Development of a latex agglutination test based on VH antibody fragment for detection of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important porcine pathogen that causes diseases in both swine and human. For rapid SS2 identification, a novel latex agglutination test (LAT) based on heavy-chain variable domain antibody (VH) was developed. Firstly, the soluble 47B3 VH antibody fragment from a phage display library, in which cysteine residues were engineered at the C-terminus, was expressed in Escherichia coli. The purified protein was then gently reduced to form monomeric soluble 47B3 VH subsequently used to coat with latex beads by means of site-specific conjugation. The resulting VH-coated beads gave a good agglutination reaction with SS2. The LAT was able to distinguish S. suis serotype 2 from serotype 1/2, which shares some common sugar residues, and showed no cross-reaction with other serotypes of S. suis or other related bacteria. The detection sensitivity was found to be as high as 1.85x106 cells. The LAT was stable at 4°C for at least six months without loss of activity. To the best of our knowledge, this is the first LAT based on a VH antibody fragment that can be considered as an alternative for conventional antibody-based LAT where VHs are the most favored recombinant antibody.

Development of a human antibody fragment directed against the alpha folate receptor as a promising molecule for targeted application.

Alpha folate receptor (FRα) is currently under investigation as a target for the treatment of patients with non-small-cell lung cancer (NSCLC), since it is highly expressed in tumor cells but is largely absent in normal tissue. In this study, a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα was enriched and selected by phage bio-planning. The positive phage clone (3A102 VH) specifically bound to FRα and also cross-reacted with FRß, as tested by ELISA. Clone 3A102 VH was then successfully expressed as a soluble protein in an E. coli shuffle strain. The obtained soluble 3A102 VH demonstrated a high affinity for FRα with affinity constants (Kaff) values around 7.77 ± 0.25 × 107 M-1, with specific binding against both FRα expressing NSCLC cells and NSCLC patient-derived primary cancer cells, as tested by cell ELISA. In addition, soluble 3A102 VH showed the potential desired property of a targeting molecule by being internalized into FRα-expressing cells, as observed by confocal microscopy. This study inspires the use of phage display to develop human VH antibody (Ab) fragments that might be well suited for drug targeted therapy of NSCLC and other FRα-positive cancer cells.