Heterogenous chemiluminescent specific binding assay
First Claim
1. In a 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 reagent means including a labeled conjugate comprising a specific binding substance coupled to a labeling substance, said reagent means and the ligand forming a binding reaction system producing a bound-phase and a free-phase of the labeled conjugate;
(b) separating said bound-phase and said free-phase; and
(c) determining said labeling substance in said bound-phase or said free-phase as a function of said ligand in said liquid medium,the improvement wherein said labeling substance is a chemiluminescent reactant and wherein said chemiluminescent label is determined in said bound-phase or said free-phase by forming the chemiluminescent reaction therein and measuring the light produced.
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Abstract
A heterogeneous specific binding assay which employs a substance having reactant activity, i.e., a reactant, in a chemiluminescent reaction as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the chemiluminescent reactant. The activity of the conjugated reactant in the chemiluminescent reaction, i.e., the production of light, is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogeneous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques. After the necessary separation of the bound- and free-phases resulting in the specific binding reaction system, the extent of binding of the labeled constituent is determined by contacting either phase with the necessary materials to form the chemiluminescent monitoring reaction and measuring the light produced.
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Citations
56 Claims
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1. In a heterogeneous specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of:
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(a) contacting said liquid medium with reagent means including a labeled conjugate comprising a specific binding substance coupled to a labeling substance, said reagent means and the ligand forming a binding reaction system producing a bound-phase and a free-phase of the labeled conjugate; (b) separating said bound-phase and said free-phase; and (c) determining said labeling substance in said bound-phase or said free-phase as a function of said ligand in said liquid medium, the improvement wherein said labeling substance is a chemiluminescent reactant and wherein said chemiluminescent label is determined in said bound-phase or said free-phase by forming the chemiluminescent reaction therein and measuring the light produced. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A heterogeneous competitive binding assay method for determining a ligand in a liquid medium, comprising the steps of:
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(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. - View Dependent Claims (13, 14, 15, 16, 17, 18)
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19. A sandwich-type heterogeneous specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of:
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(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. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27)
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28. 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. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
- bringing together in an aqueous liquid zone;
- 42. Reagent means for use in a heterogeneous competitive binding assay for determining a ligand in a liquid medium, comprising (a) an insoluble form of one of said ligand or a specific binding partner thereof, and (b) a conjugate comprising the other of said ligand and binding partner labeled with a substance which is a reactant in a chemiluminescent reaction.
- 49. Reagent means for use in a sandwich-type heterogeneous binding assay for determining a ligand in a liquid medium, comprising (a) an insoluble form of a specific binding partner of said ligand, and (b) a conjugate comprising a specific binding partner of said ligand labeled with a substance which is a reactant in a chemiluminescent reaction.
Specification