**2. Methods and materials**

The monoclonal antibody TU-20 and its scFv was purchased from Exbio, CZ. The antibody recognizes the peptide sequence ESESQGPK. ScFv TU-20 is a recombinant protein expressed in *E. col*i. (Dráberová et al., 1998)

Fig. 1. The structure of the monoclonal antibody on the base of IgG and its scFv fragment.

#### **2.1 Radioiodination of the antibody**

125I (T1/2 = 59,4 h) radioiodination of TU-20 and scFv TU-20 was performed via chloramine-T with or without stopping reaction with sodium thiosulfate agent. The ratio of an amount of TU-20 to radioactivity was 1 *μ*g to 5.5-7.0 MBq of 125I. The ratio of an amount of the fragment to radioactivity was 1 *μ*g to 1.5-2.0 MBq of 125I. 123I (T1/2 = 13,3 h) radioiodination of the fragment scFv TU-20 was performed via chloramine-T with stopping reaction with sodium thiosulfate. The ratio of an amount of the fragment to radioactivity was 1 *μ*g to 3-5 MBq 123I (Švecová et al., 2008). The structure of the radiolabeled antibody is shown in the Fig. 2.

The monoclonal antibody TU-20 was radioiodinated by using either chloramine-T or iodogen as an oxidizing agent. Iodination via chloramine-T was provided in two alternative ways: either with or without stopping a reaction by a reducing agent (Dráberová et al., 1998).

The reaction was performed under following conditions: 10 *μ*l of TU-20 (1 mg/ml) was transferred to 10 *μ*l phosphate buffer (PBS, 0,01 M, pH 7,4) in a reaction vessel and 125I radioactivity (approximately 5,36 MBq) was added. Finally, the solution of chloramine-T in PBS (0,1 mg/ml) was added to the reaction vessel. The amount of chloramine-T ranged from 0,5 to 6 *μ*g per 10 *μ*g of the antibody. After the reaction time (60 seconds), during which the 2 Will-be-set-by-IN-TECH

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

The main objective of this work is to develop a method for direct imaging of the structural

The monoclonal antibody TU-20 and its scFv was purchased from Exbio, CZ. The antibody recognizes the peptide sequence ESESQGPK. ScFv TU-20 is a recombinant protein expressed

Fig. 1. The structure of the monoclonal antibody on the base of IgG and its scFv fragment.

125I (T1/2 = 59,4 h) radioiodination of TU-20 and scFv TU-20 was performed via chloramine-T with or without stopping reaction with sodium thiosulfate agent. The ratio of an amount of TU-20 to radioactivity was 1 *μ*g to 5.5-7.0 MBq of 125I. The ratio of an amount of the fragment to radioactivity was 1 *μ*g to 1.5-2.0 MBq of 125I. 123I (T1/2 = 13,3 h) radioiodination of the fragment scFv TU-20 was performed via chloramine-T with stopping reaction with sodium thiosulfate. The ratio of an amount of the fragment to radioactivity was 1 *μ*g to 3-5 MBq 123I (Švecová et al., 2008). The structure of the radiolabeled antibody is shown in the Fig. 2.

The monoclonal antibody TU-20 was radioiodinated by using either chloramine-T or iodogen as an oxidizing agent. Iodination via chloramine-T was provided in two alternative ways: either with or without stopping a reaction by a reducing agent (Dráberová et al., 1998).

The reaction was performed under following conditions: 10 *μ*l of TU-20 (1 mg/ml) was transferred to 10 *μ*l phosphate buffer (PBS, 0,01 M, pH 7,4) in a reaction vessel and 125I radioactivity (approximately 5,36 MBq) was added. Finally, the solution of chloramine-T in PBS (0,1 mg/ml) was added to the reaction vessel. The amount of chloramine-T ranged from 0,5 to 6 *μ*g per 10 *μ*g of the antibody. After the reaction time (60 seconds), during which the

mouse and in modified organism with amyotrophic lateral sclerosis (ALS).

degradation of peripheral neurones by various types of neuropathies.

**2. Methods and materials**

in *E. col*i. (Dráberová et al., 1998)

**2.1 Radioiodination of the antibody**

reaction mixture was gently agitated, the reaction alternatively might be or not stopped with 100 *μ*l of the solution sodium thiosulfate in water (4 mg/ml) (Chizzonite et al., 1991).

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

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 20 minutes (Švecová et al., 2008).

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

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

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

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

orange and blue colors, respectively. In our case, TMB was used for colorimetric visualization.

Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies 451

Fig. 4. Equipment for optical density measurement in the ELISA settings.

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

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

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

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

unlabeled antigen for the limited constant number of binding sites on the antibody.

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

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

without the need for an intermediate spacer arm.

radio-labeled antigen.

The settlement of the procedure see in the Fig. 4.

Fig. 3. ELISA principle. A specific antigen (an antibody plays the role of the "antigen" in the case of the antibody ELISA detection) is bound to the specific antibody coated on the solid carrier (microtitration plate). Subsequently, another specific antibody (labeled by an apropriate enzyme which catalyzes the coloured and easily detectable reaction) is added to the previously bound antigen.

antibody (the 'capture' antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well.

Afterwards, an antigen is added, and, allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the 'detection' antibody) is allowed to bind to the antigen, and, therefore, the setting is described as the sandwich. The assay is then quantified by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate.

Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as "matched pairs", meaning that they can recognize separate epitopes on the antigen so they do not hinder each other's binding.

ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those that convert a colorless substrate to a colored product, e.g., pnitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2'-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3'5,5'-tetramethylbenzidine base (TMB), which yield green, 4 Will-be-set-by-IN-TECH

Fig. 3. ELISA principle. A specific antigen (an antibody plays the role of the "antigen" in the case of the antibody ELISA detection) is bound to the specific antibody coated on the solid carrier (microtitration plate). Subsequently, another specific antibody (labeled by an apropriate enzyme which catalyzes the coloured and easily detectable reaction) is added to

antibody (the 'capture' antibody) is purified and bound to a solid phase typically attached to

Afterwards, an antigen is added, and, allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the 'detection' antibody) is allowed to bind to the antigen, and, therefore, the setting is described as the sandwich. The assay is then quantified by measuring the amount of labeled second antibody

Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as "matched pairs", meaning that they can recognize separate epitopes on the antigen so they do not hinder

ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those that convert a colorless substrate to a colored product, e.g., pnitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2'-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3'5,5'-tetramethylbenzidine base (TMB), which yield green,

bound to the matrix, through the use of a colorimetric substrate.

the previously bound antigen.

the bottom of a plate well.

each other's binding.

orange and blue colors, respectively. In our case, TMB was used for colorimetric visualization. The settlement of the procedure see in the Fig. 4.

Fig. 4. Equipment for optical density measurement in the ELISA settings.
