Construction of Camelus dromedaries Immune Single Domain Antibodies Library for Development of Schistosoma mansoni Specific Nanobodies Using Phage Display Strategy

Theodor Bilharz Research Institute

Bibliographic Information

Authors: El-Kalamawy H.A.; Awwad M.H.; Diab T.M.; Okasha H.; Abdel-Kareim A.M.; Marawan M.A.; Shoulah S.A.; El-Dabaa E.

Journal: Recent Patents on Biotechnology

Publisher: Bentham Science Publishers

Publication Date: 2025

Volume / Issue: Volume 19 / Issue 1

Pages: 69–83

ISSN: 18722083

DOI: 10.2174/0118722083275669231227063413

Scopus: View on Scopus

PubMed: 39840412

Document Type: Article

Authors and Affiliations

El-Kalamawy H.A., Center of Excellence in Recombinant Biopharmaceutical Proteins, Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, Egypt; Awwad M.H., Department of Zoology, Faculty of Science, Benha University, Benha, Egypt; Diab T.M., Department of Parasitology, Theodor Bilharz Research Institute, Giza, Egypt; Okasha H., Center of Excellence in Recombinant Biopharmaceutical Proteins, Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, Egypt; Abdel-Kareim A.M., Department of Zoology, Faculty of Science, Benha University, Benha, Egypt; Marawan M.A., Department of Animal Medicine (Infectious Diseases), Faculty of Veterinary Medicine, Benha University, Benha, Egypt; Shoulah S.A., Department of Animal Medicine (Infectious Diseases), Faculty of Veterinary Medicine, Benha University, Benha, Egypt; El-Dabaa E., Center of Excellence in Recombinant Biopharmaceutical Proteins, Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute, Giza, Egypt

Abstract

Background: Schistosoma mansoni poses a considerable global public health chal-lenge. In Egypt, approximately 60% of the inhabitants in the Northern and Eastern areas of the Nile Delta are affected by this parasite, whereas the Southern region experiences a significant-ly lower infection rate of 6%. Aim: Construction of an immune phage display Nbs library based on the VHH framework for selecting S. mansoni-specific Nbs for seeking cost-effective, sensitive, and specific diagnostic tools for rapidly detecting Schistosoma mansoni. Methods: Camel was immunized using soluble adult worm antigens (SAWP) for the production of Variable domains of heavy chains of camelid heavy-chain only antibodies (VHHs), which are known as nanobodies (Nb). The PBMCs repertoires VHH sequences library have been constructed with a high percentage of insertion and right orientation using pADL-23c phagmid and M13 phage followed by three rounds of bio-panning against SAWP using phage display technique. Evaluations using polyclonal phage ELISA and other techniques have been carried out to reveal the successful enrichment of anti-SAWP Nbs (VHH) clones. Evaluation of the diagnostic potentiality of these Nbs was carried out using ELISA on human serum sam-ples confirmed for S. mansoni infection. Receiver Operator of Characteristics (ROC) curve analysis was used for discrimination between S. mansoni infection and both negative controls and the Fasciola hepatica group. Results: Using monoclonal ELISA, Nbs of 22 clones out of 24 selected clones showed binding affinity to SAWP. The cutoff values of the produced anti-S. mansoni Nbs was > 0.19, leading to 80% sensitivity, 95% specificity, and 90% accuracy. Sequence analysis of three of these Nbs with high binding affinities showed diversity in their targets, considering their CDR3 aa sequences. Conclusion: This study successfully generated a diverse phage library enriched with anti-S. mansoni VHHs. The nanobodies produced exhibit high diagnostic potential for detecting S. mansoni infection in human patients, offering a promising avenue for the development of efficient diagnostic tools. The innovative approach described herein may have potential applications for patent considerations in the field of the field of diagnostic technology. © 2025 Bentham Science Publishers.

Keywords

bio-panning; camel nanobody; phage display; polyclonal phage ELISA; Schistosoma mansoni; soluble adult worm anti-gens; Animals; Antibodies, Helminth; Antigens, Helminth; Camelus; Cell Surface Display Techniques; Egypt; Enzyme-Linked Immunosorbent Assay; Humans; Patents as Topic; Peptide Library; Schistosomiasis mansoni; Single-Domain Antibodies; Clone cells; Monoclonal antibodies; monoclonal antibody; nanobody; polyclonal antibody; helminth antibody; parasite antigen; Diagnostics tools; Heavy chain; Nanobodies; Phage-display; Polyclonal; Soluble adult worm anti-gen; agar gel electrophoresis; antibody combining site; antibody production; antibody response; antibody titer; antigen detection; Article; bacterial load; bacterium culture; binding affinity; biopanning; Bradford assay; colony forming unit; complementarity determining region; controlled study; diagnostic value; dromedary; Enterobacteria phage M13; enzyme assisted extraction; enzyme linked immunosorbent assay; flow cytometry; functional enrichment analysis; immunization; immunogenicity; infection rate; ligation; male; maximum binding capacity; molecular cloning; nested polymerase chain reaction; nonhuman; phagemid; polyacrylamide gel electrophoresis; predictive value; RNA extraction; schistosomiasis; sensitivity and specificity; sequence alignment; animal; camel; cell surface display; chemistry; diagnosis; human; immunology; parasitology; patent; procedures

Citation Information

Scopus Citations: 0

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