Comparative studies on tree pollen allergens. XII. Evaluation of direct and indirect autoradiography systems for 125I in crossed radioimmunoelectrophoresis and other immunoautoradiographic techniques.
Visualization by autoradiography of specific IgE binding in crossed radioimmunoelectrophoresis (CRIE) and other 125I-immunoautoradiography (IAR) techniques is done in two different ways; either by traditional direct autoradiography (D-ARG), where the film is exposed to the 125I-anti-IgE incubated sample at room temperature, or by indirect autoradiography (ID-ARG), applying intensifying screen, low-temperature exposure and, eventually, pre-exposure. This study confirmed that D-ARG provided the benefits of simplicity and better image resolution with the disadvantage of prolonged exposure periods. ID-ARG reduced the exposures needed to produce film image densities of 0.01 and 0.1 A540 nm (i.e. autoradiographic sensitivity (AR sigma) and autoradiographic speed (ARs] to 1/18 and 1/55 respectively of the corresponding exposures in D-ARG. The lowest detection limits for 125I in 24 h were 1.2 cpm mm-2 with the indirect and 6.8 cpm mm-2 with the direct systems investigated. The major drawbacks of ID-ARG were inferior image resolution and higher background levels, especially when pre-exposure was included.
Florvaag
E
E
Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway.
D000485:Allergens / Q000276:immunology*; D001345:Autoradiography; D007123:Immunoelectrophoresis, Two-Dimensional; D007073:Immunoglobulin E / Q000378:metabolism*; D007457:Iodine Radioisotopes; D011058:Pollen / Q000276:immunology*
D000485:Allergens; D007457:Iodine Radioisotopes; D007073:Immunoglobulin E