WO2000031537A1

WO2000031537A1 - Detection of biomolecules using phage reagents

Info

Publication number: WO2000031537A1
Application number: PCT/SE1999/002156
Authority: WO
Inventors: Björn CARLSSON; Lena Carlsson
Original assignee: Carlsson Bjoern; Lena Carlsson
Priority date: 1998-11-23
Filing date: 1999-11-22
Publication date: 2000-06-02
Also published as: AU2010600A; SE9803993D0

Abstract

A reagent consisting of a phage displaying a sequence with affinity for a target molecule of interest to be detected. The reagent may be used to replace antibodies in different immunoassays to detect target molecules of interest in biological fluids and other solutions or target molecules on cell surfaces and in tissue.

Description

DETECTION OF BIOMOLECULES USING PHAGE REAGENTS

Technical field of the invention

The present invention relates to new reagents for detection of biomolecules, especially suitable for replacement of antibodies in immunoassays. The invention also relates to use of said reagents.

Background art Antibodies are widely used as recognition molecules in immunoassays to determine the concentration of differ- ent target molecules, such as antigens, in different biological fluids. Immunoassays based on the use of antibodies have been very successful, and assays of this kind are extensively used in e.g. biotechnology and pharmaceutical research. One example of an immunoassay based on the use of antibodies is ELISA (enzyme-linked immunosor- bent assay) , which is a quantitative immunoassay in which antibodies linked to an insoluble carrier is used to capture the relevant target molecule in a test solution and enzyme-labeled second antibodies are used for detection. However, the use of antibodies has several disadvantages. For example, if the target molecule is highly conserved between species, antibodies are sometimes difficult to obtain. The supply of antibodies may also be limited and the purification can be costly and time consum- ing . Another problem is the presence of heterophilic antibodies which can affect the results of immunoassays.

Several of the technical problems related to the production of antibodies have been bypassed by the use of phage display libraries, which enables the production of synthetic antibodies.

A phage display library encodes fusion proteins of a foreign polypeptide sequence and a coat protein of a bac- teriophage. The phage is said to display the foreign protein. There are two common versions of the technique, synthetic randomized peptide libraries and cDNA expression libraries. In synthetic randomized peptide libraries, the library consists of random or designed oligonu- cleotides. In cDNA expression libraries, the library con- sist of DNA sequences encoding naturally occurring proteins, for example antibody fragments. Clones expressing oligopeptides or proteins (encoded by the random oligonu- cleotides or cDNAs) that react with a specific target molecule can then be selected. For generation of selected phage for use as replacement of antibodies in immunoassays, two general strategies have been used. In the first strategy, the phage displaying a specific sequence is then used to produce recombinant antibodies, other proteins or peptides with desired binding properties.

After selection of a phage which displays a peptide or protein with high affinity for the target molecule, the corresponding protein (by itself or as fusion protein) must be expressed in an appropriate expression sys- tern. It is known to man skilled in the art that is laborious and several problems may be encountered during the production of recombinant proteins. In addition, one major, well known problem with the phage display technique is the fact that even if the selected amino acid sequence did bind to the target molecule when it was displayed on the phage, it is not certain that it binds to the target molecule in a context other than the intact phage. Even though phage display based strategies have been used to improve and simplify the generation of recombinant pro- teins (for example recombinant antibodies) to be used for the detection of a specific target molecule, the successful selection of a phage with desired binding properties has only been a small step in this direction since the applications so far have required additional manipula- tion.

In the second strategy, phages displaying antibody fragments have been used directly as immuno-reagents . The binding epitopes on phage used as immuno-reagents have been obtained by laborious strategies based on native antibody variable domains. In addition to being time consuming this procedure also limits the possible binding epitopes to those that can be obtained from biological samples .

A lot of research efforts have been made in order to solve these problems, but no satisfying solution has been found up until now.

Summary of the invention In the research work leading to the present invention it was found that it is possible to use a phage library displaying a synthetic randomized sequence and se- lect phage with affinity for a specific target molecule as a reagent in the detection of a target molecule in biological fluids and on cells instead of using the selected DNA sequence for production of a protein or peptide (for example an antibody) or phage displaying recom- binant antibody fragments which then is used for detection of the target molecule.

Another advantage of the reagent according to the invention is that it is very easy to multiplicate . All that is required is amplification of the selected phages, with methods that are well known to man skilled in the art . To obtain new reagent with the old techniques it is necessary to produce new synthetic peptides or recombinant proteins (for example antibodies) or to use laborious strategies to express recombinant antibody fragments on the surface of the phage, which is complicated and time consuming.

Thus, the present invention relates to a new reagent comprising a phage displaying a sequence with affinity for a target molecule. The characterizing features of the invention will be evident from the following description and the appended claims . Detailed description of the invention The phage in the reagent according to the invention may be any kind of phage known to man skilled in the art, for example filamentous Escherichia coli bacteriophages such as M13, fd or fl, displaying one or multiple copies of a peptide on the surface.

The term "a phage displaying a sequence" designates to a phage to which a sequence has been coupled in such a way that the sequence is free to bind to any molecules it has affinity for.

The displayed sequence may be any sequence that can bind to a target molecule.

The target molecule to be detected by use of the reagent according to the invention may be any kind of molecule of interest to detect, such as a hormone, a receptor agonist, a receptor antagonist, an antigen, an antibody, a non-peptide molecule etc.

The target molecule to be detected may present in a, optionally diluted, biological fluid, such as blood, or be placed in a solution prior to detection, or be present in a cell or tissue.

The reagent according to the invention may be used for detection of any kind of molecules, especially bio- molecules, on condition that it is possible to find a sequence with affinity for the molecule in question.

The reagent according to the invention may e.g. be used in a modified immunoassay, wherein the reagent according to the invention replaces the traditionally used antibody. The reagent according to the invention is then added to a solution of the target molecule. The fraction of the reagent combining with target molecule is estimated using an appropriate indicator. The amount of target molecule is e.g. determined by comparison with stan- dards of known amounts.

The reagent according to the invention may also be used in a modified radioimmunoassay, wherein the reagent according to the invention replaces the traditionally used antibody. A known amount of a reagent according to the invention is then saturated with a mixture of target molecule (TM) and radioactive target molecule (TM*) so that the total amount of target molecule (TM + TM*) is in excess. When non-radioactive TM is added to the reagent along with TM* , TM and TM* compete for binding to the reagent so that less TM* will be found in the reagent- target molecule complex as the ratio TM:TM* increases. The reagent-target molecule complex is thereafter separated from free TM and free TM* , and the amount of TM can be calculated from a standard curve.

The reactant may be used in the form of a solution. However, in some cases it may be advantageous to attach the reactant to a solid carrier, such as a plate, so that the amount of target molecule bound to the carrier is proportional to the amount of test reagent.

Furthermore, the reagent may also be used as a replacement of antibodies in modified versions of fluoroim- munoassays, spin immunoassays, enzyme immunoassays etc. It is also possible to use the reagent according to the invention in a modified version of immunohistochemis- try, wherein the reagent according to the invention is used, instead of antibodies as in normal immunohistochem- istry, to detect target molecules on cell surfaces and in tissue .

As stated above the reagent according to the invention may either be used in a free from, e.g. in a solution, or in a bound form. When a bound form is desired, the reagent may be attached to a carrier, such as a plate or other insoluble particles. The attachment of the reagent to the carrier can be performed according to any appropriate method known to man skilled in the art.

The invention will now be further explained in the following example. This example is only intended to illustrate the invention and should in no way be considered to limit the scope of the invention. Example - Detection of growth hormone

In this example phages displaying recognition sites for growth hormone, GH, on the surface were used as the reagent according to the invention. The phages were selected from a polyvalent random 15-mer phage display library based on high affinity for human growth hormone. The selected phage clones were amplified using methods known to man skilled in the art. The detection method used was a modified version of a radioimmunoassay.

The reagent was added to a sample consisting of a solution of GH of an unknown concentration. Also radiola- beled GH, GH* , was added. Both GH and GH* formed complexes with the reagent . Free GH and GH* were then separated from the reagent -GH and reagent-GH* by PEG precipitation, which is a well known technique.

The concentration of GH in the sample was finally calculated by conventional methods from a standard curve obtained by serial dilutions of known amounts of GH.

Claims

1. A reagent comprising a phage displaying a sequence with affinity for a target molecule.

2. A reagent according to claim 1, wherein said target molecule is a biomolecule.

3. A reagent according to claim 1 or 2 , wherein said sequence with affinity for a target molecule is a hormone recognition site and said target molecule is a hormone.

4. Use of a reagent according to any one of the claims 1 - 3 for detection of a target molecule in a biological fluid.

5. Use of a reagent according to any one of the claims 1 - 3 in a way corresponding to the use of an an- tibody in an immunoassay.

6. Use according to claim 5, wherein said immunoassay is a radioimmunoassay.

7. Use of a reagent according to any one of the claims 1 - 3 for detection of a target molecule on a cell surface of in tissue.

8. Use of a reagent according to any one of the claims 1 - 3 in a way corresponding to the use of an antibody in immunohistochemistry.

9. Use according to any one of the claims 4 - 8, wherein said reagent is linked to an insoluble carrier.