human chl1 antibody  (Sino Biological)


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    CHL1 Matched ELISA Antibody Pair Set Human
    Description:
    Each vial contains 80 ng of recombinant CHL1 Reconstitute with 1 mL detection antibody dilution buffer After reconstitution store at 20℃ to 80℃ in a manual defrost freezer A seven point standard curve usi ng 2 fold serial dilutions in sample dilution buffer and a high standard of 2 ng mL is recommended
    Catalog Number:
    SEK10143
    Price:
    None
    Category:
    Elisa pair set
    Reactivity:
    Human
    Product Aliases:
    CALL Matched ELISA Antibody Pair Set Human, L1CAM2 Matched ELISA Antibody Pair Set Human, LICAM2 Matched ELISA Antibody Pair Set Human
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    Sino Biological human chl1 antibody
    Characterization of crude and purified serum EVs ( 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 <t>anti-CHL1,</t> anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported (8). ( 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. ( C ) SDS-PAGE analysis of EMARS products in S-500 fraction. The fractionated EMARS products from serum EVs in wild-type and EML4-ALK transgenic mouse were subjected to SDS-PAGE analysis (10% gel) with fluorescence detection. The mixture of seven fractions (fraction No.1 to No. 7), and fraction 8 alone were respectively loaded in each well.
    Each vial contains 80 ng of recombinant CHL1 Reconstitute with 1 mL detection antibody dilution buffer After reconstitution store at 20℃ to 80℃ in a manual defrost freezer A seven point standard curve usi ng 2 fold serial dilutions in sample dilution buffer and a high standard of 2 ng mL is recommended
    https://www.bioz.com/result/human chl1 antibody/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human chl1 antibody - by Bioz Stars, 2021-04
    93/100 stars

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    1) Product Images from "Bimolecule detection for Extracellular Vesicle Screening"

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    Journal: bioRxiv

    doi: 10.1101/2020.07.23.217018

    Characterization of crude and purified serum EVs ( 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 (8). ( 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. ( C ) SDS-PAGE analysis of EMARS products in S-500 fraction. The fractionated EMARS products from serum EVs in wild-type and EML4-ALK transgenic mouse were subjected to SDS-PAGE analysis (10% gel) with fluorescence detection. The mixture of seven fractions (fraction No.1 to No. 7), and fraction 8 alone were respectively loaded in each well.
    Figure Legend Snippet: Characterization of crude and purified serum EVs ( 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 (8). ( 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. ( C ) SDS-PAGE analysis of EMARS products in S-500 fraction. The fractionated EMARS products from serum EVs in wild-type and EML4-ALK transgenic mouse were subjected to SDS-PAGE analysis (10% gel) with fluorescence detection. The mixture of seven fractions (fraction No.1 to No. 7), and fraction 8 alone were respectively loaded in each well.

    Techniques Used: Purification, Expressing, Mouse Assay, Transgenic Assay, Western Blot, SDS Page, Fluorescence

    Confirmation of partner molecules identified by MS proteomics. ( A ) 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). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( B ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody (upper panel). After stripping, the membranes were re-stained with anti-THBS1 antibody (lower panel). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( C ) Western blot analysis of an independent experiment samples identical to Figure 4B with anti-SLC4A1 antibody. Arrow indicates the detected band of predicted dimer SLC4A1 protein. Asterisk indicates unknown non-specific bands. ( D, E ) Expression of SLC4A1 ( D ) and CHL1 proteins ( E ) 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.
    Figure Legend Snippet: Confirmation of partner molecules identified by MS proteomics. ( A ) 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). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( B ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody (upper panel). After stripping, the membranes were re-stained with anti-THBS1 antibody (lower panel). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( C ) Western blot analysis of an independent experiment samples identical to Figure 4B with anti-SLC4A1 antibody. Arrow indicates the detected band of predicted dimer SLC4A1 protein. Asterisk indicates unknown non-specific bands. ( D, E ) Expression of SLC4A1 ( D ) and CHL1 proteins ( E ) 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.

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

    Characterization of ELISA system for fluorescein labeled-caspase 14 and human CHL1 ( A, B ) Calibration curve of sandwich ELISA for the detection of both human CHL1 and fluorescein-labeled caspase 14. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody is summarized in ( A ). The detection of several concentrations of fluorescein-labeled caspase 14 standard using HRP-labeled anti-fluorescein antibody is summarized in ( B ).
    Figure Legend Snippet: Characterization of ELISA system for fluorescein labeled-caspase 14 and human CHL1 ( A, B ) Calibration curve of sandwich ELISA for the detection of both human CHL1 and fluorescein-labeled caspase 14. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody is summarized in ( A ). The detection of several concentrations of fluorescein-labeled caspase 14 standard using HRP-labeled anti-fluorescein antibody is summarized in ( B ).

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

    Summary of advantages for human EV screening using BiEV One of hypothetical mechanisms of BiEV differences between human healthy and lung cancer patients is summarized in this figure based on our results of no significant differences of CHL1 and caspase 14 expression levels. Due to the hypothetical mechanisms, it is considerable that our BiEV analysis is useful when it is difficult to characterize secreted cancer EVs by single molecular index ( e . g . serum CHL1 single screening and typical serum biomarker (CEA and SCC in Supplementary Table 3)). In this study, BiEV analysis may emphasize the differences using the information of bimolecular complex which is formed by association between CHL1 and caspase 14 with unknown cancer cell-specific mechanisms.
    Figure Legend Snippet: Summary of advantages for human EV screening using BiEV One of hypothetical mechanisms of BiEV differences between human healthy and lung cancer patients is summarized in this figure based on our results of no significant differences of CHL1 and caspase 14 expression levels. Due to the hypothetical mechanisms, it is considerable that our BiEV analysis is useful when it is difficult to characterize secreted cancer EVs by single molecular index ( e . g . serum CHL1 single screening and typical serum biomarker (CEA and SCC in Supplementary Table 3)). In this study, BiEV analysis may emphasize the differences using the information of bimolecular complex which is formed by association between CHL1 and caspase 14 with unknown cancer cell-specific mechanisms.

    Techniques Used: Expressing, Biomarker Assay

    Purification of EVs from serum. ( 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).
    Figure Legend Snippet: Purification of EVs from serum. ( 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).

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

    BiEV analysis for serum EVs from EML4-ALK transgenic mouse. ( 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 ) Western blot analysis of fluorescein-labeled EMARS products from serum EVs using anti-fluorescein antibody. ( C ) Western blot analysis of TSG101 in serum EVs from WT, TL, and TS using anti-TSG101 antibody. Arrows indicate the detected band of monomer TSG101, and black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( D ) Western blot analysis of indicated samples with anti-CHL1 (upper panel) and anti-α2 integrin (lower panel) antibody. Arrow indicates the detected band of each molecule.
    Figure Legend Snippet: BiEV analysis for serum EVs from EML4-ALK transgenic mouse. ( 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 ) Western blot analysis of fluorescein-labeled EMARS products from serum EVs using anti-fluorescein antibody. ( C ) Western blot analysis of TSG101 in serum EVs from WT, TL, and TS using anti-TSG101 antibody. Arrows indicate the detected band of monomer TSG101, and black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( D ) Western blot analysis of indicated samples with anti-CHL1 (upper panel) and anti-α2 integrin (lower panel) antibody. Arrow indicates the detected band of each molecule.

    Techniques Used: Transgenic Assay, Mouse Assay, Purification, Immunoprecipitation, SDS Page, Fluorescence, Western Blot, Labeling, Molecular Weight

    Characterization of ELISA system for SLC4A1 ( 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.
    Figure Legend Snippet: Characterization of ELISA system for SLC4A1 ( 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.

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

    Measurement of CHL1-caspase 14 BiEV in serum EVs from lung cancer patients. ( A ) Confirmation of partner molecules 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). ( B ) 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 Supplementary Table 3. Asterisks indicate the samples were below detection limit. ( C ) Comparison of BiEV indexes between H and LC. The BiEV indexes of LC were significantly higher than that of H ( p = 0.019) by a Mann–Whitney test. ( 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.
    Figure Legend Snippet: Measurement of CHL1-caspase 14 BiEV in serum EVs from lung cancer patients. ( A ) Confirmation of partner molecules 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). ( B ) 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 Supplementary Table 3. Asterisks indicate the samples were below detection limit. ( C ) Comparison of BiEV indexes between H and LC. The BiEV indexes of LC were significantly higher than that of H ( p = 0.019) by a Mann–Whitney test. ( 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.

    Techniques Used: Immunoprecipitation, Fluorescence, Western Blot, Labeling, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Recombinant, MANN-WHITNEY, Purification

    Measurement of CHL1-SLC4A1 BiEV in serum EVs from lung cancer patients ( A ) 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. ( B ) Comparison of serum EV CHL1 levels between H (open bar) and LC (closed bar) by using ELISA system. There were also no significant differences between them. ( C ) 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. ( D ) Confirmation of human CHL1-SLC4A1 BiEV in serum EVs. The healthy person (H) and lung cancer patients (LC) EAMRS samples were respectively applied to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-SLC4A1 antibody. Arrows indicate the detected band of human SLC4A1 proteins (including predicted dimer). ( E ) Measurement of fluorescein-labeled human SLC4A1 using sandwich ELISA system used in mouse fluorescein-labeled SLC4A1. The serum EVs from 6 H (open bar) and 6 LC (closed bar) were applied to EMARS reaction followed by ELISA measurement, respectively. The EMARS products containing fluorescein-labeled SLC4A1 were added to anti-SLC4A1 antibody-coated ELISA plates. “BiEV index (SLC4A1)” was calculated based on the value of fluorescein-labeled SLC4A1 in standard samples as described in “ Materials and methods ”.
    Figure Legend Snippet: Measurement of CHL1-SLC4A1 BiEV in serum EVs from lung cancer patients ( A ) 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. ( B ) Comparison of serum EV CHL1 levels between H (open bar) and LC (closed bar) by using ELISA system. There were also no significant differences between them. ( C ) 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. ( D ) Confirmation of human CHL1-SLC4A1 BiEV in serum EVs. The healthy person (H) and lung cancer patients (LC) EAMRS samples were respectively applied to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-SLC4A1 antibody. Arrows indicate the detected band of human SLC4A1 proteins (including predicted dimer). ( E ) Measurement of fluorescein-labeled human SLC4A1 using sandwich ELISA system used in mouse fluorescein-labeled SLC4A1. The serum EVs from 6 H (open bar) and 6 LC (closed bar) were applied to EMARS reaction followed by ELISA measurement, respectively. The EMARS products containing fluorescein-labeled SLC4A1 were added to anti-SLC4A1 antibody-coated ELISA plates. “BiEV index (SLC4A1)” was calculated based on the value of fluorescein-labeled SLC4A1 in standard samples as described in “ Materials and methods ”.

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

    Measurement of CHL1-SLC4A1 BiEV in serum EVs. ( A ) Measurement of fluorescein-labeled SLC4A1 using a sandwich ELISA. The serum EVs from 12 wild-type mice (open bar) and 18 small lung tumor-bearing EML4-ALK transgenic mice (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled SLC4A1 were added to anti-SLC4A1 antibody-coated ELISA plates. A BiEV index (SLC4A1) was calculated based on the value of fluorescein-labeled SLC4A1 in standard samples. ( B ) Comparison of BiEV indexes between WT and TS. The BiEV indexes of TS were significantly higher than that of WT ( p = 8.9 × 10 −3 ) by a Mann–Whitney test. ( C ) ROC curve for BiEV indexes. AUC was calculated as 0.782. ( D ) A scatter-plot of tumor mass versus BiEV index. Correlation between BiEV index and lung tumor mass of TS was estimated using Spearman’s rank correlation coefficient (0.389: p = 0.111). ( E ) Western blot analysis of SLC4A1 in whole-serum EVs from WT and TS. An aliquot of the serum from 12 animals in the wild-type group and 18 animals in small lung tumor-bearing EML4-ALK transgenic mice was mixed in equal proportions followed by EV purification. Arrows indicate the detected band of SLC4A1. Asterisks indicate unknown bands (predicted as non-specific or partial fragments).
    Figure Legend Snippet: Measurement of CHL1-SLC4A1 BiEV in serum EVs. ( A ) Measurement of fluorescein-labeled SLC4A1 using a sandwich ELISA. The serum EVs from 12 wild-type mice (open bar) and 18 small lung tumor-bearing EML4-ALK transgenic mice (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled SLC4A1 were added to anti-SLC4A1 antibody-coated ELISA plates. A BiEV index (SLC4A1) was calculated based on the value of fluorescein-labeled SLC4A1 in standard samples. ( B ) Comparison of BiEV indexes between WT and TS. The BiEV indexes of TS were significantly higher than that of WT ( p = 8.9 × 10 −3 ) by a Mann–Whitney test. ( C ) ROC curve for BiEV indexes. AUC was calculated as 0.782. ( D ) A scatter-plot of tumor mass versus BiEV index. Correlation between BiEV index and lung tumor mass of TS was estimated using Spearman’s rank correlation coefficient (0.389: p = 0.111). ( E ) Western blot analysis of SLC4A1 in whole-serum EVs from WT and TS. An aliquot of the serum from 12 animals in the wild-type group and 18 animals in small lung tumor-bearing EML4-ALK transgenic mice was mixed in equal proportions followed by EV purification. Arrows indicate the detected band of SLC4A1. Asterisks indicate unknown bands (predicted as non-specific or partial fragments).

    Techniques Used: Labeling, Sandwich ELISA, Mouse Assay, Transgenic Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Western Blot, Purification

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Bimolecule detection for Extracellular Vesicle Screening
    Article Snippet: In the case of membrane stripping, the membranes were treated with stripping solution (Wako Chemicals, Osaka, Japan) at room temperature for 20 min, re-blocking, and re-stained with appropriate antibody. .. ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ). .. 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).

    Direct ELISA:

    Article Title: Bimolecule detection for Extracellular Vesicle Screening
    Article Snippet: In the case of membrane stripping, the membranes were treated with stripping solution (Wako Chemicals, Osaka, Japan) at room temperature for 20 min, re-blocking, and re-stained with appropriate antibody. .. ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ). .. 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).

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    Sino Biological human chl1 antibody
    Characterization of crude and purified serum EVs ( 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 <t>anti-CHL1,</t> anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported (8). ( 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. ( C ) SDS-PAGE analysis of EMARS products in S-500 fraction. The fractionated EMARS products from serum EVs in wild-type and EML4-ALK transgenic mouse were subjected to SDS-PAGE analysis (10% gel) with fluorescence detection. The mixture of seven fractions (fraction No.1 to No. 7), and fraction 8 alone were respectively loaded in each well.
    Human Chl1 Antibody, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of crude and purified serum EVs ( 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 (8). ( 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. ( C ) SDS-PAGE analysis of EMARS products in S-500 fraction. The fractionated EMARS products from serum EVs in wild-type and EML4-ALK transgenic mouse were subjected to SDS-PAGE analysis (10% gel) with fluorescence detection. The mixture of seven fractions (fraction No.1 to No. 7), and fraction 8 alone were respectively loaded in each well.

    Journal: bioRxiv

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    doi: 10.1101/2020.07.23.217018

    Figure Lengend Snippet: Characterization of crude and purified serum EVs ( 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 (8). ( 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. ( C ) SDS-PAGE analysis of EMARS products in S-500 fraction. The fractionated EMARS products from serum EVs in wild-type and EML4-ALK transgenic mouse were subjected to SDS-PAGE analysis (10% gel) with fluorescence detection. The mixture of seven fractions (fraction No.1 to No. 7), and fraction 8 alone were respectively loaded in each well.

    Article Snippet: ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ).

    Techniques: Purification, Expressing, Mouse Assay, Transgenic Assay, Western Blot, SDS Page, Fluorescence

    Confirmation of partner molecules identified by MS proteomics. ( A ) 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). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( B ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody (upper panel). After stripping, the membranes were re-stained with anti-THBS1 antibody (lower panel). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( C ) Western blot analysis of an independent experiment samples identical to Figure 4B with anti-SLC4A1 antibody. Arrow indicates the detected band of predicted dimer SLC4A1 protein. Asterisk indicates unknown non-specific bands. ( D, E ) Expression of SLC4A1 ( D ) and CHL1 proteins ( E ) 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.

    Journal: bioRxiv

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    doi: 10.1101/2020.07.23.217018

    Figure Lengend Snippet: Confirmation of partner molecules identified by MS proteomics. ( A ) 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). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( B ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody (upper panel). After stripping, the membranes were re-stained with anti-THBS1 antibody (lower panel). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( C ) Western blot analysis of an independent experiment samples identical to Figure 4B with anti-SLC4A1 antibody. Arrow indicates the detected band of predicted dimer SLC4A1 protein. Asterisk indicates unknown non-specific bands. ( D, E ) Expression of SLC4A1 ( D ) and CHL1 proteins ( E ) 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: ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ).

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

    Characterization of ELISA system for fluorescein labeled-caspase 14 and human CHL1 ( A, B ) Calibration curve of sandwich ELISA for the detection of both human CHL1 and fluorescein-labeled caspase 14. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody is summarized in ( A ). The detection of several concentrations of fluorescein-labeled caspase 14 standard using HRP-labeled anti-fluorescein antibody is summarized in ( B ).

    Journal: bioRxiv

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    doi: 10.1101/2020.07.23.217018

    Figure Lengend Snippet: Characterization of ELISA system for fluorescein labeled-caspase 14 and human CHL1 ( A, B ) Calibration curve of sandwich ELISA for the detection of both human CHL1 and fluorescein-labeled caspase 14. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody is summarized in ( A ). The detection of several concentrations of fluorescein-labeled caspase 14 standard using HRP-labeled anti-fluorescein antibody is summarized in ( B ).

    Article Snippet: ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ).

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

    Summary of advantages for human EV screening using BiEV One of hypothetical mechanisms of BiEV differences between human healthy and lung cancer patients is summarized in this figure based on our results of no significant differences of CHL1 and caspase 14 expression levels. Due to the hypothetical mechanisms, it is considerable that our BiEV analysis is useful when it is difficult to characterize secreted cancer EVs by single molecular index ( e . g . serum CHL1 single screening and typical serum biomarker (CEA and SCC in Supplementary Table 3)). In this study, BiEV analysis may emphasize the differences using the information of bimolecular complex which is formed by association between CHL1 and caspase 14 with unknown cancer cell-specific mechanisms.

    Journal: bioRxiv

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    doi: 10.1101/2020.07.23.217018

    Figure Lengend Snippet: Summary of advantages for human EV screening using BiEV One of hypothetical mechanisms of BiEV differences between human healthy and lung cancer patients is summarized in this figure based on our results of no significant differences of CHL1 and caspase 14 expression levels. Due to the hypothetical mechanisms, it is considerable that our BiEV analysis is useful when it is difficult to characterize secreted cancer EVs by single molecular index ( e . g . serum CHL1 single screening and typical serum biomarker (CEA and SCC in Supplementary Table 3)). In this study, BiEV analysis may emphasize the differences using the information of bimolecular complex which is formed by association between CHL1 and caspase 14 with unknown cancer cell-specific mechanisms.

    Article Snippet: ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ).

    Techniques: Expressing, Biomarker Assay

    BiEV analysis for serum EVs from EML4-ALK transgenic mouse. ( 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 ) Western blot analysis of fluorescein-labeled EMARS products from serum EVs using anti-fluorescein antibody. ( C ) Western blot analysis of TSG101 in serum EVs from WT, TL, and TS using anti-TSG101 antibody. Arrows indicate the detected band of monomer TSG101, and black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( D ) Western blot analysis of indicated samples with anti-CHL1 (upper panel) and anti-α2 integrin (lower panel) antibody. Arrow indicates the detected band of each molecule.

    Journal: bioRxiv

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    doi: 10.1101/2020.07.23.217018

    Figure Lengend Snippet: BiEV analysis for serum EVs from EML4-ALK transgenic mouse. ( 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 ) Western blot analysis of fluorescein-labeled EMARS products from serum EVs using anti-fluorescein antibody. ( C ) Western blot analysis of TSG101 in serum EVs from WT, TL, and TS using anti-TSG101 antibody. Arrows indicate the detected band of monomer TSG101, and black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( D ) Western blot analysis of indicated samples with anti-CHL1 (upper panel) and anti-α2 integrin (lower panel) antibody. Arrow indicates the detected band of each molecule.

    Article Snippet: ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ).

    Techniques: Transgenic Assay, Mouse Assay, Purification, Immunoprecipitation, SDS Page, Fluorescence, Western Blot, Labeling, Molecular Weight

    Characterization of ELISA system for SLC4A1 ( 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.

    Journal: bioRxiv

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    doi: 10.1101/2020.07.23.217018

    Figure Lengend Snippet: Characterization of ELISA system for SLC4A1 ( 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: ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ).

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

    Measurement of CHL1-caspase 14 BiEV in serum EVs from lung cancer patients. ( A ) Confirmation of partner molecules 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). ( B ) 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 Supplementary Table 3. Asterisks indicate the samples were below detection limit. ( C ) Comparison of BiEV indexes between H and LC. The BiEV indexes of LC were significantly higher than that of H ( p = 0.019) by a Mann–Whitney test. ( 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.

    Journal: bioRxiv

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    doi: 10.1101/2020.07.23.217018

    Figure Lengend Snippet: Measurement of CHL1-caspase 14 BiEV in serum EVs from lung cancer patients. ( A ) Confirmation of partner molecules 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). ( B ) 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 Supplementary Table 3. Asterisks indicate the samples were below detection limit. ( C ) Comparison of BiEV indexes between H and LC. The BiEV indexes of LC were significantly higher than that of H ( p = 0.019) by a Mann–Whitney test. ( 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: ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ).

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

    Measurement of CHL1-SLC4A1 BiEV in serum EVs. ( A ) Measurement of fluorescein-labeled SLC4A1 using a sandwich ELISA. The serum EVs from 12 wild-type mice (open bar) and 18 small lung tumor-bearing EML4-ALK transgenic mice (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled SLC4A1 were added to anti-SLC4A1 antibody-coated ELISA plates. A BiEV index (SLC4A1) was calculated based on the value of fluorescein-labeled SLC4A1 in standard samples. ( B ) Comparison of BiEV indexes between WT and TS. The BiEV indexes of TS were significantly higher than that of WT ( p = 8.9 × 10 −3 ) by a Mann–Whitney test. ( C ) ROC curve for BiEV indexes. AUC was calculated as 0.782. ( D ) A scatter-plot of tumor mass versus BiEV index. Correlation between BiEV index and lung tumor mass of TS was estimated using Spearman’s rank correlation coefficient (0.389: p = 0.111). ( E ) Western blot analysis of SLC4A1 in whole-serum EVs from WT and TS. An aliquot of the serum from 12 animals in the wild-type group and 18 animals in small lung tumor-bearing EML4-ALK transgenic mice was mixed in equal proportions followed by EV purification. Arrows indicate the detected band of SLC4A1. Asterisks indicate unknown bands (predicted as non-specific or partial fragments).

    Journal: bioRxiv

    Article Title: Bimolecule detection for Extracellular Vesicle Screening

    doi: 10.1101/2020.07.23.217018

    Figure Lengend Snippet: Measurement of CHL1-SLC4A1 BiEV in serum EVs. ( A ) Measurement of fluorescein-labeled SLC4A1 using a sandwich ELISA. The serum EVs from 12 wild-type mice (open bar) and 18 small lung tumor-bearing EML4-ALK transgenic mice (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled SLC4A1 were added to anti-SLC4A1 antibody-coated ELISA plates. A BiEV index (SLC4A1) was calculated based on the value of fluorescein-labeled SLC4A1 in standard samples. ( B ) Comparison of BiEV indexes between WT and TS. The BiEV indexes of TS were significantly higher than that of WT ( p = 8.9 × 10 −3 ) by a Mann–Whitney test. ( C ) ROC curve for BiEV indexes. AUC was calculated as 0.782. ( D ) A scatter-plot of tumor mass versus BiEV index. Correlation between BiEV index and lung tumor mass of TS was estimated using Spearman’s rank correlation coefficient (0.389: p = 0.111). ( E ) Western blot analysis of SLC4A1 in whole-serum EVs from WT and TS. An aliquot of the serum from 12 animals in the wild-type group and 18 animals in small lung tumor-bearing EML4-ALK transgenic mice was mixed in equal proportions followed by EV purification. Arrows indicate the detected band of SLC4A1. Asterisks indicate unknown bands (predicted as non-specific or partial fragments).

    Article Snippet: ELISA for CHL1 proteins in serum or EVsThe mouse serum CHL1 level was measured with direct ELISA method using HRP-conjugated anti-mouse and human CHL1 antibody as described previously ( ).

    Techniques: Labeling, Sandwich ELISA, Mouse Assay, Transgenic Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Western Blot, Purification