Immunoaffinity purification is considered a subset of affinity chromatography utilizing an immunoglobulin as the biospecific ligand. The immunoglobulin can be either a polyclonal grouping of antibody molecules or a monoclonal antibody. (1)
Purification techniques target the binding of specific proteins from cell or tissue lysates to immobilized antibody groups like Protein A/G resin or Streptavidin. After a series of wash steps, the target molecule or protein is selectively eluted for analysis. Immunoaffinity applications range from clinical diagnostics to environmental monitoring.
There are several product types for immunoaffinity purification using Protein A/G or streptavidin such as prepackaged cartridges, spin columns and magnetic beads. Magnetic beads for use in isolation and separation of small molecules, proteins or antibodies are incubated with a sample. Unbound contaminants are washed from the bead and then the target bound molecules are eluted. Unlike cartridges or spin columns, magnetic bead purification methods can be automated, but still pose some problems. In addition to their costly nature, magnetic beads have been known to come loose and make their way into the injection system of the instrumentation (often LC-MS/MS), which can severely damage the instrument.
How does Tip-on-Tip SPE compare to other automated methods for immunoaffinity purification?
DPX Filtration tips utilized within the Tip-on-Tip solid phase extraction (ToT SPE) workflow provide a flexible format for the automation of immunoaffinity purification. ToT SPE can be used as an automated alternative to spin columns or cartridges. For ToT SPE, a much less expensive, agarose or silica based immunoaffinity resin is mixed with the sample solution. After an optimal incubation period, the solution is aspirated as a slurry and the resin is collected in the filtration tip. The ToT device is subsequently washed and analytes eluted by aspirating and dispensing wash and elution solvents, respectively. Wash and elution steps only take a couple of minutes. The figure below shows the comparison of our ToT SPE workflow to a traditional magnetic bead workflow. Resin can be purchased in bulk to use with ToT SPE methods, making this a cost effective alternative to magnetic beads.
Benefits of this Methodology
ToT SPE provides a fast, automated INTip solution for a variety of applications including immunoaffinity purification. ToT SPE is ideal for applications that require specific incubation conditions between resin and sample solution. ToT SPE utilizes a bottom Single Phase Filtration tip with a disperser. The disperser provides optimal dispersive solid phase extraction conditions during wash and elution steps.
Single Phase Filtration tips are compatible with a wide range of resins. The beauty in this technology is that customers don’t have to change the chemistry. Chemistry available in a spin column or cartridge format can be used with an INTip method. Customers can take an existing workflow and use pipette tips for ToT SPE to automate that workflow.
50 µm Low Retention Frit
30-50 µm Frit
Watch this workflow in action!
Tip-on-Tip methods are compatible with all Hamilton Robotics systems: Microlab Vantage, Star and Nimbus. The top tip used for ToT methods on a Hamilton system is a wide-bore conductive CO-RE tip (Compressed O-Ring Expansion). An o-ring precisely fits with a small groove found only in CO-RE tips. This lock-and-key design facilitates an air-tight seal for the utmost in accuracy, precision, and reproducibility regardless of the liquid being used. For the customer, this translates to being able to pipette even the most viscous samples.
 John Tharakan, Immunoaffinity Purification, in: Vedpal S. Malik, Erik P. Lillehoj (Eds.), Antibody Techniques, Elsevier BV, New York, 1994, pp. 327-341. https://doi.org/10.1016/B978-0-12-466460-9.50019-5