Comparison of enhanced chemiluminescence and colorimetric techniques for the immuno-detection of alpha 1-antitrypsin.
In order to detect, characterize and quantify blotted proteins, such as human alpha 1-antitrypsin (AAT), there is a need for a specific, extremely sensitive, non-radioactive and uniform revelation system applicable to diluted biological fluids and to culture supernatants of cells isolated from such fluids. We compared two immunochemical revelation systems, enhanced chemiluminescence (ECL) and colorimetric procedures, applied to human ATT, after determining their optimal conditions of performance. ECL was the most sensitive method (down to 50 pg blotted AAT), but could not be used to quantify AAT. In contrast, the colorimetric method enables quantification of blotted AAT, either simply dotted or transferred after SDS-polyacrylamide gel electrophoresis, but is not as sensitive as ECL. Using these two complementary procedures, we have been able to detect AAT in the culture supernatant of a monocytic cell line (THP-1), to characterize the different forms of AAT present in the culture supernatant of blood monocytes and to quantify both.
Venembre
P
P
Laboratoire de Biochimie A, Hôpital Bichat, Paris, France.
Seta
N
N
Boutten
A
A
Dehoux
M
M
Aubier
M
M
Durand
G
G
D002460:Cell Line; D003124:Colorimetry / Q000379:methods*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D012680:Sensitivity and Specificity; D000515:alpha 1-Antitrypsin / Q000032:analysis*
D000515:alpha 1-Antitrypsin