Method for isolating molecules, cells and other particles which are specifically bound to a large particle

A method for isolating molecules, cells and other particles which are specifically bound to a large particle. It includes: 1) incubation of the sample with one or more sets of large particles which are able to specifically bind/capture a large number of molecules, cells or other particles, 2) analysis of each individual large-capturing particle mixed with the sample, and 3) sorting the large particles containing specifically-bound molecules, cells or other particles.

EXAMPLE Material and Methods: Samples: Peripheral blood was obtained from 10 female patients diagnosed as suffering from breast cancer, prior to curative surgery. All patients included in this study showed over expression of the Her2-neu oncogene in fine-needle aspiration diagnostic samples. In all cases, a minimum of 50 mL of k3 EDTA-anticoagulated peripheral blood was obtained by venipuncture. All samples were processed within a period of 6 hours after they were obtained and they were maintained at room temperature until processed. During this period, samples were allowed to sediment. Sample preparation: All k3 EDTA-anticoagulated peripheral blood samples were centrifuged at 540 g for 5 minutes (room temperature), the supernatant (plasma) was discarded in order to eliminate the soluble Her2-neu protein which could interfere with the binding of Her2-neu&plus; epithelial cells to the large-capturing particles described below and placed in a separate tube. Afterwards, the cell pellet was resuspended in an identical volume (up to 50 mL/sample) of isotonic saline solution and gently mixed by placing the sample in an end-to-end rotator. Then the sample was incubated with a set of 200 mm beads (Control particles, Union Biometrica, Boston, Mass., USA) which had been previously coated with an anti-Her 2 neu monoclonal antibody (Becton/Dickinson Biosciences, San Jose, Caif., USA) according to well-established procedures. For that purpose the sample was passed through a chamber with a 50 mm cuper net on both its entrance and end connected to the sample stream at both sides. This chamber contained around 10 4 beads coated with the antiHer2-neu monoclonal antibody. The sample was allowed to pass through this chamber in a discontinuous way by clamping the tubes containing the sample both at the entrance and exit of the chamber. Speed of sample incubation with the beads was of around 1 mL/min. Afterwards, the depleted sample was collected in a 50 mL Falcon tube (Becton/Dickinson Biosciences, San Jose, Calif., USA), the saline washed out by centrifuging for 10 min at 540 g (room temperature) and resuspended in the plasma. In turn, the chamber containing the beads was washed by passing phosphate buffered saline (PBS) for 5 minutes. Afterwards, the end net of the chamber was opened and the beads allowed to drop into a tube where they remained in suspension in an isotonic buffer (PBS; pH&equals;7.6) (final volume of 20 mL). Then the beads were allowed to sediment by centrifuging for 5 min at 540 g and resuspended in a volume of 300 mL of PBS with 1% freshly prepared paraformaldehyde containing 80 ng/mL of L-alfa-lysophosphatidylcholine (Sigma, St Louis, Mo., USA). Immediately afterwards, 20 mL of an anti-cytokeratin 18 monoclonal antibody directly conjugated to fluorescein isothiocyanate (FITC) was added to the bead suspension and a 15 minute incubation in the darkness was performed (room temperature) to allow the monoclonal antibody to stain the Her2-neu epithelial cells bound to the large-capturing beads. Sorting of cytokeratin 18&plus; beads: After this incubation period, 300 uL of PBS containing 1% paraformaldehyde was added to the bead suspension. After a 10 minute incubation period at room temperature, 40 mL of sheath fluid (Union Biometrica) was added to the bead suspension. Then beads were resuspended by gently mixing in an end-to-end rotator. Then the bead suspension was analyzed in a COPAS flow cytometer equipped with an argon ion laser and detectors for both light scatter time of flight and green fluorescence. Beads were run at a speed of around 200 to 250 beads/second. Beads showing green fluorescence at levels higher than background fluorescence were sorted into a Petri dish while remaining beads were sent to the waste container.