ace2 buffer  (Sino Biological)

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: Schematic diagram illustrating the hypothesis underlying the study. The SARS-CoV-2 spike protein RBD binds ACE2 on the surface of potential host cells. The RBD has a partial negative image of ACE2. Some antibodies against the RBD may then have a conformation sufficiently like ACE2 to have proteolytic activity similar to the proteolytic activity of ACE2. The hypothesis further suggests that the catalytic activity of some antibodies will have a substrate specificity like ACE2, but because the negative image of RBD present antibodies is imperfect, some antibodies with proteolytic activity will have an altered substrate specificity, potentially affecting multiple proteolytic regulatory cascades.

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Activity Assay

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: SARS-CoV-2 spike RBD peptides for ACE2-like activity competition inhibition assay

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Activity Assay, Inhibition, Sequencing

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: Inhibition of exogenously added recombinant ACE2 substrate cleavage activity in undiluted EDTA-anticoagulated pooled plasma samples with and without added ZnCl 2 . ACE2 is a Zn 2+ metalloprotease. In this ACE2 spiking experiment, no ACE2 activity was detectable when exogenous ACE2 was added to the plasma, unless Zn 2+ was also added, or unless the plasma was diluted prior to the addition of the spiked-in ACE2. Adding an ACE2 inhibitor blocked the detection of the ACE2 activity in all conditions, indicating that the activity was specific to ACE2. The findings indicate that no native ACE2 activity should be detectable in the EDTA-anticoagulated plasma, so any ACE2 substrate cleavage activity observed in the EDTA-anticoagulated plasma should be due to something other than ACE2 or other Zn 2+ metalloproteases.

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Inhibition, Recombinant, Activity Assay

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: Survey of ACE2 substrate cleavage activity in plasma from 67 patients with COVID-19 sampled at about day 7 of hospitalization. The plots present the results of the fluorometric assays for ACE2 substrate cleavage activity run on the 67 patients, plus control samples. Several patients, including the 11 patients we selected for further study (4, 5, 9, 11, 13, 15, 27, 43, 44, 47, and 48) because they had among the top 20% of RFU values and had sufficient plasma sample remaining for the immunoglobulin depletion protocol, showed an ability to cleave ACE2 substrate (HDP: commercially purchased pooled healthy donor plasma; HDP + P.C: HDP with added recombinant human ACE2; HDP + P.C + I: HDP + P.C. + ACE2; inhibitor; P.P.P alone: pooled plasma from negative samples).

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Activity Assay, Recombinant

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: Scatter plot of the background corrected RFU values for the plasma samples from the 67 patients evaluated in this study. The top 20% of values are plotted in red, the middle 70% are plotted in black, and the bottom 10% are plotted in blue. The top 20% and bottom 10% were selected for the ACE2 substrate cleavage activity assays before and after immunoglobulin depletion shown in .

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Activity Assay

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: Plasma IgG concentration before and after antibody depletion for selected patients exhibiting ACE2 substrate cleavage activity in Fig. 2 , then re-assayed for ACE2 substrate cleavage activity with results shown in Fig. 4

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Concentration Assay, Activity Assay

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: ACE2 substrate cleavage activity in patients before and after immunoglobulin depletion. The figure shows ACE2 substrate cleavage activity for 11 patients (4, 5, 9, 11, 13, 15, 27, 43, 44, 47, and 48) who had among the top 20% of RFU values and for which we had sufficient plasma for immunoglobulin depletion. The data are presented before (blue points) and after (red points) immunoglobulin depletion by a 100 KDa size exclusion column followed by protein A/G bead absorption. We also show ACE2 substrate cleavage activity assays before and after immunoglobulin depletion for pooled patient plasma (PPP) negative controls and samples from three arbitrarily selected patients, 17, 64, and 65, who had among the bottom 10% of RFU values and had sufficient plasma remaining for the immunoglobulin depletion protocol. The data suggest that for the 11 patients demonstrating ACE2 substrate cleavage activity in this cohort, the ACE2 substrate cleavage activity was depleted in an immunoglobulin-associated manner.

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Activity Assay

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: Substrate cleavage specificity of patient plasma. ACE2 substrate cleavage activity specificities for 11 patients (4, 5, 9, 11, 13, 15, 27, 43, 44, 47, and 48) who had among the top 20% of RFU values were measured with ACE2 standard substrate [Mca-APK(Dnp)] and control substrate (OMNIMMP fluorogenic control). All 11 samples as well as a positive control (ACE2 P.C.), purified recombinant human ACE2 provided by the assay kit, had specific cleavage activities on Mca-APK(Dnp) but no activities on the control substrate. HDP: commercially purchased pooled healthy donor plasma as ACE2-activity negative control sample.

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Activity Assay, Positive Control, Purification, Recombinant, Negative Control

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: Plasma ACE2-like activity competitive inhibition by SARS-CoV-2 spike RBD peptides. ACE2 substrate cleavage activity for 11 patients (4, 5, 9, 11, 13, 15, 27, 43, 44, 47, and 48) who had among the top 20% of RFU values was measured by incubation with serially diluted SARS-CoV-2 spike RBD peptide pools. ACE2-like catalytic activities in plasma were competitively inhibited by the RBD peptide pool in a dose-dependent manner. AUCs of each curve from samples with individual peptide amounts were calculated based on all 16-hour RFU readouts. rhACE2: purified recombinant human ACE2 provided by the assay kit as a positive control. PPP: commercially purchased pooled healthy donor plasma as ACE2-activity negative control sample.

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Activity Assay, Inhibition, Incubation, Purification, Recombinant, Positive Control, Negative Control

Journal: mBio

Article Title: ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients

doi: 10.1128/mbio.00541-24

Figure Lengend Snippet: Correlation of ACE2 substrate cleavage activity (ACE2 activity) with anti-RBD antibody (Ab Against RBD) in patient plasma. Anti-RBD antibody concentrations in plasma samples were determined using ImmunoCAP antibody-binding assay. The negative control pooled human plasma is indicated by the red symbol. The patient’s plasma sample is indicated in green. The negative control plasma values were omitted in the calculation of the r - and P -values.

Article Snippet: We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2.

Techniques: Activity Assay, Binding Assay, Negative Control