Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Western blot analysis of CD63 EV marker in precipitated EV samples. The precipitated EVs derived using ExoQuick Solution were prepared from the serum collected from 2 mice of wild-type (WT) and EML4-ALK transgenic (TG) mouse as described in the “ Materials and methods ”. ( B ) Analysis of EMARS products obtained from EMARS reaction for crude mouse serum EVs. EMARS reaction was carried out directly in the precipitated serum EVs from two of WT and TG mice with or without HRP-conjugated CHL1 probe. The EMARS products were subsequently subjected to SDS-PAGE (10% gel) with fluorescein detection and CBB staining. ( C ) Fractionation using Sephacryl S-500 chromatography. Blue dextran (an indicator of void volume) and mouse serum (an indicator of protein elution) were used for preliminary experiments. The dotted line indicates absorbance of blue dextran. The solid line indicates protein concentration measured using a BCA protein kit. ( D ) Morphological observation of serum EVs (fraction No. 6 and No. 8) using cryo-electron microscopy. Lower panel of fraction No.6 is an enlarged view of a part of the upper panel. Scale bar; 200 nm (upper panel) and 50 nm (lower panel).

Article Snippet: In the case of the human CHL1 level in serum and serum EVs by sandwich ELISA, diluted serum or precipitated serum EVs described above were applied to the ELISA plates after coated with capture antibody, anti-human CHL1 antibody (10143-MM05; Sino Biological, Beijing, China), followed by the detection with HRP-conjugated anti-human CHL1 antibody (described above).

Techniques: Western Blot, Marker, Derivative Assay, Transgenic Assay, SDS Page, Staining, Fractionation, Chromatography, Protein Concentration, Electron Microscopy

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Protein expression of precipitated EVs. The serum collected from seven mice of wild-type (WT) and EML4-ALK transgenic mouse (TL group) was subjected to Western blot analysis with anti-CHL1, anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported. ( B ) Western blot analysis for TSG101 antigen detection in Sephacryl S-500 fractions. The fractions were concentrated with Nanosep ® centrifugal unit, and then subjected to Western blot analysis with anti-TSG101 antibody. TSG101-(Ub)n indicates ubiquitinated TSG101.

Article Snippet: In the case of the human CHL1 level in serum and serum EVs by sandwich ELISA, diluted serum or precipitated serum EVs described above were applied to the ELISA plates after coated with capture antibody, anti-human CHL1 antibody (10143-MM05; Sino Biological, Beijing, China), followed by the detection with HRP-conjugated anti-human CHL1 antibody (described above).

Techniques: Expressing, Transgenic Assay, Western Blot

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) The EMARS products for serum EVs from EML4-ALK transgenic mouse. To average experimental results over each group, an aliquot of the serum (10 μL each) from 10 animals in each group (WT), large lung tumor-bearing (TL), and small lung tumor-bearing mice (TS) was mixed in equal proportions, and then applied to EV purification and EMARS. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. The right panel indicates the same gel as the left panel, but exposed for a longer time. ( B ) Confirmation of candidate partner molecules (CD5L and PZP) with mouse CHL1 in EVs. The EMARS products of WT, TL, and TS were respectively applied to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-CD5L (left panel) antibody. After the stripping as described in the “ Materials and methods ”, the membranes were re-stained with anti-PZP antibody (right panel). Arrows indicate the detected band of CD5L and PZP proteins (including predicted dimer). Asterisk indicates unknown bands (predicted as non-specific or partial fragments). ( C, D ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody ( C ). After stripping, the membranes were re-stained with anti-THBS1 antibody ( D ). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( E ) Expression of SLC4A1 (left column) and CHL1 proteins (right column) in tumor tissues from two male and two female EML4-ALK transgenic mice. The fragments of lung cancer tissues were mashed and washed gently with PBS, and then lysed with SDS-PAGE sample buffer directly. The resulting samples were subjected to Western blot analysis with anti-SLC4A1 antibody and anti-CHL1 antibody. Arrows indicate the detected band of monomer SLC4A1 and CHL1 proteins.

Article Snippet: In the case of the human CHL1 level in serum and serum EVs by sandwich ELISA, diluted serum or precipitated serum EVs described above were applied to the ELISA plates after coated with capture antibody, anti-human CHL1 antibody (10143-MM05; Sino Biological, Beijing, China), followed by the detection with HRP-conjugated anti-human CHL1 antibody (described above).

Techniques: Transgenic Assay, Purification, Immunoprecipitation, SDS Page, Fluorescence, Western Blot, Stripping Membranes, Staining, Expressing

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Western blot analysis of TSG101 ( A ) and CD63 ( B ) in serum EVs from WT, TL, and TS using anti-TSG101 and CD63 antibody. Arrows indicate the detected band of monomer TSG101 and CD63. The black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( C ) Western blot analysis of EMARS products with anti-CHL1 antibody. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. Arrow indicates the detected band of CHL1. Asterisk indicates unknown bands (predicted as non-specific or partial fragments).

Article Snippet: In the case of the human CHL1 level in serum and serum EVs by sandwich ELISA, diluted serum or precipitated serum EVs described above were applied to the ELISA plates after coated with capture antibody, anti-human CHL1 antibody (10143-MM05; Sino Biological, Beijing, China), followed by the detection with HRP-conjugated anti-human CHL1 antibody (described above).

Techniques: Western Blot, Molecular Weight, Purification, Immunoprecipitation, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Calibration curve of sandwich ELISA for the detection of both SLC4A1 partial proteins and fluorescein-labeled SLC4A1. The detection of several concentrations of recombinant SLC4A1 partial protein using HRP-labeled anti-SLC4A1 antibody which is prepared using Zenon system is summarized in ( A ). The detection of several concentrations of self-made standard materials containing fluorescein-labeled SLC4A1 using HRP-labeled anti-fluorescein antibody is summarized in ( B ). ( C ) Comparison of serum CHL1 levels between wild-type (WT) and small tumor-bearing EML4-ALK transgenic (TS) mice by using previously established ELISA system for CHL1 measurement. There were no significant differences between them.

Article Snippet: In the case of the human CHL1 level in serum and serum EVs by sandwich ELISA, diluted serum or precipitated serum EVs described above were applied to the ELISA plates after coated with capture antibody, anti-human CHL1 antibody (10143-MM05; Sino Biological, Beijing, China), followed by the detection with HRP-conjugated anti-human CHL1 antibody (described above).

Techniques: Sandwich ELISA, Labeling, Recombinant, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Calibration curve of sandwich ELISA for the detection of both human CHL1. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody. ( B ) Comparison of serum CHL1 levels between H (open bar) and LC (closed bar) by using ELISA system for human CHL1 measurement. There were no significant differences between them. ( C ) Comparison of CHL1-expressing EVs between H (open bar) and LC (closed bar). The serum EVs were purified by using ExoQuick Solution followed by ELISA measurement of CHL1 levels. There were also no significant differences between them.

Article Snippet: In the case of the human CHL1 level in serum and serum EVs by sandwich ELISA, diluted serum or precipitated serum EVs described above were applied to the ELISA plates after coated with capture antibody, anti-human CHL1 antibody (10143-MM05; Sino Biological, Beijing, China), followed by the detection with HRP-conjugated anti-human CHL1 antibody (described above).

Techniques: Sandwich ELISA, Recombinant, Labeling, Enzyme-linked Immunosorbent Assay, Expressing, Purification

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) EMARS products purified from serum EVs of healthy person (H) and lung cancer (LC) patients. Fifty microliters of mouse serums was collected from the H and LC groups, and utilized in EV purification followed by EMARS reactions. To average experimental results over each group, an aliquot of the serum (10 μL each) from 5 H and 5 LC was mixed each in equal proportions. The EMARS products were subjected to SDS-PAGE analysis with fluorescence detection. ( B ) Confirmation of caspase 14 as a partner molecule with CHL1 identified by MS proteomics. The H and LC samples were applied respectively to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-caspase 14 antibodies. Arrows indicate the detected band of caspase 14 proteins (including predicted dimer). ( C ) Measurement of fluorescein-labeled caspase 14 using a sandwich ELISA. Serum EVs from 12 H (open bar) and 12 LC (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled caspase 14 were added to anti-caspase 14 antibody-coated ELISA plates. “BiEV index (caspase 14)” was calculated based on the value of fluorescein-labeled recombinant caspase 14 made by fluorescein-labeling regent. The values are shown as the average of three independent ELISA experiments using the same samples. The detail data of H and LC persons is provided in Table S3. Asterisks indicate the samples were below detection limit. ( D ) ROC curve for BiEV indexes. The AUC was calculated as 0.811. ( E ) Western blot analysis of caspase 14 in whole-serum EVs from H and LC. An aliquot of the serum (2 μL each) from 12 persons in H and LC was mixed in equal proportions followed by EV purification with precipitation protocol. Arrows indicate the detected band of caspase 14.

Article Snippet: In the case of the human CHL1 level in serum and serum EVs by sandwich ELISA, diluted serum or precipitated serum EVs described above were applied to the ELISA plates after coated with capture antibody, anti-human CHL1 antibody (10143-MM05; Sino Biological, Beijing, China), followed by the detection with HRP-conjugated anti-human CHL1 antibody (described above).

Techniques: Purification, SDS Page, Fluorescence, Immunoprecipitation, Western Blot, Labeling, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Western blot analysis of CD63 EV marker in precipitated EV samples. The precipitated EVs derived using ExoQuick Solution were prepared from the serum collected from 2 mice of wild-type (WT) and EML4-ALK transgenic (TG) mouse as described in the “ Materials and methods ”. ( B ) Analysis of EMARS products obtained from EMARS reaction for crude mouse serum EVs. EMARS reaction was carried out directly in the precipitated serum EVs from two of WT and TG mice with or without HRP-conjugated CHL1 probe. The EMARS products were subsequently subjected to SDS-PAGE (10% gel) with fluorescein detection and CBB staining. ( C ) Fractionation using Sephacryl S-500 chromatography. Blue dextran (an indicator of void volume) and mouse serum (an indicator of protein elution) were used for preliminary experiments. The dotted line indicates absorbance of blue dextran. The solid line indicates protein concentration measured using a BCA protein kit. ( D ) Morphological observation of serum EVs (fraction No. 6 and No. 8) using cryo-electron microscopy. Lower panel of fraction No.6 is an enlarged view of a part of the upper panel. Scale bar; 200 nm (upper panel) and 50 nm (lower panel).

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Western Blot, Marker, Derivative Assay, Transgenic Assay, SDS Page, Staining, Fractionation, Chromatography, Protein Concentration, Cryo-Electron Microscopy

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Protein expression of precipitated EVs. The serum collected from seven mice of wild-type (WT) and EML4-ALK transgenic mouse (TL group) was subjected to Western blot analysis with anti-CHL1, anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported. ( B ) Western blot analysis for TSG101 antigen detection in Sephacryl S-500 fractions. The fractions were concentrated with Nanosep ® centrifugal unit, and then subjected to Western blot analysis with anti-TSG101 antibody. TSG101-(Ub)n indicates ubiquitinated TSG101.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Expressing, Transgenic Assay, Western Blot, Membrane

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) The EMARS products for serum EVs from EML4-ALK transgenic mouse. To average experimental results over each group, an aliquot of the serum (10 μL each) from 10 animals in each group (WT), large lung tumor-bearing (TL), and small lung tumor-bearing mice (TS) was mixed in equal proportions, and then applied to EV purification and EMARS. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. The right panel indicates the same gel as the left panel, but exposed for a longer time. ( B ) Confirmation of candidate partner molecules (CD5L and PZP) with mouse CHL1 in EVs. The EMARS products of WT, TL, and TS were respectively applied to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-CD5L (left panel) antibody. After the stripping as described in the “ Materials and methods ”, the membranes were re-stained with anti-PZP antibody (right panel). Arrows indicate the detected band of CD5L and PZP proteins (including predicted dimer). Asterisk indicates unknown bands (predicted as non-specific or partial fragments). ( C, D ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody ( C ). After stripping, the membranes were re-stained with anti-THBS1 antibody ( D ). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( E ) Expression of SLC4A1 (left column) and CHL1 proteins (right column) in tumor tissues from two male and two female EML4-ALK transgenic mice. The fragments of lung cancer tissues were mashed and washed gently with PBS, and then lysed with SDS-PAGE sample buffer directly. The resulting samples were subjected to Western blot analysis with anti-SLC4A1 antibody and anti-CHL1 antibody. Arrows indicate the detected band of monomer SLC4A1 and CHL1 proteins.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Transgenic Assay, Purification, Immunoprecipitation, SDS Page, Fluorescence, Western Blot, Stripping Membranes, Staining, Expressing

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Western blot analysis of TSG101 ( A ) and CD63 ( B ) in serum EVs from WT, TL, and TS using anti-TSG101 and CD63 antibody. Arrows indicate the detected band of monomer TSG101 and CD63. The black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( C ) Western blot analysis of EMARS products with anti-CHL1 antibody. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. Arrow indicates the detected band of CHL1. Asterisk indicates unknown bands (predicted as non-specific or partial fragments).

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Western Blot, Molecular Weight, Ubiquitin Proteomics, Purification, Immunoprecipitation, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Calibration curve of sandwich ELISA for the detection of both SLC4A1 partial proteins and fluorescein-labeled SLC4A1. The detection of several concentrations of recombinant SLC4A1 partial protein using HRP-labeled anti-SLC4A1 antibody which is prepared using Zenon system is summarized in ( A ). The detection of several concentrations of self-made standard materials containing fluorescein-labeled SLC4A1 using HRP-labeled anti-fluorescein antibody is summarized in ( B ). ( C ) Comparison of serum CHL1 levels between wild-type (WT) and small tumor-bearing EML4-ALK transgenic (TS) mice by using previously established ELISA system for CHL1 measurement. There were no significant differences between them.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Sandwich ELISA, Labeling, Recombinant, Comparison, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Calibration curve of sandwich ELISA for the detection of both human CHL1. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody. ( B ) Comparison of serum CHL1 levels between H (open bar) and LC (closed bar) by using ELISA system for human CHL1 measurement. There were no significant differences between them. ( C ) Comparison of CHL1-expressing EVs between H (open bar) and LC (closed bar). The serum EVs were purified by using ExoQuick Solution followed by ELISA measurement of CHL1 levels. There were also no significant differences between them.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Sandwich ELISA, Recombinant, Labeling, Comparison, Enzyme-linked Immunosorbent Assay, Expressing, Purification

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) EMARS products purified from serum EVs of healthy person (H) and lung cancer (LC) patients. Fifty microliters of mouse serums was collected from the H and LC groups, and utilized in EV purification followed by EMARS reactions. To average experimental results over each group, an aliquot of the serum (10 μL each) from 5 H and 5 LC was mixed each in equal proportions. The EMARS products were subjected to SDS-PAGE analysis with fluorescence detection. ( B ) Confirmation of caspase 14 as a partner molecule with CHL1 identified by MS proteomics. The H and LC samples were applied respectively to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-caspase 14 antibodies. Arrows indicate the detected band of caspase 14 proteins (including predicted dimer). ( C ) Measurement of fluorescein-labeled caspase 14 using a sandwich ELISA. Serum EVs from 12 H (open bar) and 12 LC (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled caspase 14 were added to anti-caspase 14 antibody-coated ELISA plates. “BiEV index (caspase 14)” was calculated based on the value of fluorescein-labeled recombinant caspase 14 made by fluorescein-labeling regent. The values are shown as the average of three independent ELISA experiments using the same samples. The detail data of H and LC persons is provided in Table S3. Asterisks indicate the samples were below detection limit. ( D ) ROC curve for BiEV indexes. The AUC was calculated as 0.811. ( E ) Western blot analysis of caspase 14 in whole-serum EVs from H and LC. An aliquot of the serum (2 μL each) from 12 persons in H and LC was mixed in equal proportions followed by EV purification with precipitation protocol. Arrows indicate the detected band of caspase 14.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Purification, SDS Page, Fluorescence, Immunoprecipitation, Western Blot, Labeling, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Recombinant