Molecular probes for biomolecular recognition are of great importance in the fields of chemistry, biology, medical sciences and in biotechnology as well. These probes have been used in studies of biological functions and in ultrasensitive detection of biological factors responsible for many diseases (1). On the other front, the developments of nontoxic and biocompatible magnetic particles have been disclosed for biological applications since mid-1980s (2).

Recently, magnetic particles have attracted growing interest as high performance biomaterial which is used for transport and separation of cells or cell parts (2, 3), MRI (4), hyperthermia (5) and drug delivery (6). Bio-logical samples such as blood, serum, cell suspensions and cell lysates are allowed to be exposed to specific ligand-coupled particles, and the captured molecules or cells are then rapidly separated using magnetic fields (3, 7, 8).

Magnetic particles conjugated with anti tumor monoclonal antibodies provide a new approach to identify tumor cells.

Antibodies labeled with magnetic nano-particles give magnetic signals on exposure to a magnetic field. Iron oxide particles are usually coated with different organic shells including dextran, albumin or polyethylene glycol. Coated nanoparticles can be manufactured with a variety of functional groups (such as amino, aldehyde, hydroxyl, sulfate and carboxyl groups) on their surfaces. Considering these properties, we used super paramagnetic iron oxide (nanomag-D-SPIO 20nm) with COOH group on the surface, for conjugation to a humanized anti her2/neu monoclonal antibody (Herceptin) as a cancer targeting antibody.

Nanomag-D-SPIO 20 nm nanoparticles (surface COOH) and MACS separator with MS columns were purchased from Micromod (Miltenyi Biotech GmbH, Germany). The breast carcinoma cell lines SKBR-3 and T47D were obtained from Pasteur Institute of Iran. Other reagents and chemicals were obtained from Merck and Sigma.

We used 20nm nanoparticles (a magnetic core covered with dextran) with carboxyl group for conjugation to Herceptin as a cancer targeting antibody. The final products of conjugation were suspensions without precipitate and the amount of immobilized antibody was 20–36 µg Ab/mg nanoparticles (Figure 1).

The amount of iron conjugated was determined by potassium thiocyanate method using FeCl3-6H2O as a reference (Figure 2).

The SKBR3 and T47D cell lines showed high levels of her2/neu expression, after incubation with Herceptin-FITC (Figure 3), but not with the isotype control antibody (data not shown).

These results confirmed the presence of antigen on the cell surface. Specific binding of the conjugates to the cells can be determined by using electron microscopy, MRI imaging and iron staining (13, 18, 19).

In this study after incubation of the cells with conjugated nanoparticles, the blue stain induced by iron staining showed the accumulation of conjugated nanoparticles on the cell surface (Figure 4).

Polymer-coated magnetic particles with particle sizes 5–500 nm have been employed in medicine or biotechnology for many years (16). In this study the 20 nm nanoparticles were coupled via their surface carboxyl group to the amino groups on the Herceptin antibody using the EDC method (10, 17).

After conjugation, the amount of immobilized antibody was approximately 20 µg/mg magnetite. However by increasing concentration of antibody during the process, the efficiency of the conjugation did not improve. In other studies the efficiency of conjugation has been reported as 5–20 µg Ab/mg particles (9, 18). Conjugated nanoparticles bound specifically to the her2/neu antigen.

Iron staining, confirmed the presence of nanoparticles on the cell surface. In the present study we showed specific binding of Herceptin-nanomagnetic particle conjugates to her2/neu over expressing cells, suggesting a future application of Herceptin-magnetite for MR imaging of breast cancer.