Due to the high demand of customers requesting low endotoxin antibody productions over the last year, we have tried many of the commercially available endotoxin removal products on the market. Most of these products are primarily based on immobilized Polymixin B attached to a cross-linked, beaded, agarose affinity support. The accompanying literature states they can effectively remove high levels of endotoxin in one pass with minimal antibody loss. Polymixin B resin works by neutralizing the biological activity of endotoxin, which binds to the Lipid A portion of the lipoploysaccharide. I found these Polymixin-B based products to be effective in neutralizing endotoxin levels by ~ 75-80%, however we were seeing antibody losses of greater then 50% at times. Discouraged with these results, I discovered a new endotoxin removal product made by Associates of Cape Cod that worked remarkably well. They have developed a new endotoxin removal system based on an endotoxin-specific binding protein from the horseshoe crab,
Limulus polyphemus. The kit consists of microcentrifuge tubes that contain the Endotoxin Neutralizing Protein (ENP), which is linked to 65micron diameter silica beads. ENP is an 11.8 kD protein purified from the horseshoe crab that has strong binding characteristics to most strains of endotoxin except Vibrio sp.
End-x endotoxin removal resin is very easy to use and comes complete with instructions. The entire process can be completed in an hour and should be done under a laminar flow hood to limit any additional bacterial contamination into the process. To begin with, the samples are first tested to identify their endotoxin load so you can monitor their bacterial reduction as they are incubated on the End-x beads. Once tested, samples are transferred into the End-x tubes and then placed on a lab bench rocker and rotated end to end for 1.0 hour at 4°C. Most of the endotoxin that was contained in the sample will be bound to the resin within the first hour of incubation. After incubation, the tubes are spun in a centrifuge for 2-3 minutes to separate the supernatant from the resin. The supernatant is then carefully transferred away from the resin into one of the pyrogen free receiver tubes enclosed with the kit while the endotoxin remains attached to the resin. Samples are tested once again by preferred method to monitor for endotoxin levels. Once tested, samples can be placed back into the tubes for additional incubation if the endotoxin levels are still too high. The one advantage I found with the End-x tubes is they can be used repeatedly with the same samples until the limit of the tubes has been reached, i.e. 1ug or ~ 12,000 EU for the 2.0 mL tubes and 10ug or ~ 120,000 EU for the 50.0 mL tubes. The tubes have a very high capacity for endotoxin and can be challenged several times over before the tubes maximum capacity is reached. I found the End-x beads to successfully reduce the endotoxin load in our samples by a combined 91% with only a combined 16% product loss over three separate studies.
The price of the End-x endotoxin removal tubes was very reasonable with the smaller tubes priced at $125.00 for six of the 2.0 mL tube sets and each tube has a capacity to remove 1ug of endotoxin. The advantage of purchasing the smaller tube is that if you are working with volumes of less then 2.0 mL per sample, it allows you to remove endotoxin from six different samples without cross contaminating. If you are working with larger volumes and don’t mind doing more then one incubation, you can purchase the larger tube set which holds 50.0 mL. The larger 50.0 ml set costs $175.00 for one of the tubes, which has a capacity to remove 10ug of endotoxin. Additionally, they also offer a naturally derived form of the ENP, which is available in 1.0 mg and 10.0 mg quantities. This protein can be purchased separately and attached to a larger volume of affinity resin that might work better for your specific application.
Overall, we were very pleased with the reproducible results that we saw from Associates of Cape Cod End-x endotoxin removal columns. You can efficiently remove endotoxin from small volumes within an hour and not see the large sample losses that were common with other manufacturers brands. We found these End-x columns to work great in our lab for the purpose they serve and I would recommend this endotoxin removal product to others.
Thomas Sewell
R&D Applications Specialist
Maine Biotechnology Services