mouse anti human mpo monoclonal antibody  (Bio-Techne corporation)


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    Bio-Techne corporation mouse anti human mpo monoclonal antibody
    A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial <t>MPO-DNA</t> complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, <t>IgG2b</t> isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.
    Mouse Anti Human Mpo Monoclonal Antibody, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human mpo monoclonal antibody/product/Bio-Techne corporation
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti human mpo monoclonal antibody - by Bioz Stars, 2024-09
    95/100 stars

    Images

    1) Product Images from "ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo"

    Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0250265

    A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial MPO-DNA complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, IgG2b isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.
    Figure Legend Snippet: A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial MPO-DNA complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, IgG2b isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

    For assessment of A)—C) serially diluted calibrator and D) 1:2 diluted plasma, the following modifications of the initial MPO-DNA complex ELISA protocol were applied: Microwells were coated with A) 1 - 5 μg/ml rabbit anti-human MPO polyclonal antibody (no. 07-496-I, EMD Millipore), B) 4 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam) or C) and D) 1 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam), 1 μg/ml mouse IgG2a kappa monoclonal isotype control (no. 010-001-332, Rockland Immunochemicals) or were left uncoated. For MPO-DNA complex detection A) peroxidase-conjugated mouse anti-DNA monoclonal antibody (clone MCA-33, as described for the initial ELISA protocol), B) 0.031 – 2 μg/ml HRP-labeled mouse anti-human MPO monoclonal antibody (no. NBP2-41406H, Bio-Techne) or C) and D) 0.5 μg/ml biotinylated mouse anti-human MPO monoclonal antibody (no. HM2164BT, HyCult Biotech) followed by 0.5 μg/ml streptavidin-HRP were applied.
    Figure Legend Snippet: For assessment of A)—C) serially diluted calibrator and D) 1:2 diluted plasma, the following modifications of the initial MPO-DNA complex ELISA protocol were applied: Microwells were coated with A) 1 - 5 μg/ml rabbit anti-human MPO polyclonal antibody (no. 07-496-I, EMD Millipore), B) 4 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam) or C) and D) 1 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam), 1 μg/ml mouse IgG2a kappa monoclonal isotype control (no. 010-001-332, Rockland Immunochemicals) or were left uncoated. For MPO-DNA complex detection A) peroxidase-conjugated mouse anti-DNA monoclonal antibody (clone MCA-33, as described for the initial ELISA protocol), B) 0.031 – 2 μg/ml HRP-labeled mouse anti-human MPO monoclonal antibody (no. NBP2-41406H, Bio-Techne) or C) and D) 0.5 μg/ml biotinylated mouse anti-human MPO monoclonal antibody (no. HM2164BT, HyCult Biotech) followed by 0.5 μg/ml streptavidin-HRP were applied.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Labeling

    A) Microwells were coated with MPO antibody, various isotype controls or were left uncoated. Calibrator or 1:2 diluted plasma samples were analyzed according to the initial MPO-DNA complex ELISA protocol. The calibrator with a relative concentration of “0” equals blank. B) MPO antibody (Bio-Rad) and C) isotype control (IgG2b, Bio-Techne) were processed to Fab and Fc fragments. Western blot analysis after reducing SDS-PAGE (12.5% gel) of unprocessed antibodies (lane 2), papain cleaved unpurified antibodies (lane 3), purified Fab fragments (fractions 1–3, lanes 4–6) as well as Fc fragments (fractions 1–3, lanes 7–9) are depicted. Molecular weight marker (Kaleidoscope™ Prestained Standard) was applied in lane 1 and is given in kDa. Intact immunoglobulin G heavy chains (IgG H ) in lane 2 and the respective Fc and Fab fragment chains in lanes 3–9 are indicated with blue, black and green arrowheads, respectively. Please note that the immunoglobulin G light chain (IgG L ) remains unaltered by the fragmentation process and has a molecular weight close to the fragmented Fab heavy chain. Hence, the two protein bands could not be resolved and are both indicated by the green arrowhead. D) and E) MPO antibody and isotype control (for both: Fab fragment, fraction 1) were coated onto microwells and incubated with D) serially diluted calibrator or E) 1:2 diluted plasma samples. The further ELISA procedure was performed as indicated for the initial MPO-DNA complex ELISA protocol.
    Figure Legend Snippet: A) Microwells were coated with MPO antibody, various isotype controls or were left uncoated. Calibrator or 1:2 diluted plasma samples were analyzed according to the initial MPO-DNA complex ELISA protocol. The calibrator with a relative concentration of “0” equals blank. B) MPO antibody (Bio-Rad) and C) isotype control (IgG2b, Bio-Techne) were processed to Fab and Fc fragments. Western blot analysis after reducing SDS-PAGE (12.5% gel) of unprocessed antibodies (lane 2), papain cleaved unpurified antibodies (lane 3), purified Fab fragments (fractions 1–3, lanes 4–6) as well as Fc fragments (fractions 1–3, lanes 7–9) are depicted. Molecular weight marker (Kaleidoscope™ Prestained Standard) was applied in lane 1 and is given in kDa. Intact immunoglobulin G heavy chains (IgG H ) in lane 2 and the respective Fc and Fab fragment chains in lanes 3–9 are indicated with blue, black and green arrowheads, respectively. Please note that the immunoglobulin G light chain (IgG L ) remains unaltered by the fragmentation process and has a molecular weight close to the fragmented Fab heavy chain. Hence, the two protein bands could not be resolved and are both indicated by the green arrowhead. D) and E) MPO antibody and isotype control (for both: Fab fragment, fraction 1) were coated onto microwells and incubated with D) serially diluted calibrator or E) 1:2 diluted plasma samples. The further ELISA procedure was performed as indicated for the initial MPO-DNA complex ELISA protocol.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, SDS Page, Purification, Molecular Weight, Marker, Incubation

    A) and C) Serially diluted calibrator or B) and D) plasma samples diluted at 1:10, 1:30 and 1:100 (with sample diluent of the ABTS ELISA Buffer Kit) were supplemented A) and B) without (w/o) or with increasing doses (10 μg/ml, 100 μg/ml, 500 μg/ml) of protein A-purified mouse IgG from normal mouse serum or C) and D) without (w/o) or with graded doses (0.1%, 1%, 10%) of TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according the initial protocol in microwells coated with MPO antibody, isotype control or left uncoated. Note that data points of uncoated microwells overlap in 8A and C. E) Plasma was diluted 1:100 without (w/o) or with supplementation of 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according to the initial protocol in microwells coated with MPO antibody or isotype control. F) and G) Plasma of two donors at dilutions of 1:100, 1:200 and 1:400 was supplemented with 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was based on the initial protocol with microwells coated with MPO antibody or isotype control or left uncoated.
    Figure Legend Snippet: A) and C) Serially diluted calibrator or B) and D) plasma samples diluted at 1:10, 1:30 and 1:100 (with sample diluent of the ABTS ELISA Buffer Kit) were supplemented A) and B) without (w/o) or with increasing doses (10 μg/ml, 100 μg/ml, 500 μg/ml) of protein A-purified mouse IgG from normal mouse serum or C) and D) without (w/o) or with graded doses (0.1%, 1%, 10%) of TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according the initial protocol in microwells coated with MPO antibody, isotype control or left uncoated. Note that data points of uncoated microwells overlap in 8A and C. E) Plasma was diluted 1:100 without (w/o) or with supplementation of 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according to the initial protocol in microwells coated with MPO antibody or isotype control. F) and G) Plasma of two donors at dilutions of 1:100, 1:200 and 1:400 was supplemented with 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was based on the initial protocol with microwells coated with MPO antibody or isotype control or left uncoated.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay

    mouse anti human mpo monoclonal antibody  (Bio-Techne corporation)


    Bioz Verified Symbol Bio-Techne corporation is a verified supplier
    Bioz Manufacturer Symbol Bio-Techne corporation manufactures this product  
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    Structured Review

    Bio-Techne corporation mouse anti human mpo monoclonal antibody
    A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial <t>MPO-DNA</t> complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, <t>IgG2b</t> isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.
    Mouse Anti Human Mpo Monoclonal Antibody, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human mpo monoclonal antibody/product/Bio-Techne corporation
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti human mpo monoclonal antibody - by Bioz Stars, 2024-09
    95/100 stars

    Images

    1) Product Images from "ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo"

    Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0250265

    A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial MPO-DNA complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, IgG2b isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.
    Figure Legend Snippet: A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial MPO-DNA complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, IgG2b isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

    For assessment of A)—C) serially diluted calibrator and D) 1:2 diluted plasma, the following modifications of the initial MPO-DNA complex ELISA protocol were applied: Microwells were coated with A) 1 - 5 μg/ml rabbit anti-human MPO polyclonal antibody (no. 07-496-I, EMD Millipore), B) 4 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam) or C) and D) 1 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam), 1 μg/ml mouse IgG2a kappa monoclonal isotype control (no. 010-001-332, Rockland Immunochemicals) or were left uncoated. For MPO-DNA complex detection A) peroxidase-conjugated mouse anti-DNA monoclonal antibody (clone MCA-33, as described for the initial ELISA protocol), B) 0.031 – 2 μg/ml HRP-labeled mouse anti-human MPO monoclonal antibody (no. NBP2-41406H, Bio-Techne) or C) and D) 0.5 μg/ml biotinylated mouse anti-human MPO monoclonal antibody (no. HM2164BT, HyCult Biotech) followed by 0.5 μg/ml streptavidin-HRP were applied.
    Figure Legend Snippet: For assessment of A)—C) serially diluted calibrator and D) 1:2 diluted plasma, the following modifications of the initial MPO-DNA complex ELISA protocol were applied: Microwells were coated with A) 1 - 5 μg/ml rabbit anti-human MPO polyclonal antibody (no. 07-496-I, EMD Millipore), B) 4 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam) or C) and D) 1 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam), 1 μg/ml mouse IgG2a kappa monoclonal isotype control (no. 010-001-332, Rockland Immunochemicals) or were left uncoated. For MPO-DNA complex detection A) peroxidase-conjugated mouse anti-DNA monoclonal antibody (clone MCA-33, as described for the initial ELISA protocol), B) 0.031 – 2 μg/ml HRP-labeled mouse anti-human MPO monoclonal antibody (no. NBP2-41406H, Bio-Techne) or C) and D) 0.5 μg/ml biotinylated mouse anti-human MPO monoclonal antibody (no. HM2164BT, HyCult Biotech) followed by 0.5 μg/ml streptavidin-HRP were applied.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Labeling

    A) Microwells were coated with MPO antibody, various isotype controls or were left uncoated. Calibrator or 1:2 diluted plasma samples were analyzed according to the initial MPO-DNA complex ELISA protocol. The calibrator with a relative concentration of “0” equals blank. B) MPO antibody (Bio-Rad) and C) isotype control (IgG2b, Bio-Techne) were processed to Fab and Fc fragments. Western blot analysis after reducing SDS-PAGE (12.5% gel) of unprocessed antibodies (lane 2), papain cleaved unpurified antibodies (lane 3), purified Fab fragments (fractions 1–3, lanes 4–6) as well as Fc fragments (fractions 1–3, lanes 7–9) are depicted. Molecular weight marker (Kaleidoscope™ Prestained Standard) was applied in lane 1 and is given in kDa. Intact immunoglobulin G heavy chains (IgG H ) in lane 2 and the respective Fc and Fab fragment chains in lanes 3–9 are indicated with blue, black and green arrowheads, respectively. Please note that the immunoglobulin G light chain (IgG L ) remains unaltered by the fragmentation process and has a molecular weight close to the fragmented Fab heavy chain. Hence, the two protein bands could not be resolved and are both indicated by the green arrowhead. D) and E) MPO antibody and isotype control (for both: Fab fragment, fraction 1) were coated onto microwells and incubated with D) serially diluted calibrator or E) 1:2 diluted plasma samples. The further ELISA procedure was performed as indicated for the initial MPO-DNA complex ELISA protocol.
    Figure Legend Snippet: A) Microwells were coated with MPO antibody, various isotype controls or were left uncoated. Calibrator or 1:2 diluted plasma samples were analyzed according to the initial MPO-DNA complex ELISA protocol. The calibrator with a relative concentration of “0” equals blank. B) MPO antibody (Bio-Rad) and C) isotype control (IgG2b, Bio-Techne) were processed to Fab and Fc fragments. Western blot analysis after reducing SDS-PAGE (12.5% gel) of unprocessed antibodies (lane 2), papain cleaved unpurified antibodies (lane 3), purified Fab fragments (fractions 1–3, lanes 4–6) as well as Fc fragments (fractions 1–3, lanes 7–9) are depicted. Molecular weight marker (Kaleidoscope™ Prestained Standard) was applied in lane 1 and is given in kDa. Intact immunoglobulin G heavy chains (IgG H ) in lane 2 and the respective Fc and Fab fragment chains in lanes 3–9 are indicated with blue, black and green arrowheads, respectively. Please note that the immunoglobulin G light chain (IgG L ) remains unaltered by the fragmentation process and has a molecular weight close to the fragmented Fab heavy chain. Hence, the two protein bands could not be resolved and are both indicated by the green arrowhead. D) and E) MPO antibody and isotype control (for both: Fab fragment, fraction 1) were coated onto microwells and incubated with D) serially diluted calibrator or E) 1:2 diluted plasma samples. The further ELISA procedure was performed as indicated for the initial MPO-DNA complex ELISA protocol.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, SDS Page, Purification, Molecular Weight, Marker, Incubation

    A) and C) Serially diluted calibrator or B) and D) plasma samples diluted at 1:10, 1:30 and 1:100 (with sample diluent of the ABTS ELISA Buffer Kit) were supplemented A) and B) without (w/o) or with increasing doses (10 μg/ml, 100 μg/ml, 500 μg/ml) of protein A-purified mouse IgG from normal mouse serum or C) and D) without (w/o) or with graded doses (0.1%, 1%, 10%) of TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according the initial protocol in microwells coated with MPO antibody, isotype control or left uncoated. Note that data points of uncoated microwells overlap in 8A and C. E) Plasma was diluted 1:100 without (w/o) or with supplementation of 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according to the initial protocol in microwells coated with MPO antibody or isotype control. F) and G) Plasma of two donors at dilutions of 1:100, 1:200 and 1:400 was supplemented with 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was based on the initial protocol with microwells coated with MPO antibody or isotype control or left uncoated.
    Figure Legend Snippet: A) and C) Serially diluted calibrator or B) and D) plasma samples diluted at 1:10, 1:30 and 1:100 (with sample diluent of the ABTS ELISA Buffer Kit) were supplemented A) and B) without (w/o) or with increasing doses (10 μg/ml, 100 μg/ml, 500 μg/ml) of protein A-purified mouse IgG from normal mouse serum or C) and D) without (w/o) or with graded doses (0.1%, 1%, 10%) of TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according the initial protocol in microwells coated with MPO antibody, isotype control or left uncoated. Note that data points of uncoated microwells overlap in 8A and C. E) Plasma was diluted 1:100 without (w/o) or with supplementation of 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according to the initial protocol in microwells coated with MPO antibody or isotype control. F) and G) Plasma of two donors at dilutions of 1:100, 1:200 and 1:400 was supplemented with 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was based on the initial protocol with microwells coated with MPO antibody or isotype control or left uncoated.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay

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    Bio-Techne corporation mouse anti human mpo monoclonal antibody
    A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial <t>MPO-DNA</t> complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, <t>IgG2b</t> isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.
    Mouse Anti Human Mpo Monoclonal Antibody, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human mpo monoclonal antibody/product/Bio-Techne corporation
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti human mpo monoclonal antibody - by Bioz Stars, 2024-09
    95/100 stars
    		  Buy from Supplier

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    A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial MPO-DNA complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, IgG2b isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.

    Journal: PLoS ONE

    Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

    doi: 10.1371/journal.pone.0250265

    Figure Lengend Snippet: A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial MPO-DNA complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, IgG2b isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.

    Article Snippet: Alternative detection antibodies and other reagents: horseradish peroxidase (HRP)-labeled mouse anti-human MPO monoclonal antibody (clone MPO421-8B2, IgG1, RRID:AB_2827763, no. NBP2-41406H, Bio-Techne), biotinylated mouse anti-human MPO monoclonal antibody (clone 266-6K1, IgG1, RRID:AB_10234434, no. HM2164BT, HyCult Biotech, Uden, The Netherlands) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific).

    Techniques: Isolation, Enzyme-linked Immunosorbent Assay

    For assessment of A)—C) serially diluted calibrator and D) 1:2 diluted plasma, the following modifications of the initial MPO-DNA complex ELISA protocol were applied: Microwells were coated with A) 1 - 5 μg/ml rabbit anti-human MPO polyclonal antibody (no. 07-496-I, EMD Millipore), B) 4 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam) or C) and D) 1 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam), 1 μg/ml mouse IgG2a kappa monoclonal isotype control (no. 010-001-332, Rockland Immunochemicals) or were left uncoated. For MPO-DNA complex detection A) peroxidase-conjugated mouse anti-DNA monoclonal antibody (clone MCA-33, as described for the initial ELISA protocol), B) 0.031 – 2 μg/ml HRP-labeled mouse anti-human MPO monoclonal antibody (no. NBP2-41406H, Bio-Techne) or C) and D) 0.5 μg/ml biotinylated mouse anti-human MPO monoclonal antibody (no. HM2164BT, HyCult Biotech) followed by 0.5 μg/ml streptavidin-HRP were applied.

    Journal: PLoS ONE

    Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

    doi: 10.1371/journal.pone.0250265

    Figure Lengend Snippet: For assessment of A)—C) serially diluted calibrator and D) 1:2 diluted plasma, the following modifications of the initial MPO-DNA complex ELISA protocol were applied: Microwells were coated with A) 1 - 5 μg/ml rabbit anti-human MPO polyclonal antibody (no. 07-496-I, EMD Millipore), B) 4 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam) or C) and D) 1 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam), 1 μg/ml mouse IgG2a kappa monoclonal isotype control (no. 010-001-332, Rockland Immunochemicals) or were left uncoated. For MPO-DNA complex detection A) peroxidase-conjugated mouse anti-DNA monoclonal antibody (clone MCA-33, as described for the initial ELISA protocol), B) 0.031 – 2 μg/ml HRP-labeled mouse anti-human MPO monoclonal antibody (no. NBP2-41406H, Bio-Techne) or C) and D) 0.5 μg/ml biotinylated mouse anti-human MPO monoclonal antibody (no. HM2164BT, HyCult Biotech) followed by 0.5 μg/ml streptavidin-HRP were applied.

    Article Snippet: Alternative detection antibodies and other reagents: horseradish peroxidase (HRP)-labeled mouse anti-human MPO monoclonal antibody (clone MPO421-8B2, IgG1, RRID:AB_2827763, no. NBP2-41406H, Bio-Techne), biotinylated mouse anti-human MPO monoclonal antibody (clone 266-6K1, IgG1, RRID:AB_10234434, no. HM2164BT, HyCult Biotech, Uden, The Netherlands) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling

    A) Microwells were coated with MPO antibody, various isotype controls or were left uncoated. Calibrator or 1:2 diluted plasma samples were analyzed according to the initial MPO-DNA complex ELISA protocol. The calibrator with a relative concentration of “0” equals blank. B) MPO antibody (Bio-Rad) and C) isotype control (IgG2b, Bio-Techne) were processed to Fab and Fc fragments. Western blot analysis after reducing SDS-PAGE (12.5% gel) of unprocessed antibodies (lane 2), papain cleaved unpurified antibodies (lane 3), purified Fab fragments (fractions 1–3, lanes 4–6) as well as Fc fragments (fractions 1–3, lanes 7–9) are depicted. Molecular weight marker (Kaleidoscope™ Prestained Standard) was applied in lane 1 and is given in kDa. Intact immunoglobulin G heavy chains (IgG H ) in lane 2 and the respective Fc and Fab fragment chains in lanes 3–9 are indicated with blue, black and green arrowheads, respectively. Please note that the immunoglobulin G light chain (IgG L ) remains unaltered by the fragmentation process and has a molecular weight close to the fragmented Fab heavy chain. Hence, the two protein bands could not be resolved and are both indicated by the green arrowhead. D) and E) MPO antibody and isotype control (for both: Fab fragment, fraction 1) were coated onto microwells and incubated with D) serially diluted calibrator or E) 1:2 diluted plasma samples. The further ELISA procedure was performed as indicated for the initial MPO-DNA complex ELISA protocol.

    Journal: PLoS ONE

    Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

    doi: 10.1371/journal.pone.0250265

    Figure Lengend Snippet: A) Microwells were coated with MPO antibody, various isotype controls or were left uncoated. Calibrator or 1:2 diluted plasma samples were analyzed according to the initial MPO-DNA complex ELISA protocol. The calibrator with a relative concentration of “0” equals blank. B) MPO antibody (Bio-Rad) and C) isotype control (IgG2b, Bio-Techne) were processed to Fab and Fc fragments. Western blot analysis after reducing SDS-PAGE (12.5% gel) of unprocessed antibodies (lane 2), papain cleaved unpurified antibodies (lane 3), purified Fab fragments (fractions 1–3, lanes 4–6) as well as Fc fragments (fractions 1–3, lanes 7–9) are depicted. Molecular weight marker (Kaleidoscope™ Prestained Standard) was applied in lane 1 and is given in kDa. Intact immunoglobulin G heavy chains (IgG H ) in lane 2 and the respective Fc and Fab fragment chains in lanes 3–9 are indicated with blue, black and green arrowheads, respectively. Please note that the immunoglobulin G light chain (IgG L ) remains unaltered by the fragmentation process and has a molecular weight close to the fragmented Fab heavy chain. Hence, the two protein bands could not be resolved and are both indicated by the green arrowhead. D) and E) MPO antibody and isotype control (for both: Fab fragment, fraction 1) were coated onto microwells and incubated with D) serially diluted calibrator or E) 1:2 diluted plasma samples. The further ELISA procedure was performed as indicated for the initial MPO-DNA complex ELISA protocol.

    Article Snippet: Alternative detection antibodies and other reagents: horseradish peroxidase (HRP)-labeled mouse anti-human MPO monoclonal antibody (clone MPO421-8B2, IgG1, RRID:AB_2827763, no. NBP2-41406H, Bio-Techne), biotinylated mouse anti-human MPO monoclonal antibody (clone 266-6K1, IgG1, RRID:AB_10234434, no. HM2164BT, HyCult Biotech, Uden, The Netherlands) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, SDS Page, Purification, Molecular Weight, Marker, Incubation

    A) and C) Serially diluted calibrator or B) and D) plasma samples diluted at 1:10, 1:30 and 1:100 (with sample diluent of the ABTS ELISA Buffer Kit) were supplemented A) and B) without (w/o) or with increasing doses (10 μg/ml, 100 μg/ml, 500 μg/ml) of protein A-purified mouse IgG from normal mouse serum or C) and D) without (w/o) or with graded doses (0.1%, 1%, 10%) of TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according the initial protocol in microwells coated with MPO antibody, isotype control or left uncoated. Note that data points of uncoated microwells overlap in 8A and C. E) Plasma was diluted 1:100 without (w/o) or with supplementation of 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according to the initial protocol in microwells coated with MPO antibody or isotype control. F) and G) Plasma of two donors at dilutions of 1:100, 1:200 and 1:400 was supplemented with 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was based on the initial protocol with microwells coated with MPO antibody or isotype control or left uncoated.

    Journal: PLoS ONE

    Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

    doi: 10.1371/journal.pone.0250265

    Figure Lengend Snippet: A) and C) Serially diluted calibrator or B) and D) plasma samples diluted at 1:10, 1:30 and 1:100 (with sample diluent of the ABTS ELISA Buffer Kit) were supplemented A) and B) without (w/o) or with increasing doses (10 μg/ml, 100 μg/ml, 500 μg/ml) of protein A-purified mouse IgG from normal mouse serum or C) and D) without (w/o) or with graded doses (0.1%, 1%, 10%) of TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according the initial protocol in microwells coated with MPO antibody, isotype control or left uncoated. Note that data points of uncoated microwells overlap in 8A and C. E) Plasma was diluted 1:100 without (w/o) or with supplementation of 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according to the initial protocol in microwells coated with MPO antibody or isotype control. F) and G) Plasma of two donors at dilutions of 1:100, 1:200 and 1:400 was supplemented with 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was based on the initial protocol with microwells coated with MPO antibody or isotype control or left uncoated.

    Article Snippet: Alternative detection antibodies and other reagents: horseradish peroxidase (HRP)-labeled mouse anti-human MPO monoclonal antibody (clone MPO421-8B2, IgG1, RRID:AB_2827763, no. NBP2-41406H, Bio-Techne), biotinylated mouse anti-human MPO monoclonal antibody (clone 266-6K1, IgG1, RRID:AB_10234434, no. HM2164BT, HyCult Biotech, Uden, The Netherlands) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay