Table 1.

Technical protocol ¹²⁵ I labelling of BoNT-A according to IODOGEN-coated method

Step
1.	X μL 125I is added to wobbling reaction vial
2.	250 μL phosphate buffer (0.5 M, pH 7.4) is added
3.	50 μL BoNT-A/ phosphate buffer (=50 μg BoNT-A) is addeda
4.	115-X μL phosphate buffer (0.1 M, pH 6.8) is added to adjust the reaction volume to 215 μL
5.	17.5 μL freshly prepared IODOGEN/acetonitrile solution (2.5 μg/35 μL) is added into the reaction mixture (start of reaction, t = 0)
6.	At t = 45 s, another 17.5 μL IODOGEN/acetonitrile solution (2.5 μg/35 μL) is added
7.	At t = 90 s, 100 μL ascorbic acid solution (25 mg/mL, pH 5.0) is added to stop the reaction and to reduce any formed S-Cl bonds
8.	At t = 4.30 min, 10 μL BSA (50 mg/mL) is addedb
9.	At t = 5.30 min, samples are taken for ITLC for determination of the labelling efficiency
10.	At t = 10 min, 360 μL reaction mixture is transferred to a syringe connected to a filterc
11.	The reaction vial is rinsed by 640 μL phosphate buffer (0.1M, pH 6.8) and also transferred to the syringe connected to the filterc
12.	The combined solution in the syringe is filtered and purified on a PD10 column with ascorbic acid solution (5 mg/mL, pH 5.0) as eluent, collected fractions were 0.5 mL
13.	Fractions with highest amount of 125I-BoNT-A and the highest radiochemical purity are pooled
14.	Samples of the pooled 125I-BoNT-A (fractions 6, 7, 8) are taken for ITLC
15.	Final product is diluted by BSA (1 mg/mL) till a concentration of 1 μg/100 μL and stored at 20°C

t = time.

^aVial of 100 μg lyophilized BoNT-A powder is reconstituted with 100 μL phosphate buffer (0.1 M, pH 6.8).

^bFor encapsulation of the product to protect against radiation damage and to prevent absorption of material on the surface of the vials/syringes. Instead of BSA (bovine serum albumin), HSA (human serum albumin) or any other macromolecule can be used.

^cFilter of 0.2 μm (Acrodisc Gelman Sciences, Ann Arbor, MI, USA).