Methods and reagents for the rapid and efficient isolation of circulating cancer cells

• US 7,332,288 B2
• Filed: 10/11/2002
• Issued: 02/19/2008
• Est. Priority Date: 02/12/1998
• Status: Expired due to Term

• Current Assignee: Menarini Silicon Biosystems S.P.A
• Original Assignee: Immunivest Corporation
• Inventors: Liberti, Paul A., Racila, Emilian V., Rao, Galla Chandra, Uhr, Jonathan W., Terstappen, Leon W. M. M.
• Primary Examiner(s): Le; Long V.
• Assistant Examiner(s): Haq; Shafiqul
• Application Number:
  US10/269,579
• Publication Number:
  US 20030129676A1
• Time in Patent Office: 1,957 Days
• Field of Search: 435/7.1, 435/7.5, 436/526, 436/533, 530/363, 530/403
• US Class Current: 435/7.1
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B82Y 25/00 Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells
• CPC Class Codes: H01F 1/0054 Coated nanoparticles, e.g. nanoparticles coated with organic surfactant

Abstract

A highly sensitive assay is disclosed which combines immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis to detect, enumerate and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood and has a greater sensitivity than conventional PCR or immunohistochemistry by 1-2 orders of magnitude. In addition, the assay facilitates the biological characterization and staging of carcinoma cells.

Claims

1. A coated magnetic particle comprising a nanoparticle core material of magnetite, a protein base coating material of bovine serum albumin directly coated onto quasi-stable crystalline clusters of said core material and an additional streptavidin coating coupled to said base coating material, wherein a fraction of the streptavidin which is coupled to said base coating material is bound to biotinylated antibody and substantial portion of said streptavidin remaining is bound to biotin-bovine serum albumin conjugate.

2. A composition comprising a plurality of magnetic particles as claimed in claim 1, suspended in a biologically compatible aqueous medium.

3. A coated, magnetic particle within a population of coated, magnetic nanoparticles, said coated, magnetic particle comprising:

a. a fractionated nanoparticle core between 90 nm and 150 nm that provides between 83% and 93% target recovery in external magnetic gradient wherein said core comprises at least one transition metal oxide;

b. a base coating material adsorbed onto said nanoparticle core wherein said material comprises a protein directly coated onto a quasi-stable crystalline cluster of said fractionated nanoparticle core; and

c. an additional streptavidin coating coupled to said base coating material, wherein a fraction of the streptavidin which is coupled to said base coating material is bound to biotinylated antibody and substantial portion of said streptavidin remaining is bound to biotin-bovine serum albumin conjugate.

4. A coated magnetic particle comprising a nanoparticle core material having a magnetic transition metal oxide core, a protein base coating material coated on the nanoparticle core and an additional streptavidin coating coupled to said base coating material through a bifunctional linking compound, wherein a fraction of the streptavidin which is coupled to said base coating material is bound to biotinylated antibody and substantial portion of said streptavidin remaining is bound to biotin-bovine serum albumin conjugate.

5. The coated magnetic particle of claim 4 wherein said bifunctional compound is succinimidyl-propiono-dithiopyridine or sulfosuccinimidil-4-[maleimidomethyl]cyclohexane-1-carboxylate.

6. The coated magnetic particle of claim 4 wherein said magnetic transition metal oxide core is magnetite.