Immunoassay reagents and method for determining cyclosporine

Claim 1

A cyclosporine derivative of the formula: wherein R₁ is an amino acid residue of the structure R₂ is an amino acid residue of the structure R₃ is an amino acid residue of the structure R₄ is an amino acid residue of the structure R₅ is an amino acid residue of the struture wherein Z is a detectable moiety capable of producing a detectable signal, with the proviso that: when R₁ is so defined, X₁ is a linking group of 1-20 atoms excluding hydrogen, R₂ is a residue of (L)-alpha-aminobutyric acid or a residue of (L)- threonine, or a residue of (L)- threonine in which the hydroxyl group is acylated with an acyl group of 1-10 atoms or a fluoresceinyl group; R₃ is a residue of sarcosine, a residue of (D)-serine, or a residue of (D)-alanine; and R₅ is a residue of (D)-methyl-(L)-leucine; or when R₂ is so defined, X₂ is a linking group of 1-30 atoms, excluding hydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, or a derivative of MeBmt in which the hydroxyl group is acylated with an acyl group of 1-10 atoms or a fluoresceinyl moiety; R₃ is a residue of sarcosine, a residue of (D)-serine, or a residue of (D)-serine in which the hydroxyl is acylated with an acyl group of 1-10 atoms; R₄ is a residue of (D)-alanine; and R₅ is a residue of (N)-methy-(L)-lleucine; or when R₃ is so defined, X₂ is a linking group of 1-30 atoms, excluding hydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, or a derivative of MeBmt in which the hydroxyl group is acylated with an acyl group of 1-20 atoms; R₄ is a residue of (D)-alanine; and R₅ is a residue of (N)-methyl-(L)-leucine; or when R₄ is so defined, X₁ is a linking group of 1-20 atoms, excluding hydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, or a derivative of MeBmt in which the hydroxyl group is acylated with an acyl group of 1-10 atoms; R₂ is a residue of (L)-alpha-aminobutyric acid or a residue of (L)- threonine, or a residue of (L)-threonine in which the hydroxyl group is acylated with an acyl group of 1-10 atoms; and R₅ is a residue of (N)-methyl-(L)-leucine; or when R₅ is so defined, X₁ is a linking group of 1-20 atoms, excluding hydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, or a derivative of MeBmt in which the hydroxyl group is acylated with an acyl group of 1-10 atoms; R₂ is a residue of L-alpha-aminobutyric acid or a residue of (L)- threonine, or a residue of (L)-threonine in which the hydroxyl group is acylated with an acyl group of 1-10 atoms; and R₄ is a residue of (D)-alanine; and salts thereof.

Claim 10

A method for determining cyclosporine, or cyclosporine and metabolites of cyclosporine, in a test sample, said method comprising the steps of: (a) contacting said test sample with a cyclosporine derivative of the formula: wherein R₁ is an amino acid residue of the structure R₂ is an amino acid residue of the structure R₃ is an amino acid residue of the structure R₄ is an amino acid residue of the structure R₅ is an amino acid residue of the struture wherein Z is a fluorescent moiety or derivatives thereof capable of producing a detectable signal, with the proviso that: when R₁ is so defined, X₁ is a linking group of 1-20 atoms excluding hydrogen, R₂ is a residue of (L)-alpha-aminobutyric acid or a residue of (L)- Threonine, or a residue of (L)- threonine in which the hydroxyl group is acylated with an acyl group of 1-10 atoms or a fluoresceinyl group; R₃ is a residue of sarcosine, a residue of (D)-serine, or a residue of (D)-alanine; and R₅ is a residue of (N)-methyl-(L)-leucine; or when R₂ is so defined, X₂ is a linking group of 1-30 atoms, excluding hydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, or a derivative of MeBmt in which the hydroxyl group is acylated with an acyl group of 1-10 atoms or a fluoresceinyl moiety; R₃ is a residue of sarcosine, a residue of (D)-serine, or a residue of (D)-serine in which the hydroxyl is acylated with an acyl group of 1-10 atoms; R₄ is a residue of (D)-alanine; and R₅ is a residue of (N)-methyl-(L)-leucine; or when R₃ is so defined, X₂ is a linking group of 1-30 atoms, excluding hydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, or a derivative of MeBmt in which the hydroxyl group is acylated with an acyl group of 1-20 atoms; R₄ is a residue of (D)-alanine; and R₅ is a residue of (N)-methyl-(L)-leucine; or when R₄ is so defined, X₁ is a linking group of 1-20 atoms, excluding hydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, or a derivative of MeBmt in which the hydroxyl group is acylated with an acyl group of 1-10 atoms; R₂ is a residue of (L)-alpha-aminobutyric acid or a residue of (L)- threonine, or a residue of (L)-threonine in which the hydroxyl group is acylated with an acyl group of 1-10 atoms; and R₅ is a residue of (N)-methyl-(L)-leucine; or when R₅ is so defined, X₁ is a linking group of 1-20 atoms, excluding hydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, or a derivative of MeBmt in which the hydroxyl group is acylated with an acyl group of 1-10 atoms; R₂ is a residue of L- a -aminobutyric acid or a residue of (L)- threonine, or a residue of (L)-threonine in which the hydroxyl group is acylated with an acyl group of 1-10 atoms; and R₄ is a residue of (D)-alanine; and salts thereof, and an antibody capable of binding to (i) cyclosporine, or cyclosporine and metabolites of cyclosporine, and (ii) said cyclosporine derivative, to form a reaction solution therewith, said cyclosporine derivative capable of producing a detectable fluorescence polarization response to the presence of said antibody; (b) passing a plane of polarized light through the said reaction solution to obtain a fluorescence polarization response; and (c) detecting said fluorescent polarization response to said reaction solution as a function of the amount of cyclosporine, or cyclosporine and metabolites of cyclosporine, present in said test sample.