JP5487570B2 - Method for producing fusion protein of antibody and protein - Google Patents

Description

  The present invention relates to a method for producing a fusion protein of an antibody and a protein, and a fusion protein of an antibody and a protein.

  In the open sandwich method, a VH region polypeptide and a VL region polypeptide of an antibody that specifically recognizes an antigen are prepared, one of the polypeptides is labeled with a reporter molecule to form a labeled polypeptide, and the other polypeptide is labeled. In this method, an immobilized polypeptide is immobilized on a solid phase, an antigen-containing sample and a labeled polypeptide are brought into contact with the solid phase, and the amount of reporter molecule of the labeled polypeptide bound to the immobilized polypeptide is measured. The open sandwich method is an immunoassay that utilizes the phenomenon that VH and VL increase the association constant in the presence of antigen, so the interaction between VH and VL in the absence of antigen is small, and the association constant is present in the presence of antigen. It is indispensable to greatly change. In order to accurately estimate the interaction between VH and VL, it is necessary to express and purify VH and VL separately, and much time and effort have been required so far.

  As a method for examining the interaction between VH / VL without expressing and purifying VH and VL, a method called split-Fv has been reported (Patent Document 1). This method uses VH and VL as a fusion protein in each of the phage coat proteins pVII and pIX by using differently the amber (stop) codon contained in the expression vector and E. coli that does not show the amber suppressor function. The expression method and the method in which one of VH or VL is used as a fusion protein of phage coat protein pVII or pIX and the remaining VH or VL is secreted and expressed are used separately. In this method, by changing the type of E. coli with the same vector, it is possible to evaluate the affinity of the VH / VL complex for the antigen and the interaction between the VH / VL in the absence of the antigen.

  However, this method uses two coat proteins of the phage and the phage becomes unstable, so that VH / VL is not expressed on the phage, or between VH / VL expressed as a fusion protein of coat proteins pVII and pIX. The distance between the two is not sufficient for interaction, and the affinity for the antigen is reduced.

  In addition, as a method for evaluating the VH / VL interaction based on the same concept, affinity evaluation is performed with a single-chain antibody (scFv) -displayed phage, and then terminated at the VH / VL linker of scFv using a recombinant enzyme. A method for expressing VH and VL as separate components (for example, MBP fusion VL and VH-displayed phage) by inserting a sequence such as a codon by homologous recombination has been reported (Non-patent Document 1). In this method, (1) pIII, which is less toxic among phage coat proteins, is used for scFv display. (2) Since it is scFv, VH / VL always exists as a pair. The advantage is that panning can be performed. In homologous recombination at the linker part, various sequences can be inserted while maintaining the orientation by using an enzyme with high sequence specificity such as Cre recombinase. For example, fusion with MBP with high solubility and expression It is possible to secrete VH or VL as a protein. However, in the scFv, the VH / VL linker is usually about 15 amino acids, but in this method, since the two recombinase recognition sequences are present in the linker portion, the linker length is as long as about 40-45 amino acids. . Therefore, there is a problem that the display rate on the phage decreases. In addition, recombination into a vector for evaluating VH / VL interaction requires expensive recombination enzymes, and the recombination efficiency is not high.

  When producing an enzyme-labeled antibody, the most common chemical labeling method involves culturing antibody-producing hybridoma cells, purifying the antibody secreted in the culture supernatant, exchanging the buffer, and then synthesizing the conjugate with the enzyme using a crosslinking reagent. It is. However, in this method, it is necessary to highly purify the antibody before the reaction, remaining unlabeled antibody, inactivation by labeling on the antigen binding site, and the like. As another method, heterologous expression in Escherichia coli of a fusion protein of a cloned antibody fragment such as scFv or Fab and an enzyme is performed. This method enables high yields, almost 100% modification rate, site-specific modification, etc., but cloning of genes encoding heavy and light chains of antibodies, creation of expression vectors incorporating them, etc. A complicated experiment is required. Therefore, as a new method, a method of expressing an antibody fragment and enzyme fusion protein by inducing artificial trans-splicing in a hybridoma cell has been developed (Non-patent Document 2). There was a problem that the expression efficiency of the fusion protein was low.

Abstracts of the 73rd Annual Meeting of the Society of Chemical Engineers of Japan (2008), p. 366 “Efficient conversion of phage-displayed antibodies into open sandwich ELISA using Cre recombinase” 119 pages of abstracts from the 57th Annual Meeting of the Japanese Society for Biotechnology, "Expression and Analysis of Antibody Variable Region-Human Placental Alkaline Phosphatase Fusion Protein" International Publication WO 2004/0166782

  Until now, enzyme-labeled antibodies used in ELISA and the like have been used for chemical labeling methods in which antibodies purified from the culture supernatant of hybridoma cells and the like are chemically linked to a reporter enzyme using a crosslinking reagent, etc. It has been produced by a method in which a fusion protein (enzyme fusion antibody) linked to a Fab-type antibody or the like at the gene level is expressed in large quantities in microorganisms such as Escherichia coli. However, the chemical labeling method has a problem that it is necessary to highly purify the antibody before the reaction, unlabeled antibody remains, inactivation by labeling on the antigen binding site, and the like. In addition, the method for expressing an enzyme fusion antibody requires complicated experimental operations such as cloning of genes encoding the heavy and light chains of the antibody and the production of expression vectors thereof. In recent years, a method for easily preparing an enzyme fusion antibody by artificial trans-splicing in antibody-producing cells has been developed, but there is a problem that the protein expression efficiency is low. An object of the present invention is to provide a method for labeling an antibody simply and in a short time.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors induce expression of a light chain fusion protein in which a target protein is fused to the C terminus of an antibody light chain or light chain constant region domain in antibody-producing cells. As a result, it was found that the light chain fusion protein associates with the antibody heavy chain, and as a result, the antibody labeled with the target protein can be secreted outside the cell, and the present invention has been completed.

  That is, according to the present invention, a gene encoding a light chain or light chain constant region domain of an antibody and a gene encoding a protein, a fusion protein of the light chain or light chain constant region domain of the antibody and the protein There is provided a method for producing a fusion protein of an antibody and a protein, which comprises introducing an expression vector having an expression vector linked so as to be expressed into a cell that expresses the antibody or a part thereof, and expressing it.

Preferably, the protein is an enzyme or a fluorescent protein.
Preferably, the protein is alkaline phosphatase, β-galactosidase, luciferase, peroxidase.

Preferably, the cell expressing the antibody or a part thereof is a cell expressing an antibody heavy chain gene and an antibody light chain gene, or a cell expressing an antibody heavy chain gene.
Preferably, the expression vector is an expression vector in which a gene encoding a protein is linked to the 3 ′ end of a gene encoding a light chain or light chain constant region domain of an antibody.

  According to the present invention, there is further provided an antibody-protein fusion protein produced by the above-described method of the present invention, wherein the protein is bound to the antibody light chain.

Preferably, the fusion protein of the present invention comprises (i) a fusion protein comprising two antibody heavy chains and two antibody light chains having a protein on the C-terminal side,
(ii) a fusion protein comprising two antibody heavy chains, one antibody light chain having a protein on the C-terminal side and one antibody light chain,
(iii) a fusion protein comprising two antibody heavy chains and two antibody light chain constant region domains having a protein on the C-terminal side, or
(iv) a fusion protein comprising two antibody heavy chains, one antibody light chain constant region domain having a protein on the C-terminal side, and one antibody light chain;
Any of these, or especially in the case of IgM and IgA, are multimers thereof.

  The present invention further provides an immunoassay kit containing the above-described fusion protein of the present invention.

According to the present invention, a fusion protein comprising the above (iii) two antibody heavy chains and two antibody light chain constant region domains having a protein on the C-terminal side, or (iv) an antibody heavy chain 2 A fusion protein composed of an antibody light chain constant region domain having a protein on the C-terminal side and an antibody light chain and an antibody light chain in the presence of an antigen, There is provided an immunoassay method comprising detecting an interaction between an antibody and an antibody light chain.
In the above immunoassay method, preferably, antibody heavy chain and antibody light chain that interact specifically in the presence of an antigen are selected.

  According to the method of the present invention, it is possible to produce a target labeled antibody simply and in a short time simply by introducing a vector that expresses a light chain fusion protein. The method of the present invention is a highly versatile method because it can be applied to various antibodies with the same vector as long as the same target protein is labeled. Furthermore, when a fusion protein of a light chain constant region domain and an enzyme such as alkaline phosphatase is expressed, immunoassay by the open sandwich method is possible using the secreted enzyme-labeled heavy chain and light chain. The difference between the method of the present invention and the conventional chemical labeling method is that a reaction step is unnecessary and the labeling site can be specified. Also, the difference from the method of expressing the enzyme fusion antibody in E. coli is that it is not necessary to clone an antibody gene for each antibody to be labeled and newly prepare an expression vector into which the antibody gene is inserted. Compared to antibody labeling by trans-splicing, higher expression levels can be expected because quaternary structure formation at the protein level is used. When hybridoma cells secreting enzyme-labeled antibodies produced by the method of the present invention are cultured, ELISA can be directly performed using the culture supernatant. In addition, when a fusion protein of CL and enzyme is expressed, VL-CL originally associated with the heavy chain and an antibody fragment labeled with enzyme and lacking VL can be easily obtained. Can be used to perform an open sandwich ELISA using antigen-dependent changes in VH / VL interaction (FIG. 10).

Hereinafter, the present invention will be described in more detail.
In the method for producing a fusion protein of an antibody and a protein according to the present invention, an expression having a gene encoding a light chain or light chain constant region domain of the antibody and a gene encoding a protein linked to the 3 ′ end side thereof Introducing the vector into a cell expressing the antibody or part thereof, and expressing the vector.

  In the present invention, a fusion protein in which a protein of interest such as an enzyme is fused to the C-terminus of an antibody light chain (light chain variable region (VL) and light chain constant region (CL)) or light chain constant region domain (CL) By introducing a vector capable of expressing (light chain fusion protein) into an antibody-producing cell, the light chain fusion protein expressed in the cell associates with the antibody heavy chain, and as a result, the target protein is linked. Antibodies are synthesized and further secreted extracellularly (FIG. 10).

  It is also possible to change the shape of the obtained antibody depending on the shape of the light chain to be fused. For example, when a fusion protein of VL-CL and an enzyme is expressed, it is labeled with one or two molecules of the enzyme. A full-length antibody (a fusion protein described in (i) or (ii) below), and when expressing an enzyme fusion protein with CL, VL-CL derived from the introduced cell, An enzyme-labeled antibody fragment (a fusion protein described in (iii) or (iv) below) labeled with one or two molecules of enzyme and lacking VL can be obtained (FIG. 10).

That is, according to the present invention, for example, any of the following fusion proteins and multimers thereof can be produced.
(i) a fusion protein comprising two antibody heavy chains and two antibody light chains having a protein on the C-terminal side;
(ii) a fusion protein comprising two antibody heavy chains, one antibody light chain having a protein on the C-terminal side and one antibody light chain,
(iii) a fusion protein comprising two antibody heavy chains and two antibody light chain constant region domains having a protein on the C-terminal side, or
(iv) A fusion protein comprising two antibody heavy chains, one antibody light chain constant region domain having a protein on the C-terminal side, and one antibody light chain.

  The protein (protein fused to the antibody) used in the present invention is preferably a protein that can be used as a label, and for example, an enzyme or a fluorescent protein is preferable. As the enzyme, alkaline phosphatase, β-galactosidase, luciferase, peroxidase, or the like can be used. As the fluorescent protein, green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), cyan A fluorescent protein (CFP) or a red fluorescent protein (RFP) can be used, but is not limited thereto.

  In the present invention, a gene encoding a light chain or light chain constant region domain of an antibody and a gene encoding a protein are expressed so that a fusion protein between the light chain or light chain constant region domain of the antibody and the protein can be expressed. The linked expression vector is introduced into a cell that expresses the antibody or a part thereof and expressed.

  The gene encoding the protein may be linked to the 5 ′ end side of the gene encoding the light chain or light chain constant region domain of the antibody, or may be linked to the 3 ′ end side, preferably 3 ′. It can be linked to the terminal side. Further, the gene encoding the protein may be directly linked to the 5 ′ end side or 3 ′ end side of the gene encoding the light chain or light chain constant region domain of the antibody, or may be linked via an appropriate linker sequence. However, it can be preferably linked via an appropriate linker sequence. As the linker sequence, for example, Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser or Gly-Gly-Gly-Gly-Ser can be used. is not.

  The type of expression vector is not particularly limited, and a gene encoding a light chain or light chain constant region domain of the introduced antibody and a gene encoding a protein are expressed and the light chain or light chain constant region domain of the antibody is expressed. Any expression vector can be used as long as it can express a fusion protein with the protein. As an expression vector, a viral vector or a plasmid that can be autonomously propagated in a host cell or can be integrated into the host cell chromosome can be used. Viral vectors are derived from SV40, adenovirus, retrovirus, lentivirus, etc., and plasmid DNA includes plasmids derived from Escherichia coli, Bacillus subtilis, or yeast, but selection in prokaryotic cells and antibody-producing cells. It is desirable to have a marker gene.

  In the present invention, the above expression vector is introduced into a cell that expresses the antibody or a part thereof and expressed. The cell expressing the antibody or a part thereof may be a cell expressing the antibody heavy chain gene and the antibody light chain gene to produce a full-length antibody, or a cell expressing the antibody heavy chain gene. Good. Examples of cells expressing the antibody or a part thereof include bacteria (such as E. coli), yeast, animal cells (myeloma, hybridoma, COS cell, CHO cell, etc.), insect cells, etc., but animal cells (myeloma, Hybridomas, COS cells, CHO cells, etc.) are preferred.

  The method of introducing the expression vector into the cell is not particularly limited, and examples thereof include calcium phosphate method, electroporation method, spheroplast method, lithium acetate method, lipofection method, etc. It can also be carried out using the culture supernatant.

  According to the present invention, (iii) a fusion protein comprising two antibody heavy chains and two antibody light chain constant region domains having a protein on the C-terminal side, or (iv) two antibody heavy chains A fusion protein composed of one antibody light chain constant region domain having a protein on the C-terminal side and one antibody light chain, and the antibody light chain in the presence of an antigen, An immunoassay can be performed by detecting the interaction with the antibody light chain, whereby the antibody heavy chain and antibody light chain that specifically interact in the presence of the antigen can be selected. The above-described immunoassay can be performed by the open sandwich immunoassay described below.

  Proteinaceous antigens are generally measured by a method using two types of antibodies called a sandwich method. The sandwich method needs to prepare two types of antibodies that can simultaneously bind to an antigen, but has the advantage of high specificity and sensitivity. However, small molecules with a molecular weight of 1000 or less are too small to be sandwiched between two types of antibodies. That is, since a small molecule having a molecular weight of 1000 or less is a unitary antigen having only one antigenic determinant, it is difficult to sandwich between two types of antibodies. For this reason, such small molecules are usually measured by a method called a competitive method. However, the competitive method has the drawbacks that it is difficult to set conditions, the sensitivity is low, and the measurement operation requires considerable attention.

  The present inventors have reported an immunoassay called an open sandwich immunoassay as a method capable of non-competitively measuring even a small molecule without such drawbacks. This method basically uses the principle that “the variable region (antigen-binding site) of an antibody is unstable without an antigen but is stabilized when an antigen binds”. An antibody is composed of two chains, an H chain and an L chain, and each antigen binding site is called VH and VL, and these constitute a variable region Fv which is the smallest unit capable of recognizing an antigen. Recently, gene fragments encoding VH and VL can be easily cloned using phage display methods, etc., but the binding between VH and VL is non-covalent and unstable in many cases. In most cases, it is used as a single chain antibody (scFv).

  The present inventors have found that this unstable Fv may be stabilized when an antigen binds, and by using this, the antigen concentration can be measured easily and rapidly with higher sensitivity. In other words, the VL fragment was immobilized on a plate, and the VH fragment combined with phage or alkaline phosphatase was mixed with a sample containing the antigen, washed once, and then the phage or enzyme immobilized on the plate. It was found that if the amount was measured, it showed a very good correlation with the antigen amount (UEDA, H. et al. Nature Biotechnol. 14, 1714-1718 (1996)).

Furthermore, the present inventors have developed a method for easily examining whether the antibody on hand is suitable for the Serpen Sandwich method (Aburatani, T. et al., Anal. Chem. 75,
4057-4064 (2003); Hiroshi Ueda. New immunoassay that can measure small molecules non-competitively Bio Medical Quick Review Nets No.027 (2004); and Hiroshi Ueda. Measurement ". Biochemistry, 76 (7), 670-674 (2004)). Using this method (split Fv system), which is very similar to a commercially available phage antibody system, changes both the antigen-binding ability of the antibody variable region of the hybridoma on hand and the strength of the VH / VL interaction by changing the Escherichia coli that produces the phage. Can be easily examined, and antibodies with better properties can be selected.

  In the present invention, (iii) a fusion protein comprising two antibody heavy chains and two antibody light chain constant region domains having a protein on the C-terminal side, or (iv) two antibody heavy chains and C A fusion protein composed of one antibody light chain constant region domain having a protein on the terminal side and one antibody light chain is contacted with the antibody light chain in the presence of an antigen, so that the fusion protein and the antibody light chain An immunoassay can be performed by detecting an interaction with. Since a protein having a function as a label is bound to the fusion protein, the interaction between the fusion protein and the antibody light chain can be detected by detecting this protein.

Using the antibody obtained by the method of the present invention and having a weak VH / VL interaction in the absence of an antigen and a strong VH / VL interaction in the presence of an antigen, for example, the following measurement kit should be prepared. Is possible.
(1) The VL fragment is immobilized on a tube or microplate using biotin / avidin interaction or using physical adsorption.
(2) A fusion protein of a VH fragment and a reporter enzyme (for example, alkaline phosphatase) is prepared, and this is brought into contact with a solid phase on which VL is immobilized together with a sample for a certain period of time.
(3) After washing, the immobilized enzyme activity is measured and used as an index of the antigen concentration in the sample.

It is also possible to produce the following measurement kit.
(1) The VH fragment and the VL fragment are labeled with two kinds of fluorescent dyes (for example, fluorescein and rhodamine) that overlap each other in absorption and fluorescence spectra.
(2) These are mixed with the sample, and only the fluorescent dye on the short wavelength side is excited with excitation light after about 5 minutes. By measuring the fluorescence intensity derived from two types of fluorescent dyes, the fluorescence energy transfer phenomenon due to the association of VH / VL can be detected. The ratio of the two fluorescence intensities is used as an index of the antigen concentration in the sample. In this method, the antigen concentration can be measured in a short time and without a washing operation, compared to the previous method.

It is also possible to prepare the following measurement kit.
(1) VH fragment and VL fragment are expressed in E. coli as a fusion protein with two kinds of enzyme fragments (for example, LacZΔα and LacZΔω) that are not active alone or increase in activity when they are close to each other. And purify.
(2) Mix two types of fusion protein and sample, wait for a certain period of time, mix with substrate (eg, luminescent substrate Galacton Plus, Tropix, Bedford, MA), and measure the activity of the fusion protein complex in the sample. It is used as an index of the antigen concentration. This method can measure the antigen concentration much more sensitively than the previous two methods, and does not include a washing operation (Yokozeki et al., Anal. Chem. 74 (11), 2500 -2504, 2002).

  As a target to be measured by the above method, firstly, a specific protein, peptide, various hormones, narcotics, therapeutic drugs, etc. in serum in a clinical test are considered. Further, chemical substances and agricultural chemicals suspected of toxicity such as dioxin, bisphenol A, nonylphenol and the like in the environmental water are also objects to be measured by the present invention.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Experimental material vector
pSV-Vμ1: A vector that expresses anti-NP (4-hydroxy-3-nitrophenylacetic acid) IgM heavy chain in eukaryotic cells (EMBO J., 1983, 2, 1373-1378). The one assigned by Dr. Neuberger of MRC Molecular Biology Laboratory in the UK was used.
pSEAP-His: A vector that can induce SEAP-Histag expression in eukaryotic cells (Abstracts of the 57th Annual Meeting of the Biotechnology Society (2005), p. 119, presentation number 3C14-2 “Antibody variable region-human placental alkaline phosphatase fusion protein Expression and Analysis "
pUCλ: a vector having the coding site of the anti-NP antibody λ chain (J. Biochem. 1997, 122, 322-329).

Primer
VLMfeIrev: 5'-GGTCCAATTGCAGGCTGTTGTGACTCAGGAA-3 '(SEQ ID NO: 1)
CLMfeIrev: 5'-GCAACAATTGCAGCCCAAGTCTTCGCCATCAG-3 '(SEQ ID NO: 2)
C _L (G ₄ S) ₁ AflIIfor:
5'-CCTACTTAAGGACTCACCCGCGCTACCACCACCACCGGAACAGTCAGCACGGGACAA-3 '(SEQ ID NO: 3)
C _L (G ₄ S) ₂ AflIIfor:
5'-CCTACTTAAGGACTCACCCGCGCTACCACCACCACCACTTCCTCCTCCTCCGGAACAGTCAGCACGGGACAA-3 '(SEQ ID NO: 4)

Cell line
COS-1: African green monkey kidney cells transformed with SV40
J558L: An antibody-producing cell that has been mutated to express and secrete the anti-NP antibody λ chain. Purchased from The European Collection of Cell Cultures. (Proc Natl Acad Sci US A. 1983, 80, 825-829)
XL-10 gold: E. coli with the following genotypes (Stratagene Co., La Jolla, CA)
Tet ^r Δ (mcrA) 183, Δ (mcrCB-hsdSMR-mrr) 173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac, The [F'proAB, lacI ^q , ZΔM15, Tn10 (Tet ^r ) , Tn5 (Kan ^r ), Amy]

Abbreviations used in the following examples are as follows.
LB: Medium containing 1% bacto tryptone, 0.5% yeast extract, 0.5% NaCl
LBA: LB containing 100 mg / ml ampicillin
LBA plate: LB agar containing 100 mg / ml ampicillin
SOC: Medium containing 2% bactotryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 20 mM glucose, 10 mM MgCl ₂
PBS: 10 mM phosphate buffer (pH 7.6) containing 137 mM NaCl and 2.7 mM KCl
TBST: 10 mM Tris-HCl buffer (pH 7.6) containing 150 mM NaCl and 0.1% Tween-20
TAE buffer: 40 mM Tris-acetate (pH 8.3) containing 1 mM EDTA
PCIA: phenol: chloroform: isoamyl alcohol (25: 24: 1) mixture

In all experiments, water purified with milliQ (Millipore Co., Billerica, MA) was used. Hereafter, milliQ water is indicated. Unless otherwise indicated, ordinary reagents used were those of Sigma (St. Louis, MO), Nacalai Tesque (Kyoto), Wako Pure Chemical (Osaka), Kanto Chemical (Tokyo). Oligo DNA was synthesized at Texas Genomics Japan (Tokyo) or Invitrogen (Tokyo).
T3000 thermocycler (Biometra, Goettingen Germany) was used for Polymerase chain reactions (PCR), and CEQ ^™ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA) was used for DNA sequencing.

Example 1
Outline of Experiment pV _L C _L (G ₄ ) expressing a fusion protein (λ chain-SEAP fusion protein) of anti-NP antibody λ chain and human placental alkaline phosphatase (SEAP) in COS-1 cells expressing anti-NP antibody heavy chain By introducing S) ₁ SEAP-His6 or pV _L C _L (G ₄ S) ₂ SEAP-His6, a SEAP-labeled antibody in which the λ chain-SEAP fusion protein is associated with the heavy chain is secreted (Fig. 1) and cultured. It was demonstrated that immunoassay such as ELISA was possible using the supernatant.

(1) Preparation of pV _L C _L (G ₄ S) ₁ SEAP-His6, pV _L C _L (G ₄ S) ₂ SEAP-His 6 First, pUCλ λ chain gene was amplified by PCR reaction, and Wizard SV Gel and Purification was performed using PCR Clean-Up System (Promega Co., Madison, Wis.). There are two types of primer combinations, V _L MfeIrev and C _L (G ₄ S) ₁ AflIIfor, and V _L MfeIrev and C _L (G ₄ S) ₂ AflIIfor. PCR reaction conditions are as follows. A schematic view of the obtained λ chain gene fragment is shown in FIG.

Reaction solution composition
pUCλ (90 μg / ml) 1 μl
Primer (C _L (G ₄ S) ₁ AflIIfor or C _L (G ₄ S) ₂ AflIIfor) (50 μM) 1 μl
Primer (V _L MfeIrev) (50 μM) 1 μl
10x ExTaq buffer (Mg ²⁺ 20 mM) (Takara bio Inc., Otsu) 10 μl
dNTP Mixture (2.5 mM each) (Takara bio Inc.) 8 μl
5 U / μl ExTaq DNA polymerase (Takara bio Inc.) 1 μl
milliQ water 78 μl

Reaction cycle
1, 96 ℃ 2 min
2, 94 ℃ 1 min
3, 55 ℃ 1 min
4, 72 ℃ 1 min
(2 to 4 30 times)
5, 72 ℃ 5 min
6, 16 ℃ ∞

  Each of the two λ chain gene fragments was inserted into a TA cloning vector by a ligation reaction using a TA cloning kit (Invitrogen Co., Carlsbad, Calif.). After a ligation reaction at 14 ° C for 16 hours, add 1 μl of the reaction solution to 100 μl of E. coli XL10-Gold competent cell on ice and let stand for 20 minutes, then place it in a 42 ° C heat block for 45 seconds to heat. I was shocked. After leaving still on ice for 2 minutes, 200 μl of SOC medium was added and cured at 37 ° C. for 30 minutes, applied to an LBA plate, and cultured at 37 ° C. overnight. The resulting colonies were inoculated into 4 ml of LBA and cultured with shaking at 37 ° C. overnight. Plasmid DNA was extracted from E. coli collected by centrifugation with QIAquick miniprep kit (Qiagen, Hilden, Germany), and sequenced according to the protocol of Beckman Coulter.

  Two TA cloning vectors into which the λ chain gene fragment has been inserted are 1 μl AflII restriction enzyme solution (New England BioLabs, Inc., Ipswich, Mass.), 1 μl MfeI restriction enzyme solution (New England BioLabs, Inc.), 5 μl A 10x BSA solution and 5 μl 10x NEB 4 buffer (New England BioLabs, Inc.) were added and allowed to stand overnight at 37 ° C. The λ chain gene fragment cleaved by restriction enzyme treatment was electrophoresed on a 1.5% agarose gel (TAE buffer), and then a band around 400 bp or 800 bp was excised, and the Wizard SV Gel and PCR Clean-Up System (Promega Co.) and dissolved in 50 μl of milliQ water. The pSEAP-His vector was similarly treated with restriction enzyme, and then electrophoresed (TAE buffer) on 1.5% agarose gel (Agarose S, NIPPON GENE CO., LTD., Toyama). Was extracted using Wizard SV Gel and PCR Clean-Up System (Promega Co.) and dissolved in 50 μl of milliQ water.

2 μl each of the λ chain gene fragment treated with restriction enzyme and pSEAP-His were mixed, 4 μl Ligation High Ver2 (TOYOBO CO., LTD., Osaka) was added thereto, and reacted at 16 ° C. for 30 minutes. After mixing with an equal volume of PCIA to completely inactivate the ligase, collect the supernatant (74 μl) containing DNA, add 7.4 μl 3 M sodium acetate and 148 μl ethanol, and add 20 ml at −80 ° C. After standing still and centrifuging at 11,600 g at 4 ° C. for 20 minutes, the resulting precipitate was washed with 70% ethanol, dried and suspended in 20 μl of milliQ water. Subsequently, 4 μl of ligation reaction solution was added to 100 μl of DH5α competent cell, and the ligation product was introduced into the cells in the same manner as described above, and then immediately 900 μl of SOC was added and incubated at 37 ° C. for 30 minutes. It was. After that, colonies obtained by applying to LBA plates and culturing overnight at 37 ° C were cultured overnight in 4 ml of LBA, and plasmid DNA was extracted from E. coli collected by centrifugation using QIAquick miniprep kit (Qiagen). Then, sequencing was performed according to the protocol of Beckman Coulter, and the gene structure of pV _L C _L (G ₄ S) ₁ SEAP-His6 and pV _L C _L (G ₄ S) ₂ SEAP-His 6 was designed (FIG. 2). It was confirmed to have

(2) Expression of fusion protein in COS-1 cells λ chain-SEAP fusion protein expression vectors pV _L C _L (G ₄ S) ₁ SEAP-His6 and pV _L C _L (G ₄ S) ₂ SEAP-His6 Were transfected into COS-1 cells with pSV-Vμ1, each encoding an anti-NP antibody heavy chain. Transfection was performed according to the protocol using Transfection (BIO-RAD Laboratories, Inc., Hercules, CA). No vector as negative control, pscFv-SEAP (72nd year of Chemical Engineering Society) instead of pV _L C _L (G ₄ S) ₁ SEAP-His6 and pV _L C _L (G ₄ S) ₂ SEAP-His6 as positive control The same transfection procedure was carried out with the addition of J204 “Abstract and Analysis of Antibody Fusion Proteins Using Eukaryotic Alkaline Phosphatase” (2007) J204.

Transfected COS-1 cells were cultured in D-MEM medium (SIGMA-Aldrich Co., containing 10% fetal bovine serum (Invitrogen Co.) and 100 units / ml penicillin, 100 μg / ml streptomycin (GIBCO). 10 ml of St. Louis, MO) was placed in a 100 mm IWAKI dish (AGC Techno Glass, Chiba) and cultured under conditions of 37 ° C. and 5% CO ₂ . After transfection, the culture supernatant 72 to 168 hours later was collected and analyzed.

(3) Measurement of SEAP activity of culture supernatant In order to confirm the expression of the λ chain-SEAP fusion protein or the SEAP-labeled antibody, the alkaline phosphatase activity of the transfected culture supernatant of COS-1 was measured. The culture supernatant was prepared by inactivating COS-1 cell-derived alkaline phosphatase by heat treatment at 65 ° C. for 30 minutes, and then dispensing 10 μl of the medium onto a Costar 96 well white plate (Corning Inc. Corning, NY). Then, 0.9 μl of luminescent substrate CDP-star solution (Tropix Inc., Bedford, Mass.), 9 μl of 10 × Buffer, and 80 μl of milliQ water were added. Thereafter, the mixture was incubated for 30 minutes at room temperature, protected from light, and luminescence was measured using Luminescencer-JNR (ATTO Co., Tokyo).

  The results of measuring the SEAP activity of the culture supernatant are shown in FIG. When the heat inactivation treatment at 65 ° C was performed, residual alkaline phosphatase activity was confirmed in all culture supernatants transfected with the λ chain-SEAP fusion protein expression vector, and λ chain-SEAP fusion protein or SEAP-labeled antibody was It was confirmed that it was secreted.

(4) Confirmation of fusion protein by Western-Blot In order to recover SEAP-labeled antibody contained in the culture supernatant, 10 μl of Goat anti-Mouse IgM Agarose (SIGMA-Aldrich Co.) was added to 1 ml of the culture supernatant. The mixture was stirred at room temperature using a rotator for 30 minutes, and agarose beads were collected by centrifugation. The agarose beads bound with the SEAP-labeled antibody were suspended in 20 μl of 2 × SDS-PAGE loading buffer) and heat-treated at 95 ° C. for 5 minutes. 10 μl of the supernatant obtained by centrifugation was collected, 1 μl of 2-mercaptoethanol was added, and the mixture was further heated at 95 ° C. for 5 minutes. This was applied to a total amount of 12% polyacrylamide gel and run at 10 mA for 30 minutes and at 20 mA for 90 minutes. After electrophoresis, the protein was transferred from a polyacrylamide gel to a membrane Trans-Blot Transfer Medium (BIO-RAD Laboratories, Inc.) at 2.5 mA / cm ² for 30 minutes. The transferred membrane was immersed in an immunoblock and blocked overnight at 4 ° C. After blocking, the membrane was washed with TBST by shaking 3 times for 15 minutes, and the primary antibody Goat anti-Mouse λ-Light-Chain (BETHYL laboratories) diluted 5000 times with Can Get Signal Sol.1 (TOYOBO CO., LTD.) , Inc., Montgomery, TX) and shaken at room temperature for 1 hour. After washing 3 times with TBST for 15 minutes, Rabbit anti-Goat IgG (H + L): HRP diluted 10,000 times with Can Get Signal Sol.2 (TOYOBO., LTD.) As a secondary antibody Conjugate solution (DPRA Inc., Manhattan, KS) was added and shaken for 1 hour at room temperature. Finally, after washing with TBST three times for 15 minutes, 1 μl of SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc. Rockford, IL) was photographed with LAS-4000 (FUJIFILM Co., Tokyo). As a result, a band near the theoretical molecular weight of the λ chain-SEAP fusion protein was confirmed from the COS-1 culture supernatant sample transfected with the vector encoding the λ chain-SEAP fusion protein (FIG. 4). The sample used for Western-Blotting is a collection of IgM heavy chain by Goat anti-Mouse IgM Agarose, but this time the result that λ chain-SEAP fusion protein was confirmed was It indicates that the fusion protein and the heavy chain are bound and secreted.

  In addition, IgM heavy chain was added Goat anti-Mouse IgM: HRP Conjugate (DPRA Inc., Manhattan, KS) diluted 10,000 times with Can Get Signal Sol.2 to another membrane transcribed by the same method as above, Similarly, it was confirmed by taking a Western-Blotting image.

(5) ELISA
In order to confirm the antigen binding ability of the SEAP-labeled antibody (association of λ chain-SEAP fusion protein and heavy chain), ELISA was performed using a Costar 96 well white plate on which NP-BSA or BSA was immobilized. Antigen immobilization was performed by dispensing 100 μl of NP-BSA or BSA solution (1 μg / ml) per well and incubating overnight at 4 ° C. Thereafter, 200 μl of immunoblock (DS Pharma Biomedical, Osaka) diluted 5-fold with PBS was dispensed, and blocking was performed for 2 hours at room temperature or overnight at 4 ° C. Then, it was washed 4 times with PBS containing 0.1% Tween20 (PBST), and a solution containing SEAP-labeled antibody (diluted culture supernatant twice with PBS and added immunoblock to a final concentration of 5%) was added. The reaction was allowed to stand at room temperature for 1 hour. After washing 3 times with PBST, the wells were washed with 1 × CDP Buffer (Tropix Inc.), and then a reaction solution containing 0.9 μl CDP-star solution, 9 μl 10 × Buffer, 80 μl milliQ water was added. Thereafter, the mixture was incubated for 30 minutes at room temperature, protected from light, and luminescence was measured using Luminescencer-JNR. As a result, as shown in FIG. 5, a significantly stronger signal was obtained in the NP-BSA-immobilized well than the background BSA-immobilized well, and the practicality of the SEAP-labeled antibody for immunoassay was demonstrated. It was.

Example 2
Outline of Experiment pC _L (G ₄ S) ₁ SEAP expressing fusion protein of CL of λ chain and human placental alkaline phosphatase (SEAP) (CL-SEAP fusion protein) in COS-1 cells expressing heavy chain of anti-NP antibody -His6 or pC _L (G ₄ S) ₂ SEAP-His6 introduces a secreted SEAP-labeled heavy chain in which the CL-SEAP fusion protein is associated with the heavy chain (Fig. 6). Culture supernatant and anti-NP antibody It was demonstrated that an open sandwich ELISA was possible using the λ chain.

(1) Preparation of pC _L (G ₄ S) ₁ SEAP-His6 and pC _L (G ₄ S) ₂ SEAP-His 6 First, the CL gene of pUCλ was amplified by PCR reaction, and Wizard SV Gel and PCR Clean-Up System (Promega Co.). There are two types of primer combinations: C _L MfeIrev and C _L (G ₄ S) ₁ AflIIfor, C _L MfeIrev and C _L (G ₄ S) ₂ AflIIfor. PCR reaction conditions are as follows. A schematic diagram of the obtained λ chain gene fragment is shown in FIG.

Reaction solution composition
pUCλ (90 μg / ml) 1 μl
Primer (C _L (G ₄ S) ₁ AflIIfor or C _L (G ₄ S) ₂ AflIIfor) (50 μM) 1 μl
Primer (C _L MfeIrev) (50 μM) 1 μl
10x ExTaq buffer (Mg ²⁺ 20 mM) (Takara bio Inc.) 10 μl
dNTP Mixture (2.5 mM each) (Takara bio Inc.) 8 μl
5 U / μl ExTaq DNA polymerase (Takara bio Inc.) 1 μl
milliQ water 78 μl

Reaction cycle
1, 96 ℃ 2 min
2, 94 ℃ 1 min
3, 55 ℃ 1 min
4, 72 ℃ 1 min
(2 to 4 30 times)
5, 72 ℃ 5 min
6, 16 ℃ ∞

  The two λ chain gene fragments were subcloned using the TA cloning kit and sequenced in the same manner as described above.

The two types of TA cloning vectors into which the CL gene fragment was inserted were cleaved with AflII and MfeI in the same manner as described above, and ligated with the pSEAP-His vector that had been treated with the same restriction enzyme, and pC _L ( G ₄ S) ₁ SEAP-His6 and pC _L (G ₄ S) ₂ SEAP-His 6 were obtained (FIG. 7).

(2) Expression of fusion protein in COS-1 cells The prepared CL-SEAP fusion protein expression vectors pC _L (G ₄ S) ₁ SEAP-His6 and pC _L (G ₄ S) ₂ SEAP-His 6 COS-1 cells were transfected with pSV-Vμ1 encoding the antibody heavy chain, and after culture in the same manner as described above, a culture supernatant was obtained.

(3) Measurement of SEAP activity of culture supernatant
In order to confirm the expression of CL-SEAP fusion protein or SEAP-labeled heavy chain (aggregate of CL-SEAP fusion protein and heavy chain), measurement of alkaline phosphatase activity in the culture supernatant of transfected COS-1 was performed as described above. As well.
The results of measuring the SEAP activity of the culture supernatant are shown in FIG. In the culture supernatant transfected with the CL-SEAP fusion protein expression vector, residual alkaline phosphatase activity after heat treatment was confirmed, and it was confirmed that the CL-SEAP fusion protein or SEAP-labeled antibody was secreted.

(4) Confirmation of fusion protein by Western Blot As described above, SEAP-labeled heavy chain contained in the culture supernatant was collected using Goat anti-Mouse IgM Agarose, and Western blotting was performed. As a result, a band around the theoretical molecular weight was confirmed from the COS-1 culture supernatant sample transfected with the vector encoding the CL-SEAP fusion protein, and the CL-SEAP fusion protein and heavy chain were bound and secreted. (FIG. 4).

(5) Open sandwich ELISA
To confirm that an open sandwich ELISA using SEAP-labeled heavy chain is possible, add culture supernatant and anti-NP antibody λ chain to Costar 96 well white plate immobilized with NP-BSA or BSA, A tripartite complex consisting of NP-BSA, SEAP-labeled heavy chain and anti-NP antibody λ chain formed in the stationary phase was estimated. Antigen immobilization and blocking were carried out in the same manner as above, and a solution containing SEAP-labeled heavy chain (culture supernatant was diluted 2-fold with PBS and immunoblock was added to a final concentration of 5%) and anti-antigen purified from J558L. NP antibody λ chain was added and allowed to react at room temperature for 1 hour. After washing 3 times with PBST, the wells were washed with 1 × CDP Buffer, and then a reaction solution containing 0.9 μl CDP-star solution, 9 μl 10 × Buffer, 80 μl milliQ water was added. Thereafter, the mixture was incubated for 30 minutes at room temperature, protected from light, and luminescence was measured using Luminescencer-JNR. As a result, as shown in FIG. 9, a significantly stronger signal was obtained in the NP-BSA-immobilized well than in the background BSA-immobilized well, and the practicality of the SEAP-labeled heavy chain for the open sandwich ELISA was demonstrated. It was done.

FIG. 1 shows production of a SEAP-labeled antibody using a λ chain-SEAP fusion protein expression vector. FIG. 2 shows a preparation scheme of a λ chain-SEAP fusion protein expression vector. FIG. 3 shows SEAP activity in the culture supernatant. “Background” indicates the SEAP activity of the culture supernatant of COS-1 cells in which only a transfection operation was performed without adding a vector. `` ΜVC <sub>L</sub> (G <sub>4</sub> S) <sub>2</sub> SEAP '' and `` μVC _L (G ₄ S) SEAP '' are pV _L C _L (G ₄ S) ₁ SEAP-His6 and pV _L C _L (G ₄ S) ₂ SEAP, respectively. COS-1 cells transfected with -His6 and “scFv-SEAP” show the SEAP activity of the culture supernatant of COS-1 cells transfected with pscFvSEAP as a positive control. FIG. 4 shows the results of Western blotting of the culture supernatant. pV _L C _L (G ₄ S) ₁ SEAP-His6 (1), pC _L (G ₄ S) ₁ SEAP-His6 (2), pV _L C _L (G ₄ S) ₂ SEAP-His6 (3), pC The culture supernatant of COS-1 cells transfected with _L (G ₄ S) ₂ SEAP-His6 (4) was collected with Goat anti-Mouse IgM Agarose, run on SDS-PAGE, and then Goat anti-Mouse λ Light Chain It detected using. FIG. 5 shows ELISA for immobilized antigen (NP-BSA) using the culture supernatant. The notation in the figure is the same as in FIG. FIG. 6 shows preparation of a SEAP fusion heavy chain using a CL-SEAP fusion protein expression vector. FIG. 7 shows a preparation scheme of a CL-SEAP fusion protein expression vector. FIG. 8 shows SEAP activity in the culture supernatant. “Background” indicates the SEAP activity of the culture supernatant of COS-1 cells in which only a transfection operation was performed without adding a vector. `` ΜC <sub>L</sub> (G <sub>4</sub> S) <sub>2</sub> SEAP '' and `` μC _L (G ₄ S) SEAP '' transfer pC _L (G ₄ S) ₁ SEAP-His6 and pC _L (G ₄ S) ₂ SEAP-His 6, respectively. The transfected COS-1 cells and “scFv-SEAP” show the SEAP activity of the culture supernatant of COS-1 cells transfected with pscFvSEAP as a positive control. FIG. 9 shows OS-ELISA for immobilized antigen (NP-BSA) using culture supernatant and light chain. FIG. 10 shows a conceptual diagram of preparation of an enzyme-labeled antibody by the light chain fusion method. The figure shows the case of antibodies that form H2L2 heterodimers such as IgG. In the case of IgM and IgA, these are considered to be secreted as pentamers and dimers, respectively. In IgM in particular, the L-chain enzyme fusion protein binds to some of the 10 possible binding sites, allowing enzyme labeling while maximizing the original antigen specificity.

Claims (2)

(A) A gene encoding a light chain or a light chain constant region domain of an antibody and a gene encoding a protein selected from alkaline phosphatase, β-galactosidase, luciferase, or peroxidase, An expression vector having a normal domain domain and the protein fused so as to be expressed can be expressed in a cell expressing the heavy chain gene of the antibody and the light chain gene of the antibody, or the heavy chain gene of the antibody. Produced by introducing into a cell to express it,
(iii) a fusion protein comprising two antibody heavy chains and two antibody light chain constant region domains having a protein on the C-terminal side, or
(iv) a fusion protein comprising two antibody heavy chains, one antibody light chain constant region domain having a protein on the C-terminal side and one antibody light chain;
(B) an antibody light chain,
A method for immunoassay comprising detecting the interaction between the fusion protein and the antibody light chain by contacting in the presence of an antigen. The immunoassay method according to claim 1, wherein an antibody heavy chain and an antibody light chain that specifically interact in the presence of an antigen are selected.