Fluorescence immunoassays using fluorescent dyes free of aggregation and serum binding

A method for determining the presence or amount of a target analyte in a sample comprising the steps:

• (a) contacting a sample suspected of containing a target analyte with a first receptor capable of specifically recognizing said target analyte to form a complex of said target analyte and said first receptor, said first receptor being labeled with a fluorescent probe which comprises a fluorescent porphyrin or azaporphyrin that is both:
• (i) coordinated to a central metal atom and (ii) coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure;
• (b) contacting the complex with a second receptor capable of specifically recognizing said target analyte, said second receptor being bound to a solid carrier, to form a complex of said first labeled receptor, said target analyte and said second receptor bound to said solid carrier;
• (c) measuring either the amount of labeled first receptor associated with said solid carrier or the amount of unreacted labeled first receptor;
• and
• (d) relating the amount of labeled first receptor measured to the amount of labeled first receptor measured for a control sample free of said target analyte, or relating the amount of labeled first receptor measured with the amount of labeled first receptor measured in samples containing known amounts of target analyte.

A method for determining the presence or amount of a target analyte in a sample comprising the steps of:

• (a) simultaneously contacting a sample suspected of containing a target analyte with first and second receptors capable of specifically recognizing said target analyte, said first receptor being labeled with a fluorescent probe which comprises a fluorescent porphyrin or azaporphyrin that is both:
• (i) coordinated to a central metal atom and (ii) coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure and said second receptor being bound to a solid carrier, to form a complex of said first receptor, said target analyte, and said second receptor;
• (b) measuring either the amount of labeled first receptor associated with said solid carrier or the amount of unreacted labeled first receptor;
• and
• (c) relating the amount of labeled first receptor measured to the amount of labeled first receptor measured for a control sample free of said target analyte, or relating the amount of labeled first receptor measured with the amount of labeled first receptor measured in samples containing known amounts of target analyte.

A method for determining the presence or amount of a target analyte in serum comprising the steps:

• (a) contacting serum suspected of containing target analyte with a known quantity of added target analyte of analog thereof linked to a fluorescent probe wherein said fluorescent prove comprises a detectable labeled marker component, wherein said detectable labeled marker component comprises a fluorescent porphyrin or azaporphyrin that is both:
• (i) coordinated to a central metal atom and (ii) coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure;
• (b) contacting said serum with a receptor capable of specifically recognizing said target analyte;
• (c) determining the amount of fluorescent probe linked to added target analyte bound to said receptor or the amount of fluorescent probe linked to added target analyte which is not bound to said receptor;
• and
• (d) comparing said amount of bound or unbound fluorescent probe with the amount of fluorescent probe in a standard serum sample free of said target analyte or containing said target analyte in a known amount.