scholarly journals A magnetic bead immunoassay to detect human IgG reactive to SARS-CoV-2 Spike S1 RBD produced in Escherichia coli

2021 ◽  
Author(s):  
Marcelo dos Santos Conzentino ◽  
Ana C Goncalves ◽  
NIgella M Paula ◽  
Fabiane G Rego ◽  
Dalila Zanette ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Receptor Binding ◽  
High Throughput ◽  
Low Cost ◽  
Magnetic Bead ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Operating Characteristics ◽  
Human Igg ◽  
Bead Immunoassay

Immunological assays to detect SARS-CoV-2 Spike receptor binding domain antigen seroconversion in humans are important tools to monitor the levels of protecting antibodies in the population in response to infection and/or immunization. Here we describe a simple, low cost and high throughput Ni2+ magnetic bead immunoassay to detect human IgG reactive to Spike S1 RBD Receptor Binding Domain produced in Escherichia coli. A 6xHis tagged Spike S1 RBD was expressed in E. coli and purified by affinity chromatography. The protein was mobilized on the surface of Ni2+ magnetic beads and used to investigate the presence of reactive IgG in the serum obtained from pre-pandemic and COVID-19 confirmed cases. The method was validated with a cohort of 290 samples and an area under the receiver operating characteristics curve of 0.94 was obtained. The method operated with>82% sensitivity at 98% specificity and was also able to track human IgG raised in response to vaccination with Comirnaty with 85% sensitivity. The IgG signal obtained with the described method was well correlated with the signal obtained when pre fusion Spike produced in HEK cell lines were used as antigen. This novel low-cost and high throughput immunoassay may act as an important tool to investigate protecting IgG antibodies against SARS-CoV-2 in the human population.

scholarly journals Receptor Binding Domain of Escherichia coli F18 Fimbrial Adhesin FedF Can Be both Efficiently Secreted and Surface Displayed in a Functional Form in Lactococcus lactis

2004 ◽  
Vol 70 (4) ◽  
pp. 2061-2071 ◽  
Author(s):  
Agneta Lindholm ◽  
Andreas Smeds ◽  
Airi Palva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Receptor Binding ◽  
Intestinal Epithelial Cells ◽  
Surface Display ◽  
Cell Surface Display ◽  
Binding Domain ◽  
Intestinal Epithelial ◽  
Receptor Binding Domain ◽  
Amino Acid Residues

ABSTRACT Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae. In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF. In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated. Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L. lactis subsp. cremoris protein PrtP for evaluation of secretion efficiency. To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L. lactis AcmA anchor or the PrtP cell wall binding domain. An HtrA-defective L. lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins. Recombinant L. lactis clones secreting the receptor binding domain of F18 fimbriae as a fusion with the H domains of L. lactis protein PrtP were first constructed by using two different signal peptides. FedF-PrtP fusions, directed by the signal sequence of L. brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L. lactis Usp45. In the surface display systems tested, the L. lactis AcmA anchor performed significantly better, particularly in the L. lactis NZ9000ΔhtrA strain, compared to the L. lactis PrtP anchor region. Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L. lactis cells to porcine intestinal epithelial cells. These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L. lactis.

scholarly journals Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

Vaccines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1493
Author(s):  
Ahmed O. Shalash ◽  
Armira Azuar ◽  
Harrison Y. R. Madge ◽  
Naphak Modhiran ◽  
Alberto A. Amarilla ◽  
...  
Keyword(s):  
Receptor Binding ◽  
High Throughput ◽  
Vaccine Development ◽  
Competitive Elisa ◽  
Binding Domain ◽  
Side Reactions ◽  
Elisa Assay ◽  
Receptor Binding Domain ◽  
Validation Parameters ◽  
Containment Measures

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

scholarly journals Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

2021 ◽  
Author(s):  
Ahmed O. Shalash ◽  
Armira Azuar ◽  
Harrison Y. R. Madge ◽  
Naphak Modhiran ◽  
Alberto A. Amarilla ◽  
...  
Keyword(s):  
Receptor Binding ◽  
High Throughput ◽  
Vaccine Development ◽  
Competitive Elisa ◽  
Binding Domain ◽  
Side Reactions ◽  
Elisa Assay ◽  
Receptor Binding Domain ◽  
Validation Parameters ◽  
Containment Measures

Abstract This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin-converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include: 1) serum heat treatment to prevent non-specific interactions, 2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, 3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and 4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 hours for >30 serum samples; this includes the 7.5 hours of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious, time-consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2=0.975, n=25), demonstrating high sensitivity.

scholarly journals Ultra-fast, high throughput and inexpensive detection of SARS-CoV-2 seroconversion

2021 ◽  
Author(s):  
Marcelo S. Conzentino ◽  
Tatielle P. C. Santos ◽  
Khaled A. Selim ◽  
Berenike Wagner ◽  
Janette T. Alford ◽  
...  
Keyword(s):  
High Throughput ◽  
Magnetic Beads ◽  
Low Cost ◽  
Magnetic Bead ◽  
Minimal Amount ◽  
High Throughput Analysis ◽  
Major Drawback ◽  
Analytical Time ◽  
Magnetic Extraction ◽  
Bead Immunoassay

ABSTRACTA technique allowing high throughput, fast and low-cost quantitative analysis of human IgG antibodies reacting to SARS-CoV-2 antigens will be required to understand the levels of protecting antibodies in the population raised in response to infections and/or to immunization. We described previously a fast, simple, and inexpensive Ni2+ magnetic bead immunoassay which allowed detection of human antibodies reacting against the SARS-CoV-2 nucleocapsid protein using a minimal amount of serum or blood. A major drawback of the previously described system was that it only processed 12 samples simultaneously. Here we describe a manually operating inexpensive 96 well plate magnetic extraction / homogenization process which allows high throughput analysis delivering results of 96 samples in chromogenic format in 12 minutes or in fluorescent ultrafast format which takes only 7 minutes. We also show that His tag antigen purification can be performed on the fly while loading antigens to the Ni2+ magnetic beads in a process which takes only 12 min reducing the pre analytical time and cost. Finally, we show that the magnetic bead immunoassay is antigen flexible and can be performed using either Nucleocapsid, Spike or Spike RBD. The method performed with low inter and intra assay variability using different antigens and detection modes and was able to deliver >99.5% specificity and >95% sensitivity for a cohort of 203 pre pandemic and 63 COVID-19 positive samples.

scholarly journals Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

2021 ◽  
Author(s):  
A. Berndt ◽  
T. Smalley ◽  
B. Ren ◽  
A. Badary ◽  
A. Sproles ◽  
...  
Keyword(s):  
Green Algae ◽  
Receptor Binding ◽  
Large Scale ◽  
Low Cost ◽  
Binding Domain ◽  
Intact Protein ◽  
Receptor Binding Domain ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Recombinant Production

ABSTRACTRecombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated, lacking the amino terminus. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

scholarly journals Flexibility in the Receptor-Binding Domain of the Enzymatic Colicin E9 Is Required for Toxicity against Escherichia coli Cells

2004 ◽  
Vol 186 (14) ◽  
pp. 4520-4527 ◽  
Author(s):  
Christopher N. Penfold ◽  
Bryan Healy ◽  
Nicholas G. Housden ◽  
Ruth Boetzel ◽  
Mireille Vankemmelbeke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Receptor Binding ◽  
Disulfide Bond ◽  
Coiled Coil ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Immunity Protein ◽  
Mutant Proteins ◽  
Colicin E9

ABSTRACT The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.

scholarly journals The Receptor-Binding Domain of Influenza Virus Hemagglutinin Produced in Escherichia coli Folds into Its Native, Immunogenic Structure

2010 ◽  
Vol 85 (2) ◽  
pp. 865-872 ◽  
Author(s):  
R. M. DuBois ◽  
J. M. Aguilar-Yanez ◽  
G. I. Mendoza-Ochoa ◽  
Y. Oropeza-Almazan ◽  
S. Schultz-Cherry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Influenza Virus ◽  
Receptor Binding ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Influenza Virus Hemagglutinin
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