JP2006218442A - Apparatus and method for b/f separation in immunoassay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus and a method for B/F separation in immunoassay using a small, lightweight magnet by which a high gradient magnetic field can be obtained. <P>SOLUTION: There is provided a method for B/F separation in immunoassay in which an antigen-antibody reaction is performed between a solid-phase antibody (or a solid-phase antigen) and a target antigen (or antibody), and a target antigen (or antibody) and a labeled antibody (or a labeled antigen) using a magnetic material as a solid phase, and then the solid-phase antibody (or the solid-phase antigen) is B/F separated using a magnet device, wherein the solid-phase antibody (or the solid-phase antigen) is B/F separated by using a pair of magnets of which the same poles that are repellent to each other are arranged to face each other and placing a separation container in the vicinity of a space between the magnetic poles. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a target antigen (or antibody) that did not bind to a solid phase antibody (or solid phase antigen) and a labeled antibody (or labeled antigen) that did not bind to the target antigen (or antibody) in an immunoassay. ) From the reaction vessel.

  The enzyme immunoassay method (Enzyme immunoassay method, EIA method) is one of immunoassay methods for quantifying a specific antigen or antibody in a living body by utilizing a specific antigen-antibody reaction similar to “key and keyhole”. It is. Because of its extremely high detection sensitivity, it is widely used for the quantification of substances present in trace amounts in the living body, such as cancer markers, antigens of various infectious diseases, and hormones. Samples to be measured are body fluids such as blood (particularly serum), urine, saliva, spinal fluid.

  FIG. 1 shows an example of the principle of the EIA method. FIG. 1A shows an initial state of the EIA method. In the figure, 1 is a magnetic particle solid phase. For example, magnetic particles made of a ferromagnetic material are used. On the surface of the magnetic particle solid phase 1, an antibody (a kind of protein) is adsorbed to form a solid phase antibody 2. When a sample containing the antigen 3 to be measured is added thereto and reacted with the magnetic particle solid phase 1 for a certain period of time, the antigen 3 causes an antigen-antibody reaction with the solid phase antibody 2, and the surface of the magnetic particle solid phase 1. To form an antigen-antibody complex 4 as shown in FIG. The antigen in this state is referred to as a bound antigen 5 (abbreviated as Bound form, B), and the binding is not cut by washing of the magnetic particle solid phase 1. On the other hand, the antigen 3 unbound to the solid phase antibody 2 is called free form (abbreviated as F), and can be easily removed from the reaction vessel by washing the magnetic particle solid phase 1. it can. The operation of separating the bound antigen 5 and the free antigen 3 by washing is called B / F separation. After the B / F separation, only the bound antigen 5 remains in the reaction vessel as shown in FIG.

  Next, in the EIA method, an antibody (enzyme-labeled antibody) labeled with an appropriate enzyme (for example, HRP, ie, horseradish peroxidase) against the bound antigen 5 forming the antigen-antibody complex 4 6 is bound by an antigen-antibody reaction, and the bound antigen 5 is labeled with an enzyme. FIG. 1 (d) shows this situation. When enzyme-labeled antibody 6 labeled with labeling enzyme 7 is added to the reaction vessel and reacted with magnetic particle solid phase 1 for a certain period of time, enzyme-labeled antibody 6 sandwiches bound antigen 5 with solid-phase antibody 2. The enzyme-labeled antibody-antigen-antibody complex 8 is formed by sandwiching them in the shape. At this time, in the EIA method, in order to sufficiently label the bound antigen 5 bound to the solid phase antibody 2, an excess amount of the enzyme-labeled antibody 6 is typically added. Therefore, after labeling the bound antigen 5 by the antigen-antibody reaction between the bound antigen 5 and the enzyme-labeled antibody 6, the enzyme-labeled antibody 9 bound to the antigen and the extra free enzyme label not bound to the antigen. It is necessary to perform the operation of separating the antibody 6, that is, the B / F separation again. After this B / F separation, only the enzyme-labeled antibody-antigen-antibody complex 8 labeled with an enzyme remains on the surface of the magnetic particle solid phase 1 as shown in FIG. This means that as many labeled enzymes 7 as the number of bound antigens 5 remaining on the surface of the magnetic particle solid phase 1 remain.

  As shown in FIG. 1 (f), the amount of the labeling enzyme 7 bound to the surface of the magnetic particle solid phase 1 is added with a coloring reagent (for example, OPDA, that is, orthophenylenediamine) 10 that can be a substrate for the enzyme. Then, it can be quantified by reacting with the labeling enzyme 7 and measuring the amount of the coloring reagent 11 after coloring that is generated.

  In the above example, the method for quantifying an antigen using a solid phase antibody and an enzyme-labeled antibody has been described. However, the same operation is performed using a solid phase antigen and an enzyme-labeled antigen instead of the solid phase antibody and the enzyme-labeled antibody. Can be used to quantify the antibody in the sample.

  As described above, in the EIA method, B / F separation for separating an antigen (or antibody) bound to a magnetic particle solid phase by an antigen-antibody reaction and an unbound free antigen (or antibody), and an antigen B / F separation for separating the labeled antibody (or labeled antigen) bound to the antibody complex from the unbound free labeled antibody (or labeled antigen) is an important key for determining the accuracy of the measurement.

  FIG. 2 shows the configuration of a B / F separation apparatus for the magnetic separation solid phase method. In the figure, 12 is a magnetic particle solid phase. Normally, in the magnetic separation solid phase method, a magnet 14 is disposed at the position A outside the side wall of the separation container 13, the magnetic particle solid phase 12 is magnetized by applying a magnetic field, and the magnetic particle solid phase 12 is magnetically attracted. Then, they are held together on the inner wall side surface of the separation container 13. The water supply / drainage nozzle 15 is inserted into the separation container 13 and the solution containing the released antigen (or antibody) is removed by suction. Next, the magnet 14 is moved away from the position B, a new cleaning solution is injected, and the magnetic particle solid phase 12 is resuspended and cleaned. Thereafter, the magnet 14 is returned to the A position again, and the magnetic particle solid phase 12 is gathered and held on the side surface of the inner wall of the separation container 13, and the cleaning liquid is sucked and removed. This is repeated a plurality of times to perform B / F separation.

JP 7-181188 A JP-A-8-62224 JP-A-8-178931 JP-A-9-218201 JP 9-325148 A Japanese Patent Laid-Open No. 10-96732 Japanese Patent Laid-Open No. 10-123136 JP-A-11-262678 JP 11-326338 A JP 2001-91521 A Japanese National Patent Publication No. 11-502625 JP 2001-510893 A JP-T-2002-528705 JP-T-2002-531811

  By the way, the efficiency of magnetic fractionation (magnetic force F) is expressed by the equation (1): the first term particle mass [m] × acceleration and the second term particle cross section [S] × velocity × viscosity coefficient. It is given as the sum of [σ].

The magnetic moment [M] of equation (1) is given by equation (2) as the product of vacuum permeability [μ ₀ ], magnetic susceptibility [X _m ], and magnetic field [H]. Here, the magnetic permeability [μ ₀ ] and the magnetic susceptibility [X _m ] of the vacuum are constants.

  In order to magnetically separate magnetic beads having a diameter of submicron to several microns at a high speed, a magnetic circuit having a large third term, magnetic field gradient [dH / dX] in equation (1) may be designed. In order to provide a high-throughput B / F separator, it is necessary to gather magnetic particles in a short time. For that purpose, as shown by the principle of magnetic separation, a magnet with a high magnetic field gradient [dH / dX] and a high magnetic field gradient is required. That is, a high gradient magnetic field in the Z direction is required from the magnet (●) as shown in FIG.

  As an example, the relationship between the number of superposed neodymium magnets and the magnetic field strength on the magnet surface is shown in FIG. As is apparent from the figure, even if one to ten neodymium magnets are stacked, the increase rate of the surface magnetic field decreases as the number of neodymium magnets increases, and an efficient increase in magnetic field strength cannot be obtained. Therefore, a large magnet is required to configure a magnet with a high gradient magnetic field.

  In the experiment, even when ten neodymium magnets having the highest magnetic field strength available were used, a high gradient magnetic field with which the magnetic particles in the separation vessel having a liquid layer of 5 mmφ could be assembled within 5 seconds was not obtained.

  In order to solve this problem, a larger and heavier magnet is required, resulting in a new problem that the space factor is inferior. In addition, large heavy magnets are difficult to drive at high speed, and there is a high risk of causing trouble.

  In view of the above-described points, the object of the present invention is to provide a B / F separation apparatus for immunoassay using a small, lightweight and high-gradient magnetic field that allows high-throughput by collecting magnetic particles within 5 seconds. And to provide a method.

To achieve this goal,
The B / F separation device and method in the immunoassay according to the present invention include:
After using a magnetic substance as a solid phase and reacting a solid phase antibody (or solid phase antigen) with the target antigen (or antibody) by antigen-antibody reaction, an excess amount of labeled antibody (or labeled antigen) labeled with a labeling substance is added. In an immunoassay method that adds and labels an antigen (or antibody),
After the antigen-antibody reaction between the solid phase antibody (or solid phase antigen) and the target antigen (or antibody) and between the target antigen (or antibody) and the labeled antibody (or labeled antigen), the solid phase antibody ( Or solid phase antigen) using a magnetic device for B / F separation,
B / F separation of solid-phase antibody (or solid-phase antigen) is performed by using a magnet pair in which repulsive same polarities are opposed to each other and placing a separation container close to the gap between the magnetic poles. It is a feature.

  Further, the ratio of the distance between the magnetic poles arranged opposite to each other (gap distance) and the length of the magnet in the opposite axial direction is 0.001 to 0.5.

  In addition, a plurality of magnets in which repulsive same poles are arranged to face each other are continuously arranged according to the moving direction of the separation container, and B / F separation is performed at once by the plurality of separation containers.

In addition, using a magnetic substance as a solid phase, a solid phase antibody (or solid phase antigen) and an antigen of interest (or antibody) are reacted with an antigen, and then an excessive amount of labeled antibody (or labeled antigen) labeled with a labeling substance is used. In the immunoassay method in which the antigen (or antibody) is labeled by adding
After the antigen-antibody reaction between the solid phase antibody (or solid phase antigen) and the target antigen (or antibody) and between the target antigen (or antibody) and the labeled antibody (or labeled antigen), the solid phase antibody ( Or solid phase antigen) using a magnetic device for B / F separation,
The magnet is surrounded by a yoke from the opposite end surface of the separation container to the opposite surface side of the separation container to suppress the leakage magnetic field, and the periphery of the separation container side end surface of the magnet is surrounded by the tip of the yoke to concentrate the magnetic flux. B / F separation of solid phase antibodies (or solid phase antigens) is performed using a magnet device.

  In the magnet device, the end surface of the magnet opposite to the separation container and the end of the yoke surrounding the magnet separation container side end face have the same polarity, and the magnet separation container side end surface and the magnet This is characterized in that it has a reverse polarity to the tip of the yoke surrounding the periphery of the separation container side end surface.

According to the B / F separation device and method for immunoassay of the present invention,
After using a magnetic substance as a solid phase and reacting a solid phase antibody (or solid phase antigen) with the target antigen (or antibody) by antigen-antibody reaction, an excess amount of labeled antibody (or labeled antigen) labeled with a labeling substance is added. In an immunoassay method that adds and labels an antigen (or antibody),
After the antigen-antibody reaction between the solid phase antibody (or solid phase antigen) and the target antigen (or antibody) and between the target antigen (or antibody) and the labeled antibody (or labeled antigen), the solid phase antibody ( Or when solid phase antigen) is B / F separated using a magnetic device,
B / F separation of solid-phase antibody (or solid-phase antigen) is performed by using a magnet pair in which repulsive same polarities are opposed to each other and placing a separation container close to the gap between the magnetic poles. Because it features
It is possible to provide a B / F separation apparatus and method using a magnet that collects magnetic particles within 5 seconds and enables high throughput, and is small and light and can obtain a high gradient magnetic field.

In addition, using a magnetic substance as a solid phase, a solid phase antibody (or solid phase antigen) and an antigen of interest (or antibody) are reacted with an antigen, and then an excessive amount of labeled antibody (or labeled antigen) labeled with a labeling substance is used. In the immunoassay method in which the antigen (or antibody) is labeled by adding
After the antigen-antibody reaction between the solid phase antibody (or solid phase antigen) and the target antigen (or antibody) and between the target antigen (or antibody) and the labeled antibody (or labeled antigen), the solid phase antibody ( Or solid phase antigen) using a magnetic device for B / F separation,
The magnet is surrounded by a yoke to suppress the leakage magnetic field, and the solid phase antibody (or solid phase antigen) is subjected to B / F separation using a magnet device in which the periphery of the magnet is surrounded by the yoke and the magnetic flux is concentrated. Because it is characterized by
It is possible to provide a B / F separation apparatus and method using a magnet that collects magnetic particles within 5 seconds and enables high throughput, and is small and light and can obtain a high gradient magnetic field.

  Embodiments of the present invention will be described below with reference to the drawings. FIG. 5 shows an embodiment of a B / F separation apparatus and method in immunoassay according to the present invention. In this embodiment, the B / F separation magnet is configured by making the N pole and N pole of the magnet or the S pole and S pole face each other. The direction of magnetic flux is perpendicular to the opposing axis of the magnet and is the direction of the arrow along the gap surface of the opposing magnet. The magnetic field strength at the center point of the gap sandwiched between the opposing surfaces of the magnet is theoretically doubled. Also, when the magnets are arranged to face each other and the back surfaces are connected by a yoke, the magnetic field strength at the center point theoretically doubles. The ratio between the distance (gap distance) between the magnets arranged opposite to each other and the length of the magnet in the opposite axial direction is kept at 0.001 to 0.5, more preferably 0.01 to 0.05.

  A configuration example of the counter magnet and the separation container is shown in FIG. A is a neodymium magnet, B is a magnet holder, C is a separation container, and D is a magnetic particle. The separation container C is disposed in parallel to the opposing axes of the two neodymium magnets A and close to the gap. The inner diameter of the separation container C is 5 mm, the length of the magnet A is 10 mm, and the gap distance is 0.5 mm. When a 0.5 mm gap was used as the magnetic field generation source for B / F separation, the measured value of the magnetic field gradient (liquid layer 5 mmφ) was 1.5 times that when a single magnet was used. The time was 3 seconds.

  In the experiment in which the magnetic particles D are assembled using the same separation container C, the electromagnet is turned on for 6 seconds when five neodymium magnets are arranged alone, and for 5 seconds when a current of 1600 ampere turns is passed using the electromagnet. Even when a current of 2000 ampere turns is used, an assembly time of 3.5 seconds is required, so the effect of this embodiment is great.

  FIG. 7 shows another embodiment of the B / F separation apparatus and method in the immunoassay according to the present invention. In this embodiment, a trapezoidal neodymium magnet A-2 with the tip of a pyramid flattened flat is placed on a rectangular neodymium magnet A-1, and the periphery of the magnet is separated from the opposite end surface of the separation container. Surrounding by the yoke E toward the facing surface side, while suppressing the leakage magnetic field, the tip end surface of the neodymium magnet A-2 and the tip end surface of the yoke E are matched, and the periphery of the separation container side end surface of the neodymium magnet A-2 Is surrounded by the tip of the yoke E, and the magnetic flux is concentrated by the yoke E.

  The polarity of the magnetic pole at this time is the same polarity as the end surface of the magnet opposite to the separation container side and the tip of the yoke surrounding the magnet separation container side end surface, and the magnet separation container side end surface, It arrange | positions so that it may become reverse polarity with the front-end | tip of the yoke surrounding the circumference | surroundings of the separation container side end surface of a magnet. That is, based on FIG. 7, the yoke E side of the neodymium magnet A-1 is the north pole, the opposite side of the neodymium magnet A-1, that is, the neodymium magnet A-2 side is the south pole, and the neodymium magnet A-2 neodymium magnet. The A-1 side is the N pole, the neodymium magnet A-2 separation container C side is the S pole, and the tip of the yoke E surrounding the separation container C side periphery of the neodymium magnet A-2 is the N pole. These magnetic poles may have the N pole and the S pole reversed.

  By configuring such a magnetic circuit, the leakage magnetic field from the neodymium magnets A-1 and A-2 is suppressed, and the magnetic flux is concentrated on the separation container C side of the neodymium magnet A-2 at the tip of the yoke E. did. By this method, the measured value of the magnetic field gradient (liquid phase 5 mm) obtained was 1.2 to 1.3 times that when a single magnet was used.

  The separation container C is disposed at a position close to the tip of the neodymium magnet A-2 so as to face the tip of the neodymium magnet A-2 where the magnetic flux is concentrated at the tip of the yoke E. At this time, since the tip surface of the neodymium magnet A-2 and the tip surface of the yoke E are matched in advance, the distance between the neodymium magnet A-2 and the separation container C can be minimized. Thereby, the magnetic particles D can be easily assembled on the inner wall surface of the separation container C.

  The neodymium magnet A-1 may be a columnar shape instead of a rectangular parallelepiped. Further, the neodymium magnet A-2 may not have a trapezoidal shape in which the tip of the pyramid is flattened, but may have a pole piece shape in which the tip of the cone is flattened.

  FIG. 8 shows another embodiment of the B / F separation apparatus and method in the immunoassay according to the present invention. In the present embodiment, a plurality of magnets in which repulsive same poles are arranged opposite to each other at both ends of the magnet are continuously arranged according to the moving direction of the separation container C, and are fixed by one magnet holder B-2. It has a configuration. B / F separation is usually repeated a plurality of times. Therefore, B / F separation is performed each time the separation container C sequentially advances from position a → position b → position c → position d. By setting it as such a structure, the multiple separation container C can be B / F-separated at once, and a B / F separation apparatus with a high space factor can be provided.

  The moving method of the counter magnet and the separation container C may be such that the magnet holder B-2 side fixing the counter magnets arranged at equal intervals may be moved, but conversely, the separation container C side arranged at equal intervals. You may make it move.

  In the example of FIG. 8, the counter magnet and the separation container C are arranged in a plane, but this is a reaction rotary table in which several tens to several hundreds of separation containers are arranged in the circumferential direction, or The counter magnet holder may be arranged concentrically with the separation container along the outer periphery or inner periphery of the disc-shaped B / F separation unit on which several separation containers are placed. By adopting a configuration in which the separation container group and the counter magnet group can rotate relative to each other, an automatic immunological analyzer equipped with an effective B / F separation mechanism can be provided.

  In addition, as for the arrangement | positioning of the north-pole and south pole of the opposing magnet of FIG. 8, as long as the same pole is made to oppose, either pole may be the head.

  Widely applicable to automatic immunoassay devices and methods.

It is a figure which shows the measurement principle of EIA method. It is a figure which shows an example of the conventional B / F separation apparatus. It is a figure which shows the relationship between a separation container and a magnetic field direction. It is a figure which shows the relationship between the number of neodymium magnets piled up, and the magnetic field intensity of the magnet surface. It is a figure which shows one Example of the B / F separation apparatus concerning this invention. It is an example of composition of a counter magnet and a separation container of a B / F separation device concerning the present invention. It is a figure which shows another Example of the B / F separation apparatus concerning this invention. It is a figure which shows another Example of the B / F separation apparatus concerning this invention.

Explanation of symbols

1: solid phase (magnetic bead solid phase), 2: solid phase antibody, 3: antigen not bound to solid phase antibody, 4: antigen-antibody complex, 5: antigen bound to solid phase antibody, 6: antigen- Enzyme-labeled antibody not bound to antibody complex, 7: labeled enzyme, 8: enzyme-labeled antibody-antigen-antibody complex, 9: enzyme-labeled antibody bound to antigen-antibody complex, 10: coloring reagent (before color development) 11: Coloring reagent (after color development), 12: Magnetic particle solid phase, 13: Separation container, 14: Magnet, 15: Water supply / drainage nozzle, A: Magnet, B: Magnet holder, C: Separation container, D: Magnetic particle, E: York

Claims (10)

After using a magnetic substance as a solid phase and reacting a solid phase antibody (or solid phase antigen) with the target antigen (or antibody) by antigen-antibody reaction, an excess amount of labeled antibody (or labeled antigen) labeled with a labeling substance is added. In an immunoassay method that adds and labels an antigen (or antibody),
After the antigen-antibody reaction between the solid phase antibody (or solid phase antigen) and the target antigen (or antibody) and between the target antigen (or antibody) and the labeled antibody (or labeled antigen), the solid phase antibody ( Or solid phase antigen) using a magnetic device for B / F separation,
B / F separation of solid-phase antibody (or solid-phase antigen) is performed by using a magnet pair in which repulsive same polarities are opposed to each other and placing a separation container close to the gap between the magnetic poles. A B / F separator for immunoassay. 2. The immunoassay according to claim 1, wherein a ratio of a distance (gap distance) between the opposed magnetic poles to a length of the magnet in the opposite axis direction is 0.001 to 0.5. B / F separator. 3. A plurality of magnets in which repulsive same poles are arranged to face each other are continuously arranged in accordance with the moving direction of the separation container, and B / F separation is performed at a time by the plurality of separation containers. The B / F separation apparatus in the immunoassay described. After using a magnetic substance as a solid phase and reacting a solid phase antibody (or solid phase antigen) with the target antigen (or antibody) by antigen-antibody reaction, an excess amount of labeled antibody (or labeled antigen) labeled with a labeling substance is added. In an immunoassay method that adds and labels an antigen (or antibody),
After the antigen-antibody reaction between the solid phase antibody (or solid phase antigen) and the target antigen (or antibody) and between the target antigen (or antibody) and the labeled antibody (or labeled antigen), the solid phase antibody ( Or solid phase antigen) using a magnetic device for B / F separation,
The magnet is surrounded by a yoke from the opposite end surface of the separation container to the opposite surface side of the separation container to suppress the leakage magnetic field, and the periphery of the separation container side end surface of the magnet is surrounded by the tip of the yoke to concentrate the magnetic flux. A B / F separation device for immunoassay, wherein a solid phase antibody (or solid phase antigen) is subjected to B / F separation using a magnet device. In the magnet device, the end surface of the magnet opposite to the separation container side and the tip of the yoke surrounding the periphery of the magnet separation container side end have the same polarity, and the magnet separation container side end surface is separated from the magnet. 5. The B / F separation apparatus for immunoassay according to claim 4, wherein the B / F separation apparatus has an opposite polarity to a tip of a yoke surrounding the periphery of the container side end surface. After using a magnetic substance as a solid phase and reacting a solid phase antibody (or solid phase antigen) with the target antigen (or antibody) by antigen-antibody reaction, an excess amount of labeled antibody (or labeled antigen) labeled with a labeling substance is added. In an immunoassay method that adds and labels an antigen (or antibody),
After the antigen-antibody reaction between the solid phase antibody (or solid phase antigen) and the target antigen (or antibody) and between the target antigen (or antibody) and the labeled antibody (or labeled antigen), the solid phase antibody ( Or solid phase antigen) using a magnetic device for B / F separation,
B / F separation of solid-phase antibody (or solid-phase antigen) is performed by using a magnet pair in which repulsive same polarities are opposed to each other and placing a separation container close to the gap between the magnetic poles. B / F separation method in immunoassay characterized. The ratio of the distance (gap distance) between the magnetic poles arranged opposite to each other and the length of the magnet in the opposite axis direction is 0.001 to 0.5. B / F separation method. 8. A plurality of magnets in which repulsive same poles are arranged to face each other are continuously arranged in accordance with the moving direction of the separation container, and B / F separation is performed at once by the plurality of separation containers. The B / F separation method in the immunoassay described. After using a magnetic substance as a solid phase and reacting a solid phase antibody (or solid phase antigen) with the target antigen (or antibody) by antigen-antibody reaction, an excess amount of labeled antibody (or labeled antigen) labeled with a labeling substance is added. In an immunoassay method that adds and labels an antigen (or antibody),
After the antigen-antibody reaction between the solid phase antibody (or solid phase antigen) and the target antigen (or antibody) and between the target antigen (or antibody) and the labeled antibody (or labeled antigen), the solid phase antibody ( Or solid phase antigen) using a magnetic device for B / F separation,
The magnet is surrounded by a yoke from the opposite end surface of the separation container to the opposite surface side of the separation container to suppress the leakage magnetic field, and the periphery of the separation container side end surface of the magnet is surrounded by the tip of the yoke to concentrate the magnetic flux. A B / F separation method in an immunoassay, wherein a solid phase antibody (or solid phase antigen) is subjected to B / F separation using a magnet device. In the magnet device, the end surface of the magnet opposite to the separation container side and the tip of the yoke surrounding the periphery of the magnet separation container side end have the same polarity, and the magnet separation container side end surface is separated from the magnet. 10. The B / F separation method for immunoassay according to claim 9, wherein the polarity is opposite to that of the yoke tip surrounding the periphery of the container side end surface.

Images