Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay

Abstract

Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4–13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2

“…The complete sequences of N, S, and ORF3a proteins of SARS-CoV-2 were retrieved from GenBank for the first deposited sequence of a patient from Wuhan, China (MN908947.3) through access to the Uniprot database ( : accessed 27 January 2020). Microarrays of peptides and a pool of human patient sera were used to map linear B cell epitopes using an Auto-Spot Robot ASP-222 (Intavis Bioanalytical Instruments AG, Köln, Germany) according to a previous SPOT synthesis protocol [ 14 , 15 , 16 ]. Consecutive reactive peptides ( Figure S1 ) were interrogated by their sequence overlap and amino acid composition to define epitopes that were subsequently analyzed by a PIR-peptide match ( ; accessed 18 March 2020) and BLAST homology search ( ; accessed 12 March 2020).…”

“…The following are available online at , Figure S1: Plots of signal intensities of peptides from SARS-CoV-2 proteins S, N, and ORF3a synthesized by SPOT-synthesis methodology and revealed with anti-human IgG alkaline phosphatase to the procedure previously described [ 14 , 15 , 16 ]. Figure S2: SDS-PAGE (10%) analysis of Dx-SARS2-RDB purified by affinity chromatography.…”

SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites

“…A library of 15 amino acid peptides with a 5-residues overlap was designed to represent the entire coding region of RBD (aa 319-541), NTD (aa 1-303), and partial sequence of haptoglobin (121-127; 226-234; 253-267; 283-289; 318-327) and alpha-S1-casein (31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Peptides were synthesized onto cellulose membranes using an Auto-Spot Robot ASP-222 (Intavis Bioanalytical Instruments, AG, Köln, Germany) [69,70]. Briefly, membranes containing the synthetic peptides were washed with TBS-T and then blocked with TBS-T containing 1.5% BSA under agitation for 2 h at room temperature.…”

A Pencil-Lead Immunosensor for the Rapid Electrochemical Measurement of Anti-Diphtheria Toxin Antibodies

“…Blood samples were collected from diphtheria/tetanus/pertussis (DTP)-vaccinated volunteers with no evidence of acute infection or known history of whooping cough or DIPH [ 38 ].…”