|
Application Notes
|
MagSIGNAL beads consist of many ferromagnetic grains with random oriented magnetic moments, encapsulated in a non-magnetic silica shell. The size of the ferromagnetic grains is 10-15 nm. Due to the small size and random orientation of the magnetic moments, the beads behave superparamagnetically and the magnetic moment without a magnetic field is 0 (demonstrated in figure 1 below).
|
|
Comment
|
Magnetic immunoassays (MIA) use magnetic beads as labels instead of conventional enzymes (ELISA), fluorophores or luminescent molecules. These assays involve the specific binding of an antibody with a conjugated magnetic label to its antigen. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte (virus, toxin, bacteria, cardiac marker,etc.) quantity in the initial sample.
Magnetic beads are not affected by reagent chemistry or photobleaching and are therefore stable over time. Because measurements are based on nonlinear magnetic signals, the contribution of linear magnetic signals from materials such as sample matrix and consumables are eliminated. The technology makes magnetic immunoassays possible in a variety of formats such as conventional lateral flow test (replacing gold labels with magnetic labels), microfluidic applications and biochips.
|
|
Protocol
|
MagSIGNAL are superparamagnetic beads with a mean diameter of 300 nm, which can be used as a detection label in magnetic immunoassays, or as solid support phase in immunoassays with other readout techniques.
MagSIGNAL-COOH 300 is intended for covalent antibody coupling for specific analyte detection. Other applications include protein coupling in combination with the use of a specific antibody - for instance, streptavidin coupling and a biotinylated antibody.
|
|
Reagent Preparation
|
MES Buffer: 0.1 M 2-(N-Morpholino)ethanesulfonic acid (MES), pH 5.5
PBS Buffer: 0.01 M PBS pH 7.4
Blocking Buffer: 0.1 M Tris Hydroxymethylaminoethane (TRIS buffer)
PBST Buffer: 0.01 M PBS pH 7.4 with 0.05% Tween 20
Storing B
|
|
Assay Procedure
|
For covalent coupling of amino- group containing ligands such as antibodies, proteins or low molecular substances to MagSIGNAL-COOH, the carbodiimide method can be applied.
Carbodiimides react with the terminal carboxylate-groups from the magnetic beads to highly reactive O-acylisourea derivatives and react readily with amino-groups of the ligands, as presented in Figure 1 below.
Coupling protocol
The following protocol describes the coupling of biomolecules on 1 mL (10 mg) beads. It can be scaled up by adjusting volumes of required reagents.
1.Resuspend MagSIGNAL-COOH 300 by vortexing. Transfer 1 mL from the storage bottle to a reaction container.
2.Wash the beads:Place the reaction container in a magnetic separator and wait until the separation is completed. Discard the buffer and resuspend in 1 mL MES Buffer. Repeat this step once more.
3.Resuspend the beads in 0.50 ml MES Buffer containing 20 mg EDC and 20 mg NHS (freshly prepared!) and incubate on a shaker for 15 minutes at room temperature.
4.Place the reaction container in a magnetic separator, wait until the separation is completed, and discard the supernatant. Resuspend the beads in 1 mL MES Buffer.
5.Repeat step 4 once more. The beads now contain activated COOH groups that can bind proteins and other amine containing molecules.
6.Place the reaction container in a magnetic separator, wait until the separation is completed, and discard the supernatant.
7.Add ligands to the activated beads and add PBS Buffer to a final coupling volume of 0.5 mL. Incubate on a shaker for 2 hours at room temperature.
8.Place the reaction container in a magnetic separator, wait until the separation is completed, and discard the supernatant.
9.Wash the beads 3 x with 1 mL PBST Buffer, and discard the supernatant
10.Resuspend the particles in Blocking Buffer. Incubate on a shaker for 2 hours at room temperature.
11.Wash the particles 3 x with 1 mL PBST Buffer and 3 x with 1 mL PBS Buffer. Discard the supernatant.
12.Resuspend the beads in Storing Buffer and store at 2-8 °C.
|
|
Restrictions
|
For Research Use only
|
