Heterogenous chemiluminescent specific binding assay

A heterogeneous competitive binding assay method for determining a ligand in a liquid medium, comprising the steps of:

• (a) contacting said liquid medium with (1) an insoluble form of one of said ligand or a specific binding partner thereof, and (2) a conjugate comprising the other of said ligand and binding partner labeled with a substance which is a reactant in a chemiluminescent reaction, to form a reaction mixture comprising a bound-phase and a free-phase of said labeled conjugate,
• (b) separating said bound-phase and said free-phase,
• (c) forming said chemiluminescent reaction in one of said bound-phase and said free-phase, and
• (d) measuring the light produced as a function of said ligand in said liquid medium.

A sandwich-type heterogeneous specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of:

• (a) contacting said liquid medium with (1) an insoluble form of a specific binding partner to said ligand, and (2) a labeled conjugate comprising a specific binding partner to said ligand labeled with a substance which is a reactant in a chemiluminescent reaction, to form a bound-phase comprising said labeled conjugate bound to ligand bound to said insoluble binding partner and a free-phase comprising said labeled conjugate not so bound,
• (b) separating said bound-phase and said free-phase,
• (c) forming said chemiluminescent reaction is one of said separated phases, and
• (d) measuring the light produced as a function of the ligand in the medium under assay.

A heterogeneous method of determining the presence of a ligand in a medium suspected of containing said ligand which comprises:

• bringing together in an aqueous liquid zone:
• (1) said medium;
• (2) a chemiluminescent labeled ligand;
• and (3) a binding partner having sites capable of bonding to said ligand and said chemiluminescent labeled ligand;
• the concentration of said binding partner being such that there are insufficient binding sites to combine with all of said labeled ligand present in the three component mixture;
• and analyzing in said zone for the effect of said medium on the amount of chemiluminescent labeled ligand bound to said binding partner by physically separating free chemiluminescent labeled ligand from chemiluminescent labeled ligand bound to said binding partner and measuring chemiluminescence in either of the separated free and bound phases.