High sensitivity immunoassay method

A time-resolved fluoroimmunoassay (TR-FIA) method for detecting a cytokine in a biological fluid sample, comprising:

• subjecting the biological fluid sample to a heat treatment under non-denaturing temperature conditions of about 55° C. for the cytokine;
• forming a composite by (a) binding a first antibody, including a portion bound to a solid phase and a region bindable to a cytokine, to a solid phase;
• (b) adding the sample containing the cytokine;
• (c) binding a second antibody, including a region bindable to the cytokine and a portion to which biotin is bound, to the cytokine;
• (d) adding a conjugate including streptoavidin or avidin and a fluorescent structural portion capable of being complexed with a lanthanoid metal ion;
• and (e) adding a lanthanoid metal ion to bind the conjugate of (d), the composite being formed on the solid phase;
• and
• measuring fluorescence of the fluorescent structural portion which has been complexed with the lanthanoid metal ion,
• wherein the method comprises a step of washing after each of steps (a) to (c);
• and
• wherein the cytokine is a cytokine belonging to the chemokine family, and
• wherein the fluorescent structural portion is represented by General Formula (I):
• R—Ar—C(═O)—CH2—C(═O)—CnF2n—X (I)
• (where R is a residue which is a functional group capable of forming a covalent bond with a protein;
• Ar is a hydrocarbon group having a conjugated double bond system;
• n is an integer equal to or greater than 1;
• and X is a fluorine atom or a group represented by General Formula (II):
• —C(═O)—CH2—C(═O)—Ar—R (II).