22-hACE2-DCg-G

rhACE2-IgFc protein from mammalian CHO cells, in glycerol stock

Description: The extracellular part of human recombinant angiotensin-coverting enzyme 2 (aa 17-740), a SARS-COV-2 virus receptor, was expressed as a secreted dimeric protein in mammalian (hamster) CHO cells, fused to human IgG Fc tag (rhACE2-IgFc). UniProtKB sequence accession number: Q9BYF1 (ACE2_HUMAN). rhACE2-IgFc was purified from cell culture medium under native (non-denaturing) conditions using convenient chromatographic techniques. Final product is formulated in glycerol stock for long-term storage in liquid form at -20°C.

Form: formulated in glycerol stock solution (50%) in PBS (pH 7.4).

Storage: Shipped on dry ice (shipment cost to Europe: 160 €). Store at -20ºC in liquid form (solution does not freeze at this temperature).

Concentration: 1.0 mg/ml.

Purity: >93 % as determined by SDS-PAGE.

Image: SDS-PAGE showing rhACE2-IgFc product 22-hACE2-DCg-G at approximately 115 kDa (3 μg/lane).

Applications (tested): ELISA, other immunoassays, biochemical and biophysical analysis, electrophoretic analysis.

Reactivity of rhACE2-IgFc protein (product 22-hACE2-DCg-G) with HRP-labeled SARS-CoV-2 Spike glycoprotein (Baltymas product 20-S2S-TCg-F) in ELISA:

Inhibition of rhACE2-IgFc protein (product 22-hACE2-DCg-G) reactivity with HRP-labeled SARS-CoV-2 S proteins (Baltymas products 20-S2S-TCg-F  and 22-S2SOM1-TCg-F [link]) by various sera in ELISA:

Figure: ELISA plate wells were coated with rhACE2-IgFc protein and probed with SARS-CoV-2 Spike glycoproteins (initial Wuhan sequence or Omicron variants) labeled with HRP. Before application to wells, the Spike protein samples were incubated with either PBS or indicated blood sera. Percentages above columns indicate signal inhibition compared to negative control (buffer). Blood serum of mouse immunized with SARS-CoV-2 Spike glycoprotein Receptor Binding Domain (Baltymas product 20-S2RBD-RHs-F ) was used as a positive control. Dashed lines indicate cut-offs. Serum sample was considered as inhibition positive if below the cut-off line.

Reactivity of rhACE2-IgFc protein (product 22-hACE2-DCg-G) in surrogate SARS-CoV-2 Neutralizing Antibody detection ELISA using COVID-19 positive and negative sera:

Table: Investigation of blood serum samples (N=99) using ELISA test with rhACE2-IgFc protein (product 22-hACE2-DCg-G from glycerol stock). A total of 99 blood serum samples were tested for the ability to inhibit ACE2 and S protein reactivity. Blood samples were collected during February-April 2022 by the time the first wave of Omicron variant infections appeared. All samples were tested for anti-SARS-CoV-2 S antibody positivity using ELISA (UAB Imunodiagnostika, “SARS-CoV-2 S IgG QUANT B ELISA”), which employs SARS-CoV-2 trimeric S protein (UAB Baltymas, product 20-S2S-TCg-G). Ninety-five samples were positive (anti-S IgG 13.39-14353.45 BAU/ml). cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (Genscript, Cat. No. 00847) was used as a reference test to detect samples containing neutralizing antibodies against SARS-CoV-2. The results obtained by our in-house ELISA format of rhACE2-IgFc-coated wells and HRP-labeled SARS-CoV-2 S protein variants (see examples of interaction inhibition by sera shown in the Figure above) were compared to the reference test as shown in the Table. Cut-off values of signal inhibition were determined using ROC analysis. The same results with the panel of sera were obtained by both tests for inhibition/neutralization of the interaction between ACE2 and initial Wuhan sequence Spike or RBD proteins (~88% positive). Whereas, considerably higher number of serum samples were negative (~26%) when testing inhibition of interaction between Omicron Spike and ACE2.

Note: Such rhACE2-IgFc protein construct maintains extracellular part of hACE2 in a dimeric state and provides increased stability to the protein with potential to neutralize SARS-CoV-2 (Ref. 1). hACE2 is a homodimer (Ref. 2), therefore rhACE2-IgFc construct preserves this state to be explored in functional and immuno-assays.

Unit/Price:

References:

1. Lei C, Qian K, Li T, Zhang S, Fu W, Ding M, Hu S: Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig. Nat Commun. 2020 Apr 24; 11(1):2070. doi: 10.1038/s41467-020-16048-4. Full text. Supplementary (Aa sequence).
2. Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q: Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020 Mar 27; 367(6485):1444-1448. doi: 1126/science.abb2762.