**Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies**

Veronika Kocurová

*Nuclear Physics Institute, Academy of Sciences of the Czech Republic, 250 68 Rež near Prague ˇ Czech Republic*

#### **1. Introduction**

446 Gel Electrophoresis – Advanced Techniques

Zhang, Y. & Talalay, P. (1994). Anticarcinogenic activities of organic isothiocyanates: chemistry and mechanisms. *Cancer Research,* Vol. 54, No. 7S, pp. 1976s-1981s. Zhang, Y.; Tang, L. & Gonzalez, V. (2003). Selected isothiocyanates rapidly induce growth

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Neurodegeneration is the leading term for the progressive loss of the neuron structure, including death of neurons. Many neurodegenerative diseases including the specific diseases such as Parkinson's, Alzheimer's, and Huntington's occur as a result of the neurodegenerative processes. As research progresses, many similarities appear which relate these diseases to one another on a sub-cellular level. Discovering these similarities offers hope for therapeutic advances that could ameliorate many diseases simultaneously. There are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death followed by an apoptosis. Apoptosis is a form of the programmed cell death in the multicellular organisms. It is one of the main types of the programmed cell death, and, last but not least, involves a series of the biochemical reactions leading to a characteristic cell morphology changes, and, finally, death. In according to the previously mentioned knowledge, there is a necessity to develop an imaging method which describes these cellular changes. The principal goal of the investigation monoclonal antibodies and their fragments is to examine the possibility of developing of an imaging radiotracer that would be specific for cytoskeleton of destructed dendrites and neuronal bodies. One of the suitable fitting marker, specific for neuronal tissue, performs anti III *β*-tubulin (bTcIII) antibody - TU-20 with molecular weight 150 kDa and its scFv fragment with molecular weight 27.7 kDa. The scFv fragment of TU-20 was synthesized for its higher mobility through tissue and vascular barriers. Biochemical characteristics (especially immunoaffinity) of the specific binding substance - anti III *β*-tubulin scFv fragment - is preserved, and, moreover, the biological availability is much better than in case of the whole antibody. See the structure in the Fig. 1.

To examine this hypothesis, it is necessary to radiolabel both substances with 125I and 123I. The next step is chemical analysis and, furthermore, biochemical properties are extensively investigated. The quality control, performed by gel filtration, electrophoresis, ELISA testing determines adequate properties of the radiolabeled substances for further studies.

Affinity coupling and RIA analytic methods occur under development with focusing on specifics of the antibody and its fragment behavior. *In vitro* experiment shows an extent of the preserved binding specifity of the species by incubation of the both radiolabeled substances with mice brain slices followed by an autoradiography.

Fig. 2. Radiolabeled monoclonal antibody. Radiotracer is bound to the antibody structure via

Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies 449

reaction mixture was gently agitated, the reaction alternatively might be or not stopped with

Iodination tubes, for both methods, were prepared in the same way. 100 *μ*l of iodogen dissolved in chloroform (10 - 500 *μ*g/ml) was given in a glass tube and chloroform was evaporated under a slow stream of nitrogen. The prepared iodination tubes were used immediately. The procedure for the direct method consisted in adding 10 *μ*l of TU-20 (1 mg/ml) into the reaction tube with 50 *μ*l of phosphate buffer (PB, 0,05 M, pH 8,5) and an

The indirect method was performed in two steps. Firstly, radioactivity in PB was added into the tube coated with iodogen. After 15 minutes an activated iodide was withdrawn, transferred into the vessel containing 10 *μ*l of the antibody and the mixture was agitated for

Radioiodination of the fragment scFv TU-20 was performed via chloramine-T without stopping reaction with thiosulfate as described previously for TU-20. In both cases, at the end of labeling, the reaction mixture was loaded on the top of a BSA-blocked polyacrylamide desalting column with an exclusion limit 6 kDa. Fractions were eluted with 0,1 % BSA in PBS

The immunoreactivity of the radiolabeled monoclonal antibody TU-20 was determined by an enzyme linked immunosorbent assay (ELISA) using the commercial set for detection of mouse anti - *β* III tubulin antibodies from VIDIA, CZ. One of the most useful of the immunoassays is the two antibody sandwich ELISA. This assay is used to determine the antigen concentration in unknown samples. This ELISA is fast and accurate, and if a purified antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample.

The sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies. To utilize this assay, one

100 *μ*l of the solution sodium thiosulfate in water (4 mg/ml) (Chizzonite et al., 1991).

equal amount of Na125I around 5,4 MBq. Reaction time was 15 minutes.

and measured for radioactivity. (Hamilton, 2002), (Katsetos, 2003).

The principle of ELISA testing is shown in the Fig. 3.

**2.2 Immunoreactivity testing by enzyme linked immunosobent assay (ELISA)**


20 minutes (Švecová et al., 2008).

The *in vivo* biodistribution confirmes behavior of elimination of the radiolabeled TU-20 and scFv from mice. The bi-exponential model for two-phase clearance to determine short phase half-life t1/2*<sup>α</sup>* and long phase half-life t1/2*<sup>β</sup>* values is used. For comparative study, a transgene population G93A1 Gur was chosen to show different behavior of the substances in normal mouse and in modified organism with amyotrophic lateral sclerosis (ALS).

The main objective of this work is to develop a method for direct imaging of the structural degradation of peripheral neurones by various types of neuropathies.
