Quantitative detection method of SARS-CoV-2 antibody
A quantitative detection method, sars-cov-2 technology, which is applied in the general structural detail detection of gas analyzers, detection of programmed cell death, immunoassay, etc., can solve the problems that cannot truly reflect the level of new coronavirus antibodies, inconsistencies, etc.
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Embodiment 1
[0053] Experimental setup: The proteins used in this example were obtained by expressing human HEK293T cultured cells and Escherichia coli respectively, using recombinant SARS-CoV-2S protein S1RBD domain, recombinant SARS-CoV-2S protein S2 domain, recombinant SARS-CoV-2S protein S2 domain, and recombinant SARS-CoV-2S protein respectively. CoV-2S nucleocapsid (N) protein, P24 protein coated microtiter plate, making SARS-CoV-2S protein S1RBD domain-coated microtiter plate, SARS-CoV-2S protein S2 domain-coated microtiter plate , N protein-coated microtiter plate, P24 protein-coated microtiter plate. The microtiter plates prepared above were incubated with recombinant human ACE2, respectively.
[0054]Experimental results: if figure 1 As shown, by comparing the binding of microtiter plates coated with different recombinant proteins expressed from cells from different sources to ACE2, no matter the recombinant protein expressed from human HEK293T cultured cells or the recombinant ...
Embodiment 2
[0056] Experimental setup: A fragment of ACE2, which is the expressed region Gln18-Ser740 (extracellular domain) (SEQ ID NO.1), was isolated from human AVE2 with the accession number: Q9BYF1. The recombinant ACE2 protein is prepared by using the protein polypeptide fragment, and the above-mentioned recombinant ACE2 protein is coated on the microwell surface of different microtiter plates (96-well ACE2 microwell plate) according to different doses. HIVP24 protein was coated on the microwell surface of the microtiter plate as a control. Add HRP-coupled S1RBD protein (recombinant protein expressed by human HEK293T cultured cells) into the microwell titer plate and incubate. After incubation, the microtiter plate was washed with washing solution to remove the S1RBD protein that was not immobilized on the surface of the microwell, and the chromogenic substrates 3,3',5,5'-tetramethylbenzidine, The reaction termination solution was 0.2M sulfuric acid. The absorbance of the reaction...
Embodiment 3
[0059] Experimental setup: Asn343 glycosylation site on S1RBD is essential for binding to ACE2: SARS-CoV-2 S protein has 22 putative glycosites, depending on NXS / T, X≠P The presence of motif sequences (Zhanget et al., (2020) Mol. Cellular Proteomics). Among these sites, N331 and N343 are located on the RBD and have been shown to be n-glycosylated when mutated to glutamine, which can significantly reduce the infectivity of the virus (Li et al., (2020) Cell182:1284- 1294). In order to determine whether these residues play a role in the ACE2 / S1RBD molecular interaction during the binding process, three mutated S1 RBD recombinants were prepared using the S1RBD protein polypeptide having the amino acid sequence shown in SEQ ID NO.2: N343Q, N331Q and N343Q / N331Q double mutations. These mutants and wild-type S1 RBD were incubated with ACE2 to measure ACE2 / S1 RBD binding activity.
[0060] Experimental results: if Figure 4 As shown, the binding force of ACE2 / S1RBD was basically l...
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