Functional Epitope Core Motif of the Anaplasma marginale Major Surface Protein 1a and Its Incorporation onto Bioelectrodes for Antibody Detection

Abstract

Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale , causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequence SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences' variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized: one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.

Electropolymerization of hydroxyphenylacetic acid isomers and the development of a bioelectrode for the diagnosis of bacterial meningitis

“…3.4, seems to facilitate the permeability of the ions while not compromising the system of conjugated pi electrons. The stability is linked to the ability to retain the conjugated system of pi electrons, configuring one of the essential properties of these matrices for sensory applications [13,14]. Functionalized GE were investigated in the presence of a anionic probe K 3 Fe(CN) 6 /K 4 Fe(CN) 6 (Fig.…”

“…These electrodes, after functionalization, have been used as platforms for the study of the hybridization of DNA nucleotides [6,11,12]. The classes of hydroxyphenylacetic acid polymer derivatives have been used to functionalize GE immunosensors [13,14].…”

Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

“…Since its first description (36) and introduction into laboratory practice (37), phage display technology has proven to be useful in selecting specific peptides that are highly reactive against many pathogen targets. Its biological applications have ranged from use in studies of vaccine candidates for rabies (38), hepatitis C (39), E. coli (40), Plasmodium vivax (23), and Schistosoma japonicum (41) to use with diagnostic antigens for the detection of antibodies to pneumonia (42), neurocysticercosis (43)(44)(45), equine viral arteritis (46), rabies (47), and anaplasmosis (48). These phage-displayed peptides, also called mimotopes, have shown either linear or conformational recognition with high affinity to paratope sequences of antibodies.…”

Theranostic applications of phage display to control leishmaniasis: selection of biomarkers for serodiagnostics, vaccination, and immunotherapy

“…Thus, it has been successfully applied to identify mimotopes used to diagnose malaria (22) (23) , toxoplasmosis (24) (25) , hepatitis A (26) , neurocysticercosis (27) , strongyloidiasis (28) , thyroid cancer (29) , Chagas' disease (30) , and bovine anaplasmosis (31) , and to develop vaccine candidates against cysticercosis (32) , herpes simplex virus infection (33) , cancer (34) , taeniasis (35) , hepatitis B (36) , trichinellosis (37) , Alzheimer's disease (38) , and bovine anaplasmosis (39) . Phage display technology has been also instrumental in the selection of therapeutic agents to treat various cancers such as glioblastoma, melanoma, leukemia, and prostate and thyroid cancers (40) (41) .…”