Immunochemiluminometric assays based on acridinium labels with a microtiter plate luminometer.

Inununoassays based on chemilunlinescent acridinium salts are highlysensitive, robust, and simple (1) but have not been demonstrated in a microplate format because of the lack of suitable luminometers with “on-board” reagent injection systems necessary for quantifying rapidchemiluminescent reactions. We report our initial studies on a two-site immunochemiluminometric assay (IcMA) for thyrotropin (TSH) in bloodspot discs used to screen for neonatal hypothyroidism. The assay combines the use of acridinium-labeled monoclonal antibodies and polyclonal antibody-coated microwells. We quantified emission with eithera Luminoskan luminometer (Labsystems Ltd., Basingstoke, U.K., or ICN Flow Ltd., Rickmansworth, U.K.) or a ML1000 luminometer (Dynatech Ltd., Billmghurst, U.K.). These machines were equipped with four and three reagent injectors, respectively. We coated white microtiter plate wells (Dynatech, M11912W) with sheep anti-hTSH IgG (Scottish Antibody Production Unit, Carluke, Lanarkshire, U.K.). We punched 3-mm-diameter blood spot discs into the wells and added 100 L of acridinium-labeled monoclonal anti-hTSH antibody solution (Magic Lite’TM; Ciba Corning, Medfield, MA 02052). We incubated the plates at 37 #{176}C for 1 h, and then washed the wells three times with phosphate-buffered saline (phosphate 0.05 mol/L, pH 7.4; NaCl 0.15 mol/L) containing, per liter, 1 mL of Tween 20. We then introduced the plates into the measuring chamber of the luminometer, which was programmed to inject successive 100-FL volumes ofperoxide and alkali(MagicLiteReagents 1 and 2) with a 0.1-s delay between injections. Emission intensity was integrated over 2 a after the second injection and was recorded as relative light units. The dose-response curve for the assay is shown in Figure 1. The intra-assay precision (CV) forreplicate determinations (n = 10 at each concentration) at 3.6, 6.3, 26.8, and 43.0 milli-int. unitsfL was 7.5%, 14.3%, 16.2%, and 5.8%, respectively. The precision of measurement is thus adequate in the clinically important region (‘-15 milli-int. unitsfL) between euthyreid neonates and neonates with congenital primary hypothyroidism. Correlation with our routine “in-house” immunoradiometric assay (IRMA) was good (r = 0.990, n = 16): ICMA = 1.O4IRMA + 1.9 milli-int. units/L. 200 250 300