Apparatus and methods for magnetic separation featuring external magnetic means

A method for separating magnetic particles from a fluid containing same, said method comprising:

• a) introducing said fluid into a non-magnetic container having a peripheral wall and an open top;
• b) positioning said container adjacent to a magnetic means producing a magnetic field gradient in said fluid, said magnetic field being stronger in said fluid adjacent to said wall than in the fluid most distant from said wall, said magnetic field being produced by positioning a plurality of magnets facing the container and around the periphery of said container such that said magnets face the container with alternating polarity producing a substantially uniform magnetic field along a portion of said peripheral wall;
• c) collecting a substantially uniform layer of said particles on a broad portion of said peripheral wall in accordance with said substantially uniform magnetic field along said peripheral wall;
• and
• d) removing said fluid from said container separately from the magnetic particles adhering to said wall while said container is positioned adjacent said magnetic means.

A method for determining the presence or quantity of an immunoreactive ligand in a test medium suspected of containing said ligand, said method comprising:

• a) contacting said test medium with a quantity of colloidal magnetic particles comprising a first receptor thereon which specifically binds to said ligand, and with a second, enzyme-linked receptor which specifically binds to said ligand, under conditions causing binding of both said receptors to said ligand, resulting in magnetic particles bearing both enzyme and ligand;
• b) introducing said test medium into a non-magnetic container having a peripheral wall;
• c) positioning said container adjacent to a magnetic means for generating a high gradient magnetic field in said test medium, said magnetic field being stronger in said test medium adjacent to said wall than in said test medium most distant from the wall and operative upon said magnetic particles to cause said particles to adhere to said wall, said magnetic means comprising a plurality of magnets facing the container and positioned around at least a portion of the periphery of said container;
• d) removing said test medium from said container while said magnetic particles remain adhered to the wall of said container;
• e) washing said magnetic particles while said particles remain adhered to said wall to remove unbound substances therefrom;
• and
• f) 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.

A method for separating a bioentity from a test sample within a non-magnetic vessel, said bioentity selected from the group consisting of cells, cell components, and viruses, having a characteristic determinant, said method comprising:

• a) introducing into said test sample a reagent which specifically birds to said determinant, in an amount sufficient to bind a portion of said reagent to said determinant and to provide a remaining portion of excess reagent in said test sample;
• b) introducing into said test sample, in the presence of said excess reagent, a multivalent capture agent which specifically binds to said determinant-bound reagent, thereby to form a bioentity-reagent-capture agent complex, at least one of said determinant-bound reagent and said capture agent conjugated to colloidal magnetic particles;
• and
• c) collecting said complex from said test sample upon the interior periphery of the vessel under the influence of a high gradient magnetic field.

A method for separating a biological cell having a characteristic determinant from a test medium, said method comprising:

• a) introducing into said medium a monoclonal antibody which specifically birds to said determinant, in an amount sufficient to bind said monoclonal antibody to said determinant and to provide excess monoclonal antibody in said test sample;
• b) introducing into said test sample, in the presence of said excess monoclonal antibody, colloidal magnetic particles conjugated to anti-Fc which specifically birds to said determinant-bound monoclonal antibody, thereby to form a cell-monoclonal antibody-magnetic anti-Fc conjugate;
• and
• c) separating said conjugate from said medium under the influence of a high gradient magnetic field.

A method for separating bioentity from a test sample within a non-magnetic vessel, said bioentity selected from the group consisting of cells, cell components, and viruses, having a characteristic determinant, said method comprising:

• a) introducing into said test sample a reagent which specifically birds to for said determinant, in an amount sufficient to bind a portion of said reagent to said determinant and to provide a remaining portion of excess reagent in said test sample;
• b) introducing into said test sample, in the presence of said excess reagent, a multivalent capture agent which specifically binds to said determinant-bound reagent, thereby to form a bioentity-reagent-capture agent complex, at least one of said determinant-bound reagent and said capture agent conjugated to colloidal magnetic particles;
• and
• c) collecting said complex from said test sample upon a collecting surface under the influence of a high gradient magnetic field and in the presence of said excess.

A method for magnetically separating a target substance from a non-magnetic test medium, comprising the steps of:

• a) placing the test medium in a container having a peripheral wall;
• b) attaching said target substance to colloidal magnetic particles in suspension having a binding agent thereon which specifically binds with said target substance, resulting in target substance-bearing magnetic particles;
• c) producing in said test medium a high gradient magnetic field in which the magnetic field is stronger in the test medium adjacent to said wall of the container than in the test medium more distant from the wall and is operative upon the magnetic particles to attract said magnetic particles toward said wall and cause such particles to be adhered to a broad portion of said wall, and
• d) controlling the quantity of magnetic particles in said suspension relative to the surface area of said broad portion of said wall and controlling the orientation of the exposed surface area relative to said magnetic means, thereby to cause adherence of said particles to said broad portion of said wall in a substantially single layer to substantially prevent entrapment of interfering substances.

A method for determining the presence or quantity of an immunoreactive ligand in a test medium suspected of containing said ligand, said method comprising:

• a) placing the test medium into a container having a peripheral wall;
• b) contacting said test medium with a quantity of colloidal magnetic particles having a first receptor thereon which specifically binds to said ligand;
• c) contacting said test medium with a second receptor which specifically binds to said ligand, said second receptor having a label with a detectable characteristic;
• d) producing in said test medium a high gradient magnetic field in which the magnetic field is stronger in the test medium adjacent to said wall of the container than in the test medium more distant from the wall and is operative upon the magnetic particles attached to said ligand to attract said magnetic particles toward said wall;
• e) collecting said magnetic particles on a broad portion of said wall;
• f) removing said test medium from said container while said magnetic particles remain adhered to the wall of said container;
• g) washing said magnetic particles while said particles remain adhered to said wall to substantially remove unbound substances therefrom;
• and
• h) detecting the detectable characteristic of said second receptor bound to said ligand, as a measure of the presence or quantity of said ligand in said test sample.