阅读说明：本技术 一种抗体或抗体组合物的制备方法 (Preparation method of antibody or antibody composition ) 是由 叶绪权 王旭 于 2020-04-24 设计创作，主要内容包括：本发明属于生物制品技术领域,具体的说是一种抗体或抗体组合物的制备方法；该方法包括如下步骤：S1：血清提取；S2：初步沉淀；S3：二次沉淀；S4：IgG抗体分离：分离罐内部设置有上层沉淀锅和下层沉淀锅；上层沉淀锅通过密封圈滑动设置在分离罐内；下层沉淀锅通过密封圈固定设置在分离罐内；分离罐设置有搅拌进料装置,且搅拌进料装置用于对上层沉淀锅内的血清进行高速搅拌；下层沉淀锅内设置有沉淀分离机构,且沉淀分离机构通过连接轴与搅拌进料装置连接；下层沉淀锅与上层沉淀锅之间连接有吸液管,且吸液管上安装有抽吸泵；下层沉淀锅上连接有进液管,且进液管端部伸出分离罐；本发明提高了血清中的抗体能够通过酒精沉淀法进行高效制备。(The invention belongs to the technical field of biological products, in particular to a preparation method of an antibody or an antibody composition; the method comprises the following steps: s1: extracting serum; s2: preliminary precipitation; s3: secondary precipitation; s4: IgG antibody separation: an upper layer sedimentation pot and a lower layer sedimentation pot are arranged in the separation tank; the upper layer sedimentation pot is arranged in the separation tank in a sliding way through a sealing ring; the lower layer sedimentation pot is fixedly arranged in the separation tank through a sealing ring; the separation tank is provided with a stirring and feeding device, and the stirring and feeding device is used for stirring the serum in the upper-layer precipitation pot at a high speed; a sedimentation separating mechanism is arranged in the lower layer sedimentation pot and is connected with the stirring and feeding device through a connecting shaft; a liquid suction pipe is connected between the lower layer sedimentation pot and the upper layer sedimentation pot, and a suction pump is arranged on the liquid suction pipe; a liquid inlet pipe is connected to the lower layer sedimentation pot, and the end part of the liquid inlet pipe extends out of the separation tank; the invention improves the high-efficiency preparation of the antibody in the serum by an alcohol precipitation method.)

1. A method of producing an antibody or antibody composition, comprising: the method comprises the following steps:

s1: serum extraction: the method comprises the following steps of immediately storing blood extracted from livestock raising into a centrifugal tube for sealing, transferring the blood into a low-temperature chamber, storing the centrifugal tube into a sample bin of a centrifugal machine through a centrifugal test tube rack, setting parameters of the centrifugal machine, setting the rotating speed at 3500-4500 rpm, and setting the centrifugal time at 4-8 minutes; after the centrifugal machine stops working, taking out the centrifugal tube from the sample bin, wherein the lower layer of the centrifugal tube is a blood cell layer in a sediment mode, and the upper layer of the centrifugal tube is a serum layer;

s2: primary precipitation: injecting the blood clear liquid extracted in the step S1 into an upper layer precipitation pot (2) through a draft tube (55) arranged on a separation tank (1), adding 3 times of distilled water under high-speed stirring in the circumferential direction of a conical stirring tube (57) of a stirring and feeding device (5), adjusting the pH value to 7.7, and cooling to 0 ℃; then adding pre-cooled-25 deg.C alcohol, stirring at high speed to obtain alcohol with final concentration of 20%, and maintaining at-5 deg.C; so that precipitate A is generated in the upper layer precipitation pot (2);

s3: secondary precipitation: sucking out supernatant solution in the upper layer precipitation pot (2) through a stirring and feeding device (5) to obtain a residual precipitate A; filling 25 times of volume of 0.15-20 mol/L NaCl cold solution into the lower-layer precipitation pot (3) through a liquid inlet pipe (65), then introducing the precipitate A precipitated in the upper-layer precipitation pot (2) into the lower-layer precipitation pot (3) to enable the precipitate A to be suspended in 14mol/L NaCl cold solution, then adding 0.06mol/L acetic acid into the lower-layer precipitation pot (3) through the liquid inlet pipe (65) to adjust the pH value to 5.1, and standing to generate a precipitate B and a supernatant solution;

s4: IgG antibody separation: sucking the supernatant solution in the lower layer precipitation pot (3) into the upper layer precipitation pot (2) through a pipette (64), adjusting the pH of the supernatant to 7.4, adding cold-25 ℃ alcohol until the final concentration is 25%, maintaining the temperature at minus 5 ℃, and then standing the obtained C precipitate, wherein the C precipitate contains 90-98% IgG antibody; then purifying the IgG antibody in the C precipitate by a gel filtration method;

wherein an upper-layer precipitation pot (2) and a lower-layer precipitation pot (3) are arranged in the separation tank (1); the upper-layer sedimentation pot (2) is arranged in the separation tank (1) in a sliding manner through a sealing ring (4); the lower-layer sedimentation pot (3) is fixedly arranged in the separation tank (1) through a sealing ring (4); the separation tank (1) is provided with a stirring and feeding device (5), and the stirring and feeding device (5) is used for stirring the serum in the upper-layer sedimentation pot (2) at a high speed; a precipitation separation mechanism (6) is arranged in the lower layer precipitation pot (3), and the precipitation separation mechanism (6) is connected with the stirring and feeding device (5) through a connecting shaft (61); a suction pipe (64) is connected between the lower layer sedimentation pot (3) and the upper layer sedimentation pot (2), and a suction pump is arranged on the suction pipe (64); the lower layer sedimentation pot (3) is connected with a liquid inlet pipe (65), and the end part of the liquid inlet pipe (65) extends out of the separation tank (1).

2. The method of claim 1, wherein the antibody or antibody composition is prepared by: the stirring and feeding device (5) comprises a rotary cavity shaft (51), a driving gear (52), a driven gear (53), a driving motor (54), a guide pipe (55), a cavity rotating disc (56) and a conical stirring pipe (57); the rotary cavity shaft (51) is rotatably arranged in the separation tank (1), and the bottom end of the rotary cavity shaft (51) is positioned in the upper-layer sedimentation pot (2); a driven gear (53) is mounted at the top end of the rotating cavity shaft (51); the driving gear (52) is rotatably arranged at the top end of the separation tank (1) through a rotating column, and the driving gear (52) is meshed with the driven gear (53); the output end of the driving motor (54) is connected with the driving gear (52), and the driving motor (54) is fixedly arranged above the separation tank (1); the outer wall of the rotating cavity shaft (51) is provided with a mounting hole (511), a guide pipe (55) is mounted in the rotating cavity shaft (51), and the top end of the guide pipe (55) is communicated with the injection pipe through a bearing; a cavity rotating disc (56) is fixedly arranged at the bottom end of the rotating cavity shaft (51), and a plurality of liquid guide holes (561) are uniformly formed in the circumferential direction of the bottom end of the cavity rotating disc (56); the bottom end of the draft tube (55) is communicated with the cavity rotating disc (56); a conical stirring pipe (57) is arranged in each liquid guide hole (561), and a hydraulic control valve is arranged on each conical stirring pipe (57); a rotating cavity is formed in the bottom end of the upper-layer sedimentation pot (2), and a sealing plate (7) is rotatably arranged in the rotating cavity through a rotating connecting shaft mechanism (8); a plurality of diversion holes (71) are formed in the bottom end of the upper layer sedimentation pot (2) and the sealing plate (7), and the diversion holes (71) formed in the upper layer sedimentation pot (2) and the diversion holes (71) formed in the sealing plate (7) are arranged in a rotating and staggered mode.

3. The method of claim 2, wherein the antibody or antibody composition is prepared by: the rotary connecting shaft mechanism (8) comprises a connecting shaft column (81), a cross-shaped inserting block (82) and an electric telescopic rod (83); the connecting shaft column (81) is fixedly connected to the sealing plate (7), and the connecting shaft column (81) is rotatably connected with the upper-layer sedimentation pot (2); a cross-shaped inserting groove (811) is formed in the upper end face of the connecting shaft column (81); the electric telescopic rod (83) is fixedly arranged in the rotary cavity shaft (51), and a cross-shaped insertion block (82) is fixed at the telescopic end of the electric telescopic rod (83); the cross plug-in block (82) is in plug-in fit with the cross plug-in groove (811).

4. The method of claim 3, wherein the antibody or antibody composition is prepared by: the precipitation separation mechanism (6) comprises a connecting shaft (61), a rotary arc-shaped cover (62) and a flow guide cover (63); the top end of the connecting shaft (61) is fixedly connected to the top end of the connecting shaft column (81), and the bottom end of the connecting shaft (61) is rotatably connected into the lower-layer sedimentation pot (3); a rotary arc cover (62) is fixedly arranged on the connecting shaft (61), and the rotary arc cover (62) is rotatably arranged in the lower-layer settling pan (3); the air guide sleeve (63) is fixedly arranged at the bottom end of the upper-layer sedimentation pot (2), and the bottom end of the air guide sleeve (63) is positioned above the rotary arc-shaped cover (62).

5. The method of claim 4, wherein the antibody or antibody composition is prepared by: a plurality of arc-shaped guide plates (9) are fixedly arranged on the connecting shaft (61), and a plurality of glass stirring strips (10) are arranged on two side walls of each arc-shaped guide plate (9); the arc-shaped guide plate (9) is positioned in the rotating arc-shaped cover (62).

6. The method of claim 5, wherein the antibody or antibody composition is prepared by: the pipette (64) is positioned on the inner side wall of the rotating arc-shaped cover (62), and an elastic scraping strip (11) is arranged on the side wall of the pipette (64); the elastic scraping strip (11) is in rotating friction contact with the rotating arc-shaped cover (62).

Technical Field

The invention belongs to the technical field of biological products, and particularly relates to a preparation method of an antibody or an antibody composition.

Background

Serum antibodies were among the first products of antibody medicine and were only prepared in small scale in the laboratory for scientific research purposes. Fibrinogen is one of the main components that distinguish plasma from serum, and is a hexamer with molecular weight of 340kDa formed by connecting a, beta and gamma chains through 29 pairs of disulfide bonds, and is used as a receptor of platelet membrane glycoprotein IIb/IIIa to participate in activated aggregation of platelets and in third-stage coagulation under the action of thrombin and other components of animal body.

With the rapid development of the intensification of the breeding industry, the failure of immunity, the sporadic outbreak of major epidemic diseases of animals, the clinical prohibition of antiviral drugs in veterinarians and the mixed infection caused by primary viral diseases provide market space for serum, immunoglobulin, albumin and the like with the specificity of various epidemic diseases of animals. At present, the most common methods for extracting and preparing serum antibodies are a freeze-thawing method, a cold alcohol precipitation method and a natural precipitation method, and the first two methods have low cost.

Although the existing cold alcohol precipitation method is low in cost, because the alcohol precipitation method needs to carry out precipitation operation on serum solution for multiple times and needs to control the temperature, concentration and Ph of alcohol, a plurality of precipitation devices are mostly adopted to carry out mutual transfer conversion precipitation through a flow guide pipe and a transfer device, and then the speed of extracting antibodies in the serum solution and the quality of extraction are easily poor.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides a preparation method of an antibody or an antibody composition, which is mainly used for solving the problems that although the existing cold alcohol precipitation method is low in cost, the alcohol precipitation method needs to carry out precipitation operation on a serum solution for multiple times, and the temperature, concentration and mixed solution Ph of alcohol need to be controlled, multiple precipitation devices are mostly adopted to carry out mutual transfer and conversion precipitation through a flow guide pipe and a transfer device, so that the phenomena of poor antibody extraction speed and poor extraction quality in the serum solution are easily caused.

The technical scheme adopted by the invention for solving the technical problems is as follows: the invention relates to a method for preparing an antibody or an antibody composition, which comprises the following steps:

s1: serum extraction: the method comprises the following steps of immediately storing blood extracted from livestock raising into a centrifugal tube for sealing, transferring the blood into a low-temperature chamber, storing the centrifugal tube into a sample bin of a centrifugal machine through a centrifugal test tube rack, setting parameters of the centrifugal machine, setting the rotating speed at 3500-4500 rpm, and setting the centrifugal time at 4-8 minutes; after the centrifugal machine stops working, taking out the centrifugal tube from the sample bin, wherein the lower layer of the centrifugal tube is a blood cell layer in a sediment mode, and the upper layer of the centrifugal tube is a serum layer; the centrifugal tube is used for high-degree centrifugal operation, so that blood in the centrifugal tube can be fully scattered, blood cells and serum in the blood can be conveniently subjected to static layering operation, and the yield, the efficiency and the quality of the obtained serum are high;

s2: primary precipitation: injecting the blood clear liquid extracted in the step S1 into an upper layer precipitation pot through a draft tube arranged on a separation tank, adding 3 times of distilled water under high-speed stirring in the circumferential direction of a conical stirring tube of a stirring and feeding device, adjusting the pH to 7.7, and cooling to 0 ℃; then adding pre-cooled-25 deg.C alcohol, stirring at high speed to obtain alcohol with final concentration of 20%, and maintaining at-5 deg.C; so that A sediment is generated in the upper layer sedimentation pot; the extracted serum is subjected to preliminary precipitation through the upper-layer precipitation pot, so that most of immunoglobulin in the serum can be subjected to high-speed stirring separation in alcohol at the temperature of-20 ℃, most of immunoglobulin in the serum can be precipitated, and the serum and the alcohol are both solution mixed components, and are stirred at a high speed along the circumferential direction of the upper-layer precipitation pot through the conical stirring pipe, so that the serum and alcohol mixed solution can generate a vortex shape at the central position of the upper-layer precipitation pot, the mixing effect of the alcohol solution and the serum is further increased, and the precipitation effect of the immunoglobulin is further increased;

s3: secondary precipitation: sucking out supernatant solution in the upper layer precipitation pot through a stirring and feeding device, and remaining precipitate A; filling 0.15-20 mol/L NaCl cold solution with the volume 25 times that of the lower layer precipitation pot through a liquid inlet pipe, then guiding the precipitate A precipitated in the upper layer precipitation pot into the lower layer precipitation pot to enable the precipitate A to be suspended in 14mol/L NaCl cold solution, then adding 0.06mol/L acetic acid into the lower layer precipitation pot through the liquid inlet pipe to adjust the pH value to 5.1, and standing to generate B precipitate and supernatant solution; introducing the solution precipitated in the upper-layer precipitation pot into the lower-layer precipitation pot, directly transferring the precipitated A precipitate in the separation pot to the lower-layer precipitation pot for secondary precipitation, adjusting the pH to 5.1 by using cold NaCl solution and 0.06mol/L acetic acid added in the lower-layer precipitation pot, and performing secondary precipitation operation on the A precipitate through the lower-layer precipitation pot to ensure that the supernatant solution comprises most of IgA, IgM and IgG;

s4: IgG antibody separation: sucking the supernatant solution in the lower layer precipitation pot into the upper layer precipitation pot through liquid absorption, adjusting the pH of the supernatant to 7.4, adding cold-25 ℃ alcohol until the final concentration is 25%, maintaining the final concentration at-5 ℃, and then standing the obtained C precipitate, wherein the C precipitate contains 90-98% IgG antibody; then purifying the IgG antibody in the C precipitate by a gel filtration method; sucking the supernatant solution in the lower layer precipitation pot into the cleaned upper layer precipitation pot through a pipette, and adjusting the concentration and the temperature of alcohol in the supernatant in the step S1 and separating and purifying other impurities in the supernatant in the step S1 because the supernatant solution in the step S1 contains a large amount of alcohol and distilled water; adding the separated and purified alcohol into the supernatant solution generated in the step S3 in the upper layer precipitation pot sucked by a pipette, highly stirring the supernatant solution generated in the step S3 by a stirring and feeding device to generate a C precipitate, and purifying the C precipitate by a gel filtration mode to form IgG antibody;

wherein an upper layer sedimentation pot and a lower layer sedimentation pot are arranged in the separation tank; the upper layer sedimentation pot is arranged in the separation tank in a sliding manner through a sealing ring; the lower layer sedimentation pot is fixedly arranged in the separation tank through a sealing ring; the separation tank is provided with a stirring and feeding device, and the stirring and feeding device is used for stirring the serum in the upper-layer precipitation pot at a high speed; a sedimentation separating mechanism is arranged in the lower layer sedimentation pot and is connected with the stirring and feeding device through a connecting shaft; a liquid suction pipe is connected between the lower layer sedimentation pot and the upper layer sedimentation pot, and a suction pump is installed on the liquid suction pipe; the lower layer sedimentation pot is connected with a liquid inlet pipe, and the end part of the liquid inlet pipe extends out of the separation tank;

when the serum antibody preparation device works, when the serum antibody needs to be prepared, firstly, the serum is put into an upper layer precipitation pot through a stirring and feeding device, then, distilled water is mixed with the serum in the upper layer precipitation pot through the stirring and feeding device, when the stirring and feeding device is used for stirring the serum and the distilled water in the upper layer precipitation pot at a high speed, an operator rotates and puts alcohol with the temperature of minus DEG C into the upper layer precipitation pot through the stirring and feeding device, so that the alcohol, the serum and the distilled water are mixed under the high-speed stirring, when the alcohol concentration is percent, the solution in the upper layer precipitation pot is subjected to standing precipitation, the A precipitate enters a lower layer precipitation pot, and the pH of the A precipitate in the lower layer precipitation pot is adjusted through NaCl solution and acetic acid added into the lower layer precipitation pot through a liquid inlet pipe, so that the B precipitate and an upper clear solution are generated; sucking the supernatant solution into an upper layer precipitation pot through a liquid suction pump, and stirring and precipitating the supernatant solution added into the upper layer precipitation pot through alcohol to form a C precipitate, wherein the C precipitate contains 90-98% IgG antibody; through the matching of an upper-layer precipitation pot and a lower-layer precipitation pot arranged in the separation tank, the upper-layer precipitation pot can precipitate a serum solution to generate multiple types of immunoglobulin, and can precipitate a supernatant solution generated after secondary precipitation to form a% to% IgG antibody; the lower-layer precipitation pot can perform secondary precipitation on the A precipitate after primary precipitation, so that IgA and IgM in the A precipitate are separated from IgG antibodies; furthermore, various antibodies contained in the serum can be precipitated and separated through the upper-layer precipitation pot and the lower-layer precipitation pot, so that the antibodies in the serum solution can be sealed in the separation tank for preparation; the precipitate obtained after the serum solution is primarily precipitated does not need to be transferred to another precipitation device by an operator, so that the phenomenon that the temperature and the pH of the precipitate change in a large range after the precipitate is transferred is reduced, and the high-efficiency preparation operation of the antibody in the serum by an alcohol precipitation method is improved; when the antibody in the existing serum is extracted by adopting an alcohol precipitation method, because the alcohol precipitation method needs to carry out precipitation operation on a serum solution for multiple times and needs to control the temperature, the concentration and the Ph of alcohol, a plurality of precipitation devices are mostly adopted to carry out mutual transfer conversion precipitation through a guide pipe and a transfer device, and then the extraction speed and the extraction quality of the antibody in the serum solution are influenced.

Preferably, the stirring and feeding device comprises a rotary cavity shaft, a driving gear, a driven gear, a driving motor, a guide pipe, a cavity rotating disc and a conical stirring pipe; the rotating cavity shaft is rotatably arranged in the separation tank, and the bottom end of the rotating cavity shaft is positioned in the upper-layer settling pan; a driven gear is mounted at the top end of the rotating cavity shaft; the driving gear is rotatably arranged at the top end of the separation tank through a rotating column, and the driving gear is meshed with the driven gear; the output end of the driving motor is connected with the driving gear, and the driving motor is fixedly arranged above the separation tank; the outer wall of the rotating cavity shaft is provided with a mounting hole, a flow guide pipe is mounted in the rotating cavity shaft, and the top end of the flow guide pipe is communicated with the injection pipe through a bearing; the bottom end of the rotating cavity shaft is fixedly provided with a cavity rotating disc, and a plurality of liquid guide holes are uniformly formed in the circumferential direction of the bottom end of the cavity rotating disc; the bottom end of the flow guide pipe is communicated with the cavity rotating disc; a conical stirring pipe is arranged in each liquid guide hole, and a hydraulic control valve is arranged on each conical stirring pipe; a rotating cavity is formed in the bottom end of the upper-layer sedimentation pot, and a sealing plate is rotatably arranged in the rotating cavity through a rotating connecting shaft mechanism; a plurality of flow guide holes are formed in the bottom end of the upper layer sedimentation pot and the sealing plate, and the flow guide holes in the upper layer sedimentation pot and the flow guide holes in the sealing plate are arranged in a rotating and staggered mode;

when the device works, when serum solution in the upper-layer precipitation pot needs to be stirred at a high speed, an operator can firstly inject the serum solution into the guide pipe through the injection pipe, the serum solution in the guide pipe can flow into the plurality of conical stirring pipes through the cavity rotating disc and then enter the upper-layer precipitation pot, then distilled water is injected into the upper-layer precipitation pot through the mode, the control unit controls the driving motor to rotate, the driving motor can drive the rotating cavity shaft to rotate, the rotating cavity shaft can drive the cavity rotating disc and the conical stirring pipes arranged in the circumferential direction of the cavity rotating disc to stir at a high speed in the upper-layer precipitation pot, the operator can inject alcohol into the guide pipe through the injection pipe when the cavity rotating disc rotates at a high speed, then the alcohol is injected into serum mixed liquid stirred at a high speed through the conical stirring pipes rotating at a high speed, and after the serum solution and the alcohol are mixed, the upper layer precipitation pot can precipitate the serum mixed solution to form precipitate A and supernatant solution; an operator connects an air pump outside the separating tank to the injection pipe, then sucks the supernatant solution into the cavity rotating disc through the conical stirring pipe, and then seals the conical stirring pipe through the hydraulic control valve; the sealing plate is rotated through the rotating connecting shaft mechanism, so that the flow guide holes in the sealing plate are aligned with the flow guide holes in the upper-layer settling pan, the sediment A in the upper-layer settling pan flows into the lower-layer settling pan through the flow guide holes, and then secondary settling operation is carried out through the lower-layer settling pan; the high-speed stirring is carried out along the circumferential direction of the upper-layer sedimentation pot through the conical stirring pipe, so that the serum and alcohol mixed solution can generate a vortex shape at the central position of the upper-layer sedimentation pot, the mixing effect of the alcohol solution and the serum is further improved, and the sedimentation effect of the immunoglobulin is further improved; when current agitating unit stirs liquid solution, stir along the central point of (mixing) shaft mostly for liquid mixture is when mixing the stirring, and the liquid solution in liquid mixture's the outer lane is difficult to high-efficient intensive mixing, influences serum solution's intensive mixing and deposits.

Preferably, the rotary connecting shaft mechanism comprises a connecting shaft column, a cross-shaped insertion block and an electric telescopic rod; the connecting shaft column is fixedly connected to the sealing plate and is rotatably connected with the upper-layer sedimentation pot; the upper end surface of the connecting shaft column is provided with a cross-shaped splicing groove; the electric telescopic rod is fixedly arranged in the rotating cavity shaft, and a cross-shaped insertion block is fixed at the telescopic end of the electric telescopic rod; the cross plug block and the cross plug groove are mutually plugged and matched; when the device works, when supernatant solution in the upper-layer settling pan is sucked into the cavity rotating disc, the driving motor stops working, the control unit controls the electric telescopic rod to extend out, the electric telescopic rod drives the cross insertion block to be inserted into the cross insertion groove, then the driving motor rotates, and the connecting shaft is driven to rotate by rotating the cavity shaft, so that the flow guide holes formed in the sealing plate are aligned with the flow guide holes formed in the upper-layer settling pan, and then A sediments in the upper-layer settling pan flow into the lower-layer settling pan through the flow guide holes; after A precipitate in the upper layer sedimentation pot is discharged, the driving motor rotates to drive the diversion holes in the sealing plate to be staggered with the diversion holes in the upper layer sedimentation pot, and then the upper layer sedimentation pot is sealed, and the electric telescopic rod contracts to enable the cross insertion block to be separated from the cross insertion groove.

Preferably, the precipitation separation mechanism comprises a connecting shaft, a rotating arc-shaped cover and a flow guide cover; the top end of the connecting shaft is fixedly connected to the top end of the connecting shaft column, and the bottom end of the connecting shaft is rotatably connected into the lower-layer settling pan; a rotary arc cover is fixedly arranged on the connecting shaft and is rotatably arranged in the lower-layer settling pan; the air guide sleeve is fixedly arranged at the bottom end of the upper-layer sedimentation pot, and the bottom end of the air guide sleeve is positioned above the rotating arc-shaped cover; when the device works, after the A sediment falls into the lower-layer settling pan through the diversion holes, the operator can adjust the pH of the A sediment in the lower-layer settling pan through NaCl solution and acetic acid added into the lower-layer settling pan through the liquid inlet pipe, then the control unit controls the electric telescopic rod to extend out, the cross insertion block is inserted into the cross insertion groove, the driving motor can drive the connecting shaft column to rotate through the rotating cavity shaft, the connecting shaft column can drive the rotating arc cover to rotate through the connecting shaft, the rotating arc cover is arranged in the lower-layer settling pan, and then the A sediment falls into the rotating arc cover, the rotating centrifugal force of the rotating arc cover rotating at high speed can drive the A sediment, NaCl solution and acetic acid mixed solution to upwards form arc rolling along the inner wall of the rotating arc cover, so that the mixed solution on the circumference of the rotating arc cover can roll to the central position of the rotating arc cover, further increasing the mixing operation of the sediment A in the lower-layer precipitation pot, NaCl solution and acetic acid, rapidly reducing the Ph of the sediment A and rapidly forming a sediment B; the kuppe that sets up can be convenient for flow out in the deposit of lower floor to A deposit and fall into the lower floor through the kuppe rapidly and deposit the pot.

Preferably, a plurality of arc-shaped guide plates are fixedly arranged on the connecting shaft, and a plurality of glass stirring strips are arranged on two side walls of each arc-shaped guide plate; the arc-shaped guide plate is positioned in the rotating arc-shaped cover; when the device works, when the connecting shaft rotates, the rotating centrifugal force of the rotating arc-shaped cover drives the mixed solution to turn over, the mixed solution falls into the upper surface of the arc-shaped guide plate, so that the arc-shaped guide plate can play a role in guiding the turned mixed solution, and the mixed solution falling into the upper surface of the arc-shaped guide plate can be centrifugally thrown into the rotating arc-shaped cover under the high-speed rotation of the arc-shaped guide plate, so that the efficient mixing contact effect of the liquid mixed solution is improved; the glass stirring bar can mix and stir the mixed solution in the rotating arc cover under the rotation of the arc guide plate; the arc-shaped guide plate provided by the invention can be suitable for rapid mixing and dissolving of liquid mixed solution, and can be used for rolling and mixing the mixed solution under the coordination of the rotating arc-shaped cover.

Preferably, the pipette is positioned on the inner side wall of the rotating arc-shaped cover, and an elastic scraping strip is arranged on the side wall of the pipette; the elastic scraping strip is in rotating friction contact with the rotating arc cover; the during operation, when rotating the arc cover and rotating, the pipette of the inside inner wall setting of fixed pipette that sets up can rotate along the inner wall that rotates the arc cover and scrape and get, can prevent effectively that when mixed solution under the high-speed centrifugal force that rotates the arc cover, when mixed solution rolls along the inside that rotates the arc cover, immunoglobulin in the mixed solution bonds on the inner wall that rotates the arc cover, and then the influence rotates the arc cover and carries out high quality extraction operation to the antibody in the A precipitate.

The invention has the following beneficial effects:

1. the lower-layer precipitation pot can be used for carrying out secondary precipitation on the A precipitate after primary precipitation, so that IgA and IgM in the A precipitate are separated from IgG antibodies; furthermore, various antibodies contained in the serum can be precipitated and separated through the upper-layer precipitation pot and the lower-layer precipitation pot, so that the antibodies in the serum solution can be sealed in the separation tank for preparation; further reducing the phenomenon that the temperature and Ph of the precipitate change in a large range after the transfer, and further improving the efficient preparation operation of the antibody in the serum by an alcohol precipitation method.

2. According to the invention, the serum and alcohol mixed solution can generate a vortex shape at the central position of the upper-layer sedimentation pot by high-speed stirring along the circumferential direction of the upper-layer sedimentation pot through the conical stirring pipe, so that the mixing effect of the alcohol solution and the serum is increased, and the sedimentation effect of immunoglobulin is increased.

Drawings

The invention will be further explained with reference to the drawings.

FIG. 1 is a flow chart of a preparation method of the present invention;

FIG. 2 is a cross-sectional view of a separator tank of the present invention;

FIG. 3 is an enlarged view of a portion of FIG. 2 of the present invention;

FIG. 4 is an enlarged view taken at A of FIG. 2 in accordance with the present invention;

in the figure: the device comprises a separation tank 1, an upper-layer sedimentation pot 2, a lower-layer sedimentation pot 3, a sealing ring 4, a stirring and feeding device 5, a rotary cavity shaft 51, a mounting hole 511, a driving gear 52, a driven gear 53, a driving motor 54, a guide pipe 55, a cavity rotating disc 56, a liquid guide hole 561, a conical stirring pipe 57, a sedimentation and separation mechanism 6, a connecting shaft 61, a rotary arc-shaped cover 62, a guide cover 63, a liquid suction pipe 64, a liquid inlet pipe 65, a sealing plate 7, a guide hole 71, a rotary connecting shaft mechanism 8, a connecting shaft column 81, a cross insertion groove 811, a cross insertion block 82, an electric telescopic rod 83, an arc-shaped guide plate 9, a glass stirring bar 10 and an elastic scraping bar 11.

Detailed Description

A method for producing an antibody or an antibody composition according to an embodiment of the present invention will be described below with reference to fig. 1 to 4.

As shown in fig. 1 to fig. 4, the method for preparing an antibody or an antibody composition according to the present invention comprises the following steps:

s1: serum extraction: the method comprises the following steps of immediately storing blood extracted from livestock raising into a centrifugal tube for sealing, transferring the blood into a low-temperature chamber, storing the centrifugal tube into a sample bin of a centrifugal machine through a centrifugal test tube rack, setting parameters of the centrifugal machine, setting the rotating speed at 3500-4500 rpm, and setting the centrifugal time at 4-8 minutes; after the centrifugal machine stops working, taking out the centrifugal tube from the sample bin, wherein the lower layer of the centrifugal tube is a blood cell layer in a sediment mode, and the upper layer of the centrifugal tube is a serum layer; the centrifugal tube is used for high-degree centrifugal operation, so that blood in the centrifugal tube can be fully scattered, blood cells and serum in the blood can be conveniently subjected to static layering operation, and the yield, the efficiency and the quality of the obtained serum are high;

s2: primary precipitation: injecting the blood clear liquid extracted in the step S1 into the upper layer precipitation pot 2 through a draft tube 55 arranged on the separation tank 1, adding 3 times volume of distilled water under high-speed stirring in the circumferential direction of a conical stirring tube 57 of a stirring and feeding device 5, adjusting the pH to 7.7 and cooling to 0 ℃; then adding pre-cooled-25 deg.C alcohol, stirring at high speed to obtain alcohol with final concentration of 20%, and maintaining at-5 deg.C; so that the sediment A is generated in the upper layer sedimentation pot 2; the extracted serum is primarily precipitated through the upper-layer precipitation pot 2, so that most of immunoglobulin in the serum can be stirred and separated at a high speed in alcohol at the temperature of-20 ℃, most of immunoglobulin in the serum can be precipitated, and the serum and the alcohol are both solution mixed, and the mixed solution of the serum and the alcohol can be stirred at a high speed along the circumferential direction of the upper-layer precipitation pot 2 through the conical stirring pipe 57, so that the vortex-shaped mixed solution of the serum and the alcohol can be generated at the central position of the upper-layer precipitation pot 2, the mixing effect of the alcohol solution and the serum is further increased, and the precipitation effect of the immunoglobulin is further increased;

s3: secondary precipitation: sucking out the supernatant solution in the upper layer precipitation pot 2 through a stirring and feeding device 5, and remaining precipitate A; filling 25 times of volume of 0.15-20 mol/L NaCl cold solution into the lower-layer precipitation pot 3 through the liquid inlet pipe 65, then introducing the precipitate A precipitated in the upper-layer precipitation pot 2 into the lower-layer precipitation pot 3 to enable the precipitate A to be suspended in 14mol/L NaCl cold solution, then adding 0.06mol/L acetic acid into the lower-layer precipitation pot 3 through the liquid inlet pipe 65 to adjust the pH value to 5.1, and standing to generate a precipitate B and a supernatant solution; introducing the solution precipitated in the upper precipitation pot 2 into the lower precipitation pot 3, directly transferring the precipitated A precipitate in the separation tank 1 into the lower precipitation pot 3 for secondary precipitation, adjusting the pH to 5.1 by using cold NaCl solution and 0.06mol/L acetic acid added in the lower precipitation pot 3, and performing secondary precipitation operation by using 3-DEG A precipitate in the lower precipitation pot to ensure that the supernatant solution comprises most of IgA, IgM and IgG;

s4: IgG antibody separation: sucking the supernatant solution in the lower layer precipitation pot 3 into the upper layer precipitation pot 2 through a pipette 64, adjusting the pH of the supernatant to 7.4, adding cold-25 ℃ alcohol until the final concentration is 25%, maintaining the temperature at-5 ℃, and then standing the obtained C precipitate, wherein the C precipitate contains 90-98% IgG antibody; then purifying the IgG antibody in the C precipitate by a gel filtration method; sucking the supernatant solution in the lower layer precipitation pot 3 into the cleaned upper layer precipitation pot 2 through a pipette 64, and adjusting the concentration and temperature of alcohol in the supernatant solution in the step S1 and separating and purifying other impurities in the supernatant solution in the step S1 because the supernatant solution in the step S1 contains a large amount of alcohol and distilled water; adding the separated and purified alcohol to the supernatant solution produced in the step S3 in the upper precipitation pot 2 sucked by the pipette 64, highly stirring the supernatant solution produced in the step S3 by the stirring and feeding device 5 to produce a C precipitate, and purifying the C precipitate by gel filtration to form IgG antibodies;

wherein, an upper layer sedimentation pot 2 and a lower layer sedimentation pot 3 are arranged in the separation tank 1; the upper layer sedimentation pot 2 is arranged in the separation tank 1 in a sliding way through a sealing ring 4; the lower layer sedimentation pot 3 is fixedly arranged in the separation tank 1 through a sealing ring 4; the separation tank 1 is provided with a stirring and feeding device 5, and the stirring and feeding device 5 is used for stirring the serum in the upper-layer sedimentation pot 2 at a high speed; a precipitation separation mechanism 6 is arranged in the lower layer precipitation pot 3, and the precipitation separation mechanism 6 is connected with the stirring and feeding device 5 through a connecting shaft 61; a liquid suction pipe 64 is connected between the lower layer sedimentation pot 3 and the upper layer sedimentation pot 2, and a suction pump is arranged on the liquid suction pipe 64; the lower layer sedimentation pot 3 is connected with a liquid inlet pipe 65, and the end part of the liquid inlet pipe 65 extends out of the separation tank 1;

when in work, when the antibody in the serum needs to be prepared, the serum is firstly put into the upper layer sedimentation pot 2 through the stirring and feeding device 5, then the distilled water is mixed with the serum in the upper layer sedimentation pot 2 through the stirring and feeding device 5, when the stirring and feeding device 5 stirs the serum and the distilled water in the upper layer sedimentation pot 2 at high speed, the operator rotationally puts the alcohol with the temperature of minus 20 ℃ into the upper layer sedimentation pot 2 through the stirring and feeding device 5, so that the alcohol, the serum and the distilled water are mixed under high-speed stirring, when the alcohol concentration is 20%, the solution in the upper layer precipitation pot 2 is subjected to standing precipitation, the precipitate A enters the lower layer precipitation pot 3, the pH of the precipitate A in the lower-layer precipitation pot 3 is adjusted by NaCl solution and acetic acid added into the lower-layer precipitation pot 3 through a liquid inlet pipe 65, so that a precipitate B and a supernatant solution are generated; sucking the supernatant solution into the upper layer precipitation pot 2 through a liquid suction pump, and stirring and precipitating the supernatant solution added into the upper layer precipitation pot 2 through alcohol to form a C precipitate, wherein the C precipitate contains 90-98% IgG antibody; through the matching of an upper-layer precipitation pot 2 and a lower-layer precipitation pot 3 arranged in the separation tank 1, the upper-layer precipitation pot 2 can precipitate serum solution to generate most types of immunoglobulin, and can precipitate supernatant solution generated after secondary precipitation to form 90% -98% IgG antibody; the lower layer precipitation pot 3 can carry out secondary precipitation on the A precipitate after primary precipitation so as to separate IgA and IgM in the A precipitate from IgG antibodies; furthermore, various antibodies contained in the serum can be precipitated and separated through the upper-layer precipitation pot 2 and the lower-layer precipitation pot 3, so that the antibodies in the serum solution can be prepared in the separation tank 1 in a sealed manner; the precipitate obtained after the serum solution is primarily precipitated does not need to be transferred to another precipitation device by an operator, so that the phenomenon that the temperature and the pH of the precipitate change in a large range after the precipitate is transferred is reduced, and the high-efficiency preparation operation of the antibody in the serum by an alcohol precipitation method is improved; when the antibody in the existing serum is extracted by adopting an alcohol precipitation method, because the alcohol precipitation method needs to carry out precipitation operation on a serum solution for multiple times and needs to control the temperature, the concentration and the pH of alcohol and a mixed solution, a plurality of precipitation devices are mostly adopted to carry out mutual transfer conversion precipitation through a guide pipe 55 and a transfer device, and then the extraction speed and the extraction quality of the antibody in the serum solution are influenced.

As an embodiment of the present invention, the stirring and feeding device 5 includes a rotary cavity shaft 51, a driving gear 52, a driven gear 53, a driving motor 54, a guide pipe 55, a cavity rotary disk 56 and a conical stirring pipe 57; the rotary cavity shaft 51 is rotatably arranged in the separation tank 1, and the bottom end of the rotary cavity shaft 51 is positioned in the upper-layer sedimentation pot 2; a driven gear 53 is mounted at the top end of the rotating cavity shaft 51; the driving gear 52 is rotatably arranged at the top end of the separation tank 1 through a rotating column, and the driving gear 52 is meshed with the driven gear 53; the output end of the driving motor 54 is connected with the driving gear 52, and the driving motor 54 is fixedly arranged above the separation tank 1; the outer wall of the rotating cavity shaft 51 is provided with a mounting hole 511, a guide pipe 55 is mounted in the rotating cavity shaft 51, and the top end of the guide pipe 55 is communicated with the injection pipe through a bearing; a cavity rotating disc 56 is fixedly arranged at the bottom end of the rotating cavity shaft 51, and a plurality of liquid guide holes 561 are uniformly formed in the circumferential direction of the bottom end of the cavity rotating disc 56; the bottom end of the guide pipe 55 is communicated with the cavity rotating disc 56; a conical stirring pipe 57 is arranged in each liquid guide hole 561, and a hydraulic control valve is arranged on each conical stirring pipe 57; a rotating cavity is formed in the bottom end of the upper-layer sedimentation pot 2, and a sealing plate 7 is rotatably arranged in the rotating cavity through a rotating connecting shaft mechanism 8; a plurality of flow guide holes 71 are formed in the bottom end of the upper layer sedimentation pot 2 and the sealing plate 7, and the flow guide holes 71 formed in the upper layer sedimentation pot 2 and the flow guide holes 71 formed in the sealing plate 7 are arranged in a rotating and staggered manner;

when the high-speed stirring device works, when the serum solution in the upper-layer sedimentation pot 2 needs to be stirred at a high speed, an operator can firstly inject the serum solution into the guide pipe 55 through the injection pipe, the serum solution in the guide pipe 55 can flow into the plurality of conical stirring pipes 57 through the cavity rotating disc 56 and then enter the upper-layer sedimentation pot 2, then distilled water is injected into the upper-layer sedimentation pot 2 through the mode, the control unit controls the driving motor 54 to rotate, the driving motor 54 can drive the rotating cavity shaft 51 to rotate, the rotating cavity shaft 51 can drive the cavity rotating disc 56 and the conical stirring pipes 57 arranged in the circumferential direction of the cavity rotating disc 56 to stir at a high speed in the upper-layer sedimentation pot 2, when the cavity rotating disc 56 rotates at a high speed, the operator can inject alcohol into the guide pipe 55 through the injection pipe, and then inject the alcohol into the serum mixed solution stirred at a high speed through the conical stirring pipes 57 rotating at a high speed, after the serum solution and the alcohol are mixed, the upper layer precipitation pot 2 can precipitate the serum mixed solution to form a precipitate A and a supernatant solution; an operator connects an air pump outside the separation tank 1 to the injection pipe, then sucks the supernatant solution into the cavity rotating disc 56 through the conical stirring pipe 57, and then seals the conical stirring pipe 57 through the hydraulic control valve; the sealing plate 7 is rotated through the rotating connecting shaft mechanism 8, so that the guide holes 71 on the sealing plate 7 are aligned with the guide holes 71 in the upper-layer sedimentation pot 2, the sediment A in the upper-layer sedimentation pot 2 flows into the lower-layer sedimentation pot 3 through the guide holes 71, and then secondary sedimentation operation is carried out through the lower-layer sedimentation pot 3; the high-speed stirring is carried out along the circumferential direction of the upper-layer sedimentation pot 2 through the conical stirring pipe 57, so that the mixed solution of the serum and the alcohol can generate a vortex shape at the central position of the upper-layer sedimentation pot 2, the mixing effect of the alcohol solution and the serum is further increased, and the sedimentation effect of the immunoglobulin is further increased; when current agitating unit stirs liquid solution, stir along the central point of (mixing) shaft mostly for liquid mixture is when mixing the stirring, and the liquid solution in liquid mixture's the outer lane is difficult to high-efficient intensive mixing, influences serum solution's intensive mixing and deposits.

As an embodiment of the present invention, the rotary coupling mechanism 8 includes a coupling post 81, a cross plug-in block 82 and an electric telescopic rod 83; the connecting shaft column 81 is fixedly connected to the sealing plate 7, and the connecting shaft column 81 is rotatably connected with the upper-layer sedimentation pot 2; the upper end surface of the connecting shaft column 81 is provided with a cross-shaped inserting groove 811; the electric telescopic rod 83 is fixedly arranged in the rotating cavity shaft 51, and a cross-shaped insertion block 82 is fixed at the telescopic end of the electric telescopic rod 83; the cross plug block 82 is in plug fit with the cross plug groove 811; when the device works, when supernatant solution in the upper-layer sedimentation pot 2 is sucked into the cavity rotating disc 56, the driving motor 54 stops working, the control unit controls the electric telescopic rod 83 to extend out, the electric telescopic rod 83 drives the cross insertion block 82 to be inserted into the cross insertion groove 811, then the driving motor 54 rotates, and can drive the connecting shaft 61 to rotate by rotating the cavity shaft 51, so that the guide holes 71 formed in the sealing plate 7 are aligned with the guide holes 71 formed in the upper-layer sedimentation pot 2, and then the sediment A in the upper-layer sedimentation pot 2 flows into the lower-layer sedimentation pot 3 through the guide holes 71; after A sediment in the upper sedimentation pot 2 is discharged, the driving motor 54 rotates to drive the diversion hole 71 on the sealing plate 7 to be dislocated with the diversion hole 71 on the upper sedimentation pot 2, so that the upper sedimentation pot 2 is sealed, and the electric telescopic rod 83 contracts, so that the cross plug-in block 82 is separated from the cross plug-in groove 811.

As an embodiment of the present invention, the precipitation separation mechanism 6 includes a connection shaft 61, a rotation arc-shaped cover 62, and a guide cover 63; the top end of the connecting shaft 61 is fixedly connected to the top end of the connecting shaft column 81, and the bottom end of the connecting shaft 61 is rotatably connected into the lower-layer sedimentation pot 3; a rotary arc cover 62 is fixedly arranged on the connecting shaft 61, and the rotary arc cover 62 is rotatably arranged in the lower-layer settling pan 3; the air guide sleeve 63 is fixedly arranged at the bottom end of the upper-layer sedimentation pot 2, and the bottom end of the air guide sleeve 63 is positioned above the rotating arc-shaped sleeve 62; when the device works, after the A sediment falls into the lower-layer sedimentation pot 3 through the diversion hole 71, an operator can adjust the Ph of the A sediment in the lower-layer sedimentation pot 3 through NaCl solution and acetic acid added into the lower-layer sedimentation pot 3 through the liquid inlet pipe 65, then the control unit controls the electric telescopic rod 83 to extend out, the cross insertion block 82 is inserted into the cross insertion groove 811, the driving motor 54 rotates to drive the connecting shaft column 81 to rotate through rotating the cavity shaft 51, the connecting shaft column 81 drives the rotating arc cover 62 to rotate through the connecting shaft 61, the rotating arc cover 62 is arranged in the lower-layer sedimentation pot 3, then the A sediment falls into the rotating arc cover 62, the rotating centrifugal force of the rotating arc cover 62 rotating at a high speed can drive the A sediment, NaCl solution and acetic acid mixed solution to upwards form arc rolling along the inner wall of the rotating arc cover 62, so that the mixed solution on the circumference of the rotating arc cover 62 can roll to the central position of the rotating arc cover 62, the mixing operation of the A precipitate, NaCl solution and acetic acid in the lower-layer precipitation pot 3 is increased, the Ph of the A precipitate can be quickly reduced, and the B precipitate can be quickly formed; the air guide sleeve 63 is convenient for the sediment A to flow out of the sediment in the lower layer and fall into the sedimentation pot 3 in the lower layer through the air guide sleeve 63.

As an embodiment of the invention, a plurality of arc-shaped guide plates 9 are fixedly arranged on the connecting shaft 61, and a plurality of glass stirring strips 10 are arranged on two side walls of the arc-shaped guide plates 9; the arc-shaped guide plate 9 is positioned in the rotating arc-shaped cover 62; when the device works, when the connecting shaft 61 rotates, the rotating centrifugal force of the rotating arc-shaped cover 62 drives the mixed solution to turn over, the mixed solution falls into the upper surface of the arc-shaped guide plate 9, so that the arc-shaped guide plate 9 can guide the turned mixed solution, and the mixed solution falling into the upper surface of the arc-shaped guide plate 9 can be centrifugally thrown into the rotating arc-shaped cover 62 under the high-speed rotation of the arc-shaped guide plate 9, so that the efficient mixing contact effect of the liquid mixed solution is improved; the glass stirring strip 10 can mix and stir the mixed solution in the rotating arc-shaped cover 62 under the rotation of the arc-shaped guide plate 9; the arc-shaped guide plate 9 provided by the invention can be suitable for rapid mixing and dissolving of liquid mixed solution, and can be used for rolling and mixing the mixed solution under the coordination of the rotation of the arc-shaped cover 62.

As an embodiment of the present invention, the pipette 64 is located on the inner side wall of the rotating arc cover 62, and the elastic wiper strip 11 is provided on the side wall of the pipette 64; the elastic scraping strip 11 is in rotating friction contact with the rotating arc-shaped cover 62; during operation, when rotating arc cover 62 and rotating, the pipette 64 of the inside inner wall setting of fixed pipette 64 that sets up can rotate along the inner wall that rotates arc cover 62 and scrape, can prevent effectively that when mixed solution under the high-speed rotation centrifugal force that rotates arc cover 62, mixed solution rolls along the inside that rotates arc cover 62, and the immunoglobulin in the mixed solution bonds on the inner wall that rotates arc cover 62, and then the influence rotates the antibody of arc cover 62 in to the A precipitate and carries out high quality extraction operation.

The specific working process is as follows:

when in work, when the antibody in the serum needs to be prepared, the serum is firstly put into the upper layer sedimentation pot 2 through the stirring and feeding device 5, then the distilled water is mixed with the serum in the upper layer sedimentation pot 2 through the stirring and feeding device 5, when the stirring and feeding device 5 stirs the serum and the distilled water in the upper layer sedimentation pot 2 at high speed, the operator rotationally puts the alcohol with the temperature of minus 20 ℃ into the upper layer sedimentation pot 2 through the stirring and feeding device 5, so that the alcohol, the serum and the distilled water are mixed under high-speed stirring, when the alcohol concentration is 20%, the solution in the upper layer precipitation pot 2 is subjected to standing precipitation, the precipitate A enters the lower layer precipitation pot 3, the pH of the precipitate A in the lower-layer precipitation pot 3 is adjusted by NaCl solution and acetic acid added into the lower-layer precipitation pot 3 through a liquid inlet pipe 65, so that a precipitate B and a supernatant solution are generated; sucking the supernatant solution into the upper layer precipitation pot 2 through a liquid suction pump, and stirring and precipitating the supernatant solution added into the upper layer precipitation pot 2 through alcohol to form a C precipitate, wherein the C precipitate contains 90-98% IgG antibody; through the matching of an upper-layer precipitation pot 2 and a lower-layer precipitation pot 3 arranged in the separation tank 1, the upper-layer precipitation pot 2 can precipitate serum solution to generate most types of immunoglobulin, and can precipitate supernatant solution generated after secondary precipitation to form 90% -98% IgG antibody; the lower layer precipitation pot 3 can carry out secondary precipitation on the A precipitate after primary precipitation, so that IgA and IgM in the A precipitate are separated from IgG antibodies.

In the description of the present invention, it is to be understood that the terms "center", "front", "rear", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate orientations or positional relationships based on those shown in the drawings, and are used only for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore, should not be taken as limiting the scope of the present invention.

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.