TITLE: A protocol for the isolation of IgE antibodies from human plasma using magnetic beads
AUTHORS: Gudrun Baersch

[DESCRIPTION]
A protocol for the isolation of IgE antibodies from human plasma using magnetic beads

Human IgE antibodies are also present in plasma and there are many situations where isolation of these antibodies is essential. The concentration of such antibodies can vary in many allergic diseases.  Magnetic beads can be the material of choice to isolate such antibodies. The method is quite simple. It consists of 4 steps: 1. attachment of the antibodies to the magnetic beads 2. washing to remove the impurities 3. elution 4. neutralization of the solution containing the antibodies.

It is highly advisable to use magnetic racks that are designed to fit the microtubes exactly so that liquids can be removed without distrubing the bead pellet.

The advantage of the magnetic bead isolation method is that it is very fast and can be used for both small and large plasma volumes. Used plasma can be collected and used for further protein isolations. Used beads can be regenerated also.

We have developed a protocol for the isolation of IgE antibodies, which will be presented here.

If the user wants to isolate the antibodies for sterile purposes, he should work under laminar flow.


[BEFORE_START]
Please read manual.
Prepare the chemicals to be used.

[GUIDELINES]
Ethical guidelines may apply, please check before starting experiments. 

[STEPS]
1. Preparatory work:
Label the tubes and calculate the chemicals required. 
2. The user must first wash the specified quantity of magnetic beads (Tube A) in 1000µl buffer solution (Tube B). For this purpose, 1000µl buffer solution is added to the beads, the tube is placed in the magnetic rack and the liquid is pipetted off after 2 minutes while the magnetic beads remain in the tube. Resuspend the beads in corresponding amount of buffer solution (30µl Tube B). 
3. Performing of the isolation:
The user should use 30µl magnetic beads for the isolation of IgE from 2000µl plasma (or corresponding ratios). The user should use the plasma in a 1:4 dilution (add 1500µl buffer solution to 500µl plasma (Tube B). It is also possible the cell cultures containing IgE to be used instead of plasma.

4. Leave the mixture at room temperature for 30 minutes in dark. Shake occasionally.
5. Place the tube in the Genekam magnetic rack for 2 minutes. During this step, the magnetic beads are attracted towards the magnet (Fig. 1, 2).
6. Carefully remove the liquid with a pipette tip without distrubing the magnetic pellet and collect it in a new tube alternatively one can tilt the magnetic rack to drop the fluid in waste collecting container while keeping the magnetic pellet in the tube (Fig. 1). (This is only possible, if user is using Genekam magnetic rack as the tubes fits very tightly in this rack.)
7. Two washing steps: 
To do this, add 1000µl of buffer solution (Tube B) to the beads, place the tube in the magnetic rack and pipette off the liquid after 2 minutes while the magnetic beads remain in the tube.
8. Elution step:
To do this, add 100µl of elution buffer (Tube C) to the beads and incubate for 10 minutes. Place the tube in the magnetic rack and collect the liquid in a fresh sample tube after 2 minutes. Finally, add 25µl of stop solution (Tube D).
9. The isolated IgE antibodies should be confirmed using ELISA or other methods.
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