Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis.

B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning experiments. There are both manual and semi-automated panning selections using polystyrene plastic, magnetic beads, and nitrocellulose as the immobilizing solid surface. Magnetic nanoparticles allow for improved antigen binding due to their large surface area. The Kingfisher Flex magnetic particle processing system was originally designed to aid in RNA, DNA, and protein extraction using magnetic beads. However, the system can be programmed for antibody phage display panning. The automation allows for a reduction in human error and improves reproducibility in between selections with the preprogrammed movements. The system requires minimum human intervention to operate; however, human intervention is needed for post-panning steps like phage rescue. In addition, polyclonal and monoclonal ELISA can be performed using the semi-automated platform to evaluate the selected antibody clones. This chapter will summarize the suggested protocol from the panning stage till the monoclonal ELISA evaluation. Other than this, important notes on the possible optimization and troubleshooting are also included at the end of this chapter.

Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis (MTB) and remains as a key public health problem worldwide. Most of MTB clinical strains are resistant to rifampicin (RIF), the first-line anti-tuberculosis drug. RIF resistance to MTB is due to mutations that mainly found in RIF resistance-determining region (RRDR) in drug target gene, RNA polymerase β subunit (rpoB). Therefore, the aim of the study is to extend the identification of variants in rpoB gene and to elucidate the effect of variants to the RIF resistance. Four of the strains, MTBR1/09, MTBR2/09, MTBR3/09 and MTB221/11 were subjected to drug susceptibility test (DST). All of the strains sequenced and submitted to the National Center for Biotechnology Information Sequence Read Archive were analyzed to identify the variants in the rpoB gene. The identified new variants were then subjected to docking to examine the drug-protein interactions. DST analysis revealed MTBR1/09, MTBR2/09 and MTBR3/09 were resistant to the RIF drug, while MTB221/11 was a presumptive susceptible strain. Two new variants were observed, the deletion (Phe433_Met434delinsLeu in MTBR1/09) and missense (Lys37Arg in MTBR3/09) variants. Meanwhile, the His445Leu, Ser450Leu, Asp103Asp, Ala1075Ala were reported variants. Docking of RIF to MTBR1/09 and MTBR3/09 mutant models revealed the RIF bound to the RIF binding site at different binding affinity and conformation. Concurrently, the new variants caused the RIF to bind to the different active site and neighboring residues. Findings from DST and docking analyses indicate that new variants potentially disturb the RIF inhibition in RpoB mutant proteins, and thus might be responsible to cause the RIF resistance.