Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-04-01
2001-11-06
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S350000
Reexamination Certificate
active
06312916
ABSTRACT:
This application is a CPA which claims priority of foreign applications 196 37 718.8 filed on Sep. 16, 1996 and 196 13 053.0 filed on Apr. 1, 1996.
DESCRIPTION
The present invention concerns muteins of avidin and streptavidin with a reduced binding affinity for biotin as well as their use as interference elimination reagents in methods for the determination of an analyte e.g. in diagnostic tests such as immunoassays and nucleic acid hybridization assays. In addition the invention concerns the use of muteins of avidin and streptavidin as systems that can be regenerated for binding biotin for example for the analysis of biotinylated molecules, for investigating receptor-ligand interactions as well as for the affinity purification of biotinylated molecules.
In detection methods for the determination of analytes such as immunoassays and nucleic acid hybridization assays the analytes are often determined by means of high affinity interaction between the partners of a specific binding pair. A typical example for a specific binding pair is the avidin/streptavidin-biotin complex. When using the avidin/streptavidin-biotin binding pair its high binding affinity is used. In this process a solid phase coated with avidin/streptavidin is for example used to which a biotinylated complex of analyte and specific receptor can bind. In other test formats avidin/streptavidin can also be used in a soluble form.
However, apart from specific interactions side reactions often also occur such as for example undesired interactions and unspecific binding reactions between the test components and additional components present in the sample or on the solid phase. In particular other substances present in the sample often bind to immobilized or soluble avidin and streptavidin and thereby cause false positive or false negative test results. Furthermore these interactions can also cause an increase in the background signal and an increased scattering of the signals which decreases the sensitivity and specificity of the respective test.
Various attempts have been made to reduce these unspecific interactions. Thus it is for example known that various carbohydrate components and various proteins, protein mixtures or protein fractions and their hydrolysates can reduce unspecific interactions between the test components and the analyte in immunoassays (Robertson et al., J. of Immun. Med. 26 (1985) 195; EP-A-260 903; U.S. Pat. No. 4,931,385). However, the use of such carbohydrate and protein components has the disadvantage that components contained therein can cause additional interferences in the test. Enzymatically produced hydrolysates can in addition be contaminated by proteases used in their production and as a rule do not have a uniform quality since the cleavage is difficult to control. Such protease impurities can attack test components and already in small amounts lead to impairment of test function and storage stability.
Furthermore the use of chemically modified proteins especially of succinylated or acetylated proteins (U.S. Pat. No. 5,051,356; EP-A-0 525 916) has also been described to reduce unspecific interactions. However, it is not possible with these substances to avoid many of the false positive or false negative results in tests for antibodies from serum.
In order to avoid unspecific interactions it has additonally been proposed that ultrafine particles be added to the test reagents with a maximum average size of 0.2 &mgr;m which are formed in such a way that they bind to interfering components and capture them (EP 0 163 312). However, this requires a special preparation of these ultrafine particles and in addition the type of unspecific factors present in the sample must be known.
In DE-A-44 07 423 and DE-A-44 34 093 it has been proposed that interferences which occur due to unspecific interactions between sample components and a streptavidin-coated solid phase be eliminated by means of a pre-reaction. The pre-reaction is advantageously carried out on a solid phase which is as similar as possible to the active streptavidin-coated solid phase but to which the sample molecules cannot bind specifically. In contrast unspecific components also bind to the inactive solid phase and can therefore be removed.
According to DE-A-44 07 423 streptavidin can be inactivated by covalent derivatization or covalent modification. However, a disadvantage is that this requires a time-consuming subsequent chemical modification. Moreover, chemical derivatization can change the region around the active centre of the native streptavidin in an undesired manner which reduces the interference elimination effects and may even lead to additional interfering interactions. Interference by unspecific interactions which occur at the biotin binding pocket cannot be eliminated by covalent modifications.
The avidin/streptavidin-biotin system is the subject matter of several investigations due to the strong, non-covalent affinity of the binding partners (K
A
about 10
15
l/mol). The high binding affinity has mainly been attributed to interactions between tryptophan residues of streptavidin and biotin. However, a significant decrease of the binding affinity of streptavidin variants to iminobiotin could be achieved by modifying the tryptophan residues (Chilkoti et al., Proc. Natl. Acad. Sci. USA 92 (1995) 1754-1758, Sano and Cantor, Proc. Natl. Acad. Sci. USA 92 (1995) 3180-3184). However, it was not possible to unequivocally demonstrate a reduction of the binding affinity to biotin (cf. Chilkoti et al., Supra, FIGS. 1A and B). Such variants are therefore unsuitable for use as interference elimination reagents since they can also specifically react with biotinylated test components due to their still very high binding affinity.
The object of the present invention was therefore to provide a reagent by means of which interfering influences on detection methods for the determination of an analyte e.g. immunoassays or nucleic acid hybridization assays can be reduced.
This object is achieved according to the invention by a polypeptide capable of binding to biotin selected from muteins of avidin and streptavidin wherein the mutein (a) differs from the native polypeptide by at least one amino acid and (b) has a binding affinity to biotin of less than 10
10
l/mol.
The binding affinity for the reaction
streptavidin/biotin complex
streptavidin+biotin
is about 10
15
l/mol. The streptavidin/biotin system used as a capture system is thus provided with one of the strongest known non-covalent interactions between a protein and a ligand. Surprisingly it was found that a polypeptide is obtained by substitution of one or several amino acids of streptavidin or avidin which on the one hand can be renatured when produced recombinantly and on the other hand enables the binding affinity to biotin to be reduced to <10
10
l/mol, furthermore the muteins according to the invention preferably having a structure which corresponds to the structure of the active polypeptide. The muteins according to the invention preferably have a high immunological cross-reactivity with the native polypeptide. In addition it is preferable that they are able to dimerize or tetramerize. Surprisingly the muteins like native streptavidin or avidin are able to bind to interfering sample components as they can occur in biological samples e.g. in body fluids such as serum, plasma, whole blood etc. despite the reduced binding affinity to biotin.
Due to their special properties the muteins according to the invention can be used for various applications. A lowering of t he binding affinity is synonymous to lowering the interactions between biotin and the muteins. Muteins according to the invention can be designed in such a way that they under no circumstances bind to biotin or that a relatively loose reversible binding is present. Since the spatial structure of the muteins is preferably not significantly changed in comparison to the native polypeptide, interactions with other substances are not affected. A reagent is obtained in this way which corresponds in its spatial struc
Brandstetter Hans
Deger Arno
Engh Richard
Kopetzki Erhard
Muller Rainer
Carlson Karen Cochrane
Mitra Rita
Roche Diagnostics GmbH
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