A COMPETITIVE DUAL-LABEL TIME-RESOLVED IMMUNOFLUOROMETRIC ASSAY FOR SIMULTANEOUS DETECTION OF CARBONIC ANHYDRASE-I AND ANHYDRASE-II IN CEREBROSPINAL-FLUID

Carbonic anhydrase (CA) is functionally an important enzyme in the cen tral nervous system (CNS) where it is involved in the control of acid- base balance and regulation of the production of cerebrospinal fluid ( CSF). Isoenzyme II (CAII) is the most widely distributed CA in the CNS being specifically present in CNS glial tissue and therefore it is ex pected to be leaked to CSF in degenerative CNS diseases. A competitive dual-labeled time-resolved immunofluorometric assay was developed for simultaneous quantification of human CAI (HCA I) and II (HCA II) in C SF. HCA I was measured to determine the blood contamination in the sam ples. This solid-phase immunoassay is based on competition between eur opium (EU(3+))- or samarium (Sm3+)-labeled antigen and the sample anti gens for polyclonal rabbit antibodies which are attached to microtiter -plate wells precoated with sheep anti-rabbit IgG. The subsequent immu noassay, including the separation of free and bound HCA I and II, requ ires only one incubation step, after which an enhancement solution dis sociates Sm3+ and EU(3+) ions from the labeled HCA I and II, respectiv ely, into a solution where they form highly fluorescent chelates. Spec tra of the fluorescent chelates in the microtitration strip wells were run on time-resolved fluorometers equipped with filters for Eu3+ (613 nm) and Sm3+ (643 nm), the fluorescence from each sample being invers ely proportional to the concentration of antigens. The detection limit of the HCA II assay was 0.3 mu g/l and that of the HCA I assay was 5. 2 mu g/l. The intra- and inter-assay imprecisions (C.V.s) were 8.0% an d 8.8% for HCA I and 6.3% and 4.8% for HCA II, respectively. The analy tical recovery ranged from 96 to 110% for HCA I and from 95 to 108% fo r HCA II. The concentration of HCA II derived from brain tissue presen t in the CSF of hydrocephalic children varied between 1.0 and 35.9 mu g/l (n = 25).