**3. Conclusion**

TU-20 and its scFv were labeled with 125I and 123I by chloramine-T (with average yield 0,72 and 0,50, resp.). Radiochemical purity and stability was revealed by gel filtration (decrease to 80 % and 50 % in two months, resp.) Fragmentation of the labeled antibody and its fragment was estimated by bis-tris gel electrophoresis followed by silver staining and autoradiography (over 95 % of radioactivity bound in the substances).

Affinity coupling and RIA adaptation for the specific conditions showed 10-30 % preserved immunoreactivity of the labeled compounds. Otherwise, these methods carry out quite high discrepancy and it will be neccessary to provide further optimising search.

In vitro studies performed on mice brain slices confirmed several important assumptions. The antibody is preferentially bound in the layer of Purkinje cells in the cerebellum. SPECT camera in vivo experiment deals with these results: activity bound in scFv is primarily distributed to the thyroid gland and digestive tract, then passes quickly through kidneys.

Distribution images of the labeled TU-20 provides ambigous because the substance is accumulated in the chest and ventral part and image resolution do not afford more detailed biodistribution identification. However, it is known from previous biodistribution preparative study that activity is distributed in lung, heart, liver, stomach and colon in first 6 h.

In vivo experiments were focused on investigation of the blood clearance and organ distribution of the radiolabeled TU-20 and scFv fragment in mice. Let´s show especially the results from scFv biodistribution study in preference. It was verified that the major part of activity, according to the amount of the labeled scFv fragment, was eliminated from blood during 2-3 hours. Minor part of activity, according to the amount of the labeled scFv fragment (0,5 - 1,0 %), was kept in the blood for some days. The value t1/2*<sup>α</sup>* for 125I-labeled scFv fragment was calculated as 2,3 h and the t1/2*<sup>β</sup>* was estimated as 62,4 h. The half-life for overall elimination of Na125I from blood was 4,5 h.

In comparison, we found that the 125I-labeled scFv fragment uptake in thyroid gland appeared much lower than for Na125I, as expected. The t1/2*<sup>α</sup>* value for 123I-labeled scFv fragment was calculated as 1,4 h, but the long phase elimination half-life t1/2*<sup>β</sup>* was not estimated due to short half-life of the isotope 123I. The radiolabeled scFv fragment passed in general through the digestive tract (stomach and intestine) and finally was eliminated through kidneys in preference.

TU-20 and ScFv TU-20 showed suitable properties for further investigation in animals which are genetically modified mutants with the ALS (Amyotrophic Lateral Sclerosis). Comparing biodistribution experiments in modified organism confirmed expected behavior. The most significant biodistribution differences occured in the area of the limbs and caudal part of spinal cord and spine.

Finally, as I can summarize, TU-20 and its scFv fragment were successfully labeled with radioiodine 123I and 125I, and, subsequently, the biochemical and analytical characteristics were investigated. Biological properties of the radiolabeled TU-20 and its scFv were evaluated in vivo by biodistribution studies.

The expected behavior of biomolecules during their elimination was observed. Furthermore, the elimination parametres were calculated. 125I-labeling of the TU-20 and its scFv is very suitable for investigation of the radiolabeled antibody fragment behavior and properties due to the long 125I half-life. On the other hand, 123I-labeling of the scFv fragment TU-20 is intended for practical imaging at SPECT camera.

In summary, TU-20 shows better immunospecific behavior in organism together with slower kinetics, on the other hand, scFv TU-20 reveales worse immunospecific characteristics in combination with much faster kinetics.
