Details

Autor(en) / Beteiligte

• Caro, R.A.
• Ciscato, V.A.
• De Giacomini, S.M.V.
• Quiroga, S.
• Radicella, R.

Titel

Labeling of proteins with 125I and experimental determination of its specific activity

Ist Teil von

• The International journal of applied radiation and isotopes, 1975-09, Vol.26 (9), p.527,IN5,531-530,IN6,532

Ort / Verlag

United States: Elsevier B.V

Erscheinungsjahr

1975

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• The purpose of the present work consists in the standardization of the labeling technique of proteins with 125I and the control of the obtained products, principally their specific activities, in order to utilize them correctly in radioimmunoassays. The quantities of Chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3·6 and 9·6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. In fact, radioiodine not bound to the protein remains in the zone corresponding to the labeled protein, when analyzed by electrophoresis. This complication did not occur when chromatographic analysis was carried out. The knowledge of the real specific activity is important since it is necessary for the determination of the mass of protein which must be put into reaction from the absolute activity of the labeled protein. For these reasons the yields and the corresponding specific activities were determined from ascending chromatograms obtained with 70% methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labeled with the proposed method, the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 ± 4. The non-specific combinations were always smaller than 5%. The possibility of determining the actual specific activity by simple chromatography, shown in the present work, should be considered as one more step towards a methodological standardization of radioimmunoassay procedures.