**2.3 Immunoaffinity testing by radioimmunoassay (RIA)**

In radioimmunoassay, a fixed concentration of radio-labeled antigen in trace amounts is incubated with a constant amount of antiserum such that the total antigen binding sites on the antibody are limited such that the only 30–50 % of the total radio-labeled antigen may be bound in the absence of the antigen. When unlabeled antigen, either as standard or test sample, is added to this system, there is competition between radio-labeled antigen and unlabeled antigen for the limited constant number of binding sites on the antibody.

The amount of radio-labeled antigen bound to antibody decreases as the concentration of unlabeled antigen increases. Following optimal incubation condition e.g. buffer, pH, time and temperature, radio-labeled antigen bound to antibody is separated from unbound radio-labeled antigen.

RIA analytic method was developed in two modifications of surface of the reactive vessel.

#### **2.4 Immunoaffinity separation affinity coupling (AC)**

Affinity coupling was develop by use the basic matrix activated Sepharose 4 Fast Flow by Pierce which was modified specific binding octapeptide (Vijayalakshmi, 1992). Activated media enable successful, convenient immobilization of ligands without the need for complex chemical syntheses or special equipment. The Sepharose matrix provides a wide range of high-capacity media with a variety of coupling chemistries for fast, easy, and safe immobilization through a chosen functional group. The principle is to immobilize the antibodies or other large proteins containing -NH2 groups by coupling them to the matrix without the need for an intermediate spacer arm.

direction of the separation) is created. The bigger sharpness of the gel zones of the molecules

Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies 453

The choice of pH of the used buffer by polymerization process, and, also the division of the molecules by the classical PAGE, because, the suitable buffer ensures the sufficient differences in the specific charge of the assorted parts of the protein mixture. The acid proteins require slightly alkaline or neutral pH (the molecules moves to the anode) and alkaline proteins require a slightly acid pH (the molecules migrate to the cathode) (Bernard et al., 1979).

The perfectly suitable modification of the PAGE electrophoresis is an arrangement in the –sodium dodecyl sulfate (abbrev. SDS, or NaDS), which makes an ability of the proteins to bind the SDS in amount of abouti 1,4 mg per 1 mg of the protein by means of the hydrofobic reaction. SDS carries a huge negative charge which enables to equalize the charge of the molecules, and, those, move in one direction in the electrophoretic gel in accordance of the molecular size. The complex SDS-protein unifies either the charge density, or, conformation

The mobility of the SDS-protein complex in the polyacrylamide gel is proportional to the logarithm of the molecular weight of an appropriate protein, which enables the gel calibration (Rédei, 2008). It is quiet convenient that the examined samples are adjusted before the whole

First, an appropriate buffer is added (e.g. TrisHCL) and SDS so that we have the same

of the similar size is ensured in this arrangement.

Fig. 6. Polymerization Process of the Structure Networking.

on the surface of the complex, see the structure in the Fig. 7.

**2.5.3 SDS-PAGE electrophoresis**

homogennous reaction setting.

process.

The correct choice of an activated medium is dictated by both the group available in the ligand molecule, and by the nature of the binding reaction with the substance to be purified. To ensure minimal interference with the normal binding reaction, immobilization should be attempted through the least critical region of the ligand (Haugland, 1995).

#### **2.5 Stability testing by electrophoresis**

Mostly used variation of the electrophoresis for the intention of the quality control of the radiolabeled substances is SDS-PAGE formation of the electrophoresis. It concerns of zone electrophoresis in gel in surface placement. The mixture of the substances is analyzed by division in accordance to the molecular weight.

#### **2.5.1 Polymerization of the polyacrylamide gel**

Polyacrylamide gel is prepared to the form by polymerization of the basic monomer acrylamide (CH2=CH-CO-NH2; abbrev. AA) and N,N'-methylen-bis-akrylamid (CH2=CH-CO-NH-CH2-NH-CO=CH-CH2; abbrev. BIS) which is implemented to the polymere randomly and might covalently bind two linear chains of the polyacrylamide. Ammonium persulfate (abbrev. APS) is used as the initiative reactant and N,N'-tetramethylendiamine (abbrev. TEMED) as the catalyzer, see the Fig. 5.

The inhibitor of the reaction is oxygen, and, therefore, the gel must be protected against the oxygen atmosphere. The polymerization has the radical and exothermic process, and, therefore, the cooling is neccessary during the whole polymerization. The ratio of AA:BIS is crucial for the gel mechanic and separation characteristics. The suggested ratio is ranging of about 40:1 (from 20:1 up to 100:1) (Jones, 2004).

Fig. 5. The Free radical Polymerization of the Acrylamide Initiated on the Addition of the Ammonium Persulfate which Forms the Free Reactive Radicals in the Water.

#### **2.5.2 PAGE electrophoresis**

PAGE separation could be conducted in the gel with the same content of the acrylamide in two different following gels, so called Laemmli electrophoresis, when the first gel contents lower percentage of the acrylamide and it is intended to the concentration of the sample at the begining of the separation (so called the concentration gel). The bigger sharpness of the zones in the gel is provided by means of the lower pH (of about two degree) against the surrounding setting. The itself separation takes place in the following part of the gel with the higher density (so called the separation gel). (Laemmli, 1970) The structure of the polymerization process see in the Fig. 6.

Other variation performs the creation of the gradient gel, where the concentration gradient of the polyacrylamide (from the part with lowe density to the part of higher density, in the direction of the separation) is created. The bigger sharpness of the gel zones of the molecules of the similar size is ensured in this arrangement.

The choice of pH of the used buffer by polymerization process, and, also the division of the molecules by the classical PAGE, because, the suitable buffer ensures the sufficient differences in the specific charge of the assorted parts of the protein mixture. The acid proteins require slightly alkaline or neutral pH (the molecules moves to the anode) and alkaline proteins require a slightly acid pH (the molecules migrate to the cathode) (Bernard et al., 1979).

#### **2.5.3 SDS-PAGE electrophoresis**

6 Will-be-set-by-IN-TECH

The correct choice of an activated medium is dictated by both the group available in the ligand molecule, and by the nature of the binding reaction with the substance to be purified. To ensure minimal interference with the normal binding reaction, immobilization should be

Mostly used variation of the electrophoresis for the intention of the quality control of the radiolabeled substances is SDS-PAGE formation of the electrophoresis. It concerns of zone electrophoresis in gel in surface placement. The mixture of the substances is analyzed by

Polyacrylamide gel is prepared to the form by polymerization of the basic monomer acrylamide (CH2=CH-CO-NH2; abbrev. AA) and N,N'-methylen-bis-akrylamid (CH2=CH-CO-NH-CH2-NH-CO=CH-CH2; abbrev. BIS) which is implemented to the polymere randomly and might covalently bind two linear chains of the polyacrylamide. Ammonium persulfate (abbrev. APS) is used as the initiative reactant

The inhibitor of the reaction is oxygen, and, therefore, the gel must be protected against the oxygen atmosphere. The polymerization has the radical and exothermic process, and, therefore, the cooling is neccessary during the whole polymerization. The ratio of AA:BIS is crucial for the gel mechanic and separation characteristics. The suggested ratio is ranging of

Fig. 5. The Free radical Polymerization of the Acrylamide Initiated on the Addition of the

PAGE separation could be conducted in the gel with the same content of the acrylamide in two different following gels, so called Laemmli electrophoresis, when the first gel contents lower percentage of the acrylamide and it is intended to the concentration of the sample at the begining of the separation (so called the concentration gel). The bigger sharpness of the zones in the gel is provided by means of the lower pH (of about two degree) against the surrounding setting. The itself separation takes place in the following part of the gel with the higher density (so called the separation gel). (Laemmli, 1970) The structure of the polymerization process see

Other variation performs the creation of the gradient gel, where the concentration gradient of the polyacrylamide (from the part with lowe density to the part of higher density, in the

Ammonium Persulfate which Forms the Free Reactive Radicals in the Water.

and N,N'-tetramethylendiamine (abbrev. TEMED) as the catalyzer, see the Fig. 5.

attempted through the least critical region of the ligand (Haugland, 1995).

**2.5 Stability testing by electrophoresis**

division in accordance to the molecular weight.

**2.5.1 Polymerization of the polyacrylamide gel**

about 40:1 (from 20:1 up to 100:1) (Jones, 2004).

**2.5.2 PAGE electrophoresis**

in the Fig. 6.

The perfectly suitable modification of the PAGE electrophoresis is an arrangement in the –sodium dodecyl sulfate (abbrev. SDS, or NaDS), which makes an ability of the proteins to bind the SDS in amount of abouti 1,4 mg per 1 mg of the protein by means of the hydrofobic reaction. SDS carries a huge negative charge which enables to equalize the charge of the molecules, and, those, move in one direction in the electrophoretic gel in accordance of the molecular size. The complex SDS-protein unifies either the charge density, or, conformation on the surface of the complex, see the structure in the Fig. 7.

The mobility of the SDS-protein complex in the polyacrylamide gel is proportional to the logarithm of the molecular weight of an appropriate protein, which enables the gel calibration (Rédei, 2008). It is quiet convenient that the examined samples are adjusted before the whole process.

First, an appropriate buffer is added (e.g. TrisHCL) and SDS so that we have the same homogennous reaction setting.

Fig. 8. Electrophoresis of the TU-20 and scFv TU-20 in the Gradient Gel by the

Autoradiography: 1. [125I]TU-20 (–); 2. [125I]scFv TU-20 (–). .

conditions

plate by means of the AIDA software.

into the membrane by means of electrophoresis.

**2.5.6 Immunoblotting**

Non-Reductive (–) and Reductive (+) Conditions Figure A) Gel Coloured by Coomassie Blue: 1. Molecular Marker; 2. TU-20 (–); 3. TU-20 (+); 4. scFv TU-20 (–); 5. scFv TU-20 (+). Figure B)

Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies 455

Fig. 9. Autoradiography of electrophoresis SDS-PAGE 125I-TU-20 (all lines) by non-reductive

125I-TU-20 59237,2 46,7 BSA(I) 53228,4 42,0 BSA(II) 14319,4 11,3 Table 1. Autoradiographical interpretation of SDS-PAGE of 125I TU-20 by non-reductive conditions. An autoradiographical visualization of the SDS-PAGE gel (which contains the radiolabeled antibody by non-reductive conditions) after developing on the luminiscent

**Peak Integral Density in PSL** % **Ratio of Peak**

Western Blot transfers the proteins, closed into the gel matrix, into the nitrocellulose membrane for further purposes of investigation after finishing of electrophoresis. Western Blot (Immunoblotting), used for the protein detection, transferes the proteins from the gel

Second, the glycerol is added, because it makes the settings in the gel more dense, so that the samples fill the sample holes properly and do not swirl. Glycerol also decreases the electroendoosmosis and makes the movement and distribution of the proteins even better.

Third, the bromophenol blue is added as the protein movement indicator. Fourth, dithiothreitol (abbrev. DTT) could be added to cleave the proteins to make an analysis more suitable. The samples could be also denatured in the hot water by the temperature of about 65 ◦C.

Fig. 7. The Structural Formulae of the Substances in the SDS-PAGE.

#### **2.5.4 Visualization and radiodetection in electrophoresis**

The proteins can be visualized directly in gel after electrophoresis proceeding, or, subsequently Western Blot technique could be processed and detection is performed in the membrane where the proteins are transferred from gel. Adsorption of the pigment is used for visualization.

#### **2.5.5 Staining in electrophoresis**

A Silver Staining shows another alternative for dying of the proteins in gel. The silver ion is insoluble and colourless, and, distinguishes the places with protein and without proteins in the polyacrylamide gel (formation of the silver complexes with alkaline or sulphuric proteins).

After this procedure, the silver ions are reduced by formaldehyde into the form of the metal silver which is perfectly visible and insoluble. The amount of proteins, which could be visualized by this procedure, ranges from the hundreds of picograms to the units nanograms.

Another staining, which si possible for this purpose of detection, is dying by means of Coomassie Blue which is less sensitive (of about 50 times), but it has another advantage that Coomassie Blue is bound to the protein in the stechiometry ratio, and, therefore, it represents a quantitative densitometry detection (maximum absorbance ranges from 560 nm to 575 nm), see the Fig. 8. An autoradiography may be used as an alternative for detection in gel of the radiolabeled compounds. The differences between electrophoresis by non-reductive (see Fig. 9 and Tab. 1 ) and reductive conditions (see Fig. 10 and Tab. 2 ) are shown below.

Fig. 8. Electrophoresis of the TU-20 and scFv TU-20 in the Gradient Gel by the Non-Reductive (–) and Reductive (+) Conditions Figure A) Gel Coloured by Coomassie Blue: 1. Molecular Marker; 2. TU-20 (–); 3. TU-20 (+); 4. scFv TU-20 (–); 5. scFv TU-20 (+). Figure B) Autoradiography: 1. [125I]TU-20 (–); 2. [125I]scFv TU-20 (–). .

Fig. 9. Autoradiography of electrophoresis SDS-PAGE 125I-TU-20 (all lines) by non-reductive conditions


Table 1. Autoradiographical interpretation of SDS-PAGE of 125I TU-20 by non-reductive conditions. An autoradiographical visualization of the SDS-PAGE gel (which contains the radiolabeled antibody by non-reductive conditions) after developing on the luminiscent plate by means of the AIDA software.

#### **2.5.6 Immunoblotting**

8 Will-be-set-by-IN-TECH

Second, the glycerol is added, because it makes the settings in the gel more dense, so that the samples fill the sample holes properly and do not swirl. Glycerol also decreases the electroendoosmosis and makes the movement and distribution of the proteins even better. Third, the bromophenol blue is added as the protein movement indicator. Fourth, dithiothreitol (abbrev. DTT) could be added to cleave the proteins to make an analysis more suitable. The samples could be also denatured in the hot water by the temperature of about

The proteins can be visualized directly in gel after electrophoresis proceeding, or, subsequently Western Blot technique could be processed and detection is performed in the membrane where the proteins are transferred from gel. Adsorption of the pigment is used for

A Silver Staining shows another alternative for dying of the proteins in gel. The silver ion is insoluble and colourless, and, distinguishes the places with protein and without proteins in the polyacrylamide gel (formation of the silver complexes with alkaline or sulphuric proteins). After this procedure, the silver ions are reduced by formaldehyde into the form of the metal silver which is perfectly visible and insoluble. The amount of proteins, which could be visualized by this procedure, ranges from the hundreds of picograms to the units nanograms. Another staining, which si possible for this purpose of detection, is dying by means of Coomassie Blue which is less sensitive (of about 50 times), but it has another advantage that Coomassie Blue is bound to the protein in the stechiometry ratio, and, therefore, it represents a quantitative densitometry detection (maximum absorbance ranges from 560 nm to 575 nm), see the Fig. 8. An autoradiography may be used as an alternative for detection in gel of the radiolabeled compounds. The differences between electrophoresis by non-reductive (see Fig.

9 and Tab. 1 ) and reductive conditions (see Fig. 10 and Tab. 2 ) are shown below.

Fig. 7. The Structural Formulae of the Substances in the SDS-PAGE.

**2.5.4 Visualization and radiodetection in electrophoresis**

65 ◦C.

visualization.

**2.5.5 Staining in electrophoresis**

Western Blot transfers the proteins, closed into the gel matrix, into the nitrocellulose membrane for further purposes of investigation after finishing of electrophoresis. Western Blot (Immunoblotting), used for the protein detection, transferes the proteins from the gel into the membrane by means of electrophoresis.

] ]

(b).

mice brain slice.

Fig. 11. Gel electrophoresis analysis of [ 125I]TU-20 – autoradiography (a) and silver staining

Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies 457

Fig. 12. [125I]TU-20 autoradiographical - Figure A) ,and, immunohistochemical visualization of the bound radiolabeled antibody in the mice brain slice - Figure B) image of the coronal

Fig. 13. Autoradiography visualization - Figure A), and, visualization of the

labeled mice brain slices by means of the software AIDA.

1D-interpretation of the bound radiolabeled antibody in the mice brain slice - Figure B) of the

Fig. 10. Autoradiography of electrophoresis SDS-PAGE of 125I-TU-20 and 125I-scFv TU-20 by reductive conditions. An autoradiographical visualization of the SDS-PAGE gel (which contains the radiolabeled antibody and fragment by reductive conditions) after developing on the luminiscent plate by means of the AIDA software.


Table 2. Autoradiographical interpretation of SDS-PAGE of 125I-scFv TU-20 (upper line) and 125I TU-20 (bottom line) by reductive conditions.

The particular proteins are subsequently indentified by the appropriate radiolabbeled antibodies (labeled by enzymatic reaction, or, by the radiolabeling reaction with 125I). The proteins bound into the membrane could be submitted to the non-specific staining, or, as an alternative, to the autoradiography. After drying, the membrane is stored with much better results than dried gel.

When the electrophoresis with all its instruments and alternatives is used as a quality control method of the radiolabeled antibodies, the following parametres were proved and chosen for this setting as the most suitable. Stability of the radiolabeled TU-20 and its scFv TU-20 was investigated on 4 - 12 % Bis-Tris gel electrophoresis.

Protein bands were visualized by staining the gels with Silver Stain Plus. 125I-labeled scFv fragment was processed by autoradiography exposing plate BAS-SR 2025, and finally developed by BAS-1800II. Autoradiographs were evaluated by AIDA 2.0 software, see the Fig. 11.

#### **2.6 Immunohistochemistry testing**

Preserved binding properties of the radiolabeled MAb or scFv for neuronal tissue were confirmed by the method of double labeling. It is based on the immunohistochemistry and autoradiography of the brain tissue slices. The 50 *μ*m thick brain slices from the wild type mouse (C57B/6/J) were incubated with the radiolabeled TU-20. The second incubation was performed with anti-mouse IgG polyclonal antibody conjugated with horseradish peroxidase (Sigma-Aldrich, USA). Afterwards, the immunohistochemistry was finalized by staining with 3,3' – diaminobenzidine (DAB) that revealed the neuronal structure, see the Fig. 12 and 13.

10 Will-be-set-by-IN-TECH

Fig. 10. Autoradiography of electrophoresis SDS-PAGE of 125I-TU-20 and 125I-scFv TU-20 by reductive conditions. An autoradiographical visualization of the SDS-PAGE gel (which contains the radiolabeled antibody and fragment by reductive conditions) after developing

125I-TU-20 63445,2 98,1 BSA(I) 1228,8 1,9 125I-scFv TU-20 77988,6 97,6 BSA(I) 1909,7 2,4 Table 2. Autoradiographical interpretation of SDS-PAGE of 125I-scFv TU-20 (upper line) and

The particular proteins are subsequently indentified by the appropriate radiolabbeled antibodies (labeled by enzymatic reaction, or, by the radiolabeling reaction with 125I). The proteins bound into the membrane could be submitted to the non-specific staining, or, as an alternative, to the autoradiography. After drying, the membrane is stored with much better

When the electrophoresis with all its instruments and alternatives is used as a quality control method of the radiolabeled antibodies, the following parametres were proved and chosen for this setting as the most suitable. Stability of the radiolabeled TU-20 and its scFv TU-20 was

Protein bands were visualized by staining the gels with Silver Stain Plus. 125I-labeled scFv fragment was processed by autoradiography exposing plate BAS-SR 2025, and finally developed by BAS-1800II. Autoradiographs were evaluated by AIDA 2.0 software, see the

Preserved binding properties of the radiolabeled MAb or scFv for neuronal tissue were confirmed by the method of double labeling. It is based on the immunohistochemistry and autoradiography of the brain tissue slices. The 50 *μ*m thick brain slices from the wild type mouse (C57B/6/J) were incubated with the radiolabeled TU-20. The second incubation was performed with anti-mouse IgG polyclonal antibody conjugated with horseradish peroxidase (Sigma-Aldrich, USA). Afterwards, the immunohistochemistry was finalized by staining with 3,3' – diaminobenzidine (DAB) that revealed the neuronal structure, see the Fig. 12 and 13.

**Peak Integral Density in PSL** % **Ratio of Peak**

on the luminiscent plate by means of the AIDA software.

125I TU-20 (bottom line) by reductive conditions.

investigated on 4 - 12 % Bis-Tris gel electrophoresis.

**2.6 Immunohistochemistry testing**

results than dried gel.

Fig. 11.

Fig. 11. Gel electrophoresis analysis of [ 125I]TU-20 – autoradiography (a) and silver staining (b).

Fig. 12. [125I]TU-20 autoradiographical - Figure A) ,and, immunohistochemical visualization of the bound radiolabeled antibody in the mice brain slice - Figure B) image of the coronal mice brain slice.

Fig. 13. Autoradiography visualization - Figure A), and, visualization of the 1D-interpretation of the bound radiolabeled antibody in the mice brain slice - Figure B) of the labeled mice brain slices by means of the software AIDA.

Fig. 15. [123I]scFv biodistribution in normal mice.

1, 2 and 3 h.

h.

Fig. 16. [123I]scFv TU-20 SPECT camera images –biodistribution study in kinetic intervals 0,5,

Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies 459

Fig. 17. 123I]-TU-20 SPECT camera images –biodistribution study in kinetic intervals 1, 2, 3, 6

Transgene population G93A1 Gur was used for comparative study to show different behavior of the substances in normal mouse and in modified organism with amyotrophic lateral

**2.9 In vivo biodistribution testing in genetically modified mice**

Fig. 14. [125I]scFv biodistribution in normal mice.

#### **2.7 In vivo preparative biodistribution testing in normal mice**

The in vivo biodistribution was carried out with the male normal mice - wild type C57B/6/J. Biodistribution studies were performed following an i.v. injection. The main focus is intended for scFv fragment due to its better mobility in organism. 125I-labeled scFv fragment, for comparison with the biodistribution of Na125I, was applied in amount of 50 kBq/50 *μ*l. 123I-labeled scFv fragment was injected in amount of 200 kBq/50 *μ*l.

Mice were sacrificed at designated times points in groups by 3 animals. The kinetic time intervals were: 3, 6, 12, 24, 48, 72, 144 hours for 125I-labeled scFv TU-20 fragment and 0,5, 1, 2, 3, 6, 12 hours for 123I-labeled scFv fragment.

Blood and major organs (included thyroid gland, kidneys, lung, heart, brain, spleen, muscle, fat, skin, gallbladder, testicles, stomach, liver, small intestine, and colon) were removed, weighed, and counted in a gamma scintillation counter to determine the % ID/g (percentage of injected dose per gram) for each radiolabeled substance.

The biodistribution figures are shown below, see the Fig. 14 and 15.

Blood clearance data for 125I-labeled scFv fragment were obtained by analyzing blood samples by using a 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.

#### **2.8 In vivo SPECT imaging biodistribution testing**

[ 123I]scFv TU-20 and [123I]TU-20 behavior in mice (wild type C57B/6/J ) was observed by use of the SPECT camera. Kinetic intervals were 0.5, 1, 2, 3 h by [123I]scFv TU-20 - see the Fig. 16 and 1, 2, 3, 6 h by [123I]TU-20 - see the Fig. 17.

Fig. 15. [123I]scFv biodistribution in normal mice.

12 Will-be-set-by-IN-TECH

The in vivo biodistribution was carried out with the male normal mice - wild type C57B/6/J. Biodistribution studies were performed following an i.v. injection. The main focus is intended for scFv fragment due to its better mobility in organism. 125I-labeled scFv fragment, for comparison with the biodistribution of Na125I, was applied in amount of 50 kBq/50 *μ*l.

Mice were sacrificed at designated times points in groups by 3 animals. The kinetic time intervals were: 3, 6, 12, 24, 48, 72, 144 hours for 125I-labeled scFv TU-20 fragment and 0,5, 1, 2,

Blood and major organs (included thyroid gland, kidneys, lung, heart, brain, spleen, muscle, fat, skin, gallbladder, testicles, stomach, liver, small intestine, and colon) were removed, weighed, and counted in a gamma scintillation counter to determine the % ID/g (percentage

Blood clearance data for 125I-labeled scFv fragment were obtained by analyzing blood samples by using a bi-exponential model for two-phase clearance to determine short phase half-life

123I]scFv TU-20 and [123I]TU-20 behavior in mice (wild type C57B/6/J ) was observed by use of the SPECT camera. Kinetic intervals were 0.5, 1, 2, 3 h by [123I]scFv TU-20 - see the Fig. 16

Fig. 14. [125I]scFv biodistribution in normal mice.

3, 6, 12 hours for 123I-labeled scFv fragment.

t1/2*<sup>α</sup>* and long phase half-life t1/2*<sup>β</sup>* values.

[

**2.8 In vivo SPECT imaging biodistribution testing**

and 1, 2, 3, 6 h by [123I]TU-20 - see the Fig. 17.

**2.7 In vivo preparative biodistribution testing in normal mice**

of injected dose per gram) for each radiolabeled substance.

The biodistribution figures are shown below, see the Fig. 14 and 15.

123I-labeled scFv fragment was injected in amount of 200 kBq/50 *μ*l.

Fig. 16. [123I]scFv TU-20 SPECT camera images –biodistribution study in kinetic intervals 0,5, 1, 2 and 3 h.

Fig. 17. 123I]-TU-20 SPECT camera images –biodistribution study in kinetic intervals 1, 2, 3, 6 h.

#### **2.9 In vivo biodistribution testing in genetically modified mice**

Transgene population G93A1 Gur was used for comparative study to show different behavior of the substances in normal mouse and in modified organism with amyotrophic lateral

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

Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies 461

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

This work was supported by the projects No. E!3177 - DIAGIM (1P040E167) and E!2510 - NEUROTUB (0E91) of EUREKA, and by the project No. IBS1048301 of Grant Agency CAS.

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combination with much faster kinetics.

**4. Acknowledgement**

**5. References**

sclerosis (ALS). Biodistribution kinetic intervals were3h(125I-scFv) and 6 h (125I-TU-20). (Heiman-Patterson T.D., 2005) (Naini A., 2007)
