Patent ID: 6204068
Filing Date: 2001-03-20
Classification: G01N,Y10S

Abstract:
A biospecific multiparameter assay method for simultaneous measurement of one or several analytes M.sub.k (k.gtoreq.1) in the same reaction solution, said method comprising the steps of:allowing each sample analyte molecule M.sub.k to react with at least two biospecific probes Ab(j) (j=1,2) labelled with fluorescent labels, to give complex Ab(k,1)+M.sub.k +Ab(k,2);stopping the reaction by adding a diluent;focusing a laser beam and photon detectors tuned to the excitation and emission wavelengths, respectively, on the diluted solution; andregistering the fluorescent signals and their time domains in the form of single photons from single analyte molecules moving through the focal point, characterized in thatthe first biospecific probe is labelled with a combination of fluorescent labels D.sub.k (K.sub.K.ltoreq.1), and the second probe is labelled with a fluorescent label F, all labels having different detectable signals and the signals from the complexes Ab.sup.Dk (k,1)+M.sub.k +AB.sup.F (k,2) are separated from the background signals and from the signals of other molecules by applying auto-correlation in the time domain and cross-correlation with regard to signals obtained from different labels, and the correlated signals are classified according to D.sub.k in order to determine the number of analyte molecules M.sub.k and their concentration in the sample, and thatthe excitation of the labels D.sub.k and F is performed by two-photon excitation.