Apparatus and methods for magnetic separation

• US 5,200,084 A
• Filed: 09/26/1990
• Issued: 04/06/1993
• Est. Priority Date: 09/26/1990
• Status: Expired due to Term

• Current Assignee: Janssen Diagnostics, LLC (Johnson & Johnson)
• Original Assignee: Immunicon Corporation
• Inventors: Liberti, Paul A., Feeley, Brian P., Gohel, Dhanesh I.
• Primary Examiner(s): Dawson, Robert A.
• Assistant Examiner(s): Savage, Matthew O.
• Application Number:
  US07/588,662
• Time in Patent Office: 923 Days
• Field of Search: 210/222, 210/223, 210/695, 252/408.1, 435/7.9, 435/7.92, 436/18, 436/177, 436/526, 514/557
• US Class Current: 210/695
• CPC Class Codes: B03C 1/002 High gradient magnetic separation
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/025 High gradient magnetic separators
• CPC Class Codes: B03C 1/032 Matrix cleaning systems
• CPC Class Codes: B03C 1/0332 using permanent magnets
• CPC Class Codes: B03C 1/034 characterised by the matrix elements
• CPC Class Codes: B03C 1/035 Open gradient magnetic separators, i.e. separators in which the gap is unobstructed, characterised by the configuration of the gap
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: G01N 2333/70589 CD45
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: G01N 35/026 having blocks or racks of reaction cells or cuvettes
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

Magnetic separation apparatus and methods for separating colloidal magnetic particles from a non-magnetic test medium in which the magnetic particles are suspended. The separator comprises a container holding the non-magnetic test medium, one or more magnetic wires disposed substantially within the test medium in the container and an external magnet for producing a magnetic field gradient within the test medium. According to the method of the invention, the container holding the test medium is positioned in the separator, producing a magnetic field gradient operative to cause the magnetic particles to be attracted to the areas surrounding the magnetized wires and to adhere to the wires. The non-magnetic test medium is separated from the magnetized particles, which may conveniently be washed while adhered to the wires, and subjected to further analysis, preferably while on the wires. The apparatus and method are useful in separating various target substances from test media by means of substances coated on the magnetic particles which bind specifically to the target substance. A modified buffer solution, when added to the test medium, reduces non-specific binding of the magnetic colloid particles to cells.

Claims

1. A magnetic separator used to separate magnetic particles from a non-magnetic test medium in which said magnetic particles are suspended, the separator comprising:

a) a non-magnetic container having an opening for receiving said test medium and defining a separation chamber;

b) one or more magnetic wires, each having a longitudinal axis, positioned within the container and effective in said test medium to intensify a magnetic field gradient generated in the test medium by an applied magnetic field, said one or more wires each being in the form of a loop having spaced apart ends so that the surfaces of said wires do not form capillaries, pockets, or interstices which might entrap non-magnetic components of the test medium, and a carried connected to said ends of said wires disposed outside of said separation chamber; and

c) magnetic means having poles confronting each other for generating magnetic flux lines extending between the poles transverse to the longitudinal axis of said wires to generate a magnetic field gradient operative upon the magnetic particles within the test medium to attract said magnetic particles toward the magnetized wires and cause such particles to be adhered to the wires.

2. A magnetic separator as claimed in claim 1, wherein said magnetic wires are disposed within said container with sufficient rigidity to resist substantial deformation when passed into or out of a magnetic field of 4-15 kGauss.

3. A magnetic separator as claimed in claim 1, wherein said one or more magnetic wires have a diameter of between about 0.8 mm and about 3.0 mm.

4. A magnetic separator as claimed in claim 1, wherein said wires are coated with a coating material capable of facilitating the removal of said particles from the surface of said wires.

5. A magnetic separator as claimed in claim 1, wherein said wire is in the shape of two substantially parallel semicircular loops having respective ends attached to said carrier.

6. A magnetic separator as claimed in claim 5, wherein said magnetic means comprise confronting magnets and the carrier is non-magnetic and is adapted to be mounted upon the confronting magnets.

7. A magnetic separator as claimed in claim 6, wherein said confronting magnets generate a field of about 7.0 to about 8.5 kGauss.

8. A magnetic separator as claimed in claim 1, wherein the separator also comprises a particle collector used to collect the magnetic particles remaining adhered to the magnetized wire after the container holding the test medium is removed from the wire, said particles being adapted to be separated from the wire and transferred to said particle collector.

9. A magnetic separator used to separate magnetic particles from a non-magnetic test medium in which said magnetic particles are suspended, the separator comprising:

a) a non-magnetic container having an opening for receiving said test medium and defining a separation chamber;

b) one or more magnetic wires, each having a longitudinal axis, positioned within the container and effective in said test medium to intensify a magnetic field gradient generated in the test medium by an applied magnetic field, and a carrier for said wires disposed outside of said separation chamber; and

c) magnetic means having pole pieces confronting each other for generating magnetic flux lines extending between the pole pieces transverse to the longitudinal axis of said wires to generate a magnetic field gradient operative upon the magnetic particles within the test medium to attract said magnetic particles toward the magnetized wires and cause such particles to be adhered to the wires, wherein said non-magnetic container comprises a microtiter well.

10. A magnetic separator used to separate magnetic particles from a non-magnetic test medium in which said magnetic particles are suspended, the separator comprising:

a) a non-magnetic container having an opening for receiving said test medium and defining a separation chamber;

b) one or more magnetic wires, each having a longitudinal axis, positioned within the container and effective in said test medium to intensify a magnetic field gradient generated in the test medium by an applied magnetic field, and a carrier for said wires disposed outside of said separation chamber; and

c) magnetic means having pole pieces confronting each other for generating magnetic flux lines extending between the pole pieces transverse to the longitudinal axis of said wires to generate a magnetic field gradient operative upon the magnetic particles within the test medium to attract said magnetic particles toward the magnetized wires and cause such particles to be adhered to the wires, wherein said non-magnetic container comprises a capillary tube.

11. A magnetic separator used to separate magnetic particles from a non-magnetic test medium in which said particles are suspended, the separator comprising:

a) a non-magnetic container with an open top capable of receiving the test medium;

b) a wire screen both adapted to be held in a fixed position within the container and formed into a triangularly shaped body having a central cavity and open at both ends; and

c) magnetic means for applying a magnetic field substantially transverse to the longitudinal axis of the wires in said wire screen to generate a magnetic field gradient operative upon the magnetic particles within the test medium to attract said magnetic particles toward the magnetized screen and cause such particles to be adhered to the screen.

12. A magnetic separator as claimed in claim 11, wherein the container also comprises a particle collector used to collect the magnetic particles remaining adhered to the magnetized screen after the test medium is decanted from the container holding the screen, said particles being adapted to be separated from the screen and collected in said particle collector.

13. A magnetic separator as claimed in claim 12, wherein the screen is coated with a coating material capable of facilitating the removal of the particles from the surface of the screen.

14. A magnetic separator as claimed in claim 11, wherein the magnetic means for applying the magnetic field comprises confronting magnets positioned on opposite sides of the non-magnetic container holding the wire screen.

15. A magnetic separator as claimed in claim 14, wherein said confronting magnets generate a field of about 7.0 to about 8.5 kGauss.

16. A magnetic separator used to separate magnetic particles from a non-magnetic test medium in which said magnetic particles are suspended, the separator comprising:

a) an array of capillary tubes, each of said tubes having an opening for receiving said test medium;

b) one or more magnetic wires disposed within the capillary tubes and substantially in contact with said test medium;

c) magnetic means for applying a magnetic field substantially transverse to the longitudinal axis of said wires to generate a magnetic field gradient operative upon the magnetic particles within the test medium to attract said magnetic particles toward the magnetized wires and cause such particles to be adhered to the wires.

17. A magnetic separator used to separate magnetic particles from a non-magnetic test medium in which said magnetic particles are suspended, the separator comprising:

a) an array of interconnected non-magnetic microtiter wells, each with an open top capable of receiving said test medium;

b) one or more magnetic wires capable of being disposed substantially within each of the microtiter wells and in contact with said test medium;

c) magnetic means for applying a magnetic field transverse to the longitudinal axis of said wires to generate a magnetic field gradient operative upon the magnetic particles within the test medium to attract said magnetic particles toward the magnetized wires and cause such particles to be adhered to the wires.

18. A magnetic separator as claimed in claim 17 wherein said wires are in the form of semicircular loops having end portions, and including a holder engaging said end portions and positioning said loops in said wells.

19. A magnetic separator as claimed in claim 17 wherein said wires comprise a wire screen formed into a hollow shape open at both ends and held in a fixed position within the container.

20. A magnetic separator as claimed in claim 19 wherein said wire screen comprises a triangularly shaped body.

21. A method for magnetically separating a target substance from a non-magnetic test medium in a magnetic separator comprising a container with an open top, magnetic wire means positioned within the container providing an exposed surface area and magnetic means for magnetizing the magnetic wire means, the method comprising the steps of:

a) contacting a quantity of magnetic particles comprising a receptor capable of binding specifically to said target substance within said test medium under conditions causing binding of said receptor to said target substance, resulting in target substance-bearing magnetic particles;

b) introducing said test medium in which said magnetic particles are suspended into said container holding said magnetic wire means; and

c) positioning said container holding magnetic wire means and said test medium having the suspension of magnetic particles therein adjacent to said magnetic means in order to generate in said test medium a magnetic field gradient operative upon the magnetic particles within the test medium to attract said magnetic particles to the exposed surface area of the magnetized wire and cause such particles to be adhered to said exposed surface, and

d) controlling the quantity of magnetic particles introduced into the container relative to the exposed surface area of said wire means, and controlling the orientation of said exposed surface area causing said particles to adhere to the surface area in a substantially single layer corresponding in thickness to about the size of said target-substance bearing magnetic particles.

22. A method as claimed in claim 21 including the step of maintaining the magnetic particles adhered to the magnetic wire means within said container while removing the non-magnetic test medium from the container.

23. A method as claimed in claim 22 including the step of washing the magnetic particles adhered to the magnetic wire means to remove residual test medium from the magnetic particles and from the magnetic wire means.

24. A method as claimed in claim 23 including the step of removing from the container the magnetic wire means with the magnetic particles adhered thereto, immersing the magnetic wire means into a resuspension medium and subjecting said magnetic wire means to conditions causing resuspension of said magnetic particles in said resuspension medium.

25. A method for determining a target substance in a test medium suspected of containing said target substance, said method comprising:

a) contacting said test medium with a quantity of colloidal magnetic particles comprising or adapted to comprise a receptor capable of binding specifically to said target substance, under conditions causing binding of said target substance to said receptor, resulting in target substance-bearing magnetic particles;

b) introducing said test medium into a separation chamber containing a magnetic field gradient intensifier comprising filamentary material having a magnetic field applied thereto, whereby said magnetic particles are caused to adhere to said filamentary material;

c) removing said test medium from said separation chamber;

d) treating said magnetic particles while adhered to said filamentary material for removal of interferring substances; and

e) determining the target substance by analysis of said magnetic particles or said removed test medium.

26. A method according to claim 25 wherein said filamentary material comprises at least one ferromagnetic wire which is disposed within said separation chamber with sufficient rigidity to resist substantial deformation when passed into or out of a magnetic field of 4-15 KGauss.

27. A method according to claim 26, wherein said wire has a diameter from about 0.8 mm. to about 3 mm.

28. A method according to claim 25, wherein said quantity of magnetic particles is controlled, relative to the exposed surface area of said filamentary material and the orientation of said exposed surface is controlled, so as to cause said magnetic particles to adhere to said exposed surface in a substantially single layer corresponding in thickness to about the size of said target-substance bearing magnetic particles.

29. A method according to claim 25, wherein said magnetic particles comprise a transition metal oxide substantially surrounded by a biofunctional polymer having available coordination sites, in proportions rendering said magnetic particles resuspendable.

30. A method according to claim 29, wherein said transition metal oxide is magnetite and said biofunctional polymer is a protein or a polymer capable of being coupled to a protein.

31. A method according to claim 25, which is performed without resuspension of said magnetic particles.

32. A method according to claim 25, which is performed batchwise.

33. A method according to claim 25, wherein said test medium comprises a detectable label whose occurrence in association with said magnetic particles, or in said removed test medium is detected in determining said target substance.

34. A method according to claim 33, wherein said test medium comprises an enzyme-linked receptor for said target substance.

35. A method according to claim 34, wherein said determination of said target substance comprises detecting the activity of said enzyme by contacting with a chromogenic substance producing a detectable color, and detecting the appearance or relative intensity of said color by reference to a standard.

36. A method according to claim 35, wherein detecting the activity of said enzyme is performed with said magnetic particles adhered to said filamentary material.

37. A method for determining an immunoreactive ligand in a test sample suspected of containing said ligand, said method comprising:

a) contacting said test sample with a quantity of colloidal magnetic particles comprising or adapted to comprise a first receptor to said ligand, and a second, enzyme-linked receptor to said ligand, under conditions causing binding of said receptors to said ligand, resulting in enzyme and target-substance bearing magnetic particles.

b) introducing said test medium into a separation chamber including at least one ferromagnetic wire disposed in said chamber with sufficient rigidity to resist substantial deformation when passed into or out of a magnetic field of 4-15 K Gauss and having a magnetic field applied thereto, thereby to produce a magnetic field gradient operative upon said magnetic particles, causing said particles to adhere to said wire;

c) washing said magnetic particles to remove unbound substances; and

d) determining the activity of said enzyme bound to said particles, as a measure of the presence or quantity of said ligand in said test sample.

38. A method as claimed in claim 37, wherein said ligand is an antigen and said first and second receptors are antibodies capable of specific interaction with said antigen.

39. A method as claimed in claim 38, wherein said antigen is a cell-surface antigen.

40. A method according to claim 37, wherein said determination of ligand comprises contacting said enzyme with a chromogenic substance producing a detectable color and detecting the appearance or relative intensity of said color by reference to a standard.

41. A method according to claim 40, wherein detecting the activity of said enzyme is performed with said magnetic particles adhered to said filamentary material.

42. A method according to claim 37, wherein said quantity of magnetic particles is controlled, relative to the exposed surface area of said filamentary material, and the orientation of said exposed surface is controlled, so as to cause said magnetic particles to adhere to said surface in a substantially single layer corresponding in thickness to about the size of said enzyme and target-substance bearing magnetic particles.