Stability testing of antibody 1A8

Code: R11B01JS

The research group of N. Leuenberger. J. Saugy, R. B. Mortensen, P. J. Schatz. S. Giraud and M. Saugy has demonstrated the detection or Hema tide™ (OMONTYS.., in anti-doping samples. This approach 1s based on the measurement of pegmesatlde in human serum/plasma samples by an assay and western blot employing specific monoclonal antibodies. This test has been implemented in WADA accredited laboratories as research test system based on ELISA (Enzyme-Linked lmmunoSorbent Assay) technique (screening method) and western blot (confirmation method}.

Actually the coated antibody of the assay is fresh coated directly before the measurement. Between different antibody batches there are variations in the geherated data of the ELISA The aim of this project Is to identfly a method to ensure antibody batches which result m reproducible data. Diflerent methods of antibody production, coating and storage of the antibody and the coated rnicroplate will be tested to establish a safe verilication procedure to detect 􀁶g1nesatide in human blood samples.

The successful development of a stable and functional coated antl-peginesatide antibody is ot

crucial importance for the availability of commercial test kits.

Main findings

The antibodies mAb anti-PEG WADA1A8 and mAb anti-Hematide WADA11F9 of the HematideTM ELISA generate in sandwich with the analyte HematideTM well differentia-ble values. The labs of Paris and Lausanne seem to have problems with the stability of the mAb anti-PEG WADA1A8 antibody. Their results are often not reproducible.

The results of this project cannot trace back these problems to the antibody. The phys-ical QC showed no changing in the structure of the protein. The antibody WADA1A8 shows a stable structure over the time of 12 months. However, the manual preparation showed the same variability in our lab when fresh prepared plates were used. If pa-rameters are fixed (see section 4.3.3) these variability cannot be find. The reason of the problems evolve from the suboptimal conditions for the ELISA and not researched backgrounds for the analyte HematideTM and the both antibodies.

The PEG linker and the anti-PEG antibody are sensitive for detergents because of homologous structures to polyethylene-glycol. In the published assay system [N. Leuenberger et al., Methods for detection and confirmation of Hema-tideTM/peginesatide in anti-doping samples, Forensic Sci. Int.(2011)] Tween 20 is used in several assay steps. Also in cell culture it is common that detergents like Kolli-phor (also known as P188/Pluronic) are added. This could influence the anti-PEG an-tibody already in cell cultivation. This omnipresent application can result in complica-tions of the ELISA. In additional experiments (section 4.3.4) the effect of the replace-ment of Tween 20 against CHAPS shows a significant improvement of the assay sys-tem.

Another possibility to improve the assay is to change the antibodies in the ELISA sandwich. It would be of advantage to use the analyte specific antibody anti-Hematide (WADA11F9) as immobilized captor-antibody. The actual coated antibody anti-PEG antibody (WADA1A8) could be engaged by polyethylene-glycol homologous struc-tures. The assay could be more specific and more stable by this change.

Also critical for the test system is the performance. The microplates should not be coated freshly by the operator. The ELISA should be as easy as possible in practice. A lot of factors are unknown yet. The influence of temperature variation, the endpoints of binding are not determined. The analyte HematideTM has to be analyzed for behav-ior.

After these analysis and optimization of the assay system there are good perspectives to create a well working, reproducible ELISA which is user-friendly and possible to produce commercially.