2.5. Post-Immunoscreening Analysis

IgE reactive cDNA clones selected for potential diagnostic value in the tertiary immunoscreening were in-vivo excised from vector λTriplEx2 and used to prepare pTriplEx2 phagemid vector. This was done by mixing 150 µL of the eluted plaque with 200 µL of E. coli BM25.8 overnight culture (supplemented with 100 µL of 1 M MgCl₂). The mixture was incubated for 30 min at 31 °C without shaking, followed by the addition of 400 µL of LB broth and further incubated for 1 h at 31 °C with 225 rpm shaking. A volume of 5 µL of cell suspension was spread with a sterile glass spreader on an LB/ampicillin agar plate and incubated at 37 °C overnight for colony formation. An isolated colony was cultured, and the plasmid was purified using QIAprep Spin Miniprep Kit (QiagenGmbH, Hilden, Germany) according to the manufacturer’s instructions.

The concentration and purity of the plasmid DNA were determined using a NanoPhotometer (Implen, München, Germany), then sent to a local scientific company (First BASE Laboratories Sdn. Bhd, Malaysia) for sequencing using vector-specific primers: 5′ TriplEx2_F (5′-TCC GAG ATC TGG ACG AGC-3′) as the forward primer and 3′TriplEx2_R (5′-TAA TAC GAC TCA CTA TAG GG-3′) as the reverse primer. Sequences were analyzed using bioinformatics search tools and other public databases for nematodes.