Acute systemic Gram-negative bacterial infections are accompanied by release of lipopolysaccharide (LPS) endotoxins into the bloodstream and an innate immune host response via the well-known toll like receptor 4 (TLR4) pathway. In this, LPS associates non-covalently with TLR4 to form an activated heterodimer (LPS/MD2/TLR4)2 complex in vivo, assisted by a coreceptor CD14. This complexation process has been illustrated ex vivo using indirect methods such as cytokine, interleukin, TNF-α measurements and by direct demonstration of sequential binding events on a surface using advanced optics. We are the first ones to carry out homogeneous self-assembly of LPS-rTLR4-MD2 conjugates in vitro in a single step, and further demonstrate the role of CD14 as a catalyst during this process. The assay comprises of LPS, MD2, CD14, and recombinant TLR4-conjugated magnetic particles co-incubated in a buffer at room temperature. The complexes are removed by magnetic separation and the extent of binding is estimated by quantifying the unbound biomolecules in the supernatant using standard biophysical techniques. Our results show that rTLR4-MD2-LPS complexes form in an hour and follow a 1:1:1 stoichiometry, in agreement with the in vivo/ex vivo studies. The assay is also highly specific; addition of known LPS-binding ligands decreased the LPS-rTLR4 complexation, allowing its use as a rapid tool for molecular inhibitor screening.

中文翻译：

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模仿TLR4-LPS途径的一步体外生物测定以及MD2和CD14共受体的作用。

急性全身革兰氏阴性细菌感染会伴随着脂多糖（LPS）内毒素的释放进入血流，并通过众所周知的收费像受体4（TLR4）途径产生先天性免疫宿主反应。在这种情况下，LPS与TLR4非共价缔合，在体内通过共受体CD14协助形成活化的异二聚体（LPS / MD2 / TLR4）2复合物。已经使用间接方法（例如细胞因子，白介素，TNF-α测量）和通过使用先进的光学器件直接证明表面上的顺序结合事件证明了这种络合过程。我们是第一个在一个步骤中体外进行LPS-rTLR4-MD2共轭物均相自组装的公司，并进一步证明了CD14在此过程中作为催化剂的作用。该测定包括LPS，MD2，CD14，在室温下在缓冲液中共孵育重组TLR4共轭磁性颗粒。通过磁分离除去复合物，并通过使用标准生物物理技术定量上清液中未结合的生物分子来估计结合程度。我们的结果表明，与体内/体外研究一致，rTLR4-MD2-LPS复合物在一个小时内形成并遵循1：1：1的化学计量比。该测定法也是高度特异性的。加入已知的LPS结合配体可减少LPS-rTLR4的络合，使其可用作分子抑制剂筛选的快速工具。我们的结果表明，与体内/体外研究一致，rTLR4-MD2-LPS复合物在一个小时内形成并遵循1：1：1的化学计量比。该测定法也是高度特异性的。加入已知的LPS结合配体可减少LPS-rTLR4的络合，使其可用作分子抑制剂筛选的快速工具。我们的结果表明，与体内/体外研究一致，rTLR4-MD2-LPS复合物在一个小时内形成并遵循1：1：1的化学计量比。该测定法也是高度特异性的。加入已知的LPS结合配体可减少LPS-rTLR4的络合，使其可用作分子抑制剂筛选的快速工具。