InvivoGen開發(fā)了一種細(xì)胞檢測(cè)方法以模擬SARS-CoV-2誘導(dǎo)的合胞體（syncytia）。合胞體是在患有嚴(yán)重COVID-19患者的肺部觀察到的現(xiàn)象。這套工具透過簡(jiǎn)單的定量和比色測(cè)定法，助您研究Spike-ACE2依賴性細(xì)胞融合（cell fusion）。
此測(cè)定是依賴轉(zhuǎn)接分子MyD88從'供體細(xì)胞'轉(zhuǎn)移到表達(dá)了NF-κB誘導(dǎo)性SEAP報(bào)告基因'受體細(xì)胞'來讀數(shù)。
?“供體”細(xì)胞：293-hMyD88細(xì)胞（轉(zhuǎn)染pUNO1-Spike質(zhì)粒后共表達(dá)Spike變異株）
?“受體”細(xì)胞：HEK-Blue??hACE2或A549-Dual??hACE2-TMPRSS2細(xì)胞
共培養(yǎng)“供體”和'受體'細(xì)胞會(huì)驅(qū)動(dòng)它們的細(xì)胞膜發(fā)生Spike-ACE2依賴性融合。在'供體'細(xì)胞中過表達(dá)的MyD88會(huì)激活信號(hào)級(jí)聯(lián)反應(yīng)，導(dǎo)致受體細(xì)胞產(chǎn)生NF-κB誘導(dǎo)性SEAP。SEAP的活性可使用檢測(cè)試劑QUANTI-Blue?溶液進(jìn)行測(cè)量。
抗體的中和能力可以用SEAP分泌量的減少來評(píng)估。如圖所示，
Anti-CoV2RBD-imd-mIgG2a阻斷了原始病毒株和P.1變異株的細(xì)胞融合，而Anti-CoV2RBD-cas-mIgG2a對(duì)相同的P.1變異株的抑制作用較弱。
?
Fig: Cell fusion inhibition assay.?293-hMyD88 cells were transfected with either the pUNO1-Spike or pUNO1-SpikeV5 plasmid and then incubated with Anti-CoV2RBD-cas-mIgG2a, Anti-CoV2RBD-imd-mIgG2a, or Anti-βGal antibodies. These cells were then co-cultured with HEK-Blue? hACE2 cells. After overnight incubation, cell fusion was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue? Solution.
相關(guān)產(chǎn)品信息
| 貨號(hào) | 名稱 | 規(guī)格 |
293-hmyd | 293-hMyD88 Cells | 3 -7 x 106?cells |
hkb-hace2 | HEK-BlueTM?hACE2 Cells | 3 -7 x 106?cells |
a549d-cov2r | A549-DualTM?hACE2-TMPRSS2 Cells | 3 -7 x 106?cells |
p1-spike-v’x’-df | pUNO1-SpikeV’X’-dfur | 20 μg |
p1-spike-v’x’ | pUNO1-SpikeV’X’ | 20 μg |
備注：dfur表示inactivated furin site
“x”指 Spike變異株的內(nèi)部編號(hào)，詳見下表表格
InvivoGen | PANGO | WHO |
Original | (D614) | - |
V1 | (G614) | - |
V2 | B.1.1.7 | Alpha |
V3 | B.1.351 | Beta |
V4 | B.1.429 | Epsilon |
V5 | P.1 | Gamma |
V6 | B.1.526 | Iota |
V7 | B.1.617.1 | Kappa |
V8 | B.1.617.2 | Delta |
V9 | C.37 | Lambda |
V10 | B.1.621 | Mu |
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