Method of determining energy transfer efficiency of a donor-acceptor pair of fluorescent dyes

Claim 1

A method of determining the energy transfer efficiency of a donor-acceptor pair of fluorescent dyes useful in fluorescent energy transfer immunoassay which comprises: (a) measuring the fluorescence and absorption spectra of donor-protein, acceptor-protein, mixture of donor-protein and acceptor-protein and donor-acceptor-protein conjugates; (b) separating the respective spectra measured in (a) into their respective donor-protein complex and acceptor-protein complex components using multiple linear regression and employing models F.sub.a-d =α·F.sub.d +β·F.sub.a +ε and A.sub.a-d =α'·A.sub.d +β'·A.sub.a +ε' where F d , F a , and F a-d are the fluorescence spectra of the donor-protein conjugate, the acceptor-protein conjugate, and the donor-acceptor-protein conjugate, respectively, α and β are the linear coefficients to be determined and ε is the error term and A d , A a , and A a-d are the absorption spectra of the donor-protein conjugate, the acceptor-protein conjugate, and the donor-acceptor-protein conjugate, respectively, α' and β' are the linear coefficients to be determined and ε' is the error term; and (c) determining the energy transfer efficiency on the basis of quenching of donor fluorescence using the equation ##EQU2## where E is energy transfer efficiency, F d is the fluorescence intensity of donor in the absence of acceptor, A d is the absorbance of donor in the absence of acceptor, F a d is the fluorescence intensity of the donor in the presence of acceptor, and A a d is the absorbance of donor in the presence of acceptor.

Claim 2

A method of determining the energy transfer efficiency of a donor-acceptor pair of fluorescent dyes useful in fluorescent energy transfer immunoassay which comprises: (a) measuring the fluorescence and absorption spectra of donor-protein, acceptor-protein, mixture of donor-protein and acceptor-protein and donor-acceptor-protein conjugates; (b) separating the respective spectra measured in (a) into their respective donor-protein complex and acceptor-protein complex components using multiple linear regression and employing models F.sub.a-d =α·F.sub.d +β·F.sub.a +ε and A.sub.a-d =α'·A.sub.d +β'·A.sub.a +ε' where F d , F a , and F a-d are the fluorescence spectra of the donor-protein conjugate, the acceptor-protein conjugate, and the donor-acceptor-protein conjugate, respectively, α and β are the linear coefficients to be determined and ε is the error term and A d , A a , and A a-d are the absorption spectra of the donor-protein conjugate, the acceptor-protein conjugate, and the donor-acceptor-protein conjugate, respectively, α' and β' are the linear coefficients to be determined and ε' is the error term; and (c) determining the energy transfer efficiency on the basis of quenching of donor fluorescence using the equation ##EQU2## where E is energy transfer efficiency, F d is the fluorescence intensity of donor in the absence of acceptor, A d is the absorbance of donor in the absence of acceptor, F a d is the fluorescence intensity of the donor in the presence of acceptor, and A a d is the absorbance of donor in the presence of acceptor.