At R&D Systems, recombinant proteins are sold in units of mass per vial (µg or mg) with a specific activity range rather than as units of activity per vial.
The biological activity of a recombinant protein is routinely measured using a bioassay, e.g. chemotaxis or cell proliferation assay, enzyme assay, or a functional ELISA. Due to a protein’s ability to exert multiple biological activities, several acceptable bioassays may exist for a particular protein and several different cell types may respond. Different cell types have different sensitivities to a particular protein. Therefore, the activity of a protein is related to the cell type as well as the bioassay employed.
The graphs below demonstrate that different concentrations of recombinant mouse IL-6 are needed to induce proliferation of two different cell types. The mass of protein required to elicit a 50% maximum response in the bioassay is called the ED50 . In these two commonly used mouse IL-6 assays, the B9 hybridoma cell line is approximately 100-fold more sensitive than the T1188.8.131.52 plasmacytoma cell line. Consequently, the same mass of IL-6 can be assigned two different units of activity.
Each new lot of protein and each new set of experimental conditions should be tested in a titration (dose response) experiment. This needs to be done when a new experiment is set up or with a new lot or source of protein.
A valid comparison between the bioactivity units of two proteins requires that the values be generated using the same bioassay. To follow a published protocol given in units or to compare to another vendor’s product sold in units, customers must examine how the author/vendor defined the unit. If the author/vendor compared their unit to the World Health Organization (WHO) standard unit, the information contained in the WHO Conversion Table may prove useful as a starting point.
We strongly encourage customers comparing different vendors’ proteins, especially when based on units of activity, to perform side-by-side titration experiments.
Bioassays are a measure of biological response to a protein; therefore, any variation in a bioassay may also lead to variable results. Such variations could include cell density, age of the cells, passage number, nutritional state, culture medium and supplements used, as well as the technique of the assay operator. Due to the variable nature of bioassays, the same protein preparation may not give the same ED50 on different occasions. The ED50 range given on R&D Systems’ protein inserts is typical for the preparation.
There are several sources of reference standards for proteins. The most common is the WHO standard from the National Institute for Biological Standards and Controls (NIBSC). Reference proteins are available for a limited number of proteins. When a standard is available, R&D Systems compares the standard with the recombinant protein in the same bioassay. The result of this comparison is published in the catalog and the International Unit (IU) equivalent conversion is also provided. The conversion between units and ED50 values is not absolute as International Units are not derived solely from bioassay activity and ED50 values can vary with slight changes in conditions. The best means of ensuring the correct dosage is to compare the new lot with the old lot in a side-by-side dose response assay in the investigator’s test system.
When a recombinant protein is an enzyme, specific activity is derived from an enzymatic assay. Each enzyme is tested for potency by cleavage of a substrate, and specific activity is expressed as picomoles/minute/microgram.