A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses

AbstractThe SARS-CoV-2 pandemic and the vaccination effort that is ongoing has created an unmet need for accessible, affordable, flexible and precise platforms for monitoring the induction, specificity and maintenance of virus-specific immune responses. Herein we validate a multiplex (Luminex-based) assay capable of detecting SARS-CoV-2-specific antibodies irrespective of host species, antibody isotype, and specimen type (e.g. plasma, serum, saliva or blood spots). The well-established precision of Luminex-based assays provides the ability to follow changes in antibody levels over time to many antigens, including multiple permutations of the most common SARS-CoV-2 antigens. This platform can easily measure antibodies known to correlate with neutralization activity as well as multiple non-SARS-CoV-2 antigens such as vaccines (e.g. Tetanus toxoid) or those from frequently encountered agents (influenza), which serve as stable reference points for quantifying the changing SARS-specific responses. All of the antigens utilized in our study can be made in-house, many in E. coli using readily available plasmids. Commercially sourced antigens may also be incorporated and newly available antigen variants can be rapidly produced and integrated, making the platform adaptable to the evolving viral strains in this pandemic.Brief SummaryA multi-antigen assay for monitoring SARS-CoV-2-specific antibodies irrespective of host species, antibody isotype, and specimen type was developed.

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Performance Comparison of a Flow Cytometry-based and Two Commercial Chemiluminescent Immunoassays for Detection and Quantification of Antibodies Binding to SARS-CoV-2 Spike Protein

10.1101/2021.04.06.21254995 ◽

2021 ◽

Author(s):

Arantxa Valdivia ◽

Fabián Tarín ◽

María Jesús Alcaraz ◽

Paula Piñero ◽

Ignacio Torres ◽

...

Keyword(s):

Flow Cytometry ◽

Performance Comparison ◽

Antibody Isotype ◽

Jurkat T Cells ◽

Specific Antibodies ◽

Clinical Sensitivity ◽

Percent Agreement ◽

Antibody Levels ◽

Roche Diagnostics ◽

Detection And Quantification

The performance of a laboratory-developed quantitative IgG/IgA flow cytometry-based immunoassay (FCI) using Jurkat T cells stably expressing full-length native S protein was compared against Elecsys® electrochemiluminiscent (ECLIA) Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), and LIAISON® SARS-CoV-2 TrimericS IgG chemiluminiscent assay (CLIA) (Diasorin S.p.a, Saluggia, IT) for detection and quantitation of SARS-CoV-2-specific antibodies. A total of 225 serum/plasma specimens from 120 acute or convalescent COVID-19 individuals were included. Overall, IgG/IgA-FCI yielded the highest number of positives (n=179), followed by IgA-FCI (n=177), Roche ECLIA (n=175), IgG-FCI (n=172) and Diasorin CLIA (n=154). Positive percent agreement between FCI and compared immunoassays was highest for Roche ECLIA, ranging from 96.1% (IgG/IgA-FCI) to 97.7% (IgG-FCI), whereas negative percent agreement was higher between FCI and Diasosin CLIA, regardless of antibody isotype. A strong correlation (Rho:0.6-0.8) was found between IgG-FCI or IgA-FCI levels and antibodies quantified by Roche ECLIA and Diasorin CLIA. The trajectory of antibody levels delineated by the different immunoassays in 22 of patients with sequential specimens (>=3) was frequently discordant, with the exception of IgG and IgA determined by FCI assay and to a lesser extent antibodies quantified by Roche ECLIA and Diasorin CLIA. The data suggest that FCI may outperform Roche ECLIA and Diasorin CLIA in terms of clinical sensitivity for serological diagnosis of SARS-CoV-2 infection.

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Ribosome Structural Organization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051670 ◽

1974 ◽

Vol 32 ◽

pp. 332-333

Author(s):

James A. Lake

Keyword(s):

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Small Subunit ◽

Structural Studies ◽

X Ray Diffraction ◽

Specific Antibodies ◽

X Ray ◽

E Coli ◽

Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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Probing structure–activity relationships in bactericidal peptide βpep-25

Biochemical Journal ◽

10.1042/bj20080506 ◽

2008 ◽

Vol 414 (1) ◽

pp. 143-150 ◽

Cited By ~ 6

Author(s):

Ruud P. M. Dings ◽

Judith R. Haseman ◽

Kevin H. Mayo

Keyword(s):

Amino Acid Residues ◽

Neutralization Activity ◽

Bacterial Membranes ◽

E Coli ◽

Structure Activity Relationships ◽

Optimal Activity ◽

Structure Activity ◽

Cationic Residues ◽

Β Sheet ◽

And Staphylococcus Aureus

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure–activity relationships in the bacterial membrane disruptor βpep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of βpep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel β-sheet structure is the bioactive conformation of βpep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, βpep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.

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Analysis of Specific Immunoglobulin G Subclass Antibodies for Serological Diagnosis of Echinococcosis by a Standard Enzyme-Linked Immunosorbent Assay

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.5.5.613-616.1998 ◽

1998 ◽

Vol 5 (5) ◽

pp. 613-616 ◽

Cited By ~ 21

Author(s):

Felix Grimm ◽

Friedrich E. Maly ◽

Jian Lü ◽

Roberto Llano

Keyword(s):

Healthy Volunteers ◽

Immunosorbent Assay ◽

Immunoglobulin G ◽

Serological Diagnosis ◽

Enzyme Linked Immunosorbent Assay ◽

Parasitic Infections ◽

Igg Subclass ◽

Specific Antibodies ◽

Specific Igg4 ◽

Antibody Levels

ABSTRACT The potential roles of specific antibodies of the different immunoglobulin G (IgG) subclasses in the serological diagnosis of cystic echinococcosis (CE) and alveolar echinococcosis (AE) were investigated by an enzyme-linked immunosorbent assay based on hydatid fluid as antigen. Specific antibodies of subclass 1 were found to be of major importance. In sera collected at the time of diagnosis (i.e., before any therapeutic intervention was initiated) they could be demonstrated in 14 of 15 sera from patients with CE and in all 12 sera from patients with AE. The most discriminatory and the most specific antibodies found in this study belonged to IgG subclass 4. Only one false-positive reaction was observed with 253 sera from healthy volunteers, and no cross-reactions occurred in 80 sera from patients with different parasitic infections. Specific IgG4 antibodies could be demonstrated in 61.0 to 66.7% (CE) or 47.6 to 66.7% (AE) of the cases. Antibody levels of IgG subclass 2 were elevated only moderately, and subclass 3 antibodies were detected in a few cases only. In addition, nonspecific reactions in sera of healthy volunteers or patients with other parasitic infections could partially be attributed to antibodies of subclasses 2 and 3.

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IMMUNOREGULATION OF BLOOD SERUM ESTRADIOL AND PROGESTERONE LEVELS IN POSTMENOPAUSAL WOMEN

Medical Immunology (Russia) ◽

10.15789/1563-0625-2019-5-869-876 ◽

2019 ◽

Vol 21 (5) ◽

pp. 869-876

Author(s):

A. N. Glushkov ◽

E. G. Polenok ◽

S. A. Mun ◽

L. A. Gordeeva ◽

V. A. Lutsenko ◽

...

Keyword(s):

Blood Serum ◽

Human Blood ◽

Solid Phase ◽

Biological Effects ◽

Serum Levels ◽

Human Blood Serum ◽

Breast Cancer Patients ◽

Specific Antibodies ◽

Healthy Women ◽

Antibody Levels

Specific antibodies against estradiol (Es) and progesterone (Pg) are known to modulate blood serum concentrations of these hormones and their biological effects after immunization of animals. It was suggested that specific IgA-Es and IgA-Pg could influence on Es and Pg levels in human blood serum. The purpose of this study was to identify the suggested correlations between serum Es and Pg and specific IgA-Es and IgA-Pg in postmenopausal healthy women (HW) and breast cancer patients (BCP). The serum levels of Es, Pg, IgA-Es and IgA-Pg were studied in 226 HW and 633 BCP by means of solid-phase immunoassay. The following results were obtained. The levels of Es in BCP (0.25 nmol/l) were higher than in HW (0.16; р < 0.0001). The levels of Pg were lower (0.79 vs 0.87; р < 0.0001), and individual Pg/Es ratios were lower (3.19 vs 6.64; р < 0.0001). Individual IgA-Pg/IgA-Es ratios correlated with decrease of Es (rs = -0.15; p = 0.029), with increase in Pg (rs = 0.38; р < 0.0001), and with increased Pg/Es ratio (rs = 0.29; р < 0.0001) in healthy women. Similar correlations were determined in BCP (correspondingly: rs = -0.14, р < 0.001; rs = 0.1, р = 0.009; rs = 0.15, р < 0.0001). The decrease of Es and increase of Pg and Pg/Es in BCP were less significant than in HW: the a quotients in regression у = ах+b (y = hormones levels and x = antibodies levels) in BCP were 3 to 4-fold lower than in HW. These peciliarities of interrelations between hormones and specific antibody levels were revealed only in ER+/PR+ BCP but not in ER+/PR- and ER-/PR- BCP. In conclusion, we have confirmed a suggestion about participation of specific antibodies in regulation of steroids levels in human blood serum. The immune regulation of hormonal status was weakened in BCP.

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Mucosal versus systemic antibody responses to SARS-CoV-2 antigens in COVID-19 patients

10.1101/2020.08.01.20166553 ◽

2020 ◽

Cited By ~ 10

Author(s):

Baweleta Isho ◽

Kento T Abe ◽

Michelle Zuo ◽

Alainna J Jamal ◽

Bhavisha Rathod ◽

...

Keyword(s):

Antibody Response ◽

Upper Airway ◽

Mucosal Immune Response ◽

Antibody Responses ◽

Igg Antibodies ◽

Blood Draw ◽

Neutralization Activity ◽

Surrogate Measure ◽

Antibody Levels ◽

Negative Controls

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the mucosal immune response and its relationship to systemic antibody levels. Since SARS-CoV-2 initially replicates in the upper airway, the antibody response in the oral cavity is likely an important parameter that influences the course of infection, but how it correlates to the antibody response in serum is not known. Here, we profile by enzyme linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor binding domain (RBD) in serum (n=496) and saliva (n=90) of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Whereas anti-CoV-2 IgA and IgM antibodies rapidly decayed, IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. In a surrogate neutralization ELISA (snELISA), neutralization activity peaks by 31-45 days PSO and slowly declines, though a clear drop is detected at the last blood draw (105-115 days PSO). Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that systemic and mucosal humoral IgG antibodies are maintained in the majority of COVID-19 patients for at least 3 months PSO. Based on their correlation with each other, IgG responses in saliva may serve as a surrogate measure of systemic immunity.

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DOP06 Non-invasive identification of adherent-invasive E. coli in patients with Crohn’s disease

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz203.045 ◽

2020 ◽

Vol 14 (Supplement_1) ◽

pp. S044-S045 ◽

Cited By ~ 1

Author(s):

A Buisson ◽

E Vazeille ◽

X Hébuterne ◽

M Fumery ◽

B Pariente ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Unmet Need ◽

Inhibition Test ◽

Invasive Ability ◽

Ileal Mucosa ◽

Multi Centre Study ◽

E Coli ◽

Non Invasive ◽

Adhesion Inhibition

Abstract Background Medications limiting the adhesion of ‘adherent and invasive E. coli’ (AIEC) represent potential strategies to treat Crohn’s disease (CD). However, the ileal AIEC identification is a time-consuming procedure, and the number of AIEC strains which colonise ileal CD mucosa remains unknown. There is an unmet need for non-invasive biomarkers to identify patients colonised by AIEC. We aimed to evaluate non-invasive biomarker of ileal AIEC colonisation in patients with CD. Methods This prospective and multi-centre study included CD patients requiring ileocoloscopy. Saliva, serum, stools and ileal biopsies were collected. Abundance and global invasive ability of ileal or faecal E. coli were performed. Isolated E. coli were characterised as AIEC or non-AIEC on I407 epithelial cells and THP1 macrophages. The ERIC-PCR profiles of ileal E. coli were performed. Ileal E. coli/CEACAM6 interaction was analysed by a yeast aggregation test and T84 assays (CEACAM6 protein expression, adhesion inhibition test with D-mannose). Quantification of serum anti-E. coli and ileal or salivary CEACAM6 was realised by ELISA. Results Overall, 102 CD patients were enrolled in this study and 25.8% of them exhibited ileal AIEC colonisation (AIEC+). The abundance and global invasive ability of ileal mucosa-associated E. coli were higher in AIEC+ CD patients compared with CD patients without AIEC (AIEC−) (p = 0.0065 and p = 0.0007, respectively). There was no difference between faecal abundance and invasive ability of E. coli between AIEC+ and AIEC− patients. The ERIC-PCR profiles of ileal E. coli showed that CD AIEC+ were for 78% of them colonised by not more than 2 clonal AIEC strains. In addition, AIEC were able to interact with CEACAM6 by binding D-mannose residues and to induce CEACAM6 expression in T84 cells (p = 0.0009 and p = 0.0185, vs. non-AIEC; respectively). This was also supported by adhesion inhibition test. Serum anti-E. coli level was higher for CD AIEC+ (vs. CD AIEC-). Ileal CEACAM6 level were positively correlated with abundance of ileal associated E. coli in AIEC+ patients (r = 0.4000; p = 0.0362) and with salivary CEACAM6 level (r = 0.4690; p < 0.0001). The non-invasive biomarker ‘serum anti-E.coli/salivary CEACAM6’ index was higher for CD AIEC+ (p = 0.0174; vs. CD AIEC-). A cut-off value < 1.34 × 10−6 eliminated the presence of ileal AIEC with a high negative predictive value (90% CI95% [69%–95%]). Conclusion Our study reported that identification of faecal AIEC cannot replace identification of AIEC from ileal biopsies, most of AIEC infection are mono or bi-clonal (≤ 2 strains) and that non-invasive biomarker such as ‘serum anti-E.coli/salivary CEACAM6’ index could be helpful to screen CD patients for AIEC infection.

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Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry

Scientific Reports ◽

10.1038/s41598-020-78895-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

John V. Dzimianski ◽

Nicholas Lorig-Roach ◽

Sara M. O’Rourke ◽

David L. Alexander ◽

Jacqueline M. Kimmey ◽

...

Keyword(s):

Immunosorbent Assay ◽

Optical Measurements ◽

Enzyme Linked Immunosorbent Assay ◽

High Complexity ◽

Antibody Isotype ◽

Biolayer Interferometry ◽

Plasma Antibody ◽

Quantitative Results ◽

Lateral Flow Test ◽

Antibody Levels

AbstractSerological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide semi-quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated “dip-and-read” format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete semi-quantitative results are obtained in less than 20 min. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens.

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Characterization of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli K12 and other Gram-negative bacteria

Biochemical Journal ◽

10.1042/bj1230501 ◽

1971 ◽

Vol 123 (4) ◽

pp. 501-505 ◽

Cited By ~ 13

Author(s):

J. W. Dale

Keyword(s):

Escherichia Coli ◽

Host Species ◽

Host Bacterium ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

R Factor ◽

Relationship Of ◽

The Relationship

1. The amino acid composition of the β-lactamase from E. coli (R-1818) was determined. 2. The R-1818 β-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The Km values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 β-lactamases is discussed.

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REGULATION OF ANTIBODY FORMATION BY SERUM ANTIBODY

Journal of Experimental Medicine ◽

10.1084/jem.130.5.1175 ◽

1969 ◽

Vol 130 (5) ◽

pp. 1175-1186 ◽

Cited By ~ 66

Author(s):

Martin W. Graf ◽

Jonathan W. Uhr

Keyword(s):

Antibody Formation ◽

Gamma Globulin ◽

Regulatory Mechanism ◽

Serum Antibody ◽

Gel Filtration ◽

Antibody Activity ◽

Specific Antibodies ◽

Human Gamma Globulin ◽

Antibody Levels ◽

Conventional Antibody

Rabbits were injected intravenously with bovine serum albumin (BSA) and bacteriophage T2 (T2). 2–3 wk later, anti-BSA was removed from such animals by a procedure which involved exposure of removed plasma to an immunoadsorbent (125I-BSA bound to bromoacetyl cellulose) and return of the adsorbed plasma to the animal. This resulted in removal of the majority of antibody activity to BSA without affecting antibody levels to T2. 1–2 days later, anti-BSA levels began to rise, and reached peak levels usually 5 days after the removal of antibody. Antibody levels to T2 did not change. No evidence was obtained that BSA was released from the immunoadsorbent into the circulation of the rabbits. Thus, only trace amounts of radioactivity were released into the plasma; most of the radioactivity was equally coprecipitable with BSA or human gamma globulin and their specific antibodies; the released material was not demonstrated to be immunogenic in primed rabbits; and the released material did not elute with BSA on gel filtration. The results are interpreted as evidence that serum antibody acts as a regulatory mechanism for antibody formation during the conventional antibody response to a metabolizable antigen.

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