Use of intramolecularly, covalently cross-linked proteins as binding partners in immunoassays

#The present invention concerns the use of intramolecularly, covalently cross-linked proteins, and in particular, the use of covalently cross-linked reverse transcriptase (RT) from HIV as immunological binding partners in immunoassays, and to immunological test procedures for detecting an analyte in a sample in which intramolecularly, covalently cross-linked proteins are used as binding partners. It also concerns intramolecularly, covalently cross-linked reverse transcriptase from HIV and a method for producing this reverse transcriptase.

#The use of proteins as binding partners for the detection of analytes in immuno-diagnostic test procedures has been known for a long time. In all conventional immunoassays, the sample is incubated with one or more binding partners that are specific for the analyte. The binding partner or binding partners bind(s) specifically to the analyte to be detected. In the case of an antibody test, for example, in the case of an HCV infection, the sample to be examined is, for example, contacted with an HCV antigen which specifically binds the anti-HCV antibody to be detected. In an antigen test, for example, for detecting the tumour marker prostate-specific antigen (PSA), the sample is contacted with antibodies which specifically bind the PSA in the sample.

#Subsequently the analyte is detected in all immunoassays. This can, for example, be carried out by binding and subsequently detecting another binding partner provided with a detectable label which binds to the complex consisting of analyte and immunological binding partner.

#FIG. 1 shows the analysis by gel permeation chromatography of the molecular weights of RT obtained after cross-linking.

#All proteins required for immunological test procedures that are familiar to a person skilled in the art can be used as the proteins. All polypeptides can be used which could, as a result of their folding, i.e., their tertiary or quaternary structure, have a tendency to unfold, to denature, or to dissociate into various subunits under the conditions of an immunoassay. When such a structural change occurs, there is a risk that immunologically important epitopes are changed in such a manner that, for example, they are no longer specifically bound by antibodies. In the worst case, this means that an immunological test result is negative, i.e., it does not indicate the presence of the antibody to be detected because the proteins used as binding partners are denatured. These disadvantages are substantially avoided by the use of intramolecularly, covalently cross-linked proteins according to the invention.

#In particular, those intramolecularly, covalently cross-linked proteins are used which are naturally composed of several subunits. DNA or RNA polymerases, particularly the reverse transcriptase from HIV, and especially preferably the reverse transcriptase from HIV-1 are preferably used.

#The proteins can be from any desired source. The proteins to be cross-linked can be isolated from their natural source such as an organism or virus. However, the use of recombinant proteins produced by genetic engineering is preferred. A recombinant purified RT is especially preferably used which is expressed by an expression clone as described, for example, in Müller et al., J. Biol. Chem. 264/24:13975-13978 (1989).