Method for carrying out a homogeneous-immunoassay based on agglutination
AssigneeBoehringer Mannheim GmbH
InventorsArno Deger, François Guillot, Michael Berger, Dittmar Schlieper
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Abstract
The invention concerns a homogeneous immunoassay based on agglutination using an Fab′-biotin material and agglutinatable particles which carry streptavidin or avidin. The invention also concerns the Fab′-biotin which is bound or linked via linkage groups to a label compound which can electrochemiluminescence and the particles having avidin or streptavidin on their surface are magnetic.
Claims14
A method for determining an analyte in a homogeneous immunoassay, consisting essentially of:
- (i) reacting a Fab′ molecule with biotin, wherein said Fab′ molecule is derived from an IgG molecule and has from 1-3 free SH groups in its hinge region and said biotin is either coupled to said free SH groups via a functional group attached to said biotin or via a spacer group coupled to biotin via a functional group, wherein the spacer group comprises an alkylene chain of from 4 to 16 carbon atoms to obtain an IgG derived Fab′-biotin conjugate or an IgG derived, Fab′-spacer biotin conjugate,
- (ii) adding said conjugate to a sample together with an agglutinatole particle coated with avidin or streptavidin, and
- (iii) determining agglutination as a determination of said analyte in said sample.
Method for determining an analyte in a sample via a homogeneous immunoassay, comprising:
- (1) forming a composition of:
- (a) the sample,
- (b) a conjugate of an Fab′ fragment and biotin, wherein said conjugate is produced by reducing an F(ab′)2 fragment having 1 to 3 free SH groups on its hinge region and reacting the Fab′ fragment with biotin having a functional group suitable for the binding or wherein the Fab′ is derivatized by a functional group wherein biotin is bound via the functional group and a spacer to at least one of said free SH groups of Fab′ fragment to form a biotinylated Fab′ fragment,
- (c) one substance selected from the group consisting of:
- (i) an analyte of interest or an analog of an analyte of interest, and,
- (ii) a binding partner or the analyte, wherein (i) and (ii) are linked to a label compound capable of being induced to electroluminescence, and
- (d) a plurality of particles having avidin or streptavidin on their surfaces,
- (2) inducing said label compound to electrochemiluminescence and measuring the luminescence emitted by the composition to determine the presence of said analyte of interest in the sample.
A method of determining an analyte in a homogeneous immunoassay consisting essentially of:
- (i) reacting a Fab′ molecule with biotin, wherein said Fab′ molecule is derived from an IgG molecule and has from 1-3 free SH groups in its hinge region, and said biotin is either coupled to said free SH groups via a functional group attached to said biotin or via a spacer group coupled to biotin via a functional group, wherein the spacer group compresses an alkylene chain of from 4 to 16 carbon atoms to obtain an IgG derived Fab′-biotin conjugate or an IgG derived, Fab′ spacer biotin conjugate,
- (ii) adding said conjugate to a sample together with an agglutinatable particle coated with avidin or streptavidin prepared by reacting avidin or streptavidin with a bifunctional compound having an N-hydroxysuccinimide group and an arylazide group under alkaline conditions of less than pH 9.5 in aqueous solution, to form a complex of said bifunctional compound and avidin or streptavidin, removing excess bifunctional compound, adding a latex particle to said aqueous solution, and irradiating said aqueous solution with a photon, to bind said complex to said latex particle, and
- (iii) determining agglutination as a determination of said analyte in said sample.
Dependencies are inferred from the claim text.