SIMULTANEOUS REACTION ASSAY FOR DIFFERENTIALLY DETECTING

MULTIMERIC FORM

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to methods for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide and immunoassay kits therefor.

DESCRIPTION OF THE RELATED ART

A multimerization of polypeptides constituting proteins has been generally known to be required for the function of proteins. However, the multimeric forms often cause diseases or disorders in some proteins. In particular, a protein exists as a monomer in normal conditions and is converted to a multimer (or aggregate form) in abnormal conditions (e.g., by the conversion to a misfolding form) .

It has been well established that proteins that are misfolded and ultimately aggregated (or accumulated), i.e., that are not in their functionally relevant conformation are devoid of normal biological activity. The failure to fold correctly, or to remain correctly folded, gives rise to many different types of biological malfunctions and hence, to many different forms of diseases (Massimo Stefani, et al . , J. MoI. Med. 81:678-699(2003); and Radford SE, et al . , Cell. 97:291- 298(1999)). Many diseases ultimately result from the presence in a living system of protein molecules with structures that are incorrect, i.e., that differ from those in normally functioning organisms.

For instance, the diseases or disorders associated with abnormal aggregation or misfolding of proteins include

Alzheimer's disease, Creutzfeldt-Jakob disease, Spongiform encephalopathies, Parkinson's disease, Huntington' s disease, Amyotrophic lateral sclerosis, Serpin deficiency, emphysema, cirrhosis, Type II Diabetes, primary systemic amyloidosis, secondary systemic amyloidosis Fronto-temporal dementias, senile systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid angiopathy and haemodialysis- related amyloidosis.

Early diagnosis of the aggregation-associated diseases has been intensively studied. However, there has not been suggested any process and approach to differentially detect multimeric (aggregating) forms from their monomeric (normal) forms.

Sporadic, variant, iatrogenic, and familial Creutzfeldt- Jakob diseases, kuru, Familial Fatal insomnia, and Gerstmann- Straussler-Scheinker syndrome in humans, scrapie in sheep and goats, feline spongiform encephalopathy in cat, mink spongiform encephalopathy, Chronic Wasting disease in deer, elk, and moose, and bovine spongiform encephalopathy in cattle are the fatal neurodegenerative diseases, resulting in transmissible spongiform encephalopathies (TSE) (Prusiner S. B. Proc. Natl. Acad. Sci. USA 95:13363-13383(1998); and Hope J. Curr. Opin. Genet. Dev. 10, 568-57(2000)). Abnormal isoform or the scrapie form of prion protein (PrP ^Sc ) has been strongly suggested to the main culprit of TSE (Caughey B. Trends Bϊochem. Sci. 26:235-42(2001)).

The normal form of the prion protein (PrP ^c ) , contains both an α-helical and a flexibly disordered portion and exists as a monomeric form (Zahn, R., et al . , Proc. Natl. Acad. Sci. USA 97:145-150(2000)), where the scrapie form (PrP ^Sc ) has highly β- sheet conformations and exists as a multimeric (aggregating) or at least dimer forms (Caughey, B., et al., J. Biol. Chem. 273: 32230-35 (1998) ). The conformational _. change from α-helical to

β-sheet conformations is the central event of the disease that seems to be responsible for its neuropathology.

While PrP ^c is protease sensitive (PrP ^sen ) , PrP ^Sc is partially resistant to proteolysis (PrP ^res ) and prone to form high- molecular-weight aggregates (Bolton D. C. Lancet, 358:164-5 (2001)). This latter feature makes it difficult to analyze the conformational transition that leads to the formation of prP ^res or to characterize it.

The method of proteinase K (PK) digestion has been used to discriminate the resistance of its various forms of PrP

(scrapie form) by digesting the cellular form, leaving only the scrapie form to be detected in ELISA. However, the PK digestion method is being questioned. PrP conformation, concentration, tissue antibodies, digestion time and buffers could influence the PK sensitivity, which significantly reduces the reliability of the PK digestion method.

Therefore, there remains a need to develop a novel approach for differentially detecting multimeric form (e.g., scrapie form of PrP) from their monomeric forms (e.g., cellular form of PrP) with much higher reliability and convenience.

Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION Under such circumstances, the present inventors have made intensive research to develop a novel method for differentially detecting multimeric (aggregating) forms from monomeric forms

of multimer-forming polypeptides, and as a result, developed a novel immunoassay approach.

Accordingly, it is an object of this invention to provide a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample.

It is another object of this invention to provide a kit for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample. It is still another object of this invention to provide a use of (a) an entrapping antibody-affinity molecule conjugate; and (b) an indicative antibody for the manufacture of a kit for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample.

Other objects and advantages of the present invention will become apparent from the detailed description to follow and together with the appended claims and drawings .

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 schematically represents the process of a specific embodiment of this invention.

Fig. 2 represents the results of the present method depending on the amount of protease K and treatment time with protease K. The experiments used the varying concentrations of protease inhibitors. RLU denotes relative luminescence unit.

Fig. 3 represents the results of the present method depending on the amount of protease K. The experiments used the constant concentration of protease inhibitors and the constant treatment time (1 hr) with protease K.

Fig. 4 represents the results of the present method depending on the amount of protease K and treatment time with

protease K. The experiments used the constant concentration of protease inhibitors.

Fig. 5 represents that the present method using protease inhibitors successfully generates differential signals between scrapie and normal PrP plasma samples.

Fig. 6 represents that the present method using protease inhibitors successfully generates differential signals between scrapie and normal PrP plasma samples.

Figs. 7a and 7b represent the effect of protease inhibitors on the binding of normal PrP with antibodies.

Fig. 8 demonstrates that protease inhibitors increase the signal intensity in a process for detecting the scrapie form of PrP using entrapping antibodies bound to a solid support, a magnetic bead.

DETAILED DESCRIPTION OF THIS INVETNION

In one aspect of this invention, there is provided a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample, which comprises the steps of: (a) obtaining the biological sample to be analyzed; (b) preparing an entrapping antibody-affinity molecule conjugate by binding an affinity molecule to the surface of the entrapping antibody, wherein the entrapping antibody recognizes an epitope on the multimer-forming polypeptide and is not bound to a solid support; (c) preparing an indicative antibody, wherein an epitope recognized by the indicative antibody is present at a position in the multimer-forming polypeptide to cause a steric hindrance by the entrapping antibody bound to its epitope to prevent the binding of the indicative antibody to the multimer- forming polypeptide; (d) contacting the entrapping antibody- affinity molecule conjugate and the indicative antibody to the

biological sample in a three dimensional manner, thereby forming an entrapping antibody-affinity molecule-multimeric form-indicative antibody complex; and (e) isolating the entrapping antibody-affinity molecule-multimeric form- indicative antibody complex by use of a counterpart affinity molecule to the affinity molecule on the entrapping antibody; and (f) detecting the formation of the entrapping antibody- affinity molecule-multimeric form-indicative antibody complex. In another aspect of this invention, there is provided a kit for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample, which comprises: (a) an entrapping antibody- affinity molecule conjugate wherein the entrapping antibody recognizes an epitope on the multimer-forrning polypeptide and is not bound to a solid support; and (b) an indicative antibody recognizing an epitope present at a position in the multimer- forming polypeptide to cause a steric hindrance by the entrapping antibody bound to its epitope to prevent the binding of the indicative antibody to the multimer-forming polypeptide. In still another aspect of this invention, there is provided a novel use of (a) an entrapping antibody-affinity molecule conjugate; and (b) an indicative antibody for the manufacture of a kit for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample, wherein the entrapping antibody recognizes an epitope on the multimer-forming polypeptide and is not bound to a solid support; wherein the indicative antibody recognizes an epitope present at a position in the multimer-forming polypeptide to cause a steric hindrance by the entrapping antibody bound to its epitope to prevent the binding of the indicative antibody to the multimer-forming polypeptide.

The present invention is directed to a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample by immunoassay involving antigen-antibody reactions. Furthermore, the present invention uses two types of antibodies, an entrapping antibody and an indicative antibody both of which are competitive in binding to a multimer-forming polypeptide. Such competitive antibody binding occurs through steric inhibition. In particular, the entrapping antibody bound to an epitope on a multimer-forming polypeptide inhibits the indicative antibody from binding to its epitope on the multimer-forming polypeptide because of competition to binding sites on the multimer-forming polypeptide. One of the features of the present invention is to perform the immunoassay under three-dimensional contacting circumstances. In the present invention, the entrapping and indicative antibodies are ensured to have three-dimensional contacting opportunities to antigens in biological samples.

The advantages of this invention become the most prominent when the entrapping and indicative antibodies are simultaneously contacted to biological samples to be analyzed. Therefore, the process of this invention is named "SRA (simultaneous reaction assay) system". The term "simultaneous" used herein with referring to the reaction of antibodies with biological samples means that the entrapping and indicative antibodies are contacted to biological samples simultaneously. This term encompasses a situation in which the entrapping and indicative antibodies are contacted to biological samples in a short time interval so long as the two antibodies have a similar opportunity (chance) to bind to multimer-forming polypeptides in biological samples.

The term "multimer-forming polypeptide" used herein refers

to a polypeptide capable of forming an aggregation (i.e., multimer) form, particularly, following conformational change, causing a wide variety of diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease, Spongiform encephalopathies, Parkinson's disease, Huntington' s disease, Amyotrophic lateral sclerosis, Serpin deficiency, emphysema, cirrhosis, Type II diabetes, primary systemic amyloidosis, secondary systemic amyloidosis Fronto-temporal dementias, senile systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid angiopathy and haemodialysis-related amyloidosis. Therefore, the term "multimer-forming polypeptide" will be interchangeably used with the term "aggregate-forming polypeptide" .

The present method uses two types of antibodies, i.e., entrapping antibody and indicative antibody. As used herein, the term "entrapping antibody" means an antibody capable of binding to the multimer-forming polypeptide of interest in biological samples and conjugated with affinity molecules for isolating an antigen-antibody complex. For instance, when biotin is used as affinity molecules, antigens binding to the entrapping antibody-biotin conjugate may be entrapped by incubating with streptavidin-magnetic beads, after which entrapped antigens may be isolated by applying magnetic field.

The term "indicative antibody" means an antibody capable of binding to the multimer-forming polypeptide and capable of indicating the presence of the multimeric form of the multimer- forming polypeptide. In other words, the indicative antibody bound to the multimer-forming polypeptide binding to the entrapping antibody is indicative of the presence of the multimeric form, which may be readily detected by use of a signal-generating label conjugated with the indicative antibody.

By "antibody" is meant an immunoglobulin protein which is capable of binding an antigen. Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g., F(ab')2, Fab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest.

In the present invention, the epitopes specifically recognized by the entrapping antibody and indicative antibody are located at positions in multimer-forming polypeptides to cause steric hindrance (competitive binding) between antibodies to be bound to the epitopes. Preferably, the amino acid sequence of the epitope recognized by the entrapping antibody is identical to, overlapped with or adjacent to that of the epitope recognized by the indicative antibody. It would be readily understood that the entrapping antibody and indicative antibody to be bound to their epitopes induce steric hindrance or are competitive in binding where the amino acid sequence of the epitope recognized by the entrapping antibody is identical to or overlapped with that of the epitope recognized by the indicative antibody. The term "overlapped with" used herein with referring to epitopes to entrapping and indicative antibodies encompasses epitopes having completely or partially overlapped amino acid sequences. For example, the epitopes to 3E7 and T2 antibodies have amino acid sequences spanning amino acid 129-148 and 135- 140, respectively of a human prion sequence. Such eptiopes can also be described as completely overlapped epitopes. Unlikely, the epitopes to 3F4 and 308 have amino acid sequences spanning amino acid 109-112 and 111-118, respectively, of a human prion sequence. Such epitopes can be described as partially overlapped epitopes.

As to the adjacent epitopes causing steric hindrance, one epitope (e.g., epitope recognized by the entrapping antibody)

in the multimer-forming polypeptide may be located at a position apart from the other epitope (e.g., epitope recognized by the indicative antibody) so long as two antibodies are competitively bound to the adjacent epitopes. According to a more preferred embodiment, the amino acid sequence of the epitope recognized by the entrapping antibody is identical to or overlapped with that of the epitope recognized by the indicative antibody. Most preferably, the amino acid sequence of the epitope recognized by the entrapping antibody is identical to or completely overlapped with that of the epitope recognized by the indicative antibody.

The cocktail form of several types of entrapping antibodies may be used in this invention. In addition, the cocktail form of several types of indicative antibodies may be used in this invention.

Another feature of this invention is to utilize an entrapping antibody-affinity molecule rather than an entrapping antibody per se. The affinity molecule bound to entrapping antibodies is intended to isolate an entrapping antibody- affinity molecule-multimeric form-indicative antibody complex finally formed through binding to its affinity counterpart molecule. For example, biotin bound to entrapping antibodies binds to its affinity counterpart molecule, streptavidin to form a biotin-conjugated entrapping antibodies-streptavidin complex followed by isolating the complex.

According to a preferred embodiment, the affinity molecule bound to the entrapping antibody and the counterpart affinity molecule are selected from the group consisting of biotin, avidin, streptavidin, antigens, haptens, antibodies, hormones, vitamins, receptors, carbohydrates, lectins, metals, chelators, polynucleotides, cofactor or prosthetic groups, apoproteins, effecter molecules, one member of a hydrophobic interactive

pair, enzyme cofactors, enzymes, polymeric acids, polymeric bases, dyes, protein binders, peptides, protein binders and enzyme inhibitors, provided that the affinity molecule and the counterpart affinity molecule are different. For example, the affinity pair includes biotin/avidin (or streptavidin) , antigens (or haptens) /antibodies, hormones (or vitamins) /receptors, carbohydrates/lectins, metals /chelators, cofactor (or prosthetic groups) /apoproteins, and enzyme cofactors/enzymes . Most preferably, the affinity molecule bound to the entrapping antibody is biotin, avidin or streptavidin, and the counterpart affinity molecule is biotin, avidin or streptavidin. Where the affinity molecule bound to the entrapping antibody is biotin, the counterpart affinity molecule is avidin or streptavidin.

According to a preferred embodiment, the counterpart affinity molecule is bound to a solid support. Solid support conjugated with the counterpart affinity molecule may be any materials isolatable by gravity, charge or magnetic force. Most preferably, the solid phase carrier is a magnetic bead. Where the counterpart affinity molecule bound to magnetic beads is used, the step (e) for isolating an entrapping antibody- affinity molecule-multimeric form-indicative antibody complex may be carried out by applying a magnetic field to the resultant of the step (d) .

The isolation of antigen-antibody complexes by use of a pair of affinity molecule/counterpart affinity molecule induces concentration effect of antigen-antibody complexes to be detected. Preferably, the entrapping antibody and indicative antibody used in this invention are not bound to a solid support such as plates and beads . The entrapping antibody and

indicative antibody in a free form allows for contacting three- dimensionally to antigens in biological samples, ensuring much more opportunities to contact to antigens in biological samples.

According to a preferred embodiment, the indicative antibody has a label generating a detectable signal or an affinity substance. The label includes, but not limited to, an enzymatic (e.g., alkaline phosphatase, peroxidase, β- galactosidase and β-glucosidase) , a radioactive (e.g., I ¹²⁵ and

C ¹⁴ ), a fluorescent (e.g., fluorescein), a luminescent, a chemiluminescent, an electrical and a FRET (fluorescence resonance energy transfer) label. The affinity substance includes biotin. Various labels and methods for labeling antibodies are well known in the art (Harlow and Lane, eds . Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.) .

Where the radioactive label is used for indicative antibodies, the antigen-antibody complex formed may be detected by measuring radioactivity from label. Where the indicative antibody is labeled with enzymes catalyzing colorimetric reactions, the antigen-antibody complex formed may be detected by use of substrates for enzymes. For example, where the indicative antibody is labeled with alkaline phosphatase, bromochloroindolylphosphate (BCIP) , nitro blue tetrazolium (NBT) , naphthol-AS-Bl-phosphate and ECF (enhanced chemifluorescence) may be used as a substrate for color developing reactions; in the case of labeled with horseradish peroxidase, chloronaphtol, aminoethylcarbazol, diaminobenzidine, D-luciferin, lucigenin (bis-IV-methylacridinium nitrate) , resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl- 3, 7-dihydroxyphenoxazine) , TMB (3, 3, 5, 5-tetramethylbenzidine) and ABTS (2, 2-Azine-di [3-ethylbenzthiazoline sulfonate]) may be used as a substrate.

Where the indicative antibody is labeled with affinity substance, the antigen-antibody complex formed may be detected by use of its binding partner linked to label generating a detectable signal (e.g., colorimetric reaction-catalyzing enzymes) . For example, where the indicative antibody is labeled with biotin, enzyme-conjugated streptavidin may be used for detecting the antigen-antibody complex; however, in this case, a biotin/streptavidin pair should not been used as affinity molecules bound to entrapping antibodies and counterpart affinity molecules.

Labels described above make it more feasible to detect qualitatively and quantitatively the multimeric antigen- antibody complex in the step (f) . Where the indicative antibody is linked to a label, the step (f) may be carried out by measuring a signal generated from the label.

The step (d) is carried out by contacting the entrapping antibody-affinity molecule conjugate and the indicative antibody to the biological sample in a three dimensional manner. The term "three dimensional manner" used herein with describing the contacting event of two types of antibodies means that both the entrapping antibody-affinity molecule conjugate and the indicative antibody are not bound to solid supports such as plates and beads, i.e., is in the free form, and therefore both of them are able to survey three- dimensionally their epitopes on antigens for specific binding. If either the entrapping antibody-affinity molecule conjugate or the indicative antibody is bound to plates, the three- dimensional scrutiny is not longer possible. The three- dimensional contact allows for enabling the antibodies to have much more opportunities to contact to antigens in biological samples .

Preferably, the step (d) is carried out by contacting the

entrapping antibody-affinity molecule conjugate and the indicative antibody simultaneously or in a short time interval to the biological sample. More preferably, the step (d) is carried out by contacting simultaneously the entrapping antibody-affinity molecule conjugate and the indicative antibody. In such simultaneous reactions, either the entrapping antibody or indicative antibody can be firstly bound to epitopes on multimer-forming polypeptides.

If the entrapping antibody-affinity molecule conjugate is initially incubated with biological samples for a long period of time (e.g., 30 min) and then the indicative antibody is contacted to biological samples, the entrapping antibody- affinity molecule conjugate are bound to most of epitopes to the entrapping antibody in the multimeric form, giving rise to the substantially complete loss of the binding opportunity of the indicative antibody to epitopes. In contrast, where the entrapping antibody and indicative antibody are contacted to biological samples simultaneously or in a short time interval, two types of antibodies is rendered to be under competition circumstances and their binding to epitopes depends on their concentration.

Where the time interval of the contacting of the entrapping antibody-affinity molecule conjugate and the indicative antibody is more than 30 min, the signal from labeled indicative antibodies becomes negligible. Preferably, the time interval of the contacting is 0-10 min, more preferably 0-8 min, still more preferably 0-5 min, most preferably 0-3 min.

According to the present invention, the order of the contacting of the entrapping antibody-affinity molecule conjugate and the indicative antibody becomes meaningless. In other words, either the entrapping antibody-affinity molecule

conjugate or the indicative antibody can be firstly contacted to the biological sample.

According to a preferred embodiment, the entrapping antibody-affinity molecule conjugate and the indicative antibody in the step (c) are used at 5:1-1:5 mole ratio of the entrapping antibody to the indicative antibody, more preferably, 3:1-1:3, still more preferably, 2:1-1:2, most preferably about 1:1.

The present invention makes it possible to differentially detect a multimeric form from a monomeric form of any multimer- forming polypeptide. According to a preferred embodiment, the multimer-forming polypeptide includes Aβ peptide and tau protein related to Alzheimer's disease, prion related to Creutzfeldt-Jakob disease and Spongiform encephalopathies, α- synuclein related to Parkinson' s disease Ig light chains related to primary systemic amyloidosis, serum amyloid A related to secondary systemic amyloidosis, tau related to Fronto-temporal dementias, transthyretin related to senile systemic amyloidosis, transthyretin related to familial amyloid polyneuropathy, cystatin C related to hereditary cerebral amyloid angiopathy, β ₂ -microglobulin related to haemodialysis- related amyloidosis, huntingtin related to Huntington' s disease, superoxide dismutase related to Amyotrophic lateral sclerosis, serpin related to Serpin deficiency, emphysema, and cirrhosis, and amylin related to Type II Diabetes.

Most preferably, the multimer-forming polypeptide is the prion protein causing Creutzfeldt-Jakob disease and Spongiform encephalopathies .

The present invention is significantly useful in detecting a multimeric prion, i.e., PrP ^Sc formed by conformational change of prion proteins.

When the present method is applied to the prion protein

(PrP) , the monomeric form is PrP ^c (cellular or normal form of prion) and the multimeric form is PrP ^Sc (scrapie or infectious form of prion) .

One of the features of this invention is to employ antibodies which are bound to epitopes having non-repeated sequence in an antigen molecule. Unless epitopes recognized by antibodies have a non-repeated sequence, the present invention may not effectively detect a multimeric form from a monomeric form of a multimer-forming polypeptide. According to a preferred embodiment, the epitope specifically recognized by the entrapping antibody and/or the epitope specifically recognized by the indicative antibody are not repeated in the multimer-forming polypeptide.

The antibodies used in this invention could be prepared according to conventional techniques such as a fusion method (Kohler and Milstein, European Journal of Immunology, 6:511- 519(1976)), a recombinant DNA method (USP 4,816,56) or a phage antibody library (Clackson et al, Nature, 352:624-628(1991); and Marks et al, J. MoI. Biol., 222:58, 1-597(1991)). The general procedures for antibody production are described in Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1988; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991. The preparation of hybridoma cell lines for monoclonal antibody production is done by fusion of an immortal cell line and the antibody producing lymphocytes. This can be done by techniques well known in the art. Polyclonal antibodies may be prepared by injection of the antigen described above to suitable animal, collecting antiserum containing antibodies from the animal, and isolating specific antibodies by any of the known affinity techniques.

The term "biological sample" used herein is an organism- originated sample of material to be tested. The biological sample refers to any cell, tissue, or fluid from a biological source, or any other medium that can advantageously be evaluated according to this invention, including a sample drawn from human, a sample drawn from an animal, a sample drawn from food designed for human or animal consumption. Preferably, the biological sample to be tested is a body fluid sample including blood, serum, plasma, lymph, milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain homogenates) , cerebral spinal fluid (CSF) , appendix, spleen and tonsillar tissue extracts. More preferably, the biological sample is a brain homogenate, blood, serum or plasma, still more preferably blood or plasma, most preferably, plasma. According to a preferred embodiment, the present method further comprises the step of (a-2) treating the biological sample with a protease inhibitor.

The treatment of protease inhibitors enhances the binding of a multimeric form (e.g. PrP ^Sc ) with antibodies in biological samples (e.g. plasma) . Protease inhibitors interfere with the binding of a normal monomer form (e.g. PrP ^c ) with antibodies in biological samples. Therefore, protease inhibitors increases both signal intensity and differentiation that indicate the existence of a multimeric form (e.g. PrP ^Sc ) . Although the protease K treatment conventionally used for detecting PrP ^Sc may contribute to signal differentiation, signal intensity is decreased. In this respect, the protease K treatment is very likely to induce false negative data. The present invention overcomes the shortcomings of conventional techniques described above.

According to the present method using protease inhibitors, the present method can differentiate a multimeric form (e.g.

PrP ^Sc ) from monomer form (e.g. PrP ^c ) in biological samples (e.g. plasma) with no help of protease K.

As used herein, the terms "protease inhibitor" refer to any molecule or collection of molecules that are capable of interfering with the proteolytic activity of one or more proteases. For example, the protease inhibitors may be capable of inhibiting serine proteases, cysteine proteases, metalloproteases and aspartic proteases.

According to a preferred embodiment, the protease inhibitor useful in this invention is a serine protease inhibitor, more preferably, 4- (2-aminoethyl) -benzenesulfonyl fluoride, leupeptin, phenylmethylsulfonyl fluoride, aprotinin, chymostatin, antithrombin III, 3, 4-dichloroisocoumarin, tosyl-

L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, diisopropyl fluorophosphates, antipain or α2-macroglobulin, most preferably, 4- (2-aminoethyl) - benzenesulfonyl fluoride.

Preferably, the concentration of protease inhibitors is in the range of 0.05-1.0 mM, more preferably 0.1-0.8 mM and most preferably 0.3-0.7 mM.

According to a preferred embodiment, the biological sample is not treated with protease K or trypsin.

Where blood or plasma is used as a biological sample, it is advantageous that the present method further comprises the step of pretreating the biological sample with sarkosyl or Triton series (e.g., Triton X-100) detergent.

In still another aspect of this invention, there is provided a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample, which comprises the steps of: (a-1) obtaining the biological sample to be analyzed; (a-2) treating the biological sample with a protease inhibitor; (b) preparing

an entrapping antibody-affinity molecule conjugate by binding an affinity molecule to the surface of the entrapping antibody, wherein the entrapping antibody recognizes an epitope on the multimer-forming polypeptide and is not bound to a solid support; (c) preparing an indicative antibody, wherein an epitope recognized by the indicative antibody is present at a position in the multimer-forming polypeptide to cause a steric hindrance by the entrapping antibody bound to its epitope to prevent the binding of the indicative antibody to the multimer- forming polypeptide and wherein the indicative antibody has a label generating a detectable signal; (d) contacting the entrapping antibody-affinity molecule conjugate and the indicative antibody simultaneously or in a time interval of 0- 10 min to the biological sample in a three dimensional manner, thereby forming an entrapping antibody-affinity molecule- multimeric form-indicative antibody complex; and (e) isolating the entrapping antibody-affinity molecule-multimeric form- indicative antibody complex by use of a counterpart affinity molecule to the affinity molecule on the entrapping antibody, wherein a counterpart affinity molecule is bound to a magnetic bead; and (f) detecting the formation of the entrapping antibody-affinity molecule-multimeric form-indicative antibody complex.

Fig. 1 schematically represents an example of this invention using the entrapping antibody-biotin conjugates, streptavidin-magnetic beads and HRP-conjugated indicative antibodies. The entrapping antibody-biotin conjugates and the HRP-conjugated indicative antibodies are simultaneously contacted to biological samples containing PrP ^c and PrP ^Sc and both of them have opportunity to bind to their epitopes on PrP ^c or PrP ^Sc . Afterwards, the reaction resultants are incubated with streptavidin-magnetic beads and then placed under magnetic

field for isolation of an entrapping antibody-affinity molecule-multimeric form (PrP ^Sc ) -indicative antibody complex. The process of this invention produces four types of results:

(a) If the entrapping antibody-biotin conjugate is bound to an epitope on PrP ⁰ , the HRP-conjugated indicative antibody cannot be bound to the epitope occupied by the entrapping antibody-biotin conjugate. Therefore, the immune reaction resultant generates no signal indicative of the existence of PrP ^Sc . (b) If the HRP-conjugated indicative antibody is bound to an epitope on PrP ⁰ , the entrapping antibody-biotin conjugate cannot be bound to the epitope occupied by the HRP-conjugated indicative antibody. Therefore, the immune reaction resultant is not isolated by use of streptavidin-magnetic beads and magnetic field, generating no signal indicative of the existence of PrP ^Sc .

(c) If the entrapping antibody-biotin conjugate is bound to a portion of epitopes on PrP ^Sc , the HRP-conjugated indicative antibody has also opportunity to bind to empty epitopes not occupied by the entrapping antibody-biotin conjugate. Therefore, the immune reaction resultant is isolated by use of streptavidin-magnetic beads and magnetic field, generating signal indicative of the existence of PrP ^Sc .

(d) If the HRP-conjugated indicative antibody is bound to a portion of epitopes on PrP ^Sc , the entrapping antibody-biotin conjugate has also opportunity to bind to empty epitopes not occupied by the HRP-conjugated indicative antibody. Therefore, the immune reaction resultant is isolated by use of streptavidin-magnetic beads and magnetic field, generating signal indicative of the existence of PrP ^Sc .

The present invention is directed to providing a kit for

differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample. The kit may further comprise a counterpart affinity molecule, a counterpart affinity molecule-solid support conjugate, buffer, protease inhibitors and/or detergent.

In summary, the prominent advantages of the present invention will be described as follows:

(i) The SRA SYSTEM requires no multimer-specific antibodies. For example, it is not dependent on PrP ^Sc -specific antibodies. Antibodies having cross-reactivity between PrP ^Sα and PrP ⁰ can be successfully applied to the present invention for differentially detecting PrP ^Sc in biological samples;

(ii) The present invention does not need proteinase K (PK) treatment having been conventionally used for PrP ^Sc detection. The present SRA method per se exhibits a sufficient potential to discriminate PrP ^So from PrP ^c without PK treatment;

(iii) The present invention enables aggregating forms (particularly, PrP ^Sc ) in plasma samples to be detected by immunoassay. Little has been suggested about successful detection of PrP ^Sc in plasma; and,

(iv) The present invention can carried out in a convenient and speedy manner, which enables the automation of the SRA SYSTEM.

The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims .

EXAMPLES

EXAMPLE I: Effects of Assay Conditions on Differentiation of p _r p ^Sc f _rom p _r p° in SRA Procedure

UMD pooled scrapie sheep plasma (Grade A, University of

Maryland, US) and the commercial sheep normal plasma (Innovative Research Inc.) were used. The plasma was centrifuged at 1500 x g for 5 min to be used in the ELISA. The samples were prepared by diluting plasma (400 μl) with 10%

Triton X-100 (160 μl, Samchun Inc., Korea) in dH ₂ O and TBST

(840 μl, Sigma), followed by voltex and centrifugation. Proteinase K (Sigma) at final concentrations (120, 60, 30, 15 and 0 μg/ml) was added to the plasma samples and incubated at 37°C for 0.5, 1 and 2 hr. Various concentrations of protease inhibitor, 4- (2-aminoethyl) -benzenesulfonyl fluoride (2.0, 1.0, 0.5 and 0 I* in dH ₂ O) were added to the diluted plasma samples and incubated for 15 min at room temperature. The samples were centrifuged at 1500 x g for 5 min. The SRA SYSTEM set was prepared by mixing the entrapping antibody (3E7-biotin, 0.4 μg, Roboscreen Inc. Germany) and the indicative antibody (3E7-HRP, 0.4 μg, Roboscreen Inc. Germany) in TBST (200 μl) . The SRA SYSTEM set and the samples were mixed and incubated for 1 hr at

37 ^° C.

Afterwards, the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (5 μl of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.). Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 μl, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the siqnal was measured in the Perkin Elmer Vector 3 luminomitor.

As represented in Fig. 2, in the case of using a constant concentration of 0.5 itiM 4- (2-aminoethyl) -benzenesulfonyl fluoride, no treatment of protease K shows the highest signal intensity and signal differentiation. In addition, as shown in Fig. 3, 30 μg/ml of protease K,

0.5 mM 4- (2-aminoethyl) -benzenesulfonyl fluoride and 1 hr incubation time also exhibited the highest signal intensity and signal differentiation if both 4- (2-aminoethyl) -benzenesulfonyl fluoride and protease K were used. Furthermore, where all of 4- (2-aminoethyl) -benzenesulfonyl fluoride and protease K were not used, the high signal intensity and signal differentiation were also produced, even though the signal in PrP ^c plasma sample was relatively higher.

As represented in Fig. 4, the signal intensity and signal differentiation between prP ^Sc and PrP ^c were shown to be the highest in the case of treatment of 0.5 mM 4- (2-aminoethyl) - benzenesulfonyl fluoride and no treatment of protease K. This result clearly reveals that the treatment of protease K leads to the digestion of PrP ^Sc in biological samples to be analyzed and therefore reduces the sensitivity of immunoassay, resulting in the generation of false negative data. Surprisingly, it was observed that in PrP ^c plasma sample, no treatment of protease K generated the similar signal intensity to the treatment of protease K in the SRA procedure of this invention.

EXAMPLE II: Detecting Differentially PrP ^Sc from PrP ^c in the Scrapie and Normal Sheep Plasmas in SRP procedure

UMD pooled scrapie sheep plasma (Grade A, University of Maryland, US) and the commercial sheep normal plasma (Innovative Research Inc.) were used. The plasma was centrifuged at 1500 x g for 5 min to be used in the ELISA. The samples were prepared by diluting plasma (400 μl) with 10%

Triton X-IOO (160 μl, Samchun Inc., Korea) in dH ₂ O and TBST

(840 μl, Sigma), followed by voltex and centrifugation. Proteinase K (Sigma) at final concentrations (30 and 0 μg/ml) was added to the plasma samples and incubated at 37 "C for 1 hr. Various concentrations of protease inhibitor, 4- (2-aminoethyl) - benzenesulfonyl fluoride (1.0, 0.5, 0.25, 0.1 and 0 itiM in dH ₂ O) were added to the diluted plasma samples and voltexed. The SRA SYSTEM set was prepared by mixing the entrapping antibody (3E7- biotin, 0.4 μg, Roboscreen Inc. Germany) and the indicative antibody (3E7-HRP, 0.4 μg, Roboscreen Inc. Germany) in TBST (200 μl) . The SRA SYSTEM set and the samples were mixed and incubated for 1 hr at 37 ^° G. The 3E7 antibody specifically reacts with the epitope of the sequence 132-152 of sheep PrP.

Afterwards, the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (5 μl of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.) . Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 μl, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the signal was measured in the Perkin Elmer Vector 3 luminomitor. Without protease K treatment, the present method using protease inhibitors [4- (2-aminoethyl) -benzenesulfonyl fluoride] could perfectly differentiate PrP ^Sc from PrP ^c in plasma as represented in Fig. 5. Interestingly, the treatment of protease inhibitors increases both signal intensity and differentiation. As shown in Fig. 6, the SRA system of this invention using protease inhibitors successfully generates differential signals between scrapie and normal plasma samples. However, the

concentrations of protease inhibitors higher than 0.5 mM interfered with the detection of PrP ^Sc .

Accordingly, it could be appreciated that the protease inhibitor, 4- (2-aminoethyl) -benzenesulfonyl fluoride enhances the differentiation of PrP ^Sc from the normal PrP in sheep plasma. Increasing concentrations of 4- (2-aminoethyl) -benzenesulfonyl fluoride further enhance the binding of PrP ^Sc with antibodies in sheep scrapie plasma. On the other hand, increasing concentrations of 4- (2-aminoethyl) -benzenesulfonyl fluoride interfered with binding of normal PrP in normal sheep plasma, resulting in decreasing signal to the normal PrP.

Consequently, the treatment of the protease inhibitor to plasma sample increases differentiation signals between PrP ^bc and PrP ^c .

EXAMPLE III : Detecting PrP ^c in Normal Sheep Plasma in the Presence of Protease Inhibitor

Commercial sheep normal plasma (Innovative Research Inc., USA) was used. The plasma was centrifuged at 1500 x g for 5 itiin to be used in the ELISA. The samples were prepared by diluting plasma (400 μl) with 10% Triton X-100 (160 μl, Samchun Inc., Korea) in dH ₂ O and TBST (840 μl, Sigma), followed by voltex and centrifugation. Various concentrations of protease inhibitor, 4- (2-aminoethyl) -benzenesulfonyl fluoride (4.0, 2.0, 1.0, 0.5 and 0 mM in dH ₂ O) (Pefabloc, Roche] were added to the diluted plasma samples and voltexed. The Control antibody set was prepared by mixing the entrapping antibody (ICSM-35-biotin, 0.1 μg, D-Gen Inc., UK) and the indicative antibody (T2-HRP, 0.1 μg, NIAH, Japan) in TBST (200 μl) . The Control antibody set and the samples were mixed and incubated for 1 hr at 37 ^° C . The ICSM-35 antibody specifically reacts with human PrP ^c and PrP ^Sc in both native and denatured conformations. The antibody also

reacts with sheep, mouse, hamster and bovine PrP ^c and PrP ^Sc . The epitope is defined within the sequence 93-102 of human PrP. The T2 antibody (Hiroko Hayashi, et al., J. Vet. Med. Sci. _r 66 (6) : 515 (2004) ) specifically reacts with the epitope of the sequence 135-140 of human PrP or the sequence of 139-144 of sheep PrP.

Afterwards, the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (5 μl of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.) . Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 μl, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the signal was measured in the Perkin Elmer Vector 3 luminomitor.

As shown in Fig. 7a, increasing concentrations of protease inhibitor interfered with the binding of normal PrP with antibodies. However, the concentrations of protease inhibitor higher than 0.5 mM interfered with the detection of PrP ^Sc as shown in Fig. 7b.

EXAMPLE IV: Evaluation of Protease Effect on Other Antibody Sets and Variation of Antibody Molar Ratio

UMD pooled scrapie sheep plasma (Grade A, University of

Maryland, US) and the commercial sheep normal plasma

(Innovative Research Inc.) were used. The plasma was centrifuged at 1500 x g for 5 min to be used in the ELISA. The samples were prepared by diluting plasma (400 μl) with 10%

Triton X-100 (160 μl, Samchun Inc., Korea) in dH-O and TBST

(840 μl, Sigma), followed by vortex and centrifugation. 4- (2-

aminoethyl) -benzenesulfonyl fluoride (0.5 mM in dH ₂ O) were added to the diluted plasma samples and vortexed. The SRA SYSTEM set was prepared by mixing the capturing antibody (3E7- biotin, 2.4, 1.6, 0.8 and 0.4 μg, Roboscreen Inc. Germany) and the indicative antibody (T2-HRP, 0.4 μg) in TBST (200 μl) . The SRA SYSTEM set and the samples were mixed and incubated for 1 hr at 37 ^° C. The 3E7 antibody specifically reacts with the epitope of the sequence 132-152 of sheep PrP. The T2 antibody specifically reacts with the epitope of the sequence 139-144 of sheep PrP.

Afterwards, the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (8- μl of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.). Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 μl, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the signal was measured at Perkin Elmer Vector 3 luminomitor.

As a result, it was observed that the 3:1-1:2 mole ratio of the entrapping antibody to the indicative antibody shows the high signal intensity and signal differentiation between PrP ^Sc and PrP ^c .

EXAMPLE V: Detecting Differentially PrP ^Sc from PrP ^c in the Scrapie and Normal Sheep Plasmas Using Combinations of Entrapping and Indicative Antibodies UMD pooled scrapie sheep plasma (Grade A, University of

Maryland, US) and the commercial sheep normal plasma

(Innovative Research Inc.) were used. The plasma was

centrifuged at 1500 x g for 5 min to be used in the ELISA. The samples were prepared by diluting plasma (400 μl) with 10% Triton X-100 (160 μl, Samchun Inc., Korea) in dH ₂ O and TBST (840 μl, Sigma) , followed by voltex and centrifugation. Protease inhibitor, 4- (2-aminoethyl) -benzenesulfonyl fluoride at 0.5 mM in dHoO was added to the diluted plasma samples and voltexed. The SRA SYSTEM set was prepared by mixing the entrapping antibody (3E7-biotin, 0.2 μg, Roboscreen Inc. Germany; and MAl-750-biotin, 0.2 μg, Bioreagents Inc. US) and the indicative antibody (T2-HRP, 0.2 μg, NIAH, Japan; 3E7-HRP, 0.2 μg, Roboscreen Inc. Germany) in TBST (200 μl) . The SRA SYSTEM set and the samples were mixed and incubated for 1 hr at 37 ^° C. The 3E7 and T2 antibody specifically reacts with the epitope of the sequence 132-152 and 139-144 of sheep PrP, respectively. The MAl-750 antibody specifically reacts with the epitope of the sequence 139-152 of sheep PrP.

Afterwards, the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (5 μl of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.) . Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 μl, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the signal was measured in the Perkin Elmer Vector 3 luminomitor.

As shown in Fig 8, the SRA system of this invention successfully using cocktail forms of antibodies generates differential signals between scrapie and normal plasma samples.

Having described a preferred embodiment of the present

invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.