Mimotope of Leptospira from phage-displayed random peptide library is reactive with both monoclonal antibodies and patients' sera.
The study aim was to use random heptapeptide library displayed by bacteriophage T7 for identifying mimotopes from 15 monoclonal antibodies (MAbs) specific to Leptospira spp., and from four leptospirosis patient sera, respectively. The bound phages, selected from fourth round of bio-panning with each antibody, were cloned by plaque isolation and the binding specificity of individual clones were confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. All together 150 phages were selected, mimotope from 86 phages (56.6%) were found to match with protein sequences of Leptospira from GenBank database. The predominant mimotopes were mimotope with sequence LTPCD that found in 27.3%, followed by TPCSK (16%), KSKKSS (4%), KTKRXAS (4%), SSKSYR (3.3%), DPNXNSF (3.3%), KSGRC (2.6%), TLINIF (2%), TPCI (2%), 1.33% each with mimotopes PKKS, PCNTKXTA, and CTKKK, and one phage each (0.66%) with mimotopes PTFGS, TNSKRK, SKSSRC, RSKRIR, VTNNTP, and CSNXSKR. Interestingly, mimotopes LTPCD, TPCSK, and TPCI were found to react with both MAb and patient's sera. The matched proteins from GenBank namely, leptospiral putative outer membrane protein (matched with mimotope PTFGS), thermolysin precursor protein (matched with mimotope TPCIXXGSAS), and hypothetical protein LIC12228 (matched with mimotope CSNXSKR), were found to locate at outer membrane of Leptospira. These phage mimotopes and matched proteins may have potential for further use as diagnostic reagent and immunogen against leptospirosis in the future. The results demonstrate that phage display technique has potential for rapidly identifying phage mimotopes that interact with leptospiral MAbs and patient's sera.
Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
Rongdej
R
Tungtrakanpoung
Pannamthip
P
Pitaksajjakul
Narisorn
N
Na-Ngarm
Wanpen
W
Chaicumpa
Pattama
P
Ekpo
Patcharin
P
Saengjaruk
Gunnar
G
Froman
Pongrama
P
Ramasoota
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000097:blood / Q000276:immunology*; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000276:immunology; D017123:Bacteriophage T7; D018388:Codon, Terminator; D003429:Cross Reactions; D004269:DNA, Bacterial / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007919:Leptospira / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007922:Leptospirosis / Q000175:diagnosis* / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D018388:Codon, Terminator; D004269:DNA, Bacterial; D000939:Epitopes; D007106:Immune Sera; D019151:Peptide Library