Oct 09, 2024
A protocol for the isolation of IgE antibodies from human plasma using magnetic beads
- Gudrun Baersch1
- 1Genekam Biotechnology AG
Protocol Citation: Gudrun Baersch 2024. A protocol for the isolation of IgE antibodies from human plasma using magnetic beads. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8j52lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 08, 2024
Last Modified: October 09, 2024
Protocol Integer ID: 109358
Keywords: IgE, Antibodies, Human Plasma, Magnetec Beads, Isolation method, Allergies
Disclaimer
Genekam Biotechnology AG
Duissernstr. 65a
47058 Duisburg
Germany
Abstract
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.
Image Attribution
Genekam IgE magnetic beads
Guidelines
Ethical guidelines may apply, please check before starting experiments.
Materials
Equipment needed
- Magnetic beads antibody isolation kit (Human IgE; full kit including all buffers, SB0238)
- Magnetic beads (sterile product) - Tube A
- Magnetic rack (Genekam Magnetic separator for different tubes: 6 x 1.5 -2ml, SB0196)
- Buffer solution -Tube B
- Elution buffer - Tube C
- Stop solution - Tube D
- Pipettor
- Pipette tips
- Lamina Flow if the user wants to produce a sterile product.
Safety warnings
-Keep the magnetic beads away from sun light.
-If the package and the bottles are damaged don´t use the kit.
-Read the material safety data sheet.
-If user wants to have sterile end product, one must work under laminar flow and with sterile instruments.
Precautions
-The kit is intended for in vitro use only.
-The kit should only be used by trained persons.
-The user must read the manual for use carefully.
-The kit should not be used after the expiry date.
-The user should work very cleanly during the removal process.
-Decontaminate the instruments regularly (once a week).
-The user should wear protective gloves and laboratory clothing.
Ethics statement
Since the user is working with human plasma, ethical standards may apply and ethics committee approval may be required.
Before start
Please read manual.
Prepare the chemicals to be used.
Preparatory work:
Label the tubes and calculate the chemicals required.
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).
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.
Leave the mixture at room temperature for 30 minutes in dark. Shake occasionally.
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).
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.)
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.
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).
The isolated IgE antibodies should be confirmed using ELISA or other methods.
Figure 1: Tube with magnetic beads in the magnetic rack
Figure 2: Scheme of antibody isolation with magnetic beads
Protocol references
Bhatia, S. (2023). A Simple, Rapid, and Highly Sensitive Magnetic Beads ELISA for Detection of SARS CoV-2 Antibodies (IgG) in Human Plasma Samples as a Point of Care Assay. Mikrobiolohichnyi Zhurnal, 85(6), 61-65. https://doi.org/10.15407/microbiolj85.06.061
Bhatia, S. Baersch, G. A simple and quick protocol for isolating human IgM antibodies from plasma using magnetic beads. Aug 14, 2024, dx.doi.org/10.17504/protocols.io.4r3l2qbbxl1y/v1
Bhatia, S. Baersch, G., Simple and fast technique for separating human mononuclear cells from small amounts of blood and buffy coat with high cell yield. Jul 18, 2024, dx.doi.org/10.17504/protocols.io.14egn6m9zl5d/v1