Competitive time-resolved immunofluorometric assay for quantifying carbonic anhydrase VI in saliva.
A competitive time-resolved immunofluorometric assay sensitive and robust enough for quantifying human salivary carbonic anhydrase isoenzyme VI (HCA VI) was developed. The solid-phase immunoassay is based on competition between Eu(3+)-labeled HCA VI and salivary HCA VI for polyclonal rabbit anti-HCA VI antibodies that are attached to microtiter plate wells precoated with sheep anti-rabbit IgG. The subsequent immunoassay including the separation of free and bound HCA VI requires only one incubation step, after which the Eu3+ of the bound labeled antigen is released into an enhancement solution. The highly fluorescent Eu chelates formed in this solution are then quantified by time-resolved fluorometry (Delfia). The time-resolution principle effectively obviates possible interferences from complex biological material such as saliva. The assay detection limit was 1.5 micrograms/L. Intra- and interassay imprecisions (CVs) were 5.1% and 5.3%, respectively. The mean analytical recovery was 93%. The mean +/- SD concentration of HCA VI in paraffin-stimulated saliva was 6.8 +/- 4.3 mg/L (n = 30) and the secretion rate was 10.2 +/- 7.9 micrograms/min. The method was useful for further investigations of the role of HCA VI in difficult matrices, e.g., saliva.
Parkkila
S
S
University of Oulu, Department of Anatomy, Finland.
Parkkila
A K
AK
Vierjoki
T
T
Ståhlberg
T
T
Rajaniemi
H
H
D002256:Carbonic Anhydrases / Q000032:analysis* / Q000378:metabolism; D015200:Fluoroimmunoassay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D007700:Kinetics; D008297:Male; D012016:Reference Values; D012463:Saliva / Q000201:enzymology* / Q000378:metabolism; D012680:Sensitivity and Specificity
D002256:Carbonic Anhydrases