96T

serum, plasma

human

This kit is for research use only. Under no circumstances should it be used for therapeutic or human diagnostic applications.

Upon receipt the HRP anti-PEG vial, the mPEG standard and the anti-PEG coated 96 well plate should be placed in a -20°C freezer until use. Do not store at lower temperatures. The remainder of the kit should be stored in a refrigerator at 4°C. The microtiter strips should be kept in a sealed bag with desiccant to minimize exposure to damp air. Test kits will remain stable for six months from the date of purchase provided that the components are stored as described above.

PEGylation of therapeutic proteins by attachment of mPEG chains slows proteolytic degradation and decreases the rate of clearance from the circulatory system, thereby increasing efficacy (refs. 1 & 2). The pharmacodynamics of mPEGylated proteins are often evaluated using an assay specific for the polypeptide chain. Such an approach requires the time consuming and expensive construction of a specific ELISA. The mPEG ELISA detects the mPEG chain and is therefore suitable for assessment of the pharmacodynamics of a range of mPEGylated biologics and mPEGs.
The format of the assay is a sandwich ELISA. It uses two anti-PEG mouse monoclonal antibodies developed at Creative Diagnostics, Inc. An antibody specific for the polyoxyethylene backbone of PEG (catalog# CABT-6-25-7) is coated on the 96-well plate and used for capture, and an antibody specific for the terminal methoxy group of mPEG (catalog# CABT-5D6-3) is conjugated to horseradish peroxidase (HRP) and used for detection.
This ELISA can be used for measurement of free mPEG (Figure 1) and mPEGylated proteins with one or more mPEG chains (Figure 2). However, it will not detect PEG chains that lack a terminal methoxy group. Sensitivity varies with mPEG chain length, therefore the mPEG or mPEGylated molecule under investigation should be used to generate a standard curve. Studies at Creative Diagnostics have demonstrated that the kit recognizes mPEG chains ≥2 kDa. The standard provided with the kit is 5 kDa mPEG-amine. It is provided so that the end user can check the performance of the kit.

Figure 1. Detection of unconjugated non-branched mPEG-amines of different molecular weights using the mPEG ELISA.

Figure 2. Detection of multi-PEGylated and mono-PEGylated BSA using the mPEG ELISA. The multi-PEGylated BSA was estimated to have an average of 5-7 20 kDa mPEG chains attached per molecule of BSA.