Monoclonal Antibodies in Immunodiagnosis & Immunotherapy

Nattokinase is a protease produced by Bacillus subtilis var. natto that exhibits various beneficial biological effects. Thus, a reliable assay to determine nattokinase levels is needed. In this study, we developed novel mouse monoclonal antibodies (mAbs) that recognize nattokinase, and created a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure nattokinase levels. The ELISA was developed using a combination of new mouse antinattokinase mAbs used as capture antibodies coated onto 96-well plates, with a peroxidase-conjugated antibody used for detection. This ELISA enabled detection of nattokinase at 1 ng/mL. We believe that the novel mAbs developed in this study will be useful in future for elucidating nattokinase function.

Neuropilin-2 (NRP2) is a cell surface receptor that plays key roles in lymphangiogenesis, but also in pathophysiological conditions such as cancer and inflammation. NRP2 targeting by efzofitimod, a novel immunomodulatory molecule, is currently being tested for the treatment of pulmonary sarcoidosis. To date, no anti-NRP2 antibodies are available for companion diagnostics. Here we describe the development and characterization of a novel NRP2 antibody. Using a variety of research techniques, that is, enzyme-linked immunoassay, Western blot, biolayer interferometry, and immunohistochemistry, we demonstrate that our antibody detects all major NRP2 isoforms and does not cross-react with NRP1. Using this antibody, we show high NRP2 expression in granulomas from sarcoidosis patient skin and lung biopsies. Our novel anti-NRP2 antibody could prove to be a useful clinical tool for sarcoidosis and other indications where NRP2 has been implicated.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest receptor tyrosine kinase family. EphB4 is essential for cell adhesion and motility during embryogenesis. Pathologically, EphB4 is overexpressed and contributes to poor prognosis in various tumors. Therefore, specific monoclonal antibodies (mAbs) should be developed to predict the prognosis for multiple tumors with high EphB4 expression, including breast and gastric cancers. This study aimed to develop specific anti-EphB4 mAbs for multiple applications using the Cell-Based Immunization and Screening method. EphB4-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/EphB4) cells were immunized into mice, and we established an anti-EphB4 mAb (clone B4Mab-7), which is applicable for flow cytometry, Western blot, and immunohistochemistry (IHC). B4Mab-7 reacted with endogenous EphB4-positive breast cancer cell line, MCF-7, but did not react with EphB4-knockout MCF-7 (BINDS-52) in flow cytometry. Dissociation constant (K_D) values were determined to be 2.9 × 10⁻⁹ M and 1.3 × 10⁻⁹ M by flow cytometric analysis for CHO/EphB4 and MCF-7 cells, respectively. B4Mab-7 detected the EphB4 protein bands from breast cancer cells in Western blot, and stained breast cancer tissues in IHC. Altogether, B4Mab-7 is very useful for detecting EphB4 in various applications.

Porcine transmissible gastroenteritis virus (TGEV) infection results in severe gastrointestinal disease manifesting vomiting, diarrhea in neonatal porcine, with extremely high mortality. Monoclonal antibody (MAb) specific to TGEV nonstructural protein (NSP)14 that contains two functional domains, exonuclease (ExoN) and methyltransferase (MTase) domains, may help elucidate the role of NSP14 in the viral life-cycle. In this study, we developed a murine MAb, designated 12F1, against TGEV NSP14 using traditional cell-fusion technique. It was shown the MAb can exclusively bind to viral NSP14, as evidenced by the results of indirect fluorescent assay and western blotting. Intriguingly, epitope screening assay shown that 12F1 targets a hinge region connecting ExoN and N7-MTase of NSP14.

CD300A is a member of the CD300 immunoglobulin (Ig)-like receptor family consisting of eight molecules in humans, all of which contain one Ig-like domain in the extracellular portion. Upon binding its ligand phosphatidylserine or phosphatidylethanolamine, CD300A mediates an inhibitory signal through the immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic portion. The CD300 family molecules are highly homologous to each other. In addition, CD300A has a single nucleotide polymorphism (rs2272111), which is a nonsense mutation encoding glutamine (CD300A^Q111) instead of arginine (CD300A^R111) at residue 111 in the Ig-like domain of CD300A. In this study, we successfully generated monoclonal antibodies (mAbs) specific to either CD300A^R111 or CD300A^Q111 or both. These mAbs are useful for the analysis of CD300A genotype by flow cytometry and the development of an antibody drug for the treatment of various diseases.