Cat. No.
MABL-3113
Application
crystallization, FACS, functional assays, inhibition, SPR, ELISA
Isotype
Engineer antibody
Species Reactivity
Human
Clone No.
NI-0101
From
Recombinant Antibody
Specificity
This antibody is specific for human TLR4 and binds the LRR 9 domain (residues 328-YN-329, 349-KLK-351); it engages both FcγRI and FcγRIIA, but not FcγRIII.
Alternative Names
hToll; CD284; Toll-like receptor 4; hu 15C1; hu15C1
UniProt
O00206
Immunogen
The original antibody was generated through a humanization process involving CDR grafting of the original mouse anti-TLR4 clone 15C1 onto selected human germline sequences and performing framework optimization.
Application Notes
The original antibody (human IgG1) was generated and used for SPR, FACS, and various functional assays. SPR determined the binding affinity of Hu 15C1 with Fcγ receptors and TLR4, showing strong binding to FcγRI (47.3 ± 1.3 nM) and FcRn (1.82 ± 0.09 nM) and moderate binding to FcγRIIA 131R (0.151 ± 0.015 µM), FcγRIIA 131H (0.311 ± 0.037 µM), and FcγRIIB (0.870 ± 0.038 µM). FACS assays evaluated Hu 15C1 binding to CHO cells expressing human TLR4 and MD-2. Functional assays included human macrophage and U937 cell assays to measure IL-6 production, confocal microscopy, and FRET analysis to visualize TLR4 and Fcγ receptor interactions, and LPS lung instillation in mice to assess cytokine and antibody levels in bronchoalveolar lavage fluid. Results indicated that Hu 15C1 enhances its inhibitory effect on TLR4 signaling through FcγRI and FcγRIIA engagement, disrupting TLR4 dimers and blocking TLR4 activation, with higher concentrations completely blocking TLR4-FcγR clustering, thus extending the duration of TLR4 inhibition (Shang et al., 2014; PMID: 24737331).
Antibody First Published
Shang et al., Selective antibody intervention of Toll-like receptor 4 activation through Fc γ receptor tethering. J Biol Chem. 2014 May 30;289(22):15309-18. PMID:24737331
Note on publication
The original publication describes the humanization and characterization of this antibody clone and a novel mechanism where anti-TLR4 antibodies are designed to tether both TLR4 and Fcγ receptors.
Size
100 μg Purified antibody.
Concentration
1 mg/ml.
Purification
Protein A affinity purified
Buffer
PBS with 0.02% Proclin 300.
Storage Recommendation
Store at 4⁰C for up to 3 months. For longer storage, aliquot and store at - 20⁰C.
