الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Schistosomiasis, caused by the parasitic blood fluke Schistosoma, is a widespread and impactful disease affecting millions of people globally. According to the World Health Organization, there are currently 251.4 million reported cases, with an additional 700 million individuals at risk of infection. In Egypt, the prevalence is notably high in the Northern and Eastern Nile Delta regions, accounting for 60%, while the Southern region reports a lower rate of 6%. Despite its significant global burden, schistosomiasis is still facing challenges such as limited diagnostic accuracy and prolonged examination times. The emergence of parasite resistance further underscores the necessity for innovative diagnostic tools.
The distinctive humoral immune response exhibited by Camelidae, featuring heavy-chain-only antibodies, has been strategically utilized by employing recombinant DNA technology. This innovative approach aims to create single-domain entities designed to specifically target antigens. These specialized entities, referred to as nanobodies, Nbs, (VHHs), present a range of unique advantages, particularly suitable for diagnostic applications. Nbs possess a simplified structure compared to traditional antibodies, making them particularly amenable to isolation and engineering through phage display and recombinant DNA technologies and enabling the generation of Nbs with a high degree of specificity and affinity for schistosome antigens.
The focal point of this study involves assessing the efficacy of the VHH immune phage display library developed against S. mansoni SAWP antigens at TBRI using the pADL-23c phagemid system and generating Schistosome-specific Nbs clones.
The initial phase of the study involved the assessment of the VHH immune library’s quality using different approaches. The development of anti- S. mansoni SAWP antibodies in the SAWP-immunized camel done using indirect ELISA demonstrated the effective generation of a significant titer of S. mansoni SAWP-specific antibodies (p = 0.0138) and consequently SAWP-specific VHH genes. Colony count and PCR for randomly selected clones from the VHH immune library revealed a moderate titer (4.5 x 105 CFU/ml) with a high percentage of insertion (90%) and right orientation (100%) of cloned VHH genes. Sequencing of one of the VHH clones revealed proper amino acids ORF starting with 1st methionine aligned with the ORF of the pADL-23C vector, which indicates the proper expression of the fusion VHHs proteins in the pADL-23c phagmid vector. Blast analysis for the ORF aa sequence showed high similarity to Camelus dromedaries VHHs. All of these parameters affirmed the high quality of the constructed VHH immune library.
Next, a high titre stock (2x 1016 PFU/ml) of the cM13 helper phage was prepared and used in the conversion of the VHH immune library to the VHH phage immune library (6.2 x 1013 virion/ml) using the phage rescue technique.
To enrich anti- S. mansoni SAWP VHH phages in the VHH phage library, three rounds of bio-panning have been used. The successful enrichment for SAWP specific clones was demonstrated by the gradual increase in the number of anti-SAWP Nbs phages after each round, using colony count and polyclonal phage ELISA. The analysis of the genetic material of the enriched phage library through agarose gel electrophoresis revealed its purity and integrity.
Subsequently, the diagnostic potentiality of the phagemid virions was demonstrated using phage ELISA on human serum samples with confirmed S. mansoni infection, human serum samples with confirmed Fasciola hepatica infection, and negative control samples. Data showed diagnostic performance with 80% sensitivity, indicating the Nbs’ ability to correctly identify true positive cases. Additionally, the specificity of the Nbs reached 95%, showcasing their capacity to accurately recognize and exclude true negative cases. The overall accuracy, considering both sensitivity and specificity, was determined to be 90%. These findings underscore the effectiveness of the anti-S. mansoni Nbs in this library for achieving reliable diagnostic outcomes.
After the completion of the third round of biopanning, 24 clones were selected for the generation of their monoclonal Nbs through periplasmic exprission. Through the implementation of monoclonal ELISA, out of the 24 clones of Nbs, 22 Nbs (91%), demonstrated a specific binding affinity to SAWP.
To gain further insights into the molecular characteristics of these Nbs, sequence analysis of VHH genes was conducted for three Nbs with the highest binding affinities. Sequence analysis highlighted diversity in their antigen targets and recognized epitopes, as demonstrated by the diversity in Complementarity Determining Region 3 (CDR3) amino acid sequences.
In conclusion, this study demonstrated the successful generation of a high-quality and diverse phage library enriched with anti-S. mansoni SAWP phagemide virions using the phage display platform developed in TBRI. Also, the Nbs in this phage library have high binding capacities for SAWP and high diagnostic potentiality for S. mansoni infection and could be used to devlop an easy and cheap national S. mansoni diagnostic kit.