A fluorescent immunosensor that lights up tumor biomarker p53 in living cells was developed based... more A fluorescent immunosensor that lights up tumor biomarker p53 in living cells was developed based on the Q-body technology. The technology was further applied to the live cell monitoring of p53 levels, and live cell sorting based on p53 expression.
Since late 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and... more Since late 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant spread of COVID-19 have given rise to a worldwide health crisis that is posing great challenges to public health and clinical treatment, in addition to serving as a formidable threat to the global economy. To obtain an effective tool to prevent and diagnose viral infections, we attempted to obtain human antibody fragments that can effectively neutralize viral infection and be utilized for rapid virus detection. To this end, several human monoclonal antibodies were isolated by bio-panning a phage-displayed human antibody library, Tomlinson I. The selected clones were demonstrated to bind to the S1 domain of the spike glycoprotein of SARS-CoV-2. Moreover, clone A7 in Fab and IgG formats were found to effectively neutralize the binding of S protein to angiotensin-converting enzyme 2 in the low nM range. In addition, this clone was successfully converted to quench-based fluore...
With the widespread application of recombinant DNA technology, many useful substances are produce... more With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O2, etc.). However, the production status of the target protein can only be known after the subsequent separation and purification process. To speed up the monitoring of the production process and screening of the higher-yield target protein variants, here we developed an antibody-based His-tag sensor Quenchbody (Q-body), which can quickly detect the C-terminally His-tagged recombinant protein produced in the culture medium. Compared with single-chain Fv-based Q-body having one dye, the Fab-based Q-body having two dyes showed a higher response. In addition, not only was fluorescence response improved but also detection sensitivity by the mutations of tyrosine to tryptophan in the heavy chain CDR region. Moreover, the effect of t...
Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) ne... more Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize hu...
The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 variable ... more The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 variable region was monitored with fluorescence resonance energy transfer (FRET) between fluorolabeled heavy chain (VH) and light chain (VL) fragments. The VH and VL fragments labeled with succinimide esters of fluorescein and rhodamine-X, respectively, were mixed in a cooled cuvette, and the change in fluorescence spectra upon antigen addition was monitored. When excited at 490 nm, significant decrease in the fluorescence at 520 nm and its increase at 605 nm were observed when an increasing amount of HEL was added to the mixture in the concentration range of 1–100 μg/mL. The assay, named open sandwich fluoroimmunoassay (FIA), is noncompetitive and homogeneous and can be conducted with one clone of antibody. With the use of appropriate antibodies, it is thought to be a quick and inexpensive alternative to the conventional laborious and/or expensive immunoassays.
Myocardial regeneration therapy using embryonic stem (ES) cells and induced pluripotent stem (iPS... more Myocardial regeneration therapy using embryonic stem (ES) cells and induced pluripotent stem (iPS) cells has been expected as a promising approach for end-staged heart failure. Recent studies including ours reported that canonical Wnt signal activation by Wnt3a in the early phase of mouse ES/iPS cell differentiation plays a pivotal role in efficient myocardial cell differentiation. Problems about the cost and the quality of recombinant Wnt3a protein, however, compromise the utility of this approach to create sufficient amounts of cardiac myocytes for cell implantation. To address these issues, we attempted to generate small-molecule-responsive artificial receptors which can activate the Wnt3a signal transduction. First, we constructed antibody/receptor chimeras, in which single-chain Fv of anti-fluorescein (FL) antibody was tethered to transmembrane/cytoplasmic domains of Wnt3a receptors, so that they could transduce the Wnt3a signal pathway in response to FL-conjugated BSA (BSA-FL) as a cognate ligand, which costs about 2000 times less than recombinant Wnt3a protein. Signal activations by these chimeric receptors were measured by luciferase reporter assay for TCF/β-catenin dependent transcription, one of the common downstream targets of canonical Wnt signal pathways. ES/iPS cells transfected with the chimeric receptors showed increased luciferase activity by BSA-FL treatment in a dose dependent manner. These activations were similar to the level when the cells are treated with 10 to 100ng/ml of recombinant Wnt3a protein. In addition, these signal activities were not perturbed by Dkk1, an antagonist of Wnt3a, suggesting that the signal transduction via chimeric receptors is independent of the Wnt3a ligand. Finally, BSA-FL treatment in the early phase of differentiation increased the efficiency for cardiac differentiation in ES/iPS cell lines stably expressing these receptors but not in the mock-expressing cell lines, exemplified by the beating ratio (BSA-FL 100% vs Control 53.6%). Application of this technology to ES/iPS cell differentiation could be a powerful tool for efficient and economical creation of cardiac myocytes for heart regeneration therapy.
The authors transfected hybridoma 2E3 cells with cDNA of mouse cyclin inhibitor p21 or p27 in pla... more The authors transfected hybridoma 2E3 cells with cDNA of mouse cyclin inhibitor p21 or p27 in plasmid pMAM-neo, aiming at increasing antibody production by controlling cell proliferation with cyclin inhibitors. The expression of the inhibitor genes was regulated by mouse mammal tumor virus (MMTV) promoter with/without addition of dexamethasone (DEX) in culture medium. Growth rate of the transfected cells was suppressed with DEX, although the transfectant grew slower than the wild type cells even without DEX, presumably because of the leaky transcription from MMTV promoter. The p27 transfectant with DEX produced 3-fold amount of antibody of the wild type or control cells in a batch during 10 days culture, and the p21 transfectant with DEX produced about 1.5-fold during 10 days. The viable stationary phase after the logarithmic growth was prolonged by five days by the expression of p27, although the cell density at the phase was about 60% of that of the wild type and control cells, and during that period the p27 transfectant accelerated antibody production. In the case of p21, the maximum number of viable p21 transfectant cells with DEX was equal to that of the wild type and control cells, and the p21 expression delayed overgrowth of transfectant cells by six days.
The quantitation of low-molecular-weight haptens has been difficult with conventional sandwich im... more The quantitation of low-molecular-weight haptens has been difficult with conventional sandwich immunoassays due to their small size. Many researchers have attempted to develop sandwich assays for haptens due to the significant advantages of the sandwich format over competitive assays including greater dynamic range, ease of automation, and sensitivity. Here we apply the open-sandwich ELISA (OS-ELISA), an immunoassay based on antigen-dependent stabilization of antibody variable regions (V(H) and V(L) domains), to hapten quantitation. Two fusion proteins, the high-affinity mutant V(H) domain from anti-4-hydroxy-3-nitrophenacetyl (NP) antibody B1-8 tethered with Escherichia coli alkaline phosphatase (V(H)(W33L)-PhoA) and the V(L) domain from the same antibody tethered with Streptococcus sp. protein G, were made. These fusion proteins when added together achieved Fv reassociation consequent to the addition of NP. Signal was generated in a direct relationship to the NP concentration with better sensitivity compared with competitive immunoassay, demonstrating this assay to be a quick noncompetitive alternative to the conventional assays for small compounds, such as environmental pollutants, drugs of abuse, and therapeutic drugs. With our previous demonstration that the OS-ELISA works well with large proteins, the OS-ELISA becomes the first practical immunoassay approach capable of quantifying any molecule regardless of their size.
In mammalian cell culture, the selection of high producers is a critical step in efficient recomb... more In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V(H) portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (V(H)-EpoR), and (ii) a V(L)-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a model recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.
We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependen... more We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependent reassociation of antibody variable domains (open sandwich bioluminescent immunoassay, OS-BLIA). The reassociation of two chimeric proteins, an antibody heavy-chain fragment (V(H))-Renilla luciferase (Rluc) and an antibody light-chain fragment (V(L))-enhanced yellow fluorescent protein (EYFP), was monitored by a bioluminescence resonance energy transfer (BRET) between the two. Upon simple mixing of the reagents with the sample, an antigen-dependent increase in BRET was observed with a measurable concentration range of 0.1 to approximately 10 microg/ml antigen hen egg lysozyme. Compared with our comparable assays based on fluorescence resonance energy transfer (FRET), a 10-fold improvement in the sensitivity was attained, probably due to a reduction in reagent concentration.
A fluorescent immunosensor that lights up tumor biomarker p53 in living cells was developed based... more A fluorescent immunosensor that lights up tumor biomarker p53 in living cells was developed based on the Q-body technology. The technology was further applied to the live cell monitoring of p53 levels, and live cell sorting based on p53 expression.
Since late 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and... more Since late 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant spread of COVID-19 have given rise to a worldwide health crisis that is posing great challenges to public health and clinical treatment, in addition to serving as a formidable threat to the global economy. To obtain an effective tool to prevent and diagnose viral infections, we attempted to obtain human antibody fragments that can effectively neutralize viral infection and be utilized for rapid virus detection. To this end, several human monoclonal antibodies were isolated by bio-panning a phage-displayed human antibody library, Tomlinson I. The selected clones were demonstrated to bind to the S1 domain of the spike glycoprotein of SARS-CoV-2. Moreover, clone A7 in Fab and IgG formats were found to effectively neutralize the binding of S protein to angiotensin-converting enzyme 2 in the low nM range. In addition, this clone was successfully converted to quench-based fluore...
With the widespread application of recombinant DNA technology, many useful substances are produce... more With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O2, etc.). However, the production status of the target protein can only be known after the subsequent separation and purification process. To speed up the monitoring of the production process and screening of the higher-yield target protein variants, here we developed an antibody-based His-tag sensor Quenchbody (Q-body), which can quickly detect the C-terminally His-tagged recombinant protein produced in the culture medium. Compared with single-chain Fv-based Q-body having one dye, the Fab-based Q-body having two dyes showed a higher response. In addition, not only was fluorescence response improved but also detection sensitivity by the mutations of tyrosine to tryptophan in the heavy chain CDR region. Moreover, the effect of t...
Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) ne... more Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize hu...
The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 variable ... more The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 variable region was monitored with fluorescence resonance energy transfer (FRET) between fluorolabeled heavy chain (VH) and light chain (VL) fragments. The VH and VL fragments labeled with succinimide esters of fluorescein and rhodamine-X, respectively, were mixed in a cooled cuvette, and the change in fluorescence spectra upon antigen addition was monitored. When excited at 490 nm, significant decrease in the fluorescence at 520 nm and its increase at 605 nm were observed when an increasing amount of HEL was added to the mixture in the concentration range of 1–100 μg/mL. The assay, named open sandwich fluoroimmunoassay (FIA), is noncompetitive and homogeneous and can be conducted with one clone of antibody. With the use of appropriate antibodies, it is thought to be a quick and inexpensive alternative to the conventional laborious and/or expensive immunoassays.
Myocardial regeneration therapy using embryonic stem (ES) cells and induced pluripotent stem (iPS... more Myocardial regeneration therapy using embryonic stem (ES) cells and induced pluripotent stem (iPS) cells has been expected as a promising approach for end-staged heart failure. Recent studies including ours reported that canonical Wnt signal activation by Wnt3a in the early phase of mouse ES/iPS cell differentiation plays a pivotal role in efficient myocardial cell differentiation. Problems about the cost and the quality of recombinant Wnt3a protein, however, compromise the utility of this approach to create sufficient amounts of cardiac myocytes for cell implantation. To address these issues, we attempted to generate small-molecule-responsive artificial receptors which can activate the Wnt3a signal transduction. First, we constructed antibody/receptor chimeras, in which single-chain Fv of anti-fluorescein (FL) antibody was tethered to transmembrane/cytoplasmic domains of Wnt3a receptors, so that they could transduce the Wnt3a signal pathway in response to FL-conjugated BSA (BSA-FL) as a cognate ligand, which costs about 2000 times less than recombinant Wnt3a protein. Signal activations by these chimeric receptors were measured by luciferase reporter assay for TCF/β-catenin dependent transcription, one of the common downstream targets of canonical Wnt signal pathways. ES/iPS cells transfected with the chimeric receptors showed increased luciferase activity by BSA-FL treatment in a dose dependent manner. These activations were similar to the level when the cells are treated with 10 to 100ng/ml of recombinant Wnt3a protein. In addition, these signal activities were not perturbed by Dkk1, an antagonist of Wnt3a, suggesting that the signal transduction via chimeric receptors is independent of the Wnt3a ligand. Finally, BSA-FL treatment in the early phase of differentiation increased the efficiency for cardiac differentiation in ES/iPS cell lines stably expressing these receptors but not in the mock-expressing cell lines, exemplified by the beating ratio (BSA-FL 100% vs Control 53.6%). Application of this technology to ES/iPS cell differentiation could be a powerful tool for efficient and economical creation of cardiac myocytes for heart regeneration therapy.
The authors transfected hybridoma 2E3 cells with cDNA of mouse cyclin inhibitor p21 or p27 in pla... more The authors transfected hybridoma 2E3 cells with cDNA of mouse cyclin inhibitor p21 or p27 in plasmid pMAM-neo, aiming at increasing antibody production by controlling cell proliferation with cyclin inhibitors. The expression of the inhibitor genes was regulated by mouse mammal tumor virus (MMTV) promoter with/without addition of dexamethasone (DEX) in culture medium. Growth rate of the transfected cells was suppressed with DEX, although the transfectant grew slower than the wild type cells even without DEX, presumably because of the leaky transcription from MMTV promoter. The p27 transfectant with DEX produced 3-fold amount of antibody of the wild type or control cells in a batch during 10 days culture, and the p21 transfectant with DEX produced about 1.5-fold during 10 days. The viable stationary phase after the logarithmic growth was prolonged by five days by the expression of p27, although the cell density at the phase was about 60% of that of the wild type and control cells, and during that period the p27 transfectant accelerated antibody production. In the case of p21, the maximum number of viable p21 transfectant cells with DEX was equal to that of the wild type and control cells, and the p21 expression delayed overgrowth of transfectant cells by six days.
The quantitation of low-molecular-weight haptens has been difficult with conventional sandwich im... more The quantitation of low-molecular-weight haptens has been difficult with conventional sandwich immunoassays due to their small size. Many researchers have attempted to develop sandwich assays for haptens due to the significant advantages of the sandwich format over competitive assays including greater dynamic range, ease of automation, and sensitivity. Here we apply the open-sandwich ELISA (OS-ELISA), an immunoassay based on antigen-dependent stabilization of antibody variable regions (V(H) and V(L) domains), to hapten quantitation. Two fusion proteins, the high-affinity mutant V(H) domain from anti-4-hydroxy-3-nitrophenacetyl (NP) antibody B1-8 tethered with Escherichia coli alkaline phosphatase (V(H)(W33L)-PhoA) and the V(L) domain from the same antibody tethered with Streptococcus sp. protein G, were made. These fusion proteins when added together achieved Fv reassociation consequent to the addition of NP. Signal was generated in a direct relationship to the NP concentration with better sensitivity compared with competitive immunoassay, demonstrating this assay to be a quick noncompetitive alternative to the conventional assays for small compounds, such as environmental pollutants, drugs of abuse, and therapeutic drugs. With our previous demonstration that the OS-ELISA works well with large proteins, the OS-ELISA becomes the first practical immunoassay approach capable of quantifying any molecule regardless of their size.
In mammalian cell culture, the selection of high producers is a critical step in efficient recomb... more In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V(H) portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (V(H)-EpoR), and (ii) a V(L)-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a model recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.
We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependen... more We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependent reassociation of antibody variable domains (open sandwich bioluminescent immunoassay, OS-BLIA). The reassociation of two chimeric proteins, an antibody heavy-chain fragment (V(H))-Renilla luciferase (Rluc) and an antibody light-chain fragment (V(L))-enhanced yellow fluorescent protein (EYFP), was monitored by a bioluminescence resonance energy transfer (BRET) between the two. Upon simple mixing of the reagents with the sample, an antigen-dependent increase in BRET was observed with a measurable concentration range of 0.1 to approximately 10 microg/ml antigen hen egg lysozyme. Compared with our comparable assays based on fluorescence resonance energy transfer (FRET), a 10-fold improvement in the sensitivity was attained, probably due to a reduction in reagent concentration.
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