human i fabp elisa kit  (Hycult Biotech)


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    Hycult Biotech human i fabp elisa kit
    Human I Fabp Elisa Kit, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    elisa kit  (Hycult Biotech)


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    Hycult Biotech elisa kit
    Elisa Kit, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    elisa kit  (Hycult Biotech)


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    Hycult Biotech elisa kit
    Elisa Kit, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    immunosorbent assay elisa kit  (Hycult Biotech)


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    Hycult Biotech immunosorbent assay elisa kit
    Immunosorbent Assay Elisa Kit, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    species lbp elisa kits  (Hycult Biotech)


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    Hycult Biotech species lbp elisa kits
    Species Lbp Elisa Kits, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    elisa kit  (Hycult Biotech)


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    Hycult Biotech elisa kit

    Elisa Kit, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis"

    Article Title: Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis

    Journal: iScience

    doi: 10.1016/j.isci.2024.109993


    Figure Legend Snippet:

    Techniques Used: Virus, Recombinant, Sensitive Assay, Enzyme-linked Immunosorbent Assay, Software, Purification

    elisa kit  (Hycult Biotech)


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    Hycult Biotech elisa kit
    Elisa Kit, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ll 37 elisa kit  (Hycult Biotech)


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    Hycult Biotech ll 37 elisa kit
    <t>LL-37</t> and hBD2 significantly reduce experimental NEC induced by Paneth cell disruption with bacterial dysbiosis P14-P16 C57Bl/6J mice were treated with dithizone and K. pneumoniae to induce NEC. Following euthanasia, ileal samples were harvested and scored for NEC-like injury. NEC injury scores of sham (gray), Dithizone (Dith) (orange), K. pneumoniae (Kleb) (green), NEC (red), hBD2 pre (light blue), hBD2 post (dark blue), hBD2 subQ (aqua), LL-37 pre (purple), and LL-37 post (plum) are shown with the dotted horizontal line indicating a NEC like injury (score of 2 or greater). Each circle represents a single animal. Mice with NEC had increased injury compared to sham, hBD2 pre, hBD2 post, hBD2 subQ, and LL-37 post (n = sham: 37, dithizone: 60, Klebsiella : 9, NEC: 99, hBD2 pre: 26, hBD2 post: 24, hBD2 subQ: 17, LL-37 pre: 25, LL-37 post: 20; p < 0.004 for all) (A). Total percentage of animals developing significant disease (score of ≥ 2) (B). On necropsy, mice with NEC (second panel) had dark discolored intestines with adhesions, while animals treated with hBD2 or LL-37(third and fourth panels) had healthier appearing intestines and less adhesions that were more similar to sham conditions (first panel). Yellow arrows indicate small intestine (C). Serum levels of IL-17a, IL-22, and TNF were quantified at time of tissue harvest (D–F). Serum levels of IL17A, IL-22, and TNF were significantly elevated in NEC compared to sham ( p = 0.0016, 0.0006, and 0.0044 respectively), but hBD2 and LL-37 treatment/prevention did not significantly alter any cytokine levels compared to NEC. P14-P16 C57Bl/6J mice were euthanized 30 min following SQ injection or oral gavage with hBD2 or oral gavage or intraperitoneal (IP) injection with LL-37. Serum and homogenized intestinal samples were obtained and quantified for hBD2 (G) and serum, homogenized intestinal samples, and intraperitoneal wash (IP Wash) samples were obtained and quantified for LL-37 (H). Intestinal homogenates from SQ treated mice had hBD2 levels >1000 pg/mL, while all other hBD2 treated samples had hBD2 levels that exceeded the detectable limit by the ELISA plate ( n = 3 animals per group). Elevation of LL-37 was detected in the IP wash of IP injected animals, but elevation was not detected in the serum or intestine in LL-37 IP injection or gavage ( n = 3 animals per group). Error bars in all figures represent SEM.
    Ll 37 Elisa Kit, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis"

    Article Title: Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis

    Journal: iScience

    doi: 10.1016/j.isci.2024.109993

    LL-37 and hBD2 significantly reduce experimental NEC induced by Paneth cell disruption with bacterial dysbiosis P14-P16 C57Bl/6J mice were treated with dithizone and K. pneumoniae to induce NEC. Following euthanasia, ileal samples were harvested and scored for NEC-like injury. NEC injury scores of sham (gray), Dithizone (Dith) (orange), K. pneumoniae (Kleb) (green), NEC (red), hBD2 pre (light blue), hBD2 post (dark blue), hBD2 subQ (aqua), LL-37 pre (purple), and LL-37 post (plum) are shown with the dotted horizontal line indicating a NEC like injury (score of 2 or greater). Each circle represents a single animal. Mice with NEC had increased injury compared to sham, hBD2 pre, hBD2 post, hBD2 subQ, and LL-37 post (n = sham: 37, dithizone: 60, Klebsiella : 9, NEC: 99, hBD2 pre: 26, hBD2 post: 24, hBD2 subQ: 17, LL-37 pre: 25, LL-37 post: 20; p < 0.004 for all) (A). Total percentage of animals developing significant disease (score of ≥ 2) (B). On necropsy, mice with NEC (second panel) had dark discolored intestines with adhesions, while animals treated with hBD2 or LL-37(third and fourth panels) had healthier appearing intestines and less adhesions that were more similar to sham conditions (first panel). Yellow arrows indicate small intestine (C). Serum levels of IL-17a, IL-22, and TNF were quantified at time of tissue harvest (D–F). Serum levels of IL17A, IL-22, and TNF were significantly elevated in NEC compared to sham ( p = 0.0016, 0.0006, and 0.0044 respectively), but hBD2 and LL-37 treatment/prevention did not significantly alter any cytokine levels compared to NEC. P14-P16 C57Bl/6J mice were euthanized 30 min following SQ injection or oral gavage with hBD2 or oral gavage or intraperitoneal (IP) injection with LL-37. Serum and homogenized intestinal samples were obtained and quantified for hBD2 (G) and serum, homogenized intestinal samples, and intraperitoneal wash (IP Wash) samples were obtained and quantified for LL-37 (H). Intestinal homogenates from SQ treated mice had hBD2 levels >1000 pg/mL, while all other hBD2 treated samples had hBD2 levels that exceeded the detectable limit by the ELISA plate ( n = 3 animals per group). Elevation of LL-37 was detected in the IP wash of IP injected animals, but elevation was not detected in the serum or intestine in LL-37 IP injection or gavage ( n = 3 animals per group). Error bars in all figures represent SEM.
    Figure Legend Snippet: LL-37 and hBD2 significantly reduce experimental NEC induced by Paneth cell disruption with bacterial dysbiosis P14-P16 C57Bl/6J mice were treated with dithizone and K. pneumoniae to induce NEC. Following euthanasia, ileal samples were harvested and scored for NEC-like injury. NEC injury scores of sham (gray), Dithizone (Dith) (orange), K. pneumoniae (Kleb) (green), NEC (red), hBD2 pre (light blue), hBD2 post (dark blue), hBD2 subQ (aqua), LL-37 pre (purple), and LL-37 post (plum) are shown with the dotted horizontal line indicating a NEC like injury (score of 2 or greater). Each circle represents a single animal. Mice with NEC had increased injury compared to sham, hBD2 pre, hBD2 post, hBD2 subQ, and LL-37 post (n = sham: 37, dithizone: 60, Klebsiella : 9, NEC: 99, hBD2 pre: 26, hBD2 post: 24, hBD2 subQ: 17, LL-37 pre: 25, LL-37 post: 20; p < 0.004 for all) (A). Total percentage of animals developing significant disease (score of ≥ 2) (B). On necropsy, mice with NEC (second panel) had dark discolored intestines with adhesions, while animals treated with hBD2 or LL-37(third and fourth panels) had healthier appearing intestines and less adhesions that were more similar to sham conditions (first panel). Yellow arrows indicate small intestine (C). Serum levels of IL-17a, IL-22, and TNF were quantified at time of tissue harvest (D–F). Serum levels of IL17A, IL-22, and TNF were significantly elevated in NEC compared to sham ( p = 0.0016, 0.0006, and 0.0044 respectively), but hBD2 and LL-37 treatment/prevention did not significantly alter any cytokine levels compared to NEC. P14-P16 C57Bl/6J mice were euthanized 30 min following SQ injection or oral gavage with hBD2 or oral gavage or intraperitoneal (IP) injection with LL-37. Serum and homogenized intestinal samples were obtained and quantified for hBD2 (G) and serum, homogenized intestinal samples, and intraperitoneal wash (IP Wash) samples were obtained and quantified for LL-37 (H). Intestinal homogenates from SQ treated mice had hBD2 levels >1000 pg/mL, while all other hBD2 treated samples had hBD2 levels that exceeded the detectable limit by the ELISA plate ( n = 3 animals per group). Elevation of LL-37 was detected in the IP wash of IP injected animals, but elevation was not detected in the serum or intestine in LL-37 IP injection or gavage ( n = 3 animals per group). Error bars in all figures represent SEM.

    Techniques Used: Disruption, Injection, Enzyme-linked Immunosorbent Assay

    LL-37 and hBD2 prevention and treatment approaches do not cause detectable alterations in the cecal microbiome Cecal samples were analyzed for microbial composition using 16S sequencing. Phylum and family level analysis showed that induction of NEC altered the microbial composition by increasing the relative amount of Proteobacteria ( p < 0.0001), and in particular Enterobacteriaceae ( p < 0.0001), while decreasing the relative amount of Firmicutes. hBD2 and LL-37 treatment (pre- or post-induction of NEC) had no impact (n = sham: 6, NEC: 9, hBD2 pre: 11, hBD2 post: 10, hBD2 subQ 5, LL-37 pre 6, LL-37 post 5, p < 0.05) [(A): Phyla, (B): Enterobacteriaceae only)]. Alpha diversity analysis via chao1 demonstrated decreases in diversity in NEC and HDP pre-treated animals (C). Principle coordinate analysis of the 16S cecal microbiome indicates animals exposed to K. pneumoniae gavage ( Klebsiella , NEC, pre hBD2, post hBD2, subQ hBD2, pre LL-37, and post LL-37) clustered together independent of HDP treatment and separate from non- Klebsiella animals (sham, dithizone) (D). Error bars in all figures represent SEM.
    Figure Legend Snippet: LL-37 and hBD2 prevention and treatment approaches do not cause detectable alterations in the cecal microbiome Cecal samples were analyzed for microbial composition using 16S sequencing. Phylum and family level analysis showed that induction of NEC altered the microbial composition by increasing the relative amount of Proteobacteria ( p < 0.0001), and in particular Enterobacteriaceae ( p < 0.0001), while decreasing the relative amount of Firmicutes. hBD2 and LL-37 treatment (pre- or post-induction of NEC) had no impact (n = sham: 6, NEC: 9, hBD2 pre: 11, hBD2 post: 10, hBD2 subQ 5, LL-37 pre 6, LL-37 post 5, p < 0.05) [(A): Phyla, (B): Enterobacteriaceae only)]. Alpha diversity analysis via chao1 demonstrated decreases in diversity in NEC and HDP pre-treated animals (C). Principle coordinate analysis of the 16S cecal microbiome indicates animals exposed to K. pneumoniae gavage ( Klebsiella , NEC, pre hBD2, post hBD2, subQ hBD2, pre LL-37, and post LL-37) clustered together independent of HDP treatment and separate from non- Klebsiella animals (sham, dithizone) (D). Error bars in all figures represent SEM.

    Techniques Used: Sequencing

    LL-37 can directly kill K pneumoniae 10031, while hBD2 has more limited antimicrobial capacity K. pneumoniae abundance was measured by PCR (A). NEC treatment significantly increased detection of K. pneumoniae ( p = 0.0002, n = 9). Pre-treatment with HDPs LL-37 and hBD2 had no effect on NEC-induced increase in K. pneumoniae ( p > 0.9999, n = 3), while post-treatment with HDPs LL-37 and hBD2 reduced K. pneumoniae to sham-levels (sham vs. Post LL-37 p = 0.1648; sham vs. Post hBD2 p = 0.8622 n = 3). Similar to oral therapeutic treatment with HDPs, SQ hBD2 decreased NEC-induced increases in K. pneumoniae toward sham levels ( p = 0.1501). K. pneumoniae (6.3 × 10 6 - 1.1 × 10 7 CFU/mL) and E. coli (2.25 × 10 6 - 1.585 × 10 7 CFU/mL) were exposed to increasing concentrations of hBD2. hBD2 decreased growth for E. coli at 80 μg/mL, and achieved a MIC at 160 μg/mL, but no concentration of hBD2 significantly inhibited growth of K. pneumoniae (n = 2.5 μg/mL: 3, 160 μg/mL: 6, and all other doses 9) (B). K. pneumoniae grown in Nutrient Broth or Tryptic Soy Broth were exposed to increasing concentrations of hBD2. hBD2 induced a sharp decline in survival at concentrations of 25 μg/mL in both media types, but complete killing of K. pneumoniae was not observed in either media or at any concentration of hBD2 tested ( n = 10 replicates for all strains and all doses) (C). In a turbidity assay, hBD2 caused significantly decreased bacterial growth for Bifidobacterium breve and Bifidobacterium adolescentis , but not in S. thermophilus (D). K. pneumoniae (5.15 × 10 6 - 1.18 × 10 7 CFU/mL) was exposed to increasing concentrations of LL-37 (E). LL-37 inhibited growth of K. pneumoniae at 25 μg/mL and completely inhibited growth at 32 μg/mL. Error bars in all figures represent SEM.
    Figure Legend Snippet: LL-37 can directly kill K pneumoniae 10031, while hBD2 has more limited antimicrobial capacity K. pneumoniae abundance was measured by PCR (A). NEC treatment significantly increased detection of K. pneumoniae ( p = 0.0002, n = 9). Pre-treatment with HDPs LL-37 and hBD2 had no effect on NEC-induced increase in K. pneumoniae ( p > 0.9999, n = 3), while post-treatment with HDPs LL-37 and hBD2 reduced K. pneumoniae to sham-levels (sham vs. Post LL-37 p = 0.1648; sham vs. Post hBD2 p = 0.8622 n = 3). Similar to oral therapeutic treatment with HDPs, SQ hBD2 decreased NEC-induced increases in K. pneumoniae toward sham levels ( p = 0.1501). K. pneumoniae (6.3 × 10 6 - 1.1 × 10 7 CFU/mL) and E. coli (2.25 × 10 6 - 1.585 × 10 7 CFU/mL) were exposed to increasing concentrations of hBD2. hBD2 decreased growth for E. coli at 80 μg/mL, and achieved a MIC at 160 μg/mL, but no concentration of hBD2 significantly inhibited growth of K. pneumoniae (n = 2.5 μg/mL: 3, 160 μg/mL: 6, and all other doses 9) (B). K. pneumoniae grown in Nutrient Broth or Tryptic Soy Broth were exposed to increasing concentrations of hBD2. hBD2 induced a sharp decline in survival at concentrations of 25 μg/mL in both media types, but complete killing of K. pneumoniae was not observed in either media or at any concentration of hBD2 tested ( n = 10 replicates for all strains and all doses) (C). In a turbidity assay, hBD2 caused significantly decreased bacterial growth for Bifidobacterium breve and Bifidobacterium adolescentis , but not in S. thermophilus (D). K. pneumoniae (5.15 × 10 6 - 1.18 × 10 7 CFU/mL) was exposed to increasing concentrations of LL-37 (E). LL-37 inhibited growth of K. pneumoniae at 25 μg/mL and completely inhibited growth at 32 μg/mL. Error bars in all figures represent SEM.

    Techniques Used: Concentration Assay


    Figure Legend Snippet:

    Techniques Used: Virus, Recombinant, Sensitive Assay, Enzyme-linked Immunosorbent Assay, Software, Purification

    fh elisa kit  (Hycult Biotech)


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    Hycult Biotech fh elisa kit
    Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations <t>of</t> <t>CPV-104,</t> CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by <t>ELISA.</t> Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.
    Fh Elisa Kit, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Moss-produced human complement factor H with modified glycans has an extended half-life and improved biological activity"

    Article Title: Moss-produced human complement factor H with modified glycans has an extended half-life and improved biological activity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2024.1383123

    Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by ELISA. Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.
    Figure Legend Snippet: Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by ELISA. Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.

    Techniques Used: Functional Assay, Activity Assay, In Vitro, Produced, Incubation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Spectroscopy

    elisa kits  (Hycult Biotech)


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    Hycult Biotech elisa kits
    Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of <t>sd-FH.</t> <t>C3</t> convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by <t>ELISA.</t> Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.
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    1) Product Images from "Moss-produced human complement factor H with modified glycans has an extended half-life and improved biological activity"

    Article Title: Moss-produced human complement factor H with modified glycans has an extended half-life and improved biological activity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2024.1383123

    Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by ELISA. Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.
    Figure Legend Snippet: Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by ELISA. Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.

    Techniques Used: Functional Assay, Activity Assay, In Vitro, Produced, Incubation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Spectroscopy

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    <t>LL-37</t> and hBD2 significantly reduce experimental NEC induced by Paneth cell disruption with bacterial dysbiosis P14-P16 C57Bl/6J mice were treated with dithizone and K. pneumoniae to induce NEC. Following euthanasia, ileal samples were harvested and scored for NEC-like injury. NEC injury scores of sham (gray), Dithizone (Dith) (orange), K. pneumoniae (Kleb) (green), NEC (red), hBD2 pre (light blue), hBD2 post (dark blue), hBD2 subQ (aqua), LL-37 pre (purple), and LL-37 post (plum) are shown with the dotted horizontal line indicating a NEC like injury (score of 2 or greater). Each circle represents a single animal. Mice with NEC had increased injury compared to sham, hBD2 pre, hBD2 post, hBD2 subQ, and LL-37 post (n = sham: 37, dithizone: 60, Klebsiella : 9, NEC: 99, hBD2 pre: 26, hBD2 post: 24, hBD2 subQ: 17, LL-37 pre: 25, LL-37 post: 20; p < 0.004 for all) (A). Total percentage of animals developing significant disease (score of ≥ 2) (B). On necropsy, mice with NEC (second panel) had dark discolored intestines with adhesions, while animals treated with hBD2 or LL-37(third and fourth panels) had healthier appearing intestines and less adhesions that were more similar to sham conditions (first panel). Yellow arrows indicate small intestine (C). Serum levels of IL-17a, IL-22, and TNF were quantified at time of tissue harvest (D–F). Serum levels of IL17A, IL-22, and TNF were significantly elevated in NEC compared to sham ( p = 0.0016, 0.0006, and 0.0044 respectively), but hBD2 and LL-37 treatment/prevention did not significantly alter any cytokine levels compared to NEC. P14-P16 C57Bl/6J mice were euthanized 30 min following SQ injection or oral gavage with hBD2 or oral gavage or intraperitoneal (IP) injection with LL-37. Serum and homogenized intestinal samples were obtained and quantified for hBD2 (G) and serum, homogenized intestinal samples, and intraperitoneal wash (IP Wash) samples were obtained and quantified for LL-37 (H). Intestinal homogenates from SQ treated mice had hBD2 levels >1000 pg/mL, while all other hBD2 treated samples had hBD2 levels that exceeded the detectable limit by the ELISA plate ( n = 3 animals per group). Elevation of LL-37 was detected in the IP wash of IP injected animals, but elevation was not detected in the serum or intestine in LL-37 IP injection or gavage ( n = 3 animals per group). Error bars in all figures represent SEM.
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    Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations <t>of</t> <t>CPV-104,</t> CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by <t>ELISA.</t> Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.
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    Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of <t>sd-FH.</t> <t>C3</t> convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by <t>ELISA.</t> Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.
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    LL-37 and hBD2 significantly reduce experimental NEC induced by Paneth cell disruption with bacterial dysbiosis P14-P16 C57Bl/6J mice were treated with dithizone and K. pneumoniae to induce NEC. Following euthanasia, ileal samples were harvested and scored for NEC-like injury. NEC injury scores of sham (gray), Dithizone (Dith) (orange), K. pneumoniae (Kleb) (green), NEC (red), hBD2 pre (light blue), hBD2 post (dark blue), hBD2 subQ (aqua), LL-37 pre (purple), and LL-37 post (plum) are shown with the dotted horizontal line indicating a NEC like injury (score of 2 or greater). Each circle represents a single animal. Mice with NEC had increased injury compared to sham, hBD2 pre, hBD2 post, hBD2 subQ, and LL-37 post (n = sham: 37, dithizone: 60, Klebsiella : 9, NEC: 99, hBD2 pre: 26, hBD2 post: 24, hBD2 subQ: 17, LL-37 pre: 25, LL-37 post: 20; p < 0.004 for all) (A). Total percentage of animals developing significant disease (score of ≥ 2) (B). On necropsy, mice with NEC (second panel) had dark discolored intestines with adhesions, while animals treated with hBD2 or LL-37(third and fourth panels) had healthier appearing intestines and less adhesions that were more similar to sham conditions (first panel). Yellow arrows indicate small intestine (C). Serum levels of IL-17a, IL-22, and TNF were quantified at time of tissue harvest (D–F). Serum levels of IL17A, IL-22, and TNF were significantly elevated in NEC compared to sham ( p = 0.0016, 0.0006, and 0.0044 respectively), but hBD2 and LL-37 treatment/prevention did not significantly alter any cytokine levels compared to NEC. P14-P16 C57Bl/6J mice were euthanized 30 min following SQ injection or oral gavage with hBD2 or oral gavage or intraperitoneal (IP) injection with LL-37. Serum and homogenized intestinal samples were obtained and quantified for hBD2 (G) and serum, homogenized intestinal samples, and intraperitoneal wash (IP Wash) samples were obtained and quantified for LL-37 (H). Intestinal homogenates from SQ treated mice had hBD2 levels >1000 pg/mL, while all other hBD2 treated samples had hBD2 levels that exceeded the detectable limit by the ELISA plate ( n = 3 animals per group). Elevation of LL-37 was detected in the IP wash of IP injected animals, but elevation was not detected in the serum or intestine in LL-37 IP injection or gavage ( n = 3 animals per group). Error bars in all figures represent SEM.

    Journal: iScience

    Article Title: Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis

    doi: 10.1016/j.isci.2024.109993

    Figure Lengend Snippet: LL-37 and hBD2 significantly reduce experimental NEC induced by Paneth cell disruption with bacterial dysbiosis P14-P16 C57Bl/6J mice were treated with dithizone and K. pneumoniae to induce NEC. Following euthanasia, ileal samples were harvested and scored for NEC-like injury. NEC injury scores of sham (gray), Dithizone (Dith) (orange), K. pneumoniae (Kleb) (green), NEC (red), hBD2 pre (light blue), hBD2 post (dark blue), hBD2 subQ (aqua), LL-37 pre (purple), and LL-37 post (plum) are shown with the dotted horizontal line indicating a NEC like injury (score of 2 or greater). Each circle represents a single animal. Mice with NEC had increased injury compared to sham, hBD2 pre, hBD2 post, hBD2 subQ, and LL-37 post (n = sham: 37, dithizone: 60, Klebsiella : 9, NEC: 99, hBD2 pre: 26, hBD2 post: 24, hBD2 subQ: 17, LL-37 pre: 25, LL-37 post: 20; p < 0.004 for all) (A). Total percentage of animals developing significant disease (score of ≥ 2) (B). On necropsy, mice with NEC (second panel) had dark discolored intestines with adhesions, while animals treated with hBD2 or LL-37(third and fourth panels) had healthier appearing intestines and less adhesions that were more similar to sham conditions (first panel). Yellow arrows indicate small intestine (C). Serum levels of IL-17a, IL-22, and TNF were quantified at time of tissue harvest (D–F). Serum levels of IL17A, IL-22, and TNF were significantly elevated in NEC compared to sham ( p = 0.0016, 0.0006, and 0.0044 respectively), but hBD2 and LL-37 treatment/prevention did not significantly alter any cytokine levels compared to NEC. P14-P16 C57Bl/6J mice were euthanized 30 min following SQ injection or oral gavage with hBD2 or oral gavage or intraperitoneal (IP) injection with LL-37. Serum and homogenized intestinal samples were obtained and quantified for hBD2 (G) and serum, homogenized intestinal samples, and intraperitoneal wash (IP Wash) samples were obtained and quantified for LL-37 (H). Intestinal homogenates from SQ treated mice had hBD2 levels >1000 pg/mL, while all other hBD2 treated samples had hBD2 levels that exceeded the detectable limit by the ELISA plate ( n = 3 animals per group). Elevation of LL-37 was detected in the IP wash of IP injected animals, but elevation was not detected in the serum or intestine in LL-37 IP injection or gavage ( n = 3 animals per group). Error bars in all figures represent SEM.

    Article Snippet: LL-37 ELISA kit , Hycult Biotech , HK321.

    Techniques: Disruption, Injection, Enzyme-linked Immunosorbent Assay

    LL-37 and hBD2 prevention and treatment approaches do not cause detectable alterations in the cecal microbiome Cecal samples were analyzed for microbial composition using 16S sequencing. Phylum and family level analysis showed that induction of NEC altered the microbial composition by increasing the relative amount of Proteobacteria ( p < 0.0001), and in particular Enterobacteriaceae ( p < 0.0001), while decreasing the relative amount of Firmicutes. hBD2 and LL-37 treatment (pre- or post-induction of NEC) had no impact (n = sham: 6, NEC: 9, hBD2 pre: 11, hBD2 post: 10, hBD2 subQ 5, LL-37 pre 6, LL-37 post 5, p < 0.05) [(A): Phyla, (B): Enterobacteriaceae only)]. Alpha diversity analysis via chao1 demonstrated decreases in diversity in NEC and HDP pre-treated animals (C). Principle coordinate analysis of the 16S cecal microbiome indicates animals exposed to K. pneumoniae gavage ( Klebsiella , NEC, pre hBD2, post hBD2, subQ hBD2, pre LL-37, and post LL-37) clustered together independent of HDP treatment and separate from non- Klebsiella animals (sham, dithizone) (D). Error bars in all figures represent SEM.

    Journal: iScience

    Article Title: Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis

    doi: 10.1016/j.isci.2024.109993

    Figure Lengend Snippet: LL-37 and hBD2 prevention and treatment approaches do not cause detectable alterations in the cecal microbiome Cecal samples were analyzed for microbial composition using 16S sequencing. Phylum and family level analysis showed that induction of NEC altered the microbial composition by increasing the relative amount of Proteobacteria ( p < 0.0001), and in particular Enterobacteriaceae ( p < 0.0001), while decreasing the relative amount of Firmicutes. hBD2 and LL-37 treatment (pre- or post-induction of NEC) had no impact (n = sham: 6, NEC: 9, hBD2 pre: 11, hBD2 post: 10, hBD2 subQ 5, LL-37 pre 6, LL-37 post 5, p < 0.05) [(A): Phyla, (B): Enterobacteriaceae only)]. Alpha diversity analysis via chao1 demonstrated decreases in diversity in NEC and HDP pre-treated animals (C). Principle coordinate analysis of the 16S cecal microbiome indicates animals exposed to K. pneumoniae gavage ( Klebsiella , NEC, pre hBD2, post hBD2, subQ hBD2, pre LL-37, and post LL-37) clustered together independent of HDP treatment and separate from non- Klebsiella animals (sham, dithizone) (D). Error bars in all figures represent SEM.

    Article Snippet: LL-37 ELISA kit , Hycult Biotech , HK321.

    Techniques: Sequencing

    LL-37 can directly kill K pneumoniae 10031, while hBD2 has more limited antimicrobial capacity K. pneumoniae abundance was measured by PCR (A). NEC treatment significantly increased detection of K. pneumoniae ( p = 0.0002, n = 9). Pre-treatment with HDPs LL-37 and hBD2 had no effect on NEC-induced increase in K. pneumoniae ( p > 0.9999, n = 3), while post-treatment with HDPs LL-37 and hBD2 reduced K. pneumoniae to sham-levels (sham vs. Post LL-37 p = 0.1648; sham vs. Post hBD2 p = 0.8622 n = 3). Similar to oral therapeutic treatment with HDPs, SQ hBD2 decreased NEC-induced increases in K. pneumoniae toward sham levels ( p = 0.1501). K. pneumoniae (6.3 × 10 6 - 1.1 × 10 7 CFU/mL) and E. coli (2.25 × 10 6 - 1.585 × 10 7 CFU/mL) were exposed to increasing concentrations of hBD2. hBD2 decreased growth for E. coli at 80 μg/mL, and achieved a MIC at 160 μg/mL, but no concentration of hBD2 significantly inhibited growth of K. pneumoniae (n = 2.5 μg/mL: 3, 160 μg/mL: 6, and all other doses 9) (B). K. pneumoniae grown in Nutrient Broth or Tryptic Soy Broth were exposed to increasing concentrations of hBD2. hBD2 induced a sharp decline in survival at concentrations of 25 μg/mL in both media types, but complete killing of K. pneumoniae was not observed in either media or at any concentration of hBD2 tested ( n = 10 replicates for all strains and all doses) (C). In a turbidity assay, hBD2 caused significantly decreased bacterial growth for Bifidobacterium breve and Bifidobacterium adolescentis , but not in S. thermophilus (D). K. pneumoniae (5.15 × 10 6 - 1.18 × 10 7 CFU/mL) was exposed to increasing concentrations of LL-37 (E). LL-37 inhibited growth of K. pneumoniae at 25 μg/mL and completely inhibited growth at 32 μg/mL. Error bars in all figures represent SEM.

    Journal: iScience

    Article Title: Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis

    doi: 10.1016/j.isci.2024.109993

    Figure Lengend Snippet: LL-37 can directly kill K pneumoniae 10031, while hBD2 has more limited antimicrobial capacity K. pneumoniae abundance was measured by PCR (A). NEC treatment significantly increased detection of K. pneumoniae ( p = 0.0002, n = 9). Pre-treatment with HDPs LL-37 and hBD2 had no effect on NEC-induced increase in K. pneumoniae ( p > 0.9999, n = 3), while post-treatment with HDPs LL-37 and hBD2 reduced K. pneumoniae to sham-levels (sham vs. Post LL-37 p = 0.1648; sham vs. Post hBD2 p = 0.8622 n = 3). Similar to oral therapeutic treatment with HDPs, SQ hBD2 decreased NEC-induced increases in K. pneumoniae toward sham levels ( p = 0.1501). K. pneumoniae (6.3 × 10 6 - 1.1 × 10 7 CFU/mL) and E. coli (2.25 × 10 6 - 1.585 × 10 7 CFU/mL) were exposed to increasing concentrations of hBD2. hBD2 decreased growth for E. coli at 80 μg/mL, and achieved a MIC at 160 μg/mL, but no concentration of hBD2 significantly inhibited growth of K. pneumoniae (n = 2.5 μg/mL: 3, 160 μg/mL: 6, and all other doses 9) (B). K. pneumoniae grown in Nutrient Broth or Tryptic Soy Broth were exposed to increasing concentrations of hBD2. hBD2 induced a sharp decline in survival at concentrations of 25 μg/mL in both media types, but complete killing of K. pneumoniae was not observed in either media or at any concentration of hBD2 tested ( n = 10 replicates for all strains and all doses) (C). In a turbidity assay, hBD2 caused significantly decreased bacterial growth for Bifidobacterium breve and Bifidobacterium adolescentis , but not in S. thermophilus (D). K. pneumoniae (5.15 × 10 6 - 1.18 × 10 7 CFU/mL) was exposed to increasing concentrations of LL-37 (E). LL-37 inhibited growth of K. pneumoniae at 25 μg/mL and completely inhibited growth at 32 μg/mL. Error bars in all figures represent SEM.

    Article Snippet: LL-37 ELISA kit , Hycult Biotech , HK321.

    Techniques: Concentration Assay

    Journal: iScience

    Article Title: Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis

    doi: 10.1016/j.isci.2024.109993

    Figure Lengend Snippet:

    Article Snippet: LL-37 ELISA kit , Hycult Biotech , HK321.

    Techniques: Virus, Recombinant, Sensitive Assay, Enzyme-linked Immunosorbent Assay, Software, Purification

    Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by ELISA. Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Moss-produced human complement factor H with modified glycans has an extended half-life and improved biological activity

    doi: 10.3389/fimmu.2024.1383123

    Figure Lengend Snippet: Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by ELISA. Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.

    Article Snippet: The concentration of CPV-104 and CPV-101 in serum samples was measured using an FH ELISA kit (HK342, Hycult Biotech) according to the manufacturer’s instructions.

    Techniques: Functional Assay, Activity Assay, In Vitro, Produced, Incubation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Spectroscopy

    Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by ELISA. Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Moss-produced human complement factor H with modified glycans has an extended half-life and improved biological activity

    doi: 10.3389/fimmu.2024.1383123

    Figure Lengend Snippet: Functional activity of the FH variants in vitro . (A) The cofactor activity of moss-produced FH variants is similar to that of sd-FH. Increasing concentrations of CPV-104, CPV-101 or sd-FH were incubated with C3b and FI. C3b cleavage products (α’68, α’46 and α’43) were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. Shown is a representative blot out of 3 independent experiments. (B) The decay acceleration activity of CPV-104 and CPV-101 is identical to that of sd-FH. C3 convertase was prepared by incubating C3b with FD and FB. The convertase was then incubated with increasing concentrations (1 nM, 5 nM, 10 nM) of CPV-104, CPV-101, sd-FH or BSA and leftover Bb fragments were measured by ELISA. Results are shown as mean ± SD of at least n = 3 independent experiments. (C) CPV-104 and CPV-101 bind with greater affinity than sd-FH to C3b. Wells were coated with C3b and then incubated with different concentrations (1 nM, 5 nM, 10 nM) of sd-FH, CPV-101, CPV-104 or BSA. Results are shown as mean ± SD of at least n = 3 independent experiments. The affinity of C3b binding was validated by SPR spectroscopy (data summarized in table show means ± SD from 3 independent measurements). (D) CPV-104 and CPV-101 inhibit TCC formation at significantly lower concentrations than sd-FH. Mg-EGTA-treated NHS was incubated with increasing concentrations of CPV-104, CPV-101 or sd-FH on LPS-coated microtiter plates to activate the alternative pathway. The quantity of C5b9 complexes was determined by ELISA. Data were normalized against untreated NHS samples. IC 50 values are shown in nM. Results are shown as mean ± SD of at least n = 3 independent experiments.

    Article Snippet: Serum samples from the mice were diluted 1:10.000 and the FH and C3 concentrations were determined using ELISA kits according to the manufacturers’ instructions (HK342 for FH, Hycult Biotech, Netherlands; ab157711 for C3, Abcam, UK).

    Techniques: Functional Assay, Activity Assay, In Vitro, Produced, Incubation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Spectroscopy