22-hACE2-DCg-F

rhACE2-IgFc protein from mammalian CHO cells, frozen

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. Then it was dialyzed against PBS, adjusted to 2.0 mg/ml concentration, filter-sterilized, aliquoted and frozen for storage.

Form: Frozen liquid in PBS (pH 7.4).

Storage: The product is shipped with dry ice (shipment cost to Europe: 160 €). Store at -80°C for the long term. After thawing may be stored for short-term at 4°C (stable at least 2 weeks at this temperature). Avoid repeated freeze/thaw cycles.

Concentration: 2.0 mg/ml.

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

Image: SDS-PAGE showing rhACE2-IgFc product 22-hACE2-DCg-F at approximately 115 kDa (1, 3 and 5 μg/lane).

Applications (tested): ELISA, other immunoassays, Western blot, SDS-PAGE.

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

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

Figure: ELISA plate wells were coated with SARS-CoV-2 Spike glycoproteins (initial Wuhan sequence or Omicron variants) and incubated with either PBS or indicated blood sera samples. Then, they were probed with rhACE2-IgFc protein labeled with HRP. 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-F labeled with HRP) 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 HRP-labeled rhACE2-IgFc protein (made from frozen product 22-hACE2-DCg-F). 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 SARS-CoV-2 Spike glycoprotein-coated wells and HRP-labeled rhACE2-IgFc protein (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. Our in-house ELISA test showed slightly higher sensitivity than the reference test in detection of neutralizing antibodies against SARS-CoV-2 initial Wuhan sequence S variant (~92% vs. ~88% of positive sera, respectively). Whereas, considerably higher number of serum samples were negative (~25%) when testing inhibition of interaction between HRP-labeled rhACE2-IgFc and Omicron Spike variant.

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:

Note: Biotinylated form of this product is available under request.

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.