Antibody cross-linked proteins providing enhanced immunoassays

Abstract

To provide a simple and inexpensive means for improving the sensitivity of immunoassay.SOLUTION: Domain-swapping type protein G dimer capable of binding to Fc region of antibody, method for detecting or quantifying target molecule in sample by immunoassay using said domain-swapping type protein G dimer, and sensitizer for immunoassay SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a domain-swapped protein G dimer capable of binding to the Fc region of an antibody, a method for detecting or quantifying a target molecule in a sample by immunoassay using the domain-swapped protein G dimer, and an immunoassay sensitizer. [Background technology]

[0002] Immunoassays are a method for detecting and quantifying test substances in samples using antigen-antibody reactions. Although immunoassays are highly specific, the test substances in biological samples are often in trace amounts, so various methods have been devised to increase detection sensitivity.

[0003] Known methods for sensitizing immunoassays include a method using biotinylated secondary antibodies (Non-Patent Document 1) and a method using dendritic amplification via biotinylated proteins (Non-Patent Document 2), but these require complex procedures. A method has also been reported in which primary antibodies are accumulated on nanofibrils bound to the Z-domain of an antibody-binding protein, thereby increasing the amount of secondary antibody bound to them (Non-Patent Document 3), but the production of nanofibrils requires approximately three days. Another method for sensitization using antibody-enzyme conjugated nanoparticles formed by biomineralization has also been reported (Non-Patent Document 4), but the preparation of nanoparticles of a size suitable for sensitization requires complex procedures. A sensitization method using a protein (8pG) in which eight antibody-binding proteins are linked in series has also been reported (Non-Patent Document 5), but the preparation of 8pG is expensive.

[0004] Protein G is a membrane protein derived from streptococci that has specific binding activity to the Fc region of antibodies (IgG), and is therefore used for antibody capture, antibody-mediated detection, etc. The inventors have developed modified extracellular domains of Protein G with improved stability and binding affinity to the Fc region. They have also reported antibody capture agents consisting of multimers in which extracellular domains of Protein G are linked in tandem (series) (Patent Documents 1 to 6). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2017-029001 [Patent Document 2] WO2015 / 050153 [Patent Document 3] WO2014 / 021240 [Patent Document 4] WO2013 / 018880 [Patent Document 5] Patent Publication No. 2015-019615 [Patent Document 6] Patent Publication No. 2015-003872 [Non-patent literature]

[0006] [Non-Patent Document 1] Lakshmipriya T, Gopinath SCB, Tang TH,2016, PLOS ONE 11(3): e0151153 [Non-patent document 2] Chu et al., Chem. Commun., 2013,49, 2397-2399 [Non-patent document 3] Rahman, M. M et al., 2021, Amyloid,28(3), 158-167. [Non-patent document 4] Su et al., Anal Chem. 2022 Apr 26;94(16):6271-6280 [Non-patent document 5] Chen et al., Anal Chem. 2019 May 30;91(13):8310-8317 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a simple and inexpensive means for improving the sensitivity of immunoassays. [Means for solving the problem]

[0008] The inventors have found that the use of domain-swapping dimers of Protein G significantly improves the sensitivity of immunoassays, and that the domain-swapping dimers have a higher sensitivity-enhancing effect than the previously known tandem-binding dimers.

[0009] The present invention is based on the above findings and provides the following [1] to

[17] . [1] A method for detecting or quantifying a target molecule in a sample by immunoassay, comprising: (1) contacting the sample with a first antibody capable of specifically binding to a target molecule and a domain-swapped Protein G dimer capable of (bivalently) binding to the Fc region of the antibody; and (2) The following step (a) or (b): (a) detecting a signal from the label of the first antibody; (b) contacting a labeled second antibody or an antigen-binding fragment thereof capable of binding to the first antibody with the sample of step (1) and detecting a signal from the label; The method comprising: The protein G may be a portion of protein G capable of binding to the Fc region of an antibody, for example, the extracellular domain of protein G, preferably the B1, B2, or B3 domain of protein G. [2] The method according to [1], wherein the immunoassay is any one selected from enzyme-linked immunosorbent assay (ELISA), Western blotting, immunostaining, and immunoprecipitation, preferably ELISA. [3] The method according to [1] or [2], wherein the domain-swapping protein G dimer is a head-to-head dimer. [4] The method according to any one of [1] to [3], wherein the domain-swapping protein G dimer has a peptide linker in loop 2 or loop 4 of the B1 domain of protein G. [5] The method according to any one of [1] to [4], wherein the peptide linker is a peptide linker consisting of 3 to 10 amino acid residues, preferably 5 to 9 amino acid residues, more preferably 6 to 8 amino acid residues, including one cysteine ​​in a polyproline sequence. [6] The method according to any one of [1] to [5], wherein Protein G has any one of the following amino acid sequences 1) to 4): 1) an amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; 2) an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, (i) an amino acid sequence in which an amino acid is substituted at at least one position selected from positions 2, 4, 17, 19, 23-25, 29, 44, 46, and 51 of SEQ ID NO: 1, and / or (ii) an amino acid sequence in which an amino acid is deleted, substituted, or added at at least one position selected from positions 21, 22, and 47-50 of SEQ ID NO: 1; 3) an amino acid sequence in which an amino acid is substituted at positions 17 and / or 29 of SEQ ID NO: 1; 4) An amino acid sequence having at least one of the following substitutions in SEQ ID NO: 1: alanine for valine at position 29, proline or glycine for valine at position 21, and proline or glycine for aspartic acid at position 22. [7] The method according to any one of [1] to [6], wherein Protein G has an amino acid sequence shown in any one of SEQ ID NOs: 1 to 12. [8] The method according to any one of [1] to [7], wherein the domain-swapped Protein G dimer capable of binding to an antibody Fc region comprises the following amino acid sequence (i) or (ii): (i) the amino acid sequence shown in SEQ ID NO: 19; (ii) An amino acid sequence having 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19. [9] A domain-swapping protein G dimer capable of (bivalently) binding to the Fc region of an antibody, the domain-swapping protein G dimer having a peptide linker in loop 2 or loop 4 of the B1 domain of protein G. More specifically, a domain-swapping Protein G dimer capable of (bivalently) binding to the Fc region of an antibody, having a peptide linker in loop 2 or loop 4 of the B1 domain of protein G; A domain-swapping protein G dimer, wherein the protein G has any one of the following amino acid sequences 1) to 4): 1) an amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; 2) an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, (i) an amino acid sequence in which an amino acid is substituted at at least one position selected from positions 2, 4, 17, 19, 23-25, 29, 44, 46, and 51 of SEQ ID NO: 1, and / or (ii) an amino acid sequence in which an amino acid is deleted, substituted, or added at at least one position selected from positions 21, 22, and 47-50 of SEQ ID NO: 1; 3) an amino acid sequence in which an amino acid is substituted at positions 17 and / or 29 of SEQ ID NO: 1; 4) An amino acid sequence having at least one of the following substitutions in SEQ ID NO: 1: alanine for valine at position 29, proline or glycine for valine at position 21, and proline or glycine for aspartic acid at position 22.

[10] The domain-swapping protein G dimer according to [9], wherein the protein G has an amino acid sequence shown in any one of SEQ ID NOs: 1 to 12.

[11] The domain-swapping protein G dimer according to [9] or

[10] , wherein the peptide linker is a peptide linker consisting of 3 to 10 amino acid residues, preferably 5 to 9 amino acid residues, more preferably 6 to 8 amino acid residues, including one cysteine ​​in a polyproline sequence.

[12] The domain-swapping protein G dimer according to any one of [9] to

[11] , wherein the peptide linker is any one selected from PPCPPP, PPPCPP, PPPCPPP, PPPCPPPP, and PPPPCPPP.

[13] The domain-swapping protein G dimer according to any one of [9] to

[12] , wherein the peptide linker is present at a position corresponding to between positions 20 and 21, 21 and 22, 22 and 23, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or 50 and 51 of SEQ ID NO: 1.

[14] A domain-swapped protein G dimer capable of binding to the Fc region of an antibody, comprising the following amino acid sequence (i) or (ii): (i) the amino acid sequence shown in SEQ ID NO: 19; (ii) An amino acid sequence having 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19.

[15] A sensitizer for immunoassays, comprising the domain-swapped protein G dimer according to any one of [9] to

[14] .

[16] Any of the above immunoassays, domain-swapped protein G dimers, or sensitizers, wherein the domain-swapped protein G dimer comprises a tag sequence.

[17] The immunoassay, domain-swapping protein G dimer, or sensitizer according to

[16] , wherein the tag sequence is one or more selected from His-tag, FLAG-tag, Spot-tag, C-tag, Strep-tag, and Avi-tag. [Effects of the Invention]

[0010] The domain-swapped protein G dimer of the present invention, when mixed with any IgG, recognizes the Fc region of the IgG through protein-protein interactions and induces one-dimensional polymerization of the IgG, thereby accumulating on the antigen and amplifying the signal. Furthermore, because the domain-swapped protein G dimer of the present invention is a small multimer, it can be produced inexpensively using an expression system using Escherichia coli. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the structure of the B1 domain of protein G. [Figure 2] FIG. 2 is a schematic diagram of the domain-swapping protein G dimer of the present invention and a conventional tandem protein G dimer. [Figure 3] FIG. 3 is a schematic diagram of an immunoassay of the present invention. [Figure 4] Figure 4 shows non-reducing SDS-PAGE of the eluted fraction after metal affinity chromatography: marker (left), variant (right). The target product is indicated by an arrow. [Figure 5] Figure 5 shows signal values ​​in the low immobilization amount region: without domain-swapping protein G dimer (white) and with domain-swapping protein G dimer (black). Error bars indicate standard deviation (SD). [Figure 6] Figure 6 shows signal values ​​in the high immobilization range: without domain-swapping protein G dimer (white) and with domain-swapping protein G dimer (black). Error bars indicate standard deviation (SD). [Figure 7] FIG. 7 shows the degree of signal enhancement in the low immobilization amount region. [Figure 8] FIG. 8 shows the degree of signal enhancement in the high immobilization range. [Figure 9] Figure 9 shows signal values ​​in the high immobilization range: without tandem protein G dimer (white) and with tandem protein G dimer (black). Error bars indicate standard deviation (SD). [Figure 10]FIG. 10 shows the increase in signal in the high immobilization amount region when tandem protein G dimer was added. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Domain-swapping protein G dimer "Protein G" is a membrane protein present in the cell membrane of streptococci, and is known to have specific binding activity to the Fc region of immunoglobulin G (IgG), a type of antibody.

[0013] The "Protein G" according to the present invention may be a portion (a domain) of Protein G, as long as it can bind to the Fc region of an antibody. Such a portion may include an extracellular domain of Protein G, such as the B1, B2, or B3 domain of Protein G. Preferably, Protein G includes the B1 domain of Protein G.

[0014] The inventors have previously prepared various stable mutants of Protein G, and such mutants may be used as Protein G. These mutants are described in JP 2017-029001, WO 2015 / 050153, JP 2015-019615, JP 2015-003872, WO 2014 / 021240, JP 2013 / 018880, JP 2009-297018, JP 2009-095322, Watanabe et al. (see below), and others, and those skilled in the art can easily obtain mutants of Protein G by following the descriptions therein.

[0015] Figure 1 shows the structure of the B1 domain of protein G. The B1 domain of protein G is an α+β protein with a symmetric fold of β1-β2-α1-β3-β4 topology. It has four loops, L1, L2, L3, and L4, that connect each secondary structure.

[0016] In the B1 domain of protein G, the following is considered. (i) Neither the surface-exposed amino acid residues in the β-strands nor the surface-exposed amino acid residues in the α-helices contribute to the intramolecular packing; (ii) the amino acid residues that form the loop structure have little effect on the structured site; (iii) amino acid residues outside the Fc binding region have little effect on Fc binding; (iv) Changes in residues close to the Fc binding region may affect Fc binding due to steric hindrance during interaction with Fc.

[0017] Considering the above (i) to (iv), substitution of amino acid residues at positions 2, 4, 17, 19, 21-25, 29, 44, and 46-51 of SEQ ID NO: 1 with other amino acid residues is expected to have little effect on the function (Fc binding) of Protein G. In particular, deletion or addition of other amino acid residues to amino acid residues in the loop portion (positions 21, 22, and 47-50 of SEQ ID NO: 1) is expected to have little effect on the structure or function (Fc binding).

[0018] Therefore, an amino acid sequence having a high sequence identity, for example, 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more, with the amino acid sequence shown in SEQ ID NO: 1, (i) an amino acid sequence in which an amino acid is substituted at at least one position selected from positions 2, 4, 17, 19, 23-25, 29, 44, 46, and 51 of SEQ ID NO: 1, and / or (ii) an amino acid sequence in which an amino acid is deleted, substituted, or added at at least one position selected from positions 21, 22, and 47-50 of SEQ ID NO: 1; It is believed that a protein having the above structure has properties and functions similar to those of the stabilized mutant of protein G (SEQ ID NO: 1) used in the examples.

[0019] In particular, positions 17 and / or 29 of SEQ ID NO: 1 are suitable as mutation sites because they have little contact between side chains. Positions 21 and 22 of SEQ ID NO: 1, into which a linker has been introduced, also have little effect on the three-dimensional structure and are thought to be suitable as mutation sites.

[0020] Regarding amino acid substitutions, residues in α-helices are preferably substituted with A, C, E, K, L, M, Q, and R, which tend to form α-helices; residues in β-strands are preferably substituted with F, I, T, V, W, and Y, which tend to form β-strands; and residues in loops are preferably substituted with D, G, H, N, P, and S, which tend to form coil structures.

[0021] Examples of suitable mutations include, in SEQ ID NO: 1, substitution of valine at position 29 with alanine, substitution of valine at position 21 with proline or glycine, and substitution of aspartic acid at position 22 with proline or glycine.

[0022] As described above, the "protein G" used in the present invention can include proteins having the amino acid sequences 1) to 4) below. 1) an amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; 2) an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, (i) an amino acid sequence in which an amino acid is substituted at at least one position selected from positions 2, 4, 17, 19, 23-25, 29, 44, 46, and 51 of SEQ ID NO: 1, and / or (ii) an amino acid sequence in which an amino acid is deleted, substituted, or added at at least one position selected from positions 21, 22, and 47-50 of SEQ ID NO: 1; 3) an amino acid sequence in which an amino acid is substituted at positions 17 and / or 29 of SEQ ID NO: 1; 4) An amino acid sequence having at least one of the following substitutions in SEQ ID NO: 1: alanine for valine at position 29, proline or glycine for valine at position 21, and proline or glycine for aspartic acid at position 22.

[0023] The amino acid sequences shown in any of SEQ ID NOs: 1 to 13 share 90% or more sequence identity with the stabilized mutant of Protein G (SEQ ID NO: 1) used in the Examples, and are expected to have similar structures and functions. SEQ ID NOs: 1 to 12 encode stabilized mutants of the B1 domain of Protein G, and SEQ ID NO: 13 encodes the wild type. DTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANEHGVDGEWTYDPETKTFTVTE (SEQ ID NO: 1) DTYKLILNGKTLKGETTTEAVDAATAEKVFKHYANEHGVHGHWTYDPETKTFTVTE (SEQ ID NO: 2) DTYKLILNGKTLKGETTTEAVDAAHAEKVFKHYANEHGVHGHWTYDPETKTFTVTE (SEQ ID NO: 3) DTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDAATKTFTVTE (SEQ ID NO: 4) DTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWCYDDATKTFTVTE (SEQ ID NO: 5) DTYKLILNGKTLKGETTTEAVDAATAEKVFKQYFNDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 6) DTYKLILNGKTLKGCTTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 7) DTYKLALNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 8) DTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYKDATKTFTVTE (SEQ ID NO: 9) DTYKLILNGKTLKGETTTCAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 10) DTYKLILNGKTLKGQTTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 11) DTYKLALNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 12) DTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 13)

[0024] The mechanism by which proteins exchange parts of their structures between molecules to form multimers is called "domain swapping." The "domain-swapped Protein G dimer" of the present invention is a bivalent Protein G dimer (having two Fc domain-binding sites) formed by the domain swapping between two Protein G molecules described above. Figure 2 shows the domain-swapped Protein G dimer of the present invention in comparison with a conventionally known tandem Protein G dimer.

[0025] "Domain-swapping protein G dimers" can be classified into head-to-head types, in which the N-terminuses are linked, and tail-to-tail types, in which the C-terminuses are linked, depending on the orientation of the two protein Gs. The domain-swapping protein G dimers used in the present invention are preferably head-to-head dimers. This is because, in tail-to-tail dimers, the two Fc-binding regions are in close proximity, which may impair Fc-binding ability due to steric hindrance, whereas, in head-to-head dimers, the two Fc-binding regions are far apart, which makes such steric hindrance less likely to occur.

[0026] The domain-swapped protein G dimer of the present invention may have a "linker" in the molecule. The linker is preferably a "peptide linker." This is because a peptide linker allows the domain-swapped protein G dimer to be easily produced by genetic engineering techniques.

[0027] Examples of "peptide linkers" include, but are not limited to, peptide linkers containing one cysteine ​​in a polyproline sequence. The length of the peptide linker is 3 to 10 amino acid residues, preferably 5 to 9 amino acid residues, and more preferably 6 to 8 amino acid residues. The position of the cysteine ​​is not particularly limited, but is preferably near the center of the peptide linker. Suitable peptide linkers include, for example, PPCPPP (SEQ ID NO: 14), PPPCPP (SEQ ID NO: 15), PPPCPPP (SEQ ID NO: 16), PPPCPPPP (SEQ ID NO: 17), and PPPPCPPP (SEQ ID NO: 18).

[0028] The linker is preferably inserted into loop 2 or loop 4 of the B1 domain of protein G. This is because inserting a linker into loop 2 or loop 4 results in the formation of a head-to-head dimer in which the two Fc-binding regions are separated, thereby avoiding the problem of steric hindrance as described above.

[0029] Specifically, the linker is preferably inserted at a position corresponding to between positions 20 and 21, 21 and 22, 22 and 23, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or 50 and 51 of SEQ ID NO: 1. It is particularly preferably inserted at a position corresponding to between positions 1 and 22 of SEQ ID NO: 1. Here, "corresponding position" means a "position corresponding to" the amino acid position of SEQ ID NO: 1 when the amino acid sequence of Protein G is aligned with SEQ ID NO: 1.

[0030] A domain-swapped protein G dimer prepared by inserting a linker (underlined) of PPPCPPP (SEQ ID NO: 16) between positions 1 and 22 of SEQ ID NO: 1: DTYKLILNGKTLKGETTTEAV PPPCPPP DAATAEKVFKQ YANEHGVDGEWTYDPETKTFTVTE (SEQ ID NO: 19) is shown in the Examples below. This domain-swapped Protein G dimer bivalently binds to the Fc region of an antibody, polymerizing and accumulating the antibody, thereby increasing the signal from the label and achieving high detection sensitivity.

[0031] As will be understood by those skilled in the art, an amino acid sequence having 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19 can also bivalently bind to the Fc region of an antibody and can be used as the domain-swapped Protein G dimer of the present invention.

[0032] The domain-swapped protein G dimer may have a sequence for solubilization, detection, purification, etc. during recombinant expression, such as a tag sequence or a sequence-specific protease recognition sequence for cleavage thereof. The tag sequence or protease recognition sequence may be located at the N-terminus or C-terminus. Tag sequences well known in the art can be used, and examples include His-tag (N- and C-terminus), FLAG-tag (N- and C-terminus), Spot-tag (N- and C-terminus), C-tag (C-terminus), Strep-tag (N- and C-terminus), and Avi-tag (N- and C-terminus). Sequence-specific protease recognition sequences include recognition sequences for Factor Xa, Thrombin, and HRV 3C Protease.

[0033] The domain-swapped protein G dimer may have a tag sequence, a sequence-specific protease recognition sequence, or a sequence encoding an enzyme for a color-developing or luminescent reaction, or may have appropriate modifications, as long as the object of the present invention is not impaired.

[0034] The domain-swapped protein G dimer of the present invention has two Fc region-binding sites, and when it comes into contact with an antibody (IgG), it can polymerize and accumulate the antibody (Figure 3), allowing for the sensitive detection of minute amounts of target molecules (antigens).

[0035] 2. Immunoassay using domain-swapped protein G dimers The present invention also provides a method for detecting or quantifying a target molecule in a sample by immunoassay using a domain-swapped protein G dimer. The method of the present invention may be either a method for detecting a signal from an antibody (primary antibody) capable of specifically binding to the target molecule (antigen) (direct method), or a method for detecting a signal from a labeled secondary antibody (which may be an antigen-binding fragment) capable of binding to the primary antibody (indirect method).

[0036] In this specification, regardless of whether the method is direct or indirect, an antibody that can specifically bind to a target molecule (antigen) is referred to as a "first antibody (primary antibody)," and an antibody (which may be an antigen-binding fragment) that can bind to the first antibody is referred to as a "second antibody (secondary antibody)."

[0037] Direct method: 1. A method for detecting or quantifying a target molecule in a sample by immunoassay, comprising: (1) contacting the sample with a first antibody capable of specifically binding to a target molecule and a domain-swapped protein G dimer capable of binding to the Fc region of the antibody; and (2) detecting a signal from the label.

[0038] Indirect method: 1. A method for detecting or quantifying a target molecule in a sample by immunoassay, comprising: (1) contacting a sample with a first antibody that specifically binds to a target molecule and a domain-swapped protein G dimer that can bind to the Fc region of the antibody; (2) contacting the sample of step (1) with a labeled second antibody or an antigen-binding fragment thereof capable of binding to the first antibody; (3) detecting a signal from the label.

[0039] The term "immunoassay" is not particularly limited as long as it uses an antibody capable of specifically binding to a target molecule, and examples thereof include enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blotting, immunostaining, and immunoprecipitation (pull-down assay). These immunoassays can be applied as either a direct method that does not use a secondary antibody, or an indirect method that uses a secondary antibody.

[0040] In ELISA, EIA, RIA, Western blotting, and immunostaining, antibody polymerization around the antigen-bound antibody increases the number of labeled antibodies per antigen, enhancing the signal. In immunoprecipitation, two mechanisms of sensitization are possible, depending on the method. When the first antibody (primary antibody) is immobilized on beads, polymerization occurs around the primary antibody immobilized on the beads in the presence of excess antibody, resulting in multivalency, which improves binding affinity and increases antigen recovery. In addition, free antibody also polymerizes, forming an insoluble precipitate while still capturing the antigen, thereby increasing antigen recovery. In the method of capturing the antigen-primary antibody by binding a secondary antibody that binds to the antigen-recognizing antibody to beads, the addition of domain-swapped Protein G dimers causes primary antibody polymerization, increasing the primary antibody recovery rate per secondary antibody. In addition, as in the previous example, free primary antibody also polymerizes, forming an insoluble precipitate while still capturing the antigen, thereby increasing antigen recovery.

[0041] The first antibody (primary antibody) is an antibody, preferably immunoglobulin G (IgG), having an Fc region so that it can be cross-linked and polymerized by a domain-swapped protein G dimer.

[0042] The second antibody (secondary antibody) may be an antibody fragment (antigen-binding fragment) as long as it is capable of binding to the first antibody (primary antibody). Examples of antigen-binding fragments include Fab, F(ab)', Fv, scFv, and sdAb. Preferably, the antigen-binding fragment is Fab.

[0043] The "label" attached to the first antibody or second antibody or antigen-binding fragment thereof can be any label known in the art, such as a fluorescent label, an enzyme, biotin (biotin / avidin), a radioactive label, a luminescent label (including bioluminescence and chemiluminescence), or a magnetic label.

[0044] Fluorescent labels include various fluoresceins, rhodamines, phycobiliproteins such as phycoerythrin and phycocyanin, FITC, TRITC, Texas Red, ATTO dyes, Alexa Fluor, DyLight dyes, Cy dyes, and Qdot; enzymes include horseradish peroxidase, alkaline phosphatase, glucose 6-phosphate dehydrogenase, and β-galactosidase; and radioactive labels include 3 H, 125 I, 35 S, 14 C. 32 P, 33 P, etc.; luminescent labels include acridinium ester, thioester, sulfonamide, luminol, isoluminol, and phenanthridinium ester; and magnetic labels include various magnetic beads, etc.

[0045] Signals from labels are detected appropriately depending on the method used. Fluorescent labels, luminescent labels, and magnetic labels are detected as signals, respectively, via fluorescence, luminescence, and magnetism. In the case of enzymes, the luminescence or color generated by reacting with a substrate is detected. In the case of biotin, detection is performed by reacting a biotin-labeled antibody with avidin labeled with a dye, enzyme, or fluorescent substance. In the immunoassay of the present invention, the signal from the label is enhanced by the accumulation of the first or second antibody around the antigen-bound antibody, allowing for sensitive detection of minute amounts of target molecules.

[0046] 3. Immunoassay enhancers The present invention also provides an immunoassay enhancer comprising the domain-swapped protein G dimer described above. The enhancer is universally applicable regardless of the type of antigen (target molecule), antibody, or immunoassay.

[0047] The immunoassay sensitizer of the present invention contains a domain-swapped protein G dimer as an essential component, and may contain reagents, buffer solutions, and the like necessary for immunoassays, as necessary. [Example]

[0048] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0049] Example 1: Preparation of Protein G dimer We modified a stabilized mutant of protein G (SEQ ID NO: 1, PDBID: 2ZW0; Watanabe et al., Biol Chem (2009) 284 pp. 12373-83) created in a previous study to generate two types of protein G dimers with different orientations: a domain-swapped dimer and a tandem dimer.

[0050] Domain-swapping protein G dimers can be oriented in two ways: head-to-head, with the N-terminus or C-terminus facing each other, or tail-to-tail. Here, we constructed a head-to-head dimer by inserting a domain-swapping-inducing sequence (hereinafter referred to as "linker"), (Pro)3-Cys-(Pro)3 (SEQ ID NO: 16), between V21 and D22 in the (L2) loop of a stabilizing mutant (PDBID: 2ZW0).

[0051] The tandem dimer was constructed by linking a stabilized mutant of Protein G (SEQ ID NO: 1, PDBID: 2ZW0) with a (Pro)6 linker (SEQ ID NO: 20). The tandem dimer is oriented tail-to-head, with the C-terminus and N-terminus linked.

[0052] The amino acid sequences of the open reading frames of the domain-swapping dimer and tandem dimer are shown below. Both dimers contained a polyhistidine tag and a thrombin recognition sequence at the C-terminus. Domain-swapping dimer MGSSHHHHHH SSGLVPRGSH MDTYKLILNG KTLKGETTTE AV PPPCPPP D AATAEKVFKQ YANEHGVDGE ​​WTYDPETKTF TVTE* (SEQ ID NO: 21) Tandem Dimer MGSSHHHHHH SSGLVPRGSH MDTYKLILNG KTLKGETTTE AVDAATAEKV FKQYANEHGV DGEWTYDPET KTFTVTE PPP PPP DTYKLIL NGKTLKGETT TEAVAATAEK VFKQYANEHG VDGEWTYDPE TKTFTVTE* (SEQ ID NO: 22) 2nd-4th place: GSS Linker 5-10: Polyhistidine tag Positions 11-20: thrombin recognition sequence 21st: Methionine, a cloning artifact 22nd: Protein G dimer (underlined part is linker)

[0053] Each protein G dimer was prepared according to the following procedure. 1) The expression vector was transformed into E. coli BL21 (DE3) and pre-cultured overnight at 37°C in LB medium containing antibiotics. 2) Add the pre-culture solution to any medium and measure the OD 600nmWhen the pH was 0.6-0.7, IPTG was added to induce expression, and the mixture was cultured overnight at 28°C. 3) The next day, the bacterial solution was centrifuged at 6000 × g for 30 minutes at 4°C to collect the bacterial cells, which were then suspended in 50 mM Tris-HCl (pH 8.0) and ultrasonically disrupted on ice. 4) After disruption, the solution was centrifuged at 12,000 x g for 30 minutes at 4°C, and the supernatant was collected. 5) Add 500 mM NaCl and 10 mM imidazole (pH 8.0) to the supernatant and load it onto a Ni-NTA column. Wash with 30 mL of wash buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole (pH 8.0)) and elute with 10 mL of elute buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 500 mM imidazole (pH 8.0)). 6) Collect several tens of μL of the eluted fraction and perform reducing and non-reducing SDS-PAGE. 7) All remaining eluted fractions were placed in a dialysis membrane with a molecular weight cutoff of 3500 and dialyzed overnight against 50 mM Tris-HCl (pH 8.0), 150 mM NaCl. 8) The dialyzed sample was subjected to size exclusion chromatography on a Superdex75 10 / 300 column, using 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, to purify the dimer fraction. 9) A portion of the purified dimer fraction was collected, the concentration was measured, and the yield was calculated.

[0054] SDS-PAGE revealed that the domain-swapped dimer was expressed in the soluble fraction of E. coli BL21(DE3) as a disulfide-linked dimer (Fig. 4). The yield was high, exceeding 20 mg / 1 L culture.

[0055] Example 2: Immunoassay using protein G dimer An experiment was conducted to determine whether the protein G dimer prepared in Example 1 can enhance the sensitivity of indirect ELISA using vascular endothelial growth factor (VEGF) as the test substance and an anti-VEGF antibody (Avastin) as the primary antibody.

[0056] Materials and measuring equipment: Test substance: VEGF121 (pET-16b, with a polyhistidine tag and Factor X recognition sequence attached to the N-terminus) The test substances were prepared according to the following procedure. 1) Transform the expression vector into E. coli BL21 (DE3) and pre-incubate overnight at 37°C in LB medium containing antibiotics. 2) Add the pre-culture solution to any medium and measure the OD 600nm When the pH was 0.6-0.7, IPTG was added to induce expression, and the mixture was cultured overnight at 28°C. 3) The next day, the bacterial solution was centrifuged at 6000 × g for 30 minutes at 4°C to recover the bacterial cells, which were then suspended in 50 mM Tris-HCl (pH 8.0) and ultrasonically disrupted on ice. 4) After disruption, centrifuge the solution at 12,000 x g for 30 minutes at 4°C and collect the pellet. 5) Add 6 M GuHCl, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl (10 mL). Shake gently and leave at room temperature overnight. 6) After leaving the sample overnight, the sample was centrifuged at 4°C, 12,000 x g, for 10 minutes, and the supernatant was collected. Imidazole (pH 8.0) [10 mM] was added to the supernatant, and the column was loaded onto a Ni-NTA column. The column was washed with 10 mL of wash buffer [50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole (pH 8.0), 6 M GuHCl], and eluted with 5 mL of elute buffer [50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 500 mM imidazole (pH 8.0), 6 M GuHCl]. The remaining eluted fractions were all placed in a dialysis membrane with a molecular weight cutoff of 3500 and dialyzed overnight against 50 mM Tris-HCl (pH 8.0), 150 mM NaCl. The external dialysis solution was replaced with the same composition and volume, and dialyzed again overnight. 7) The dialyzed sample was collected and centrifuged at 12,000 xg for 30 minutes at 4°C to collect the supernatant.

[0057] Primary antibody: Avastin for intravenous infusion 100 mg / 4 mL (Chugai Pharmaceutical Co., Ltd.) ·Secondary antibody (Peroxidase AffiniPure TM F(ab')2Fragment Goat Anti-Human IgG, F(ab')2fragment specific (min X Bov, Hrs, Ms Sr Prot), Jackson ImmunoResearch) Domain-swapping and tandem protein G dimers prepared in Example 1 ·ABTS One Component HRP Microwell Substrate(SURMODICS) Blocking agent: SuperBlock (PBS) Blocking Buffer (Thermofisher) Dilution buffer for each sample (1x PBS) Washing buffer (1x PBS-T) 96-well plate: Medisorp (Thermofisher) Absorbance plate reader (Sunrise, TECAN)

[0058] Experimental procedure (indirect ELISA) 1. Add the test substance to the plate and let it stand at 4℃ for 1 hour. 2. Remove the target molecule solution from the well, add 300 μL of blocking solution, and let stand at room temperature for 1 hour. 3. Remove the blocking solution from the wells, dilute the primary antibody to a final concentration of 1000 nM (150 μg / mL), add 100 μL of the diluted solution, and let stand at room temperature for 1 hour. 4. Add S10 to 100 μL of diluted primary antibody solution to a final concentration of 1000 nM and pipette. 1x PBS was added to the control. After preparation, let stand at room temperature for 1 hour. 5. Remove the solution from the wells and wash with PBS-T (200 μL) x 3. 6. Add 100 μL of 10,000-fold diluted secondary antibody and let stand at room temperature for 1 hour. 7. Remove the solution from the wells and wash with PBS-T (200 μL) x 3 8. Add 100 μL of ABTS One Component HRP Microwell Substrate and react at room temperature. Measure the absorbance at 405 nm using a plate reader. 9. The detection limit and signal increase were calculated based on the measured values. The detection limit was defined as the immobilization amount at which the average signal value was 3σ or more from the average blank signal, but no signal values ​​that did not meet 3σ were observed as the immobilization amount increased. The signal increase was calculated by dividing the signal value with the sensitizer by the signal value without the sensitizer for the corresponding immobilization amount of the test substance.

[0059] result In the case of domain-swapped protein G dimer, ELISA results at high and low immobilization levels of the test substance showed that the detection limit with the addition of the enhancer (0.04 ng / well) was 500-fold higher than the detection limit without the enhancer (20 ng / well) (Figures 5 and 6). The maximum signal also increased approximately 1.1-2.9-fold compared to the unimmobilized state (Figures 7 and 8). On the other hand, in the case of tandem protein G dimer, when similar ELISA was performed at high immobilization levels, the detection limit was 5 ng / well and the signal increase was approximately 1.1-1.4-fold (Figures 9 and 10). These results demonstrate that domain-swapped protein G dimer dramatically improves the sensitivity of immunoassays, demonstrating its superiority over tandem protein G dimer.

[0060] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for detecting or quantifying a target molecule in a sample by immunoassay, comprising: (1) contacting a sample with a first antibody capable of specifically binding to a target molecule and a domain-swapped protein G dimer capable of binding to the Fc region of the antibody; and (2) The following step (a) or (b): (a) the first antibody is labeled, and a signal from the label is detected; (b) contacting a labeled second antibody or an antigen-binding fragment thereof capable of binding to the first antibody with the sample of step (1) and detecting a signal from the label; The method comprising:

2. 2. The method of claim 1, wherein the immunoassay is any one selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blotting, immunostaining, and immunoprecipitation.

3. The method of claim 1, wherein the domain-swapping protein G dimer is a head-to-head dimer.

4. The method of claim 1, wherein the domain-swapped protein G dimer has a peptide linker in loop 2 or loop 4 of the B1 domain of protein G.

5. The method according to claim 4, wherein the peptide linker is a peptide linker consisting of 3 to 10 amino acid residues containing one cysteine ​​in a polyproline sequence.

6. The method according to claim 1, wherein the protein G has any one of the following amino acid sequences 1) to 4): 1) an amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; 2) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, (i) an amino acid sequence in which an amino acid is substituted at at least one position selected from positions 2, 4, 17, 19, 23-25, 29, 44, 46, and 51 of SEQ ID NO: 1, and / or (ii) an amino acid sequence in which an amino acid is deleted, substituted, or added at at least one position selected from positions 21, 22, and 47-50 of SEQ ID NO: 1; 3) an amino acid sequence in which an amino acid is substituted at positions 17 and / or 29 of SEQ ID NO: 1; 4) An amino acid sequence having at least one of the following substitutions in SEQ ID NO: 1: alanine for valine at position 29, proline or glycine for valine at position 21, and proline or glycine for aspartic acid at position 22.

7. The method of claim 1, wherein Protein G has an amino acid sequence shown in any one of SEQ ID NOs: 1 to 12.

8. The method of claim 1, wherein the domain-swapped Protein G dimer capable of binding to the Fc region of an antibody comprises the following amino acid sequence (i) or (ii): (i) the amino acid sequence shown in SEQ ID NO: 19; (ii) An amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

19.

9. A domain-swapped Protein G dimer capable of binding to an Fc region of an antibody, having a peptide linker in loop 2 or loop 4 of the B1 domain of protein G; A domain-swapping protein G dimer, wherein the protein G has any of the following amino acid sequences 1) to 4): 1) an amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; 2) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, (i) an amino acid sequence in which an amino acid is substituted at at least one position selected from positions 2, 4, 17, 19, 23-25, 29, 44, 46, and 51 of SEQ ID NO: 1, and / or (ii) an amino acid sequence in which an amino acid is deleted, substituted, or added at at least one position selected from positions 21, 22, and 47-50 of SEQ ID NO: 1; 3) an amino acid sequence in which an amino acid is substituted at positions 17 and / or 29 of SEQ ID NO: 1; 4) An amino acid sequence having at least one of the following substitutions in SEQ ID NO: 1: alanine for valine at position 29, proline or glycine for valine at position 21, and proline or glycine for aspartic acid at position 22.

10. The domain-swapped Protein G dimer according to claim 9, wherein Protein G has an amino acid sequence shown in any one of SEQ ID NOs: 1 to 12.

11. The domain-swapping protein G dimer according to claim 9, wherein the peptide linker is a peptide linker consisting of 3 to 10 amino acid residues containing one cysteine ​​in a polyproline sequence.

12. The domain-swapped protein G dimer according to claim 9, wherein the peptide linker is any one selected from PPCPPP, PPPCPP, PPPCPPP, PPPCPPPP, and PPPPCPPP.

13. 10. The domain-swapped protein G dimer according to claim 9, wherein the peptide linker is located at a position corresponding to between positions 20 and 21, 21 and 22, 22 and 23, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or 50 and 51 of SEQ ID NO:

1.

14. A domain-swapped Protein G dimer capable of binding to the Fc region of an antibody, comprising the following amino acid sequence (i) or (ii): (i) the amino acid sequence shown in SEQ ID NO: 19; (ii) An amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

19.

15. A sensitizer for immunoassays, comprising the domain-swapped protein G dimer according to any one of claims 9 to 14.

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