WO2007124593A1 - Branched peptide amplification and uses thereof - Google Patents

Abstract

The present invention relates to signal amplification in protein interaction assays and immunoassays. More specifically, the present invention is concerned with branched peptides coupled to labeled interacting partners for signal amplification. The present invention concerns methods, compositions and kits for signal amplification of protein interaction assays such as immunoassays.

Description

TITLE OF THE INVENTION

[0001] BRANCHED PEPTIDE AMPLIFICATION AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. provisional application Serial No. 60/796,586 filed on May 2, 2006, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to signal amplification in protein interaction assays and immunoassays. More specifically, the present invention is concerned with branched peptides coupled to labeled interacting partners for signal amplification.

BACKGROUND OF THE INVENTION

[0004] Development of sensitive assays for the characterization, detection and quantification of various reagents has played a key role in the evolution of life sciences. Among all techniques, immunochemistry (detection of antigens via specific antibodies) had a strong impact, both on fundamental research and applied sciences. It has allowed the introduction of sensitive and highly specific diagnostic tests for a variety of applications, ranging from the detection of pathogens to the determination of pregnancy status at home. Immunoassays are now widely accepted in medicine and are often critical in helping physicians to establish diagnostic guidelines and therapeutic strategies (Laurino et a/., 1999, Ann Clin Lab Sci 29(3): 158-66). Their use also extends to other fields such as agricultural and food industries where they can be used to detect various contaminants (Yau et al., 2003, Biotechnol Adv 2I(7):599-637).

[0005] The large variety of immunoassays available on the market has been mostly due to important technological advances. First, progress in immunology has greatly facilitated the preparation and large-scale production of antibodies with high affinity and specificity (Little et ai, 2000, Immunol Today 21_(8):364-70). Second, variations in the design of the assays have allowed the adaptation of the different tests to the nature of the samples tested, the character of the molecules to be assayed, the ease of testing, while at the same time decreasing their cost and improving their sensitivity and specificity (for example, see Stramer 2004, Vox Sang 87(Suppl 2):180-3). However, most importantly, various strategies have been developed to increase the sensitivity of immunoassays (Kricka 1994, Clin Chem 40(3):347-57). Many different approaches have also been used to enhance and visualize the signal generated in the reaction. Genome detection methodologies (Raoult et al. 2004, Nat Rev Microbiol 2(2): 151 -9) and use of new markers (Roda et al. 2004, Trends Biotechnol 22(6):295-303) represent rapidly expanding technologies useful for diagnostic purposes. However, these technologies are not as versatile as one would wish; detection of toxins and allergens and surveillance of the immune status are examples for which nucleic acid amplification technologies cannot be applied. In addition, the demand for high throughput technologies such as microarrays and low-cost point-of-care devices necessitates simpler and more sensitive methods to facilitate the specific detection of various targets.

[0006] Thus, there remains a need for new versatile and sensitive methods of detection for protein interaction assays such as immunoassays.

[0007] The present invention seeks to meet these needs and other needs.

[0008] The present description refers to a number of documents, the content of which is herein incorporated by reference in its entirety.

SUMMARY OF THE INVENTION

[0009] The present invention relates to a versatile and sensitive method of detection for protein interaction assays, which overcomes at least one drawback of the methods of the prior art.

[0010] In one embodiment, the present invention relates to a new versatile and highly sensitive detection system used in immunoassays as well as other general protein interaction assays. In another embodiment, the detection system is centered on branched peptide amplification (BPA).

[0011] Branched peptide amplification (BPA) is a new technology aimed at increasing the detection levels of various types of protein interactions such as immunoassays. Branched peptides, also known as multiple antigenic peptides (MAP) are peptides carrying multiple copies of the same epitope and represent the core of the amplification system. The MAP are designed in combination with a complementary labeled peptide which interacts with the multiple copies of the epitope presented by the MAP core. This interaction leads to a strong increase in the level of signal emitted, thereby greatly enhancing the sensitivity of detection of various types of interaction (e.g., antigen recognition by specific antibodies in immunoassays).

[0012] Thus, in an embodiment, when linked to an antigen-antibody complex and recognized by complementary peptides coupled with detectable molecules (e.g., fluorescent markers) MAP provide a system that amplifies a signal and consequently improves the sensitivity of immunoassays. The BPA technology is very versatile and therefore is not limited to immunoassays since it may also be adapted for the sensitive detection of various protein interactions, including, but not limited to enzyme-substrate interaction, ligand- receptor interaction and the like.

[0013] Thus, in a first aspect, the present invention relates to Branched Peptide Amplification (BPA) technology as a versatile tool for increasing the detection signal of a given protein interaction assay (e.g., immunoassay) thereby lowering its detection limit.

[0014] In one aspect, the branched peptides (or MAP core) of the present invention comprise: 1 ) a central core composed of a polyfunctional molecule (e.g., lysine and derivatives thereof); 2) a spacer arm or linker attached to each radiating branch of the central core; and 3) multiple copies of a first peptide Pi (of a peptide pair) which is designed to interact with a second labeled interacting peptide P₂. Each copy of the first peptide is linked to the spacer arm and radiates outwardly from the central polyfunctional core. The central polyfunctional core, together with the spacer arms are used for presenting a plurality of peptides within the same molecule, located on the branches. The branched peptides of the present invention are then used together with a second labeled peptide P₂, which interacts with the peptide Pi located on the branches of the MAP core, in order to detect a particular protein interaction.

[0015] In one embodiment, the branched peptides of the present invention are also designed to include an additional moiety (M₁) which is specific for the particular type and requirement of the assay used. This additional moiety is linked/conjugated to one end of the central polyfunctional core (see Figurei ) and is chosen such that it can interact with a target antibody or protein.

[0016] In one particular embodiment, this moiety Mi is biotin and allows the branched peptide of the present invention to interact with an antibody also linked to biotin through an avidin molecule. Thus, in one aspect, the Mi molecule interacts indirectly with the target antibody.

[0017] In another embodiment, the branched peptide of the present invention may be linked directly to an antibody (e.g., secondary antibody such as anti-human antibody, anti-mouse antibody, anti-rat antibody, etc. or any primary antibody that is specific for the target antigen) for use in an immunoassay.

[0018] In yet another embodiment, the moiety Mi may constitute a particular protein-binding domain, which is known to interact with the target molecule to be detected.

[0019] The present invention also relates to a detection method for use in a protein interaction assay comprising the use of a branched peptide, projecting a plurality of peptide P-i, together with a labeled peptide P₂ (specifically interacting with peptide Pi), wherein the branched peptide is adapted to interact with a target molecule (e.g. protein, antibody, nucleic acid) to be detected.

[0020] In accordance with the present invention, the Branched Peptide Amplification technology may also be used to detect a plurality of different target proteins in a sample. For example, a first branched peptide-labeled peptide pair (BP_1a-P_2a) can be used to detect a first protein interaction (e.g. antibody interacting with a first antigen); and a second branched peptide-labeled peptide pair (BP_1b-P_2b) can be used to detect a second protein interaction (e.g. another antibody interacting with a second antigen) in a sample. Of course, peptides P_2a and P_2b could given different labels (e.g. different fluorescent moieties) in order to specifically and distinctively detect each target molecule (e.g. each antigen, each antibody, each protein, each protein interaction, etc.) present in the sample. [0021] In an embodiment, the protein interaction assay is an immunoassay.

[0022] In another embodiment, P₁ and P₂ peptides are selected from BAP-01 , BAP-02 and BAP-03 peptide pairs.

[0023] In an embodiment, the branched peptides of the present invention comprise a plurality of identical Pi peptides. In another embodiment, the branched peptides of the present invention comprise a plurality of different Pi peptides.

[0024] In a further aspect, the present invention relates to compositions and kits for the detection of a protein interaction assay. In one embodiment the protein interaction assay is an immunoassay.

[0024] In an embodiment, the present invention concerns a composition for use in the detection of a protein interaction comprising: (A) a branched peptide comprising: i) a central core composed of a polyfunctional molecule comprising diamino carboxylic acid residues which provide the branched peptide with a plurality of amino terminal portions; ii) a spacer arm attached to the amino terminal portions of the polyfunctional molecule; and iii) a plurality of P1 peptides, wherein a P1 peptide is attached to the spacer arm and projects out of the central core, thereby presenting a plurality of peptides within the same molecule; and (B) a plurality of labeled P2 peptides, each P2 peptide being capable of specifically interacting with said P1 peptide of said branched peptide.

[0025] In another embodiment, kits of the present invention will generally comprise (1) Branched peptides coupled with a moiety Mi (e.g. biotin, antibody, etc); (2) labeled peptides (P_;>) interacting with P₁ peptides present on the branched peptides and (3) instructions for use in protein interaction assays such as immunoassays. The kit can further comprise other components such as avidin, reagents for detection of the labeled molecule as well as any other suitable components (e.g. biotinylated antibodies such as anti-human, anti-mouse, anti-rat antibodies or the like).

[0026] In one embodiment, the moiety M₁ of the branched peptides included in the kit is biotin. In another embodiment, P₁ peptides and their complementary P₂ peptides included in the kit are selected from BAP-01 ; BAP-02 and BAP-03 peptide pairs. In a further embodiment, P₂ peptides are labeled with Alexa Fluors®.

[0027] Other objects, advantages and features of the present invention will become more apparent upon reading the following non-restrictive description of specific embodiments thereof, given by way of examples only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In the appended drawings:

[0029] Figure 1 shows an embodiment of an amplification system using the BPA technology;

[0030] Figure 2 shows the amino acid sequence of the 4-branch core MAP in accordance with a first embodiment of the present invention. The spacer moiety is represented by "AGAG", the biotinylated lysine is represented by "K(biotin)" and the amino terminus of the peptides is indicated by the NH₂ groups [-AGAG-K(biotin)-NH₂ representing SEQ ID NO:"!];

[0031] Figure 3 shows the characterization by Reverse-Phase High Performance

Liquid Chromatography (RP-HPLC) of a 4-branch (A) and 8-branch (B) MAP cores in accordance with a first and second embodiment of the present invention;

[0032] Figure 4 shows the amino acid sequence of various embodiments of complementary peptides in accordance with the present invention. Linkers are shown in bold, the N-terminus of the peptides is represented by "H₂N" (at the left), the C-terminus is represented by "COOH" or "CONH₂" and the hydrophobic sequence is underlined. (A) BAP- 01 ; (B) BAP-02; and (C) BAP-03;

[0033] Figure 5 shows the purification of peptides K3_(IAAL) and E3_(IAAL) (BPA-02, Fig

5A) and peptides F and A (BPA-03, Fig. 5B) by Reverse Phase-high performance liquid chromatography (RP-HPLC). [0034] Figure 6 shows the mass spectrometry analysis of peptides K3_{( A}AL₎ and

E3₍i_AAL) (BPA-02, Fig. 6A) and peptides F and A (BPA-03, Fig. 6B);

[0035] Figure 7 shows circular dichroism spectra of the heterodimer K3_(IAAL/E3(|_AAL) coil, the K3_(IAAL), and the E3_(IAAL> peptides (BAP-02). The molar ellipticity ratio ([Θ]₂22nm/[θ]₂o8nm ratio) for K3₍IAAL)/E3₍IAAL) is 0.96. A [Θ]₂22nm/[θ]₂o8nm ratio above 0.95 reveals the presence of the heterodimeric α-helical coiled-coil;

[0036] Figure 8 shows circular dichroism spectra of the heterodimer K3_(VS_AL/E3_(VSAL)

(BAP-01 ). The molar ellipticity ratio ([θ]_222nm/[θ]₂₀8nm ratio) for K3₍I_AA_L)/E3₍I_AAL) is 0.35 (0% TFE) and 0.75 (50% TFE). A [θ]₂₂₂/[θ]₂o₈ ratio below 0.50 or above 0.95 respectively reveals the presence of a random coil or a heterodimeric α-helical coiled-coil;

[0037] Figure 9 shows the purification of Alexa Fluor® 647-labeled peptide E3₍|_AAL).

Purification was monitored through absorbance at 214, 280 and 598 nm. Position of the labeled peptide and free Alexa Fluor® are shown.

[0038] Figure 10 shows the specific binding of Alexa Fluor®-labeled E3(|_AAL) peptide to fixed K3₍|_AAL) peptide. Various amounts of K3(|_AAL> were fixed to a maleimide-activated microplate, exposed to different concentrations (0,065 to 65 μg/mL) of Alexa Fluor©-labeled E3₍|_AAL) for 30 minutes, and washed. Relative fluorescence units (RFU) were determined through reading at 678 nm following excitation at 584 nm. Diamonds = 65 μg/mL; squares = 6.5 μg/mL; triangles = 0.65 μg/mL and circles = 0.065 μg/mL.

[0039] Figure 11 shows the specific binding of FITC-labeled peptide A to fixed peptide F. Various amounts of peptide F were fixed to a microplate, exposed to 75 μg/mL FITC-labeled peptide A for 30 minutes, and washed. Relative fluorescence units (RFU) were determined through reading at 527 nm following excitation at 485 nm. Lower panel = low concentrations of labeled E3₍|_AAL); upper panel = high concentrations of labeled E3₍|_AAL);

[0040] Figure 12 shows the binding of fluorescent E3_(IAAL) to MAP-K3₍I_AAL) synthesized on plate. Various amounts of biotinylated MAP were bound to a neutravidin- coated microplate, activated and linked or not to the K3_(IAAL> peptide. After extensive washing, fluorescent E3₍|_AAL) (6,5 μg/mL) was added. After 30 min incubation and washing, relative fluorescence units (RFU) were determined through reading at 678 nm following excitation at 584 nm;

[0041] Figure 13 shows the binding of fluorescent E3_(IAAL) to MAP-K3(_IAAL) synthesized in solution. The MAP core was activated, purified and concentrated on Centricon micro-column™ (Amicon), bound to the K3₍|_AAD peptide and purified by RP-HPLC. Various amounts of biotinylated MAP-K3(IAAI_) were fixed to a neutravidin-coated plate and exposed to fluorescent E3_(IAAL) (6,5 μg/mL). After 30 min incubation and washing, relative fluorescence units (RFU) were determined through reading at 678 nm following excitation at 584 nm. Note the logarithmic scale (RFU);

[0042] Figure 14 shows the detection of antigen-antibody binding through MAP-

K3(iAAL)/fluorescent-E3, IAAL)- A sheep IgG was coated to a microplate and exposed to a specific biotinylated anti-sheep IgG. Various amounts of avidin were added, followed by MAP-K3(IAAL; and fluorescent-E3₍ι_AAL)- Relative fluorescence units (RFU) were determined through reading at 678 nm following excitation at 584 nm;

[0043] Figure 15 shows signal amplification through MAP. Various amounts of IgG sheep antibody were adsorbed to a microplate, exposed to biotinylated anti-sheep antibodies (2 μg/mL) followed by avidin (10 to 500 ng/well), various amounts of biotinylated MAP-K3(_IAAL) and exposed to Alexa Fluor®-labeled E3(I_AA_L> (15 μg/mL). Relative fluorescence units (RFU) were determined through reading at 678 nm following excitation at 584 nm. Results are shown for various levels of avidin and biotinylated MAP-K3₍|_AAD for 200 ng IgG sheep antibody (A) and for various amounts of IgG sheep antibody using 500 ng/well avidin and 7,5 μg/mL biotinylated MAP-K3₍|_AAL) (B);

[0044] Figure 16 shows the binding of biotinylated 8-branch MAP core to avidin.

Increasing amounts of biotinylated MAP-8 core were exposed to HABA-avidin and the reaction was followed by determination of the absorbance at 500 nm. The y-axis represents the variation (decrease) in absorbance;

[0045] Figure 17 shows Alexa Fluor® labeling of the 8-branch MAP core. The MAP-

8 core was labeled with Alexa Fluor® and analyzed by RP-HPLC at 214 nm (upper panel), 280 nm (second panel) and 598 nm (third panel). The nature of the solvent gradient used for the chromatography is shown on the bottom panel,

[0046] Figure 18 shows the binding of Alexa FluorΘ-labeled biotinylated 8-branch core to avidin Increasing amounts of Alexa Fluor®-labeled biotinylated MAP-8 core were exposed to HABA-avidin and the reaction was followed by measuring the absorbance at 500 nm (with peptide) As a control, the reaction was performed in the absence of MAP-8 core (without peptide), and

[0047] Figure 19 shows the detection through the 8-branch MAP Various amounts of sheep IgG antibody were absorbed to a microtiter plate and exposed to 200 ng of a biotinylated anti-sheep IgG, 500 ng of avidin and 1 μg of Alexa Fluor® MAP-8 Relative fluorescence units (RFU) were determined through reading at 678 nm following excitation at 584 nm Results are shown for 0-250 ng (A) and 0-5 ng (B) IgG sheep antibody

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0048] Branched peptides, often referred to as multiple antigenic peptides, were first designed as a way to prevent the need to conjugate single peptides to a carrier protein in order to elicit a strong immune response (Posnett et al 1988, J Biol Chem 263(4) 1719- 2588, Tarn 1988, Proc Natl Acad Sci U S A 85(15) 5409-13), a property that has been exploited to develop new vaccine strategies (Haro & Gomara 2004, Curr Protein Pept Sci 5(6) 425-33) More recently, their use has been extended to other applications such as the detection (Ndongmo et al 2004, J CIm Microbiol 42(1 1 ) 5161-9, Saravanan et al 2004, J Immunol Methods 293(1-2) 61-70, Kasubi et al 2005, J Virol Methods 125(2) 137 43) and the purification (Verdohva et al 1995, J Chromatogr B Biomed Appl 664(1 ) 175-83) of antibodies or the specific delivery of molecules into cells (Sheldon et al 1995, Proc Natl Acad Sci U S A 92(6) 2056-60) However, for all these functions, the branched peptides need to directly interact with specific molecules (antibodies or receptors) to be active Contranly, BPA technology has been designed to be universal and to not depend on specific interactions between MAP and antibodies or receptors

[0049] Fluorescence is now frequently used in immunoassays as well as other protein interaction assays The strength of the signal is proportional to the amount of fluorescent molecules bound to the target To increase the sensitivity, it is therefore advantageous to design intermediates capable of amplifying levels of bound fluorescence.

The main purpose of this work was to develop such an intermediate that could be used in a variety of protein interaction assays including immunoassays. Non-limiting examples of immunoassays include ELISA, Western blots, cytofluorometry and antibody microarrays.

[0050] Description of the proposed model of the BPA technology

[0051] Branched peptides are peptides that carry multiple copies of an epitope.

These peptides, also called MAP (multiple antigenic peptides) are synthesized as four or eight-branched peptides on a polyfunctional (e.g. polylysine) scaffold. In addition, each branch may contain multiple copies of the same epitope. These peptides represent the core of the amplification system. They are exclusively designed in combination with a complementary peptide to avoid any non-specific reactions and to ensure high affinity binding. Various lengths of branched peptide derivatives can be synthesized; preferably, from 2 to 5 copies of a basic unit (e.g., 10 amino acids/unit) are joined together through small amino acid linkers composed of small neutral amino acids such as glycine or alanine. This approach allows synthesis of branched peptides containing between 16 and 40 copies of basic unit/molecule. Each subunit of the branched peptides can be recognized by the complementary peptide, coupled to a fluorescent molecule, thereby leading to a strong increase in the level of the emitted signal. Figure 1 illustrates one possible application of the amplification system using BPA (branched peptide amplification) technology to enhance the sensitivity of antigen recognition by specific antibodies via fluorescent complementary peptides.

[0052] Nature of the branched peptide

[0053] Various branched molecules have been used to amplify the amount of labeled molecules in order to increase the sensitivity and reliability of immunoassays. These molecules include branched DNA (Urdea et al. 1993, Aids 7(Suppl 2):S11-4; Collins et al. 1997, Nucleic Acids Res 25(15):2979-84) and other water-soluble polymers such as polysaccharides, homopolymer (amino acid), natural and synthetic polypeptides and proteins, and synthetic polymers (Stanley & Lihme 1995, Am Clin Lab t4(6):22; Giovannoni et al. 2000, J Pept Res 55(3): 195-202; Lihme & Stanley 2003, US Patent 6,627,460). In the case of branched DNA, the multimeric molecule serves as an intermediate target, to bind multiple copies of a labeled specific probe. For other water-soluble polymers, the multimeric molecules are directly linked to signaling molecules, their branched nature allowing attachment of multiple copies of the signal. Currently, there is no example where branched peptides have been used as a target for the binding of multiple copies of complementary labeled peptides.

[0054] The peptides of the present invention are branched to provide multiple copies of specific sequences that interact with labeled-complementary peptides in order to enhance signal detection in protein interaction assays. Branched peptides of the present invention generally comprise 1) a central core composed of a polyfunctional molecule comprising diamino carboxylic acid residues (e.g. lysine and derivatives thereof); 2) a spacer arm or linker attached to each radiating branch of the central core; and 3) multiple copies of a first P₁ peptide (of a peptide pair) which is designed to interact with a second labeled interacting P₂ peptide. Each copy of the first peptide is linked to the spacer arm and radiates outwardly from the central polyfunctional core. The central polyfunctional core, together with the spacer arms are used for presenting a plurality of peptides within the same molecule, located on the branches. The branched peptides of the present invention are then used together with a second labeled P₂ peptide, which interacts with the P₁ peptide located on the branches of the MAP core, in order to detect a particular protein interaction.

[0055] The preferred branch-forming polyfunctional molecule is L-lysine but the use of D-lysine, polyfunctional derivatives of L-lysine, polyfunctional derivatives of D-lysine and combinations thereof may also be used in accordance with the present invention (e.g. ornithine, 1 ,2-diaminopropionic acid and 1 ,3-diamino-butyric acid, etc .. ) Alternatively, a linear peptide (20 to 50 amino acids) could serve as a backbone to which lateral peptides (one of the complementary peptides) would be grafted through activated lysines. The backbone could also be composed of polyethylene glycol (PEG) of various sizes to which multiple copies of peptides would be attached via the alcohol moieties of the PEG. In addition, non bulky, neutral amino acids (e.g. alanine, glycine, valine ) are preferably used as spacer arms or linkers. Various lengths of spacer arms may be used in accordance with the present invention. For example, the length of the spacer arm (or linker) may be between 0 and 20 residues, preferably between 3 and 20 residues, more preferably between 4 and 10 residues, most preferably between 4 and 7 residues with the particular preferred length being equivalent to 4 residues. Long spacer arms (e.g., 35 residues) are believed to provide little additional advantages since there may be too many possibilities of interference between highly flexible residues such as glycine. The optimal length of the spacer arm for use in accordance with the present invention depends on various factors such as the nature and length of Pi and P₂ peptides, the number of branches on the polyfunctional core, the nature of the labeling molecule that is attached to the P₂ peptide as well as the particular type of assays in which it is being used. Although the addition of a spacer arm is advantageous, it may not be necessary depending on the particular peptide that is attached to the polyfunctional core.

[0056] The term "spacer arm" means the residue or residues lying intermediate to the extremity of the P₁ peptide nearest to the polyfunctional core and the outermost branching point of the core. Spacer arm may also refer to the residues lying between the complementary P₂ peptide sequence and the label.

[0057] Branched peptides of the present invention contain branches which can radiate outwardly from any central core of a molecule. The central core and the spacer arm are not excessively large as to interfere with the assay. The branched peptides can be any branch molecule in which a P₁ peptide capable of interacting with a P₂ peptide occurs more than once in the molecule and appears in more than one branch. Although the term peptide implies that the molecule is made up predominantly of peptide chains, it is not necessary that the entire molecule be composed of amino acids. Non-peptide polyfunctional core and non-peptide linkers can also be used in accordance with the present invention.

[0058] The present inventors have shown that the number of branches present in the MAP core does not limit the application of the MAP as an intermediate in immunoassays or other related protein interaction assays (as demonstrated by the testing of two different configurations of multimeric peptides: 4-branch and 8-branch MAP) thereby underlining its important versatility. Thus, branched peptides of the present invention can comprise any suitable number of branches. The optimal number of branches will depend on the particular nature of the peptide pair (P₁ and P₂) used, the nature of the label as well as the kind of specific assay for which it is designed. Thus, the skilled person will appreciate that the number of N-terminal peptides (or branches) on the MAP core can be 4, 8, 16, 32, 64 or even more, with the upper limit depending on the steric hindrance and the accessibility of the branch for peptide coupling. [0059] In addition, P₁ and P₂ peptides can be of any suitable length but are preferably between 15 and 40 amino acids. Longer or shorter peptides could also be used depending once again on the steric hindrance and the accessibility of the branch peptides for coupling.

[0060] Branched peptides of the present invention as well as their complementary labeled interacting partners are synthesized using existing procedures of classical solid phase peptide synthesis.

[0061] Generally, the branched peptide molecules of the present invention will contain only one kind of a first interacting peptide (Pi) for binding to the labeled complementary peptide (P₂). However, the use of two or more kinds of P₁ peptides (and their complementary interacting partners) could also be contemplated. In addition, although technically the branched peptides of the present invention will neither have spare arms nor will they have any free branches without interacting P₁ peptides attached thereto, they are not as limited as they appear. There would normally be diminishing advantages in the construction of free branches containing branched peptides but they could nevertheless be used.

[0062] The branched peptides of the present invention are also designed to include an additional moiety (M_rsee Figure 1 ) which is chosen according to the specific requirement of the assay used. This additional moiety is linked directly or indirectly to the polyfunctional core of the branched peptides (MAP) and interacts with a target antibody, antigen or protein. Non-limiting examples of moiety M₁ that may be used in accordance with the present invention include biotin, antibodies (first or secondary), various protein binding domains (e.g., leucine zipper), substrates, oligonucleotides and other nucleic acid derivatives, etc...

[0063] Nature of the complementary peptides

[0064] A large variety of peptidic structures capable of inducing protein interactions have been described (Liddington 2004, Methods MoI Biol 261:3-14). Among these, α-helical coiled-coils can bind together at moderate to low peptide concentrations (Litowski & Hodges 2002, J Biol Chem 277(40):37272-9). Once they are formed, they dissociate only slowly. They thus appeared well suited for the BPA technology. Overall, the results obtained by the present inventors confirmed that peptides from pair BAP-01 and, mostly, peptide pair BAP- 02 could bind together in a stable manner (see Fig. 7). To show that BPA was not restricted to the use of coiled-coil interactions, an additional peptide pair (BAP-03), formed with one linear peptide and one cyclic peptide, derived from the sequence of the acetylcholine receptor and from a-bungarotoxin respectively, was prepared. The results clearly showed that this pair represents a viable alternative and could replace coiled-coil structures, extending the number of possible peptide combinations central to the BPA technology (see Fig. 1 1 ).

[0065] As used herein, the term "complementary peptide", "complementary labeled peptide", "interacting partner" or "interacting peptide" refers generally to the P₂ peptide which interacts specifically with the P₁ peptide of a P_rP₂ peptide pair. The P₁ peptide is attached to the polyfunctional MAP core and the P₂ peptide is generally labeled and provides for the detection signal.

[0066] Although the present invention has been illustrated by the use of specific

BAP-01 ; BAP-02 and BAP-03 peptide pairs, a person skilled in the art would appreciate that any type of peptide pairs can be used in accordance with the present invention provided that the interaction is (1) specific (does not generally binds to other components present in the assay) and (2) that once the two peptides interact, they dissociate only slowly. Thus, in addition to coiled-coiled structures, other small protein domains known to interact specifically and in a stable manner could also be used (e.g. leucine zipper).

[0067] Nature of the marker molecule

[0068] Recently, a new generation of fluorophores has been developed. These fluorophores, including lndocarbocyanine (Cy3) and lndodicarbocyanine (Cy5), largely used in various types of microarrays, and Alexa Fluors®, spanning the near-UV, visible and near- infrared spectrum, show greater photostability (Lichtman & Conchello 2005, Nat Methods 2(12):910-9) and appear to be well suited for immunoassays. For example, Alexa Fluors® are highly soluble in water, are insensitive to pH variations, show stronger fluorescent intensity and higher photostability as compared to other fluorophors and are compatible with most detection devices. [0069] All of the above fluorophores have been designed to be easily conjugated to various molecules through conventional chemistry. Therefore, labeling of the complementary peptides with these molecules and purification of the conjugates does not require the development of a new technology and can be achieved without difficulty as shown in the various experiments.

[0070] The conjugation to peptides and proteins of various labeling molecules, different from the fluorophores, involves reactions similar to the ones presented here. Some of these molecules are extensively used in immunoassays and include enzymes (e.g., horseradish peroxydase, alkaline phosphatase) chemiluminescent molecules and Quantum dots (Alivisatos et al. 2005, Annu Rev Biomed Eng 7:55-76). All of these molecules could be coupled to the complementary peptide and be part of the BPA technology. Thus, although Alexa Fluor® fluorophores have been used and exemplified herein, a person skilled in the art could easily customize the present method for use with other types of labels in order to fulfill specific detection needs based on the particular assay in which the present invention is used.

[0071] Non-limiting examples of other types of labeling molecules that can be used include: quantum dots, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Aminonapthalene, Benzoxadiazole, BODIPY® 493/504, BODIPY® 505/515, BODIPY® 576/589, BODIPY® FL, BODIPY® TMR, BODIPY® TR, Carboxytetramethylrhodamine, Cascade Blue®, a Coumarin, Cyanine (Cy2), Cy3, Cy5, Cy9, Dansyl Chloride, DAPI, Eosin, Erythrosin, Ethidium Homodimer II, Ethidium Bromide, Fluorescamine, Fluorescein, FTC, GFP (yellow shifted mutants T203Y, T203F, S65G/S72A), Hoechst 33242, Hoechst 33258, IAEDANS, an lndopyras Dye, a Lanthanide Chelate, a Lanthanide Cryptate, Lissamine Rhodamine, Lucifer Yellow, Maleimide, MANT, MQAE, NBD, Oregon Green® 488, Oregon Green® 514, Oregon Green® 500, Phycoerythrin, a Porphyrin, Propidium Iodide, Pyrene, Pyrene Butyrate, Pyrene Maleimide, Pyήdyloxazole, Rhodamine 123, Rhodamine 6G, Rhodamine Green, SPQ, Texas Red, TMRM, TOTO-1 , TRITC, YOYO-1 , vitamin B12, flavin-adenine dinucleotide, and nicotinamide-adenine dinucleotide.

[0072] Applications [0073] The BPA technology described herein may be used in different types of assays and thus has various applications. Different immunoassays, on a variety of solid supports, including ELISA, Western blot, microarrays or point-of-care immunotesting are examples of such applications. For certainty, while the recitation "such as an immunoassay" is used at multiple places in the application, it should be understood that in all the contexts where it used, it should not be so limited. A person skilled in the art would appreciate that additional assays may be used in accordance with the BPA technology of the present invention. Non-limiting examples include, cytofluorometry, receptor-ligand interaction analysis and protein interaction identification.

[0074] In addition, the present invention can be practiced in any way appropriate for its intended purposes. For example, the present invention can be used with samples containing appropriate extracts (e.g., body fluids, tissue samples, cells, etc) likely to contain the molecule or molecules which are to be detected (e.g. antibodies, antigen proteins, etc . ). Non-limiting examples of biological samples include blood, serum, urine, saliva, tears, milk, secretions, cell extracts, tissue extracts, stools, etc... Samples may be purified, unpurified or partially purified and/or concentrated or not.

[0075] The present invention is illustrated in further details by the following non- limiting examples.

EXAMPLES

EXAMPLE 1 : Materials and Methods

[0076] Design and synthesis of high affinity peptides. The MAP is composed of a polylysine scaffold (MAP core) that anchors linear peptides. To facilitate the chemical characterization of the synthetic molecules and to increase their versatility, it has been decided to synthesize both the MAP cores and the linear peptides separately.

[0077] Design and synthesis of Multiple Antigenic Peptide MAP cores. MAP cores were designed in such a way that they contained a biotinylated amino acid and a 15 A linker to prevent steric hindrance following attachment of the linear peptides. Figure 2 presents the sequence of the 4-branch MAP core. The amino acid lysine (K) is biotinylated. The lateral chains end by an amino group involved in the binding of the linear peptides. The 8- branch MAP core is similar to the 4-branch except for the number of branches. The MAP cores were synthesized by solid-phase peptide synthesis methodology and conventional chemistry on a peptide synthesizer and further characterized by amino acid sequencing and reverse phase HPLC.

[0078] Design and synthesis of complementary peptides. Peptides were selected according to specific criteria such as 1 ) secondary structure of the peptide, 2) low dissociation rate constant (10^~8 to 10^~10 M); and 3) length of the polypeptide chain. Three peptide pairs were derived from the scientific literature (Ruan ef a/. 1990, Proc Natl Acad Sci U S A 87(16):6156-60; Chao et al. 1996, Biochemistry 35(37):12175-85; Litowski & Hodges 2002, J Biol Chem 277(40):37272-9). One of the specific objectives was to demonstrate that BPA was not restricted to the use of a single peptide pair but could be extended to peptides with different structures and various lengths.

[0079] Three different peptide pairs were thus designed and synthesized. The first two pairs were designed based on the complementarities of coiled-coil structures. For peptide pair BPA-01 (SED ID NOs: 2 and 3), K and E peptides were slightly modified as compared to their original design (Chao ef a/. 1996, Biochemistry 35(37):12175-85). First of all, their length was limited to 3 subunits. Second, a short linker was added at the N- terminus or C-terminus of the peptides to decrease the steric hindrance and help in the addition of fluorophores. Finally, the C terminus of the E peptide was changed to an amino group to prevent detrimental reactions with the thiol group of the cysteine during the synthesis process. Figure 4 presents the various modifications for both K and E peptides, represented by K3_(VSAL) (SEQ ID NO:2) and E3_(VSA_L) (SEQ ID NO:3), respectively. The BAP- 02 peptide pair (SEQ ID NOs: 4 and 5) was designed similarly to K and E peptides of BAP- 01 except that the VSAL repeat was replaced by an IAAL repeat. Their sequences are shown in Figure 4, represented by K3_(IAAL) (SEQ ID NO:4) and E3₍|_AAL) (SEQ ID NO:5), respectively. Finally, Peptide F (SEQ ID NO:6), a 16 amino acid linear peptide derived from the acetylcholine receptor and peptide A (SEQ ID NO:7), a 15 amino acid cyclic peptide derived from α-bungarotoxin, two peptides presenting affinity for each other (Ruan ef a/. 1990, Proc Natl Acad Sci U S A 87(16):6156-60), were designed as components of peptide pair BAP-03. All peptides were synthesized by solid-phase peptide synthesis methodology using an N-alpha-(9-fluorenylmethyloxycarbonyl) (Fmoc) resin and conventional chemistry on a peptide synthesizer. [0080] Peptide characterization and purification. The amount of free sulfhydryl groups in the linear peptides, essential for the conjugation with the MAP peptides, was determined using Ellman's Reagent (Pierce). Ellman's Reagent, DTNB, reacts with a free sulfhydryl group to yield a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB). The target of DTNB in this reaction is the conjugate base (R — S-) of a free sulfhydryl group. Sulfhydryl groups may be estimated in a sample by comparison to a standard curve composed of known concentrations of a sulfhydryl-containing compound such as cysteine. O- phthalaldehyde (OPA) (Pierce) was used to determine the peptide concentration in solutions through a microassay protocol in which peptide concentrations and sample volumes were decreased. Peptides were further characterized by mass spectrometry. Finally, the various peptides were purified by reverse-phase chromatography. An association of peptides from BAP-01 and BAP-02 pairs was analyzed by circular dichroism spectroscopy while an association of peptides from BAP-03 was evaluated directly in a microplate using standard protocols where FITC-labeled peptide A was used to detect various amounts of peptide F fixed to the bottom of the plate (Fig. 11 ).

[0081] Construction of MAP peptides: the core of the amplification system. Following synthesis, purification, and characterization of various peptides, anchoring of linear peptides to the MAP cores was performed by activating the amino group at the end of each branch of the MAP core through the action of Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3^"-[2- pyridyldithioj-propionamido) hexanoate, Pierce) or Sulfo-KMUS (N-1 1 - maleimidoundecanoyloxy) sulfosuccinimide, Pierce), also reactive for the thiols present in the linear peptides, according to the manufacturer's instructions. When Sulfo-LC-SPDP was used, the processing could be monitored by spectroscopy through the production of pyhdine-2-thione, a side product of the reaction.

[0082] Fluorescence labeling of peptides. Complementary peptides were labeled with Alexa Fluor 647 maleimide according to the manufacturer's instructions (Invitrogen). Free Alexa Fluor was removed by reverse phase HPLC.

EXAMPLE 2: Synthesis and characterization of the MAP core

[0083] As stated above, to facilitate the characterization of the synthetic molecules and to increase their versatility, the MAP cores and the linear peptides were synthesized separately. Two MAP cores were prepared. Both the 4-branch and the 8-branch cores are supported through a polylysine scaffold. A schematic representation of the 4-branch MAP core is presented in Figure 2. The synthesis and the purification processes were slightly modified for the 8-branch MAP core to increase the homogeneity of the product. The analysis of both the 4-branch (Fig. 3A) and 8-branch (Fig. 3B) cores shows that the modification in the synthesis and purification strategies generated a far more standardized product, as depicted by the single major peak observed in the chromatogram (Fig. 3B).

EXAMPLE 3: Synthesis, purification and characterization of complementary peptide pairs

[0084] Three different complementary peptide pairs were synthesized (Fig. 4).

Preliminary data was obtained with BAP-01. However, due to low binding of the peptides under physiological conditions, most of the experiments were performed using BAP-02 (K3_(IAAL) and E3_(IAAL) peptides) and BAP-03 (A and F peptides) peptide pairs. These peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) (Fig. 5) and their identity was confirmed by mass spectrometry (Fig. 6). For each peptide, a single peak was obtained and the mass spectrometry analysis revealed that the molecular weight was as expected.

EXAMPLE 4: Analyzing complementary peptides by circular dichroism spectroscopy

[0085] Association of K3₍IA_AL) and E3_(IAAL) peptides (BAP-02) is mediated through their helical structure. By using circular dichroism spectroscopy, it is possible to analyze the secondary structure of the peptides. Figure 7 shows that K3 (_IAAL) and E3 (I_AAL) peptides exhibit typical α-helix spectra. Moreover, the data clearly demonstrates the formation of a heterodimeric α-helical coiled-coil complex (K3₍IA_AL/E3_(IAAL))- This is in contrast with what was obtained for the BAP-01 peptide pair for which interaction was occurring only in the presence of 50% TFE (Fig. 8). Due to the non-helical structure of the BAP-03 peptides, these were not characterized by circular dichroism spectroscopy.

EXAMPLE 5: Labeling peptide with Alexa Fluor 647 maleimide

[0086] Alexa Fluor maleimide was used to label peptide E3₍ι_AAL). Following the coupling reaction, the peptide was further purified by RP-HPLC to remove free Alexa Fluor.

As shown in Figure 9, more than 99 % of free Alexa Fluor was removed from the reaction mix. In addition, the labeled peptides could be easily recovered.

EXAMPLE 6: Binding of labeled peptide E3₍I_AAL) to peptide K3_(!AAL) (peptide pair BAP-02)

[0087] In order to demonstrate that 1 ) labeling of peptide E3<_IAA_L) with Alexa fluor was not preventing association of both complementary peptides and 2) this interaction was possible following immobilization on a solid support, various amounts of peptide K3_(IAAL) were fixed to a maleimide-activated microplate and exposed to increasing concentrations of labeled E3_(IAAU ^After washing, the fluorescence intensity was determined using a microplate fluorescence reader (Fig. 10). The data clearly demonstrates a typical dose response curve, indicative of a specific interaction, both at low (lower panel) and high (upper panel) concentrations of labeled E3_(IAA_L).

EXAMPLE 7: Binding of labeled peptide A to peptide F (peptide pair BAP-03)

[0088] In order to test binding between both of the BAP-3 peptides, peptide A was bound to a microplate in increasing concentrations and exposed to labeled peptide F. Data presented in Figure 1 1 show that these peptides can bind to each other, even without optimizing any of the conditions.

EXAMPLE 8: Construction of the MAP and recognition by the fluorescent complementary peptides

[0089] Two types of experiments were designed to analyze the binding capacity of the fluorescent E3₍IAA_L) to the K3₍|_AA_L) MAP peptide. First, the synthesis of the MAP peptides (i.e. K3_(IAAL) coupled to the MAP core) was carried out directly on a neutravidin-coated microplate. To do so, various biotinylated MAP core levels were bound to the neutravidin- coated microplate, activated and exposed to K3_(IAAL)- After the reaction, the plate was extensively washed and exposed to fluorescent E3_(IAAD- Binding of both peptides is clearly shown in Figure 12. The observed reaction is specific since only a low level of binding occurs in the absence of K3_(IAAL₎ (MAP core without K3₍I_AAL))- Second, the synthesis of the MAP peptides was performed in solution. Briefly, the MAP core was activated, purified and concentrated on Centricon® micro-column to get rid of the excess of SULFO-LC-SPDP (activator molecule). K3₍IAA_L) peptide was then added and the complex was purified by RP-

HPLC. The resulting MAP-K3_(IAAL) was fixed on a neutravidin-coated plate in different amounts and exposed to micromolar amounts of fluorescent E3_(IA_AL) peptide. A 2-log difference in the fluorescence intensity was observed between low and high amounts of MAP-K3(IAAL) showing that the system presents an amplification potential above 100-fold (Fig. 13).

EXAMPLE 9: Detection through MAP-K3(|_AAL)/fluorescent-E3(|_AAL) binding to transmit a signal in an immunoassay

[0090] Additional sets of experiments were performed to demonstrate that it is possible to use MAP-K3₍ι_AAL)/fluorescent -E3(_IAAL₎ binding to transmit a signal in a protein interaction experiment such as an immunoassay. In the first set, sheep IgG was selected as a model antigen, coated to a microplate and exposed to a specific biotinylated antibody. Various levels of avidin were then added, followed by biotinylated MAP-K3_(IAAD and by the fluorescent-E3(iAA_L) peptides (Figs. 1 and 14). The data undoubtedly demonstrates that the fluorescence emitted is directly proportional to the amount of avidin used. This data clearly demonstrates that the amplified signal is efficient in an immunoassay. A similar experiment was carried out except that various amounts of MAP-K3_(IAA_L) were added followed by the fluorescent peptide E3(I_AAL) (Fig. 15). The data demonstrates that the fluorescence levels are not only directly proportional to the levels of avidin and MAP-K3_(IAA_L) (Fig. 15A) but also to the levels of antigen coated to the plate (Fig. 15B).

EXAMPLE 10: Labeling and characterization of the 8-branch MAP peptide

[0091] In order to demonstrate the possibility of using more complex MAP, the capacity of the biotinylated 8-branch MAP core to bind avidin was first evaluated using

HABA-avidin. The HABA dye binds to avidin to produce a yellow complex which absorbs at

500 nm. Following binding to avidin, biotinylated compound will displace the HABA dye and cause the absorbance to decrease. When increasing amounts of MAP-8 (biotin) was added to HABA-avidin, the absorbance declined in a linear fashion (Fig. 16; the y-axis represents the difference in the absorbance).

[0092] To explore the possibility of using an 8-branch MAP peptide in BPA, the MAP core was labeled directly with Alexa Fluor® and purified by reverse-phase HPLC (Fig. 17). The capacity of the purified labeled MAP-8 to bind avidin was conserved since a significant decrease in the absorbance at 500 nm was observed when it was added to HABA-avidin (Fig. 18).

EXAMPLE 11 : Detection through the 8-branch MAP

[0093] Experiments were performed to demonstrate that it is possible to use an 8- branch MAP to transmit a signal when detecting a protein interaction such as in an immunoassay. Various levels of sheep IgG antibody were first coated to a microplate and exposed to a specific biotinylated antibody. Avidin was then added, followed by the fluorescent 8-branch biotinylated MAP. The data clearly demonstrates that the amount of fluorescence emitted is directly proportional to the amount of antigen (i.e. sheep IgG antibody) in the plate (Fig. 19). As can be seen in Figure 19B, the minimal level of detection is around 0,25 ng. This level is quite similar to what can be obtained in systems using a labeled antibody or labeled avidin as reporter. A stronger signal will be obtained when multiple copies of complementary peptides bind to the MAP, as described in the original design. These last experiments have been initiated to demonstrate the potential of using multiple-branch MAP. By doing so, the overall performance of such a system can be pushed further through the addition of spacers and therefore create new branches to maximize the power of the technology, as appreciated by a person of ordinary skill in the art.

[0094] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A composition for use in the detection of a protein interaction assay comprising: a) a branched peptide comprising: i) a central core composed of a polyfunctional molecule comprising diamino carboxylic acid residues which provide the branched peptide with a plurality of amino terminal portions; ii) spacer arms attached to the amino terminal portions of said polyfunctional molecule; and iii) a plurality of P1 peptides, wherein a P1 peptide is attached to a spacer arm and projects out of said central core, thereby presenting a plurality of peptides within the same molecule; and b) a plurality of P2 labeled peptides, each P2 peptide being capable of specifically interacting with said P1 peptide of said branched peptide.

2. The composition of claim 1 , wherein said branched peptide further comprises an Mi moiety which specifically interacts with a target protein.

3. The composition of claim 2, wherein said Mi moiety is biotin.

4. The composition of claim 1 , wherein said branched peptide is covalently linked to an antibody.

5. The composition of claim 1 , wherein said polyfunctional molecule is lysine.

6. The composition of claim 5, wherein said central core is (LyS)₄(LyS^(LyS) providing eight branches or (Lys)₂(Lys) providing 4 branches.

7. The composition of claim 6, wherein said spacer arm is composed of glycine residues, alanine residues or a combination thereof.

8. The composition of claim 7, wherein said spacer arm has 4 residues.

9. The composition of claim 8, wherein said spacer arm has the sequence alanine- glycine-alanine-glycine.

10. The composition of claim 9, wherein said P₁ peptide and said P₂ peptide are part of a peptide pair selected from the group consisting of: BAP-01 , BAP-02 and BAP-03.

1 1. The composition of any one of claims 1-10, wherein said P₂ labeled-peptide comprises a label selected from the group consisting of: a fluorescent moiety, an enzyme, a chemiluminescent molecule or quantum dots.

12. The composition of claim 11 , wherein said fluorescent moiety is selected from the group consisting of: Cy5, Cy3, Alexa Fluor® 555 and Alexa Fluor® 647.

13. The composition of claim 10, wherein said enzyme is selected from the group consisting of: horseradish peroxidase, alkaline phosphatase, chloramphenicol acety transferase and luciferase.

14. The composition of any one of claims 1-13, wherein said spacer arm consists of 3 to 20 residues.

15. The composition of any one of claims 1-13, wherein said spacer arm consists of 3 to 10 residues.

16. The composition of any one of claims 1-13, wherein said spacer arm consists of 4 to 7 residues.

17. The composition of claim 1 , wherein said spacer arm is absent.

18. The composition of claim 1 , wherein said spacer arm is composed of non- hydrophobic residues.

19. The composition of claim 1 , wherein said polyfunctional molecule is selected from the group consisting of: lysine, ornithine, 1 ,2-diaminopropionic acid, 1 ,3- diamino-butyric acid and polyethylene glycol molecules.

20. A kit for the detection of antigen in a sample comprising the composition of any one of claims 2 to 19 further comprising an antibody specific for the detection of a particular antigen, said antibody being adapted to interact specifically with said branched peptide.

21. The kit of claim 20, wherein said antibody and said branched peptide are biotinylated.

22. The kit of claim 21 , further comprising avidin for allowing said biotinylated antibody and said branched peptide to interact.

23. A method for amplifying and detecting the presence of an antibody in a sample comprising: a) contacting said sample with a branched peptide comprising: i) a central core composed of a polyfunctional molecule comprising diamino carboxylic acid residues which provide the branched peptide with a plurality of amino terminal portions; ii) a spacer arm attached to each amino terminal portion of said polyfunctional molecule; and iii) a plurality of P1 peptides, wherein a P1 peptide is attached to each of said spacer arms and projects out of said central core, thereby presenting a plurality of peptides within the same molecule; iv) an Mi moiety which specifically interacts with said antibody b) detecting the presence of said antibody in said sample by contacting said sample with P₂ labeled peptides which specifically interact with said P₁ peptide of said branched peptide,

wherein an antibody is present when a signal characteristic of the P₂ labeled peptide is detected.

24. A method for the detection of antibodies in a sample in which a binding reaction is performed between the antibodies and a branched peptide, said branched peptide comprising:

i) a central core composed of a polyfunctional molecule comprising diamino carboxylic acid residues which provide the branched peptide with a plurality of amino terminal portions; ii) spacer arms attached to said amino terminal portions of said polyfunctional molecule; and iii) a plurality of P1 peptides, wherein a P1 peptide is attached to a spacer arm and projects out of said central core, thereby presenting a plurality of peptides within the same molecule; wherein the detection of said antibody is performed by assessing the extent of binding between said antibody and said branched peptide by using a plurality of P₂ labeled peptides, each P₂ peptide being capable of specifically interacting with said P₁ peptide of said branched peptide and wherein said antibody and said branched peptide are capable of specifically interacting.

25. A composition for use in the detection of a protein interaction assay comprising: a) a branched peptide comprising: i) a central core composed of a polyfunctional molecule comprising diamino carboxylic acid residues which provide the branched peptide with a plurality of amino terminal portions; ii) a spacer arm attached to each amino terminal portion of said polyfunctional molecule; and iii) a plurality of P1 peptides, wherein a P1 peptide is attached to each of said spacer arms and projects out of said central core, thereby presenting a plurality of peptides within the same molecule; and b) a plurality of P2 labeled peptides, each P2 peptide being capable of specifically interacting with said P1 peptide of said branched peptide.

26. A method for amplifying and detecting the presence of an antibody in a sample comprising: a) contacting said sample with a branched peptide comprising: i) a central core composed of a polyfunctional molecule comprising diamine carboxylic acid residues which provide the branched peptide with a plurality of amino terminal portions; ii) a spacer arm attached to each amino terminal portion of said polyfunctional molecule; and iii) a plurality of P1 peptides, wherein a P1 peptide is attached to each of said spacer arms and projects out of said central core, thereby presenting a plurality of peptides within the same molecule; and iv) a M1 moiety which specifically interacts with said antibody; and b) detecting the presence of said antibody in said sample by contacting said sample with P2 labeled peptides which specifically interact with said P1 peptide of said branched peptide, wherein an antibody is present when a signal characteristic of the P₂ labeled peptide is detected.

27. A method, composition or kit for amplifying the detection of a protein interaction assay, wherein said method composition or kit is in accordance with the present invention.

28. The method, composition or kit of claim 27, wherein said protein interaction assay is an immunoassay.