is characterized in that: performing multiple rounds of filtration on serum to remove low density lipoprotein in the serum by using a plurality of devices according to any one of claims 1 to 11, wherein the filtrate is high density lipoprotein, and each round of filtration is performed by the following specific method: adding serum into the tube body from one end of the tube body, pressurizing the serum in the tube body by using a pipette gun to promote the serum to filter the filter assembly, and collecting filtrate.
14. The method for separating high-density lipoprotein in serum according to claim 13, which comprises: and (5) filtering for 2-5 times.
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| JPH02187664A (en) * | 1989-01-17 | 1990-07-23 | Dai Ichi Pure Chem Co Ltd | High-density lipoprotein fractionating agent |
| US5290703A (en) * | 1992-12-14 | 1994-03-01 | Miles, Inc. | Method for the separation of high density lipoprotein from blood samples |
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