m13ko7 helper phage Search Results


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  • 98
    New England Biolabs m13k07 helper phage
    M13k07 Helper Phage, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m13k07 helper phage/product/New England Biolabs
    Average 98 stars, based on 322 article reviews
    Price from $9.99 to $1999.99
    m13k07 helper phage - by Bioz Stars, 2020-09
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    99
    Thermo Fisher m13ko7 helper phage
    Selection and screening of antibody libraries. A: Schematic overview of the selection strategy. Low valence (LV) and high valence (HV) display were achieved by packaging with the helper phages <t>M13KO7</t> or DeltaPhage, respectively. After R1 the libraries were split into a competition branch and a thermostability branch. B: The selection outputs after R3 were screened as soluble scFvs to assess binding to target pMHC complexes and HLA-DQ2.5:CLIP2 (background) in ELISA and ratios were calculated. Each dot represents one clone. Grey dots denote unknown sequences, black dots denote unique single sequences and colors represent enriched amino acid sequences. The 107 CDR, 107 random, and 206 CDR libraries were selected in the thermostability branch, while the 206 H1 library was selected in the competition branch. C: The selection outputs after R3 were screened in phage format and are represented as in B. D: Purified and monomeric Fab fragments were tested for binding to a larger panel of HLA-DQ2.5:peptide complexes in ELISA. pMHC levels were controlled with the HLA-DQ2 antibody 2.12.E11 ( S. 4D ). Error bars illustrate mean ± SD of duplicates (n=2).
    M13ko7 Helper Phage, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m13ko7 helper phage/product/Thermo Fisher
    Average 99 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    m13ko7 helper phage - by Bioz Stars, 2020-09
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    94
    GE Healthcare helper phage m13ko7
    Characterization of selected scFv clones obtained from L3G7C-derived scFv library. (A) The specificity of anti-phOx scFv clones that derived from L3G7C was evaluated using phageELISA against different antigens, including phOx-BSA conjugate, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. After subtraction of the background binding of <t>M13KO7</t> helper phage, the binding activity of scFv clones towards different antigens was normalized against their binding towards BSA (negative control). Error bars represent SEM of three separate experiments each performed in duplicate. Difference of the means between selected clones and the parental clone (L3G7c) is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P
    Helper Phage M13ko7, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/helper phage m13ko7/product/GE Healthcare
    Average 94 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    helper phage m13ko7 - by Bioz Stars, 2020-09
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    90
    Promega m13ko7 helper phage
    Characterization of selected scFv clones obtained from L3G7C-derived scFv library. (A) The specificity of anti-phOx scFv clones that derived from L3G7C was evaluated using phageELISA against different antigens, including phOx-BSA conjugate, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. After subtraction of the background binding of <t>M13KO7</t> helper phage, the binding activity of scFv clones towards different antigens was normalized against their binding towards BSA (negative control). Error bars represent SEM of three separate experiments each performed in duplicate. Difference of the means between selected clones and the parental clone (L3G7c) is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P
    M13ko7 Helper Phage, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m13ko7 helper phage/product/Promega
    Average 90 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    m13ko7 helper phage - by Bioz Stars, 2020-09
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    93
    Antibody Design Labs Inc m13ko7 helper phage
    Characterization of selected scFv clones obtained from L3G7C-derived scFv library. (A) The specificity of anti-phOx scFv clones that derived from L3G7C was evaluated using phageELISA against different antigens, including phOx-BSA conjugate, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. After subtraction of the background binding of <t>M13KO7</t> helper phage, the binding activity of scFv clones towards different antigens was normalized against their binding towards BSA (negative control). Error bars represent SEM of three separate experiments each performed in duplicate. Difference of the means between selected clones and the parental clone (L3G7c) is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P
    M13ko7 Helper Phage, supplied by Antibody Design Labs Inc, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m13ko7 helper phage/product/Antibody Design Labs Inc
    Average 93 stars, based on 7 article reviews
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    m13ko7 helper phage - by Bioz Stars, 2020-09
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    90
    Progen Biotechnik m13ko7 helper phage
    Characterization of selected scFv clones obtained from L3G7C-derived scFv library. (A) The specificity of anti-phOx scFv clones that derived from L3G7C was evaluated using phageELISA against different antigens, including phOx-BSA conjugate, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. After subtraction of the background binding of <t>M13KO7</t> helper phage, the binding activity of scFv clones towards different antigens was normalized against their binding towards BSA (negative control). Error bars represent SEM of three separate experiments each performed in duplicate. Difference of the means between selected clones and the parental clone (L3G7c) is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P
    M13ko7 Helper Phage, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m13ko7 helper phage/product/Progen Biotechnik
    Average 90 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    m13ko7 helper phage - by Bioz Stars, 2020-09
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    88
    Stratagene helper phage m13ko7
    Characterization of selected scFv clones obtained from L3G7C-derived scFv library. (A) The specificity of anti-phOx scFv clones that derived from L3G7C was evaluated using phageELISA against different antigens, including phOx-BSA conjugate, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. After subtraction of the background binding of <t>M13KO7</t> helper phage, the binding activity of scFv clones towards different antigens was normalized against their binding towards BSA (negative control). Error bars represent SEM of three separate experiments each performed in duplicate. Difference of the means between selected clones and the parental clone (L3G7c) is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P
    Helper Phage M13ko7, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/helper phage m13ko7/product/Stratagene
    Average 88 stars, based on 8 article reviews
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    85
    Stratagene m13ko7 helper phage genome
    Effects of Ex-phage packaging on antigen-binding reactivity of scFv displayed on recombinant phage. ( A ) Determination of scFv:pIII fusion protein expression by immunoblot. Recombinant phage particles were obtained by infecting JS5 cells carrying pIGT3-hsp70 phagemid with either <t>M13KO7</t> helper phage (pIGT3/ M13KO7) (lane 1) or Ex-phage (pIGT3/ Ex-phage) (lane 2). Phage proteins were separated by loading approximately 10 10 recombinant phage into each lane of 10% SDS–PAGE, and immunoblot was carried out with anti-gIII mAb to determine the amount of scFv:pIII fusion proteins displayed on the surfaces of phage particles. Goat anti-mouse IgG antibody conjugated with HRPO was used for the secondary antibody. ( B ) Antigen-binding specificity of recombinant phage packaged with either M13KO7 or Ex-phage. BSA, lysozyme (Sigma Co.), recombinant GST or recombinant human HSP-70 were coated in microtiters, and 10 10 scFv phage packaged with either M13KO7 or Ex-phage were applied to each well for phage ELISA. The same amount of M13KO7 helper phage was used as a negative control. The bound phage were detected with anti-M13 antibody conjugated with HRPO. The binding signal was analyzed at OD 405 . ( C ) Antigen-binding sensitivity of phage particles packaged with either M13KO7 or Ex-phage. Phage ELISA was performed as described above except that serial dilutions of purified human HSP-70 fusion protein were used to coat microtiter plates.
    M13ko7 Helper Phage Genome, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m13ko7 helper phage genome/product/Stratagene
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    m13ko7 helper phage genome - by Bioz Stars, 2020-09
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    Image Search Results


    Selection and screening of antibody libraries. A: Schematic overview of the selection strategy. Low valence (LV) and high valence (HV) display were achieved by packaging with the helper phages M13KO7 or DeltaPhage, respectively. After R1 the libraries were split into a competition branch and a thermostability branch. B: The selection outputs after R3 were screened as soluble scFvs to assess binding to target pMHC complexes and HLA-DQ2.5:CLIP2 (background) in ELISA and ratios were calculated. Each dot represents one clone. Grey dots denote unknown sequences, black dots denote unique single sequences and colors represent enriched amino acid sequences. The 107 CDR, 107 random, and 206 CDR libraries were selected in the thermostability branch, while the 206 H1 library was selected in the competition branch. C: The selection outputs after R3 were screened in phage format and are represented as in B. D: Purified and monomeric Fab fragments were tested for binding to a larger panel of HLA-DQ2.5:peptide complexes in ELISA. pMHC levels were controlled with the HLA-DQ2 antibody 2.12.E11 ( S. 4D ). Error bars illustrate mean ± SD of duplicates (n=2).

    Journal: bioRxiv

    Article Title: Affinity-engineered human antibodies detect celiac disease gluten pMHC complexes and inhibit T-cell activation

    doi: 10.1101/840561

    Figure Lengend Snippet: Selection and screening of antibody libraries. A: Schematic overview of the selection strategy. Low valence (LV) and high valence (HV) display were achieved by packaging with the helper phages M13KO7 or DeltaPhage, respectively. After R1 the libraries were split into a competition branch and a thermostability branch. B: The selection outputs after R3 were screened as soluble scFvs to assess binding to target pMHC complexes and HLA-DQ2.5:CLIP2 (background) in ELISA and ratios were calculated. Each dot represents one clone. Grey dots denote unknown sequences, black dots denote unique single sequences and colors represent enriched amino acid sequences. The 107 CDR, 107 random, and 206 CDR libraries were selected in the thermostability branch, while the 206 H1 library was selected in the competition branch. C: The selection outputs after R3 were screened in phage format and are represented as in B. D: Purified and monomeric Fab fragments were tested for binding to a larger panel of HLA-DQ2.5:peptide complexes in ELISA. pMHC levels were controlled with the HLA-DQ2 antibody 2.12.E11 ( S. 4D ). Error bars illustrate mean ± SD of duplicates (n=2).

    Article Snippet: Phage libraries, beads and tubes were blocked with 5% skim milk or 2 % BSA in PBS supplemented with 0.05% tween-20 (PBS-T) prior to each round of selection.

    Techniques: Selection, Binding Assay, Enzyme-linked Immunosorbent Assay, Purification

    Characterization of selected scFv clones obtained from L3G7C-derived scFv library. (A) The specificity of anti-phOx scFv clones that derived from L3G7C was evaluated using phageELISA against different antigens, including phOx-BSA conjugate, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. After subtraction of the background binding of M13KO7 helper phage, the binding activity of scFv clones towards different antigens was normalized against their binding towards BSA (negative control). Error bars represent SEM of three separate experiments each performed in duplicate. Difference of the means between selected clones and the parental clone (L3G7c) is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P

    Journal: PLoS ONE

    Article Title: Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes

    doi: 10.1371/journal.pone.0027406

    Figure Lengend Snippet: Characterization of selected scFv clones obtained from L3G7C-derived scFv library. (A) The specificity of anti-phOx scFv clones that derived from L3G7C was evaluated using phageELISA against different antigens, including phOx-BSA conjugate, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. After subtraction of the background binding of M13KO7 helper phage, the binding activity of scFv clones towards different antigens was normalized against their binding towards BSA (negative control). Error bars represent SEM of three separate experiments each performed in duplicate. Difference of the means between selected clones and the parental clone (L3G7c) is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P

    Article Snippet: Subsequently, log-phase TG1 transformants were super-infected with M13KO7 helper phage (GE Healthcare) in a multiplicity of infection (moi) ratio of 3∶1, and phages were rescued at 30°C overnight with gentle shaking.

    Techniques: Clone Assay, Derivative Assay, Binding Assay, Activity Assay, Negative Control

    Characterization of anti-N scFv-phages. (A) Western protein analysis of scFv-phage binding to recombinant SCoV-N protein. Purified His 6 -SCoV-N recombinant protein was separated on gradient sodium dodecyl sulfate (5% - 20%) polyacrylamide gel and transferred onto a 0.2 µm nitrocellulose membrane. The stripes were probed with different anti-N scFv-phages, including L9A6B, L9A11B, L4A3A, L4A3B, L4A8B, and M13KO7 helper phage (negative control) as indicated. Purified SCoV-N protein was stained with Coomassie Blue for reference. (B) Cross-reactivity of selected anti-N scFv clones was evaluated using phageELISA against different antigens, including BSA, SCoV-N, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. Error bars represent SEM of three separate experiments each performed in duplicate. Difference between means of binding to SCoV-N protein versus to other unrelated antigens is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P

    Journal: PLoS ONE

    Article Title: Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes

    doi: 10.1371/journal.pone.0027406

    Figure Lengend Snippet: Characterization of anti-N scFv-phages. (A) Western protein analysis of scFv-phage binding to recombinant SCoV-N protein. Purified His 6 -SCoV-N recombinant protein was separated on gradient sodium dodecyl sulfate (5% - 20%) polyacrylamide gel and transferred onto a 0.2 µm nitrocellulose membrane. The stripes were probed with different anti-N scFv-phages, including L9A6B, L9A11B, L4A3A, L4A3B, L4A8B, and M13KO7 helper phage (negative control) as indicated. Purified SCoV-N protein was stained with Coomassie Blue for reference. (B) Cross-reactivity of selected anti-N scFv clones was evaluated using phageELISA against different antigens, including BSA, SCoV-N, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. Error bars represent SEM of three separate experiments each performed in duplicate. Difference between means of binding to SCoV-N protein versus to other unrelated antigens is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P

    Article Snippet: Subsequently, log-phase TG1 transformants were super-infected with M13KO7 helper phage (GE Healthcare) in a multiplicity of infection (moi) ratio of 3∶1, and phages were rescued at 30°C overnight with gentle shaking.

    Techniques: Western Blot, Binding Assay, Recombinant, Purification, Negative Control, Staining, Clone Assay

    Effects of Ex-phage packaging on antigen-binding reactivity of scFv displayed on recombinant phage. ( A ) Determination of scFv:pIII fusion protein expression by immunoblot. Recombinant phage particles were obtained by infecting JS5 cells carrying pIGT3-hsp70 phagemid with either M13KO7 helper phage (pIGT3/ M13KO7) (lane 1) or Ex-phage (pIGT3/ Ex-phage) (lane 2). Phage proteins were separated by loading approximately 10 10 recombinant phage into each lane of 10% SDS–PAGE, and immunoblot was carried out with anti-gIII mAb to determine the amount of scFv:pIII fusion proteins displayed on the surfaces of phage particles. Goat anti-mouse IgG antibody conjugated with HRPO was used for the secondary antibody. ( B ) Antigen-binding specificity of recombinant phage packaged with either M13KO7 or Ex-phage. BSA, lysozyme (Sigma Co.), recombinant GST or recombinant human HSP-70 were coated in microtiters, and 10 10 scFv phage packaged with either M13KO7 or Ex-phage were applied to each well for phage ELISA. The same amount of M13KO7 helper phage was used as a negative control. The bound phage were detected with anti-M13 antibody conjugated with HRPO. The binding signal was analyzed at OD 405 . ( C ) Antigen-binding sensitivity of phage particles packaged with either M13KO7 or Ex-phage. Phage ELISA was performed as described above except that serial dilutions of purified human HSP-70 fusion protein were used to coat microtiter plates.

    Journal: Nucleic Acids Research

    Article Title: An improved helper phage system for efficient isolation of specific antibody molecules in phage display

    doi:

    Figure Lengend Snippet: Effects of Ex-phage packaging on antigen-binding reactivity of scFv displayed on recombinant phage. ( A ) Determination of scFv:pIII fusion protein expression by immunoblot. Recombinant phage particles were obtained by infecting JS5 cells carrying pIGT3-hsp70 phagemid with either M13KO7 helper phage (pIGT3/ M13KO7) (lane 1) or Ex-phage (pIGT3/ Ex-phage) (lane 2). Phage proteins were separated by loading approximately 10 10 recombinant phage into each lane of 10% SDS–PAGE, and immunoblot was carried out with anti-gIII mAb to determine the amount of scFv:pIII fusion proteins displayed on the surfaces of phage particles. Goat anti-mouse IgG antibody conjugated with HRPO was used for the secondary antibody. ( B ) Antigen-binding specificity of recombinant phage packaged with either M13KO7 or Ex-phage. BSA, lysozyme (Sigma Co.), recombinant GST or recombinant human HSP-70 were coated in microtiters, and 10 10 scFv phage packaged with either M13KO7 or Ex-phage were applied to each well for phage ELISA. The same amount of M13KO7 helper phage was used as a negative control. The bound phage were detected with anti-M13 antibody conjugated with HRPO. The binding signal was analyzed at OD 405 . ( C ) Antigen-binding sensitivity of phage particles packaged with either M13KO7 or Ex-phage. Phage ELISA was performed as described above except that serial dilutions of purified human HSP-70 fusion protein were used to coat microtiter plates.

    Article Snippet: Amber codon (TAG) was introduced at the 5′ region of gIII of M13KO7 helper phage genome (Stratagene, USA) by site-directed mutagenesis using Mutan™-K enzyme and vector set (Takara, Japan) according to the manufacturer’s protocol using a synthetic oligonucleotide (1: 5′-TTCAACAGT CTA AGCGGAGTG-3′, the amber codon is underlined).

    Techniques: Binding Assay, Recombinant, Expressing, SDS Page, Enzyme-linked Immunosorbent Assay, Negative Control, Purification

    Mutagenesis of M13KO7 helper phage genome. ( A ) gIII DNA sequence of Ex-phage. Two codons for Glu (GAA) were substituted to amber codons (TAG) at the 5′ region of gIII of M13KO7 helper phage genome by site-directed mutagenesis using Mutan™-K kit as described in the Materials and Methods. Leader sequence of gIII is denoted by bold. Asterisks show location of the amber codons. ( B ) Plaque formation by Ex-phage. After two consecutive site-directed mutageneses, several plaques were randomly isolated, and the phage were released in LB. Two microliters of phage suspension were spotted onto top agar LB plates containing TG1 or JS5 bacterial lawn, and incubated at 37°C overnight to determine phage propagation on these E.coli strains. M13KO7 helper phage was used as a control.

    Journal: Nucleic Acids Research

    Article Title: An improved helper phage system for efficient isolation of specific antibody molecules in phage display

    doi:

    Figure Lengend Snippet: Mutagenesis of M13KO7 helper phage genome. ( A ) gIII DNA sequence of Ex-phage. Two codons for Glu (GAA) were substituted to amber codons (TAG) at the 5′ region of gIII of M13KO7 helper phage genome by site-directed mutagenesis using Mutan™-K kit as described in the Materials and Methods. Leader sequence of gIII is denoted by bold. Asterisks show location of the amber codons. ( B ) Plaque formation by Ex-phage. After two consecutive site-directed mutageneses, several plaques were randomly isolated, and the phage were released in LB. Two microliters of phage suspension were spotted onto top agar LB plates containing TG1 or JS5 bacterial lawn, and incubated at 37°C overnight to determine phage propagation on these E.coli strains. M13KO7 helper phage was used as a control.

    Article Snippet: Amber codon (TAG) was introduced at the 5′ region of gIII of M13KO7 helper phage genome (Stratagene, USA) by site-directed mutagenesis using Mutan™-K enzyme and vector set (Takara, Japan) according to the manufacturer’s protocol using a synthetic oligonucleotide (1: 5′-TTCAACAGT CTA AGCGGAGTG-3′, the amber codon is underlined).

    Techniques: Mutagenesis, Sequencing, Isolation, Incubation

    Enhancement of panning efficiency by Ex-phage package pIGT3/M13KO7 or pIGT3/Ex-phage were mixed with non-specific M13KO7 helper phage at 1:10 4 , 1:10 6 or 1:10 8 ratio, and these phage mixtures (total 10 10 phage from each diluting mixture) were applied for panning procedure with human HSP-70 protein as an antigen. Unbound phage were removed by washing with PBS–tween, and bound phage were eluted by trypsin treatment. Panning was repeated twice. Phage ELISA was performed to determine the enrichment of antigen-specific phage after each round of panning. ( A ) Percentage yield after panning. Input phage number was determined by phage ELISA, and the number of eluted phage (output) was determined by c.f.u. as described in the Materials and Methods. Percentage yield was calculated as (number of output phage/number of input phage) × 100. ( B ) Polyclonal phage ELISA on human HSP-70 protein after panning. Eluted phage from the first and the second round of panning were amplified and purified by PEG/NaCl precipitation. For polyclonal phage ELISA, 10 10 of amplified phage after each round of panning were used for ELISA. BSA was used as a negative antigen control. ( C ) Monoclonal phage ELISA on human HSP-70 protein. Forty-five E.coli colonies were randomly picked after the second round of panning, and grown up in 96-well plates in the presence of M13KO7 or Ex-phage. One hundred microliters of culture supernatant containing recombinant phage particles were used in phage ELISA. M13KO7 helper phage was used as a negative phage control (N).

    Journal: Nucleic Acids Research

    Article Title: An improved helper phage system for efficient isolation of specific antibody molecules in phage display

    doi:

    Figure Lengend Snippet: Enhancement of panning efficiency by Ex-phage package pIGT3/M13KO7 or pIGT3/Ex-phage were mixed with non-specific M13KO7 helper phage at 1:10 4 , 1:10 6 or 1:10 8 ratio, and these phage mixtures (total 10 10 phage from each diluting mixture) were applied for panning procedure with human HSP-70 protein as an antigen. Unbound phage were removed by washing with PBS–tween, and bound phage were eluted by trypsin treatment. Panning was repeated twice. Phage ELISA was performed to determine the enrichment of antigen-specific phage after each round of panning. ( A ) Percentage yield after panning. Input phage number was determined by phage ELISA, and the number of eluted phage (output) was determined by c.f.u. as described in the Materials and Methods. Percentage yield was calculated as (number of output phage/number of input phage) × 100. ( B ) Polyclonal phage ELISA on human HSP-70 protein after panning. Eluted phage from the first and the second round of panning were amplified and purified by PEG/NaCl precipitation. For polyclonal phage ELISA, 10 10 of amplified phage after each round of panning were used for ELISA. BSA was used as a negative antigen control. ( C ) Monoclonal phage ELISA on human HSP-70 protein. Forty-five E.coli colonies were randomly picked after the second round of panning, and grown up in 96-well plates in the presence of M13KO7 or Ex-phage. One hundred microliters of culture supernatant containing recombinant phage particles were used in phage ELISA. M13KO7 helper phage was used as a negative phage control (N).

    Article Snippet: Amber codon (TAG) was introduced at the 5′ region of gIII of M13KO7 helper phage genome (Stratagene, USA) by site-directed mutagenesis using Mutan™-K enzyme and vector set (Takara, Japan) according to the manufacturer’s protocol using a synthetic oligonucleotide (1: 5′-TTCAACAGT CTA AGCGGAGTG-3′, the amber codon is underlined).

    Techniques: Enzyme-linked Immunosorbent Assay, Amplification, Purification, Recombinant