Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1994-09-28
2001-08-14
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007500, C435S004000, C436S501000, C436S518000, C436S526000, C424S178100, C424S179100
Reexamination Certificate
active
06274325
ABSTRACT:
DESCRIPTION
The invention concerns a method for carrying out a homogeneous immunoassay based on agglutination in which a conjugate of an Fab′ fragment and a component K is used.
Very many substances occur in body fluids and tissues which are capable of binding to a specific binding partner and which serve as parameters for certain diseases or the state of health of the human body. These include on the one hand immunologically active proteins which have binding sites on their surface such as e.g. tumour markers, hormones or viral proteins and on the other hand, DNA fragments. Since these substances often only occur in very small amounts, methods based on immunoassays are used for their detection with which these substances can be determined very specifically and exactly. There are many variants of these methods. The various immunological methods of determination may be classified into homogeneous and heterogeneous methods. A solid phase reaction always forms part of the heterogeneous method in order to immobilize complexes which contain the substance to be detected and a labelled component and thus to separate them from unbound components. In the homogeneous method variant there is no separation of bound label and unbound label so that bound and unbound label have to be differentiated by other methods.
There are different methods for this.
A method for the detection of proteins is known from DE-OS 27 49 956 which is based on the evaluation of an agglutination reaction. In this method antibodies against the substance to be detected are bound directly to the agglutinatable particles. However, the reactivity of the antibodies can be impaired by this binding. In addition such a method of determination is susceptible to interference by rheumatoid factors.
A homogeneous method of detection is described in US patent application Ser. No. 07/396,860 (22.08.89), now U.S. Pat. No. 5,362,655, which already overcomes the disadvantages of the previously known methods. In this method the sample solution is incubated with at least two receptors which are capable of binding to one another and of which one can bind to the substance to be detected. In this method an agglutination can only take place when the substance to be detected binds to both receptors and not, however, when the receptors bind to one another or the substance to be detected binds to only one of the two receptors. Conjugates consisting of one partner of a pair which specifically bind to one another and a component K capable of specific binding to the substance to be detected are used as the receptors capable of binding to the substance to be detected. If the method described here is used for the detection of proteins which have several identical epitopes then a conjugate has to be used as the receptor capable of binding to the substance to be detected which only has a single binding site for the substance to be detected in order to prevent an unspecific agglutination.
The prior art also mentions electrochemiluminescence labels useful in detection of an analyte. In that regard EP 580 979 based on U.S. application Ser. No. 666,987, now abandoned and Ser. No. 789,113 now U.S. Pat. No. 5,238,808 filed Oct. 31, 1984 and Oct. 24, 1985 respectively (also known as EP 199804), WO 87/06706 based on U.S. application Ser. No. 858,354, now abandoned, filed Apr. 30, 1986, WO 92/14138 based on U.S. application Ser. Nos. 652,427, now abandoned and 827,269, now U.S. Pat. No. 4,953,073, filed Feb. 6, 1991 and Feb. 3, 1992 respectively, WO 90/05301 based on U.S. application Ser. No. 266,882, now abandoned, filed Nov. 3, 1988 and WO 90/11511 based on U.S. application Ser. No. 325,459, now U.S. Pat. No. 5,068,88 filed Mar. 17, 1989 provide general information about these assays and are hereby incorporated by reference.
The object of the present invention was to provide a method for carrying out homogeneous immunoassays in which unspecific agglutinations are avoided as far as possible in order that exact and reproducible results are obtained.
This object is achieved by a method for carrying out a homogeneous immunoassay based on agglutination in which a conjugate of a Fab′ fragment and a component K is used, which is characterized in that a conjugate is used which is obtained by subjecting a F(ab′)
2
fragment of an antibody of the immunoglobulin G class to reducing conditions and subsequently reacting it with the component K which either has a functional group suitable for the binding or was derivatized in a suitable manner by introduction of a functional group whereby the component K is bound via the functional group to the free SH group of the Fab′ fragment formed during the reduction and agglutinatable particles are used which carry a substance capable of binding to K.
Surprisingly it was found that unspecific agglutinations can be avoided to a large extent by using these conjugates. Methods based on agglutination immunoassays could be further improved with regard to accuracy and reproducibility by using the monovalent binding partners.
Conjugates consisting of a Fab′ fragment of an IgG antibody which is capable of specifically binding to the substance to be detected and a component K are used for the method according to the present invention. Such conjugates can be produced by treating the antibody which is to be used in each case with pepsin in a known way. In this process fragments denoted F(ab′)
2
are formed which have two paratopes and in which the two heavy chains are held together by disulfide bridges. The F(ab′)
2
fragments are then subjected to mild reducing conditions so that the intramolecular disulfides are preferentially cleaved in the hinge region but not, however, the disulfide bridges between the light and heavy chain. Therefore, the reduction is preferably carried out with a mild reagent. Cysteamine, cysteine, mercaptoethanol or boron hydride are e.g. suitable for this. In this way Fab′ fragments are obtained which have 1 to 3 free SH groups. All free SH groups are available for binding to the components used. It has, however, turned out that, because of steric hindrance, either one component is bound or only one of two or more bound components retains its activity.
A component K which is a partner of a specific binding pair is bound to these Fab′ fragments. The component K can be bound to the Fab′ fragment either via a functional group which is already present or which is introduced or which is activated, if desired. The binding either takes place directly between SH groups of the Fab′ fragment and a functional group of the component K or via a spacer.
The binding of the Fab′ fragment and component K can take place directly via the functional groups present in each case. In this form of the method the proportion of unspecific linkages is particularly small. It is also possible to use a spacer for the binding. The length of the spacer depends in this case on the position of the free SH groups which is in turn dependent on the subclass of the IgG antibody used. It was found that up to three component molecules can be bound but that because of steric hindrance only one of them is capable of binding to the partner of the specific binding pair.
Bifunctional compounds which have a functional group which is capable of binding to the SH group and a second functional group which can be the same or different and which can covalently bind to the component are suitable as the spacer. The length of the spacer depends on the position of the SH bonds and thus on the subclass of the antibody from which the fragment is derived. If the spacer is too long it has the effect that even when several SH groups are relatively close together, a binding to all components is possible since the steric hindrance is overcome. The length of the spacer is therefore dependent on the position of the SH bonds and is thus defined by the Fab′ fragment used and is in the range of 4 to 16 atoms preferably 4 to 8 atoms for Fab′ fragments of the IgG subclass I, and in the range 4 to
Berger Michael
Deger Arno
Guillot François
Schlieper Dittmar
Boehringer Mannheim GmbH
Fulbright & Jaworski LLP
Ponnaluri Padmashri
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