Production and Characterization of a Peptide-based Monoclonal Antibody Against CD44 Variant 6

Abstract

The gene that codes for the CD44 family members consists of 20 exons, nine of which encode the standard form of the molecule. The other exons can be inserted in various combinations into the membrane proximal region of the extracellular domain of the protein, giving rise to variant isoforms (CD44v). CD44 variants, especially the CD44v6, have been reported to regulate tumor invasion, progression, and metastasis of carcinomas. Producing a high affinity monoclonal antibody against human CD44v6 provides a powerful tool to monitor and trace CD44v6 function in different biological fluids. In this study, a synthetic peptide from CD44v6 was conjugated to keyhole limpet hemocyanin (KLH) and injected into BALB/c mice. Splenocytes from the immunized mice were fused with murine SP2/0 myeloma cells followed by selection of antibody producing hybridoma cells. After screening of hybridoma colonies by ELISA, high affinity antibodies were selected and purified by affinity chromatography. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibodies. Six stable hybridoma cell lines, designated as 1H1, 1H2, 2A12, 2G11, 3H3, and 3H7, were obtained. Flow cytometry and immunocytochemistry results showed that the new monoclonal antibodies recognized CD44v6 on the cell surface. This novel panel of anti-CD44v6 antibodies has the potential for investigating the role of CD44v6 in cancer pathogenesis.

Monoclonal antibody against human Tim‐3 enhances antiviral immune response

“…The monoclonal antibody development was performed using hybridoma technology . Female BALB/C mice (7 weeks old) were immunized with the Tim‐3 protein three times.…”

“…The monoclonal antibody development was performed using hybridoma technology. [34][35][36] Female BALB/C mice (7 weeks old) were immunized with the Tim-3 protein three times. On day 45, spleen cells were collected and fused with SP2/0 cells using previously described methodologies.…”

Production and Characterization of a Novel Monoclonal Antibody Against Human Sortilin

“…(9) Three BALB/c mice were immunized using peptide-KLH, according to the protocol described previously. (10) Fusion and screening Mouse myeloma SP2/0 cell line was cultured in RPMI-1640 containing 10% FBS. The feeder cells, obtained from the peritoneal cavity of a normal BALB/c mouse by RPMI-1640 injection, were plated in 96-well plates and cultured for 24 h before fusion.…”

Peptide-based Monoclonal Antibody 2D8 Against Human Sortilin

“…Hybridoma cells producing anti-sortilin antibodies were screened using indirect ELISA by coating immunizing sortilin peptide according to the method described previously. (2) The positive clones were subcloned four times using limiting dilution until the final clone was obtained.…”