biotinylated tlr2 antibody  (Miltenyi Biotec)

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Expression of Christie–Atkins–Munch–Petersen (CAMP)1 in Cutibacterium acnes ( C. acnes ) strains. C. acnes surface proteins were extracted from a five-day culture ( A , C ) in liquid RCM medium or ( B ) on solid RCM medium and separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel (50 μg). Separated proteins were transferred onto nitrocellulose membranes, which were incubated ( A , B ) with antibodies against CAMP1 or ( C ) with recombinant toll like receptor (TLR)-2. TLR-2 binding activity was detected with specific biotinylated antibodies against TLR-2, as described in the Materials and Methods. TLR-2 binding activity previously reported by Lheure et al. . Lanes 1 to 12 correspond to phylotype IA1 strains: 75150, 16351, 17248, 53468, 41103, 6919, 78910, 36862, RON, 22197, LIE and 31430, respectively. Lane 13 corresponds to phylotype IA2 strain: CHR. Lanes 14 to 22 correspond to phylotype IB strains: 14230, 12513, 22795, 47474, 27387, 25236, GUE, PIE and 54137, respectively. Lane 23 corresponds to phylotype II strain: 27647. ( D ) HaCaT cells were stimulated for 1, 4, 6 and 24 h at 37 °C, with C. acnes strains 6919 (phylotype IA1), 14230 (phylotype IB) or PIE (phylotype IB) grown in liquid or on solid medium. C-X-C motif chemokine ligand interleukin (CXCL)8/(IL)-8 protein levels were assessed by enzyme-like immunosorbent assay (ELISA). nd: non-determined.

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Expressing, Electrophoresis, Incubation, Recombinant, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Inhibition of C. acnes -induced CXCL8/IL-8 production by TLR-2 and CAMP1 antibodies. HaCaT cells were treated with ( A ) anti-TLR-2 antibody (1 μg/mL) or ( B ) anti-CAMP1 antibody (1 μg/mL) for 24 h (gray bar), and were then stimulated with various strains of C. acnes (multiplicy of infection 15 (MOI 15) (phylotype IA1, strain 6919; phylotype IB, strains PIE and 14230) for 18 h (dark bar). Control experiments correspond to C. acnes stimulation of HaCaT cells only (gray bar). CXCL8/IL-8 production was measured by ELISA. Statistical significance is indicated by ** ( p < 0.01), *** ( p < 0.001) and **** ( p < 0.0001).

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Inhibition, Infection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Specific recognition of C. acnes recombinant CAMP1 by TLR-2. ( A ) The recombinant proteins nmrCAMP1 (nm) and mrCAMP1 (m) (50 μg) were separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel and transferred onto nitrocellulose membranes, which were incubated with recombinant TLR-2, TLR-1, TLR-4 and TLR-6 (0.1 μg/mL). TLR binding activity was detected with specific biotinylated antibodies against TLR-2, TLR-1, TLR-4 and TLR-6, respectively, as described in the Materials and Methods. The arrow indicates the position of the 47 kDa band of interest. ( B ) The recombinant proteins were separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel (50 μg) with detection by Coomassie brilliant blue staining. The separated proteins were transferred onto nitrocellulose membranes, which were incubated with poly-histidine antibody or recombinant TLR-2 (0.1 μg/mL), with the detection of TLR binding activity with specific biotinylated antibodies against TLR-2. Lanes 1a and 1b contain the molecular mass markers. Arrows indicate the positions of the 30 and 47 kDa bands of interest. The peptide sequences of the 47 kDa nmrCAMP1 and mrCAMP1 and the 30 kDa protein were obtained by LC-MS/MS (in bold, the amino acid substitutions in mrCAMP1). ( C ) Immobilized nmrCAMP1 (square) and mrCAMP1 (circle) (20 μg/mL) were probed with various concentrations of TLR-1, TLR-2, TLR-4 or TLR-6 (0.016 to 16 μg/mL).

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Recombinant, Electrophoresis, Incubation, Binding Assay, Activity Assay, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Subpeptides from CAMP1 recognized by TLR-2. ( A , B , C ) The peptides derived from CAMP1 proteins: group F (positions A1-C21), group A (positions C22-E9), group C (positions E10-E13), group D (positions E14-E17), group E (positions E18-E20); a biotinylated peptide used as a positive control (position F22) and a peptide used as a negative control (position F24), were immobilized on glass plates. ( A ) The plates were incubated with TLR-2 (10 μg/mL)/anti-TLR-2 antibody (0.1 μg/mL)/HRP (0.5 μg/mL) and ( B ) with anti-TLR-2 antibody (0.1 μg/mL)/HRP (0.5 µg/mL). The arrows indicate the positions of peptides specifically recognized by TLR-2. ( D ) Immobilized rCAMP1 (20 μg/mL) proteins were probed with biotinylated TRL-2 (0.5 μg/mL) (dark bar) after pre-treatment with peptide (A14, A15, B1, B2, C1 and C3) at various concentrations (1.25, 12.5, 125, 1250 and 12,500 nM). Statistical significance is indicated by * ( p < 0.05), ** ( p < 0.01) and *** ( p < 0.001).

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Derivative Assay, Positive Control, Negative Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: In silico 3D analysis of C. acnes factor CAMP1. ( A ) Sequence alignment of CAMP factors from different species. Conservatively substituted residues are boxed and strictly conserved residues are highlighted with a red background. The figure was constructed with ESPript 3.0. Reference sequences: Cutibacterium acnes nmCAMP1: AAS92206.1; Cutibacterium acnes mCAMP1: KX581410; Streptococcus agalactiae : ZP_08649639.1; Mobiluncus curtisii : YP_003718285.1; Streptococcus pyogenes : WP_111681137.1. ( B – D ) Predicted structures of the nmCAMP1 and mCAMP1 proteins obtained by homology modeling with the trRosetta server and the images generated with PyMOL software. ( B ) Cartoon representation of CAMP1. The NTD domain is colored in yellow, the CTD domain in cyan and the linker region in red. ( C ) CAMP1 peptides involved in TLR-2 binding are highlighted: A14 in blue, A15 in cyan, B1 in yellow, B2 in orange, B3 in red, C1 in purple and C3 in pink. ( D ) Surface representation of CAMP1 peptides.

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: In Silico, Sequencing, Construct, Generated, Software, Binding Assay

Journal: PLoS ONE

Article Title: TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

doi: 10.1371/journal.pone.0167237

Figure Lengend Snippet: P . acnes surface proteins were extracted from a 5-day culture bacterial pellet and separated by electrophoresis in a 4–12% NuPAGE LDS BisTris gel (50 μg) with detection by Coomassie blue staining (A). Separated proteins were transferred onto nitrocellulose membranes, which were incubated with recombinant TLR2 (B) and TLR4 (C) (0.1 μg/ml). TLR binding activity was detected with specific biotinylated antibodies against TLR2 and TLR4, respectively, as described in the Materials and Methods. Lane 1 contains the molecular mass markers. Lanes 2 to 7 contain proteins from strains 6919, RON, CHR, GUE, TRI, and PIE, respectively. Arrows indicate the positions of the 24.5- and 27.5-kDa bands of interest.

Article Snippet: Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4 (R&D Systems, Abingdon, UK) diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing.

Techniques: Electrophoresis, Staining, Incubation, Recombinant, Binding Assay, Activity Assay

Journal: PLoS ONE

Article Title: TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

doi: 10.1371/journal.pone.0167237

Figure Lengend Snippet: Surface protein extracts were electrophoretically separated in 10% SDS-PAGE gels (13 x 13 cm), with detection by Coomassie blue staining. Proteins of interest were excised from the gel, eluted and pooled as described in the Materials and Methods. HaCaT cells were treated for 2 h with human anti-TLR2 (dashed bar) or with goat anti-IgGa antibodies (dark gray bar) and transfected by incubation for 24 h with NF-κB transcription factor (A) and CXCL8 (-173 bp) (B) inserted into a construct upstream from the luciferase gene, after which, an internal control (the Renilla luciferase expression plasmid) was added to the transfection mixture. Cell were stimulated with eluted CAMP factor 1 (50 μg/ml) for 24 h at 37°C. Relative NF-κB and CXCL8 promoter activities were determined as the ratio of firefly and Renilla luciferase activities. Pretreated HaCaT cells were stimulated by incubation with eluted CAMP factor 1 (50 μg/ml) for 24 h at 37°C, CXCL8 mRNA levels were determined by RT-qPCR (C), and CXCL8 production was measured by ELISA (D). Control experiments were performed with cells alone (dark bar) and with stimulated cells not previously treated with CAMP factor 1 (light gray bar). Data are presented as the mean ± standard deviation of two independent experiments. Statistical significance is indicated by * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4 (R&D Systems, Abingdon, UK) diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing.

Techniques: SDS Page, Staining, Transfection, Incubation, Construct, Luciferase, Control, Expressing, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLoS ONE

Article Title: TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

doi: 10.1371/journal.pone.0167237

Figure Lengend Snippet: Comparison of P . acnes inflammatory capabilities with phylogroup and CAMP factor 1 gene nucleotide sequence

Article Snippet: Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4 (R&D Systems, Abingdon, UK) diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing.

Techniques: Comparison, Binding Assay, Sequencing

Journal: PLoS ONE

Article Title: TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

doi: 10.1371/journal.pone.0167237

Figure Lengend Snippet: Phylogenetic analysis illustrating the relationship between CAMP factor 1 and 19 protein reference sequences from P . acnes (GenBank accession numbers ALT42318.1, ADE00273.1, AEE72567.1, AEW83915.1, AEW81649.1, AFU41158.1, AGJ79688.1, AID36048.1, ALT40080.1, ALT44585.1, AAS92206.1, ALT35489.1, AAT83098.1, AEH29671.1, ALT33229.1, AER06043.1, AAX14380.1, ALU23622.1, ALD69860.1), CAMP 5 factor Protein (AAV84920.1) and sequences of the 27 P . acnes strains used in this study. Phylogenetic trees were constructed by the maximum likelihood method, using PhyML3.0 , and the tree was rooted on the CAMP factor 5 protein sequence. Bootstrapping was applied to the trees (500 datasets) and bootstrap values are shown at nodes. The bar indicates the number of substitutions per site. Sequences were classified into four groups (A, B1 to B3). Squares correspond to phylotype IA 1 (red), IA 2 (yellow), IB (green), II (purple); and to CAMP1-TLR2 binding intensity with no binding as—(light gray), weak binding as +/- and + (dark gray), strong binding as ++ and +++ (black).

Article Snippet: Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4 (R&D Systems, Abingdon, UK) diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing.

Techniques: Construct, Sequencing, Binding Assay

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 1. The macrophage-stimulating activity of ESG. (A and B) Participation of the macrophage in ESG activity. CD11b+ and CD11b−cells (1 × 106 per 1 mL) derived from BALB/c PEC were incubated with saline, ESG (50–500 μg/mL) or Pam3CSK4 (TLR2 ligand; 10 ng/mL) in the presence of IFN-γ (10 ng/mL) for 48 h, then NO (A) and IL-6 (B) concentrations in the supernatants were determined. (C and D) Role of IFN-γ in the macrophage activation by ESG. CD11b+

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Incubation, Saline, Activation Assay

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 3. The activity of ESG on TLR2-expressing HEK293 cells. HEK293 cells transfected with TLR2 expression plasmid and reporter plasmids for 24 h were stimulated by either Pam3CSK4 (0–5 ng/mL), ESG (0–400 μg/mL), LPS (0–1000 ng/mL) or CpG-ODN (TLR9 ligand; 0–1000 ng/mL) at each indicated concentration for 24 h. The relative activity was calculated as the ratio of pNF-κB-Luc (firefly) activity to RL-TK (Renilla) activity. Values are the mean ± SD. Data are representative of three independent experiments (each experiment was performed in quadruplicate). **Significantly different (P < 0.01) from non-specimen group using one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Concentration Assay

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 4. The cell-stimulating activity of ESG on TLR2-deficient mice PEC. PECs derived from TLR2-deficient and WT (BALB/c) mice (2 × 105 per 200 μL) were cultured with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (TLR7 ligand; 5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/ mL). After 48 h, the culture fluid was withdrawn and the amounts of NO, IL-6 and MIP-2 in the supernatants were determined. White and black bars indicate the results for WT and TLR2KO PECs, respectively. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Values are the mean ± SD. Data are representative of three separate experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) between WT and TLR2KO by an unpaired t-test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Cell Culture, Saline

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 6. The cell-stimulating activity of ESG toward TLR2/4DKO and MyD88KO PECs. PECs (2 × 105 per 200 μL) derived from TLR2/4DKO, MyD88KO and WT mice were incubated with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/mL). After 48 h, the culture fluid was withdrawn and NO, IL-6 and MIP-2 in the supernatant was measured. The results of TLR2/4DKO and WT (C57BL/6) PECs are shown in (A)–(C), and the results of MyD88KO and WT (BALB/c) PECs are shown in (D)–(F). Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Values are the mean ± SD. Data are representative of two independent experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) between WT and KO mice by an unpaired t-test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Incubation, Saline

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 9. The binding activities of TLR2 to glycogen. (A) Bindings of ESG (100–1000 μg/mL) to TLR2 (2.5 μg/mL) were tested by ELISA. Pam3CSK4 (100 μg/mL), LPS (500 μg/mL) or imiquimod (500 μg/mL) was used as a control compound. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. (B) Dose response binding curve at the indicated concentration of TLR2 to ESG (500 μg/mL). (C) Bindings of ESG to dectin-1 (β-glucan receptor) and comparison with other glucans such as oyster glycogen (natural-source glycogen; α-1,4/1,6 glucan), soluble starch (α-1,4/ 1,6 glucan), dextran (α-1,6 glucan), SPG (soluble β-1,3/1,6 glucan) and OX-CA (particle β-1,3/1,6 glucan). All glucans, Pam3CSK4, TLR2 and dectin-1 were tested with concentrations of 1000, 100, 2.5 and 1 μg/mL, respectively. OysGly and Pam3 in the graph indicate oyster glycogen and Pam3CSK4, respectively. Data are represented as the mean ± SD from three independent experiments (each experiment was performed in quadruplicate). **Significantly different (P < 0.01) from the negative control by one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay, Comparison, Starch, Negative Control