Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry

Abstract

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG, kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

Epitope Mapping of Anti-Tiger Podoplanin Monoclonal Antibody PMab-231

“…We have previously produced mAbs against HER2 (22) or EGFR (23) using CasMab technology and mAbs against CD44, PD-L1, or CD133 using the CBIS method. (24)(25)(26)(27)(28) However, the binding epitopes of these mAbs (H 2 Mab-77 against HER2, EMab-51 against EGFR, C 44 Mab-5 against CD44, L 1 Mab-13 against PD-L1, and CMab-43 against CD133) remain to be determined because these mAbs did not react with synthesized peptides. Conversely, the binding epitopes of mAbs produced by immunizing with synthesized peptides have been easily determined using deletion mutants and point mutants of synthesized peptides.…”

Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses

“…Furthermore, LpMab-23-recognizing cancer-type podoplanin could be a novel predictor for a poor prognosis of early stage tongue cancer [ 40 ]. Recently, we also utilized a Cell-Based Immunization and Screening (CBIS) method to establish mAbs against various membrane proteins, such as CD133 [ 41 ], CD44 [ 42 ], PD-L1 [ 43 ], pig PDPN [ 34 ], horse PDPN [ 44 ], and cat PDPN [ 32 ]. Importantly, those mAbs are very useful for flow cytometry, Western blot, and immunohistochemistry.…”

Development of an anti-bear podoplanin monoclonal antibody PMab-247 for immunohistochemical analysis

“…In this study, we established PMab-247 against bPDPN, which is suitable for flow cytometry, Western blot, and immunohistochemical analyses using CBIS method. Previously, we tried to develop anti -bPDPN mAbs using CBIS method, and added Imject Alum as an adjuvant only in a first injection in the same way with our previous studies [ 14 , [24] , [25] , [26] ]. However, we could not establish anti -bPDPN mAbs, which are useful for immunohistochemical analyses (data not shown).…”

“…We employed a Cell-Based Immunization and Screening (CBIS) method [ 14 , [24] , [25] , [26] ] to develop mAbs against bPDPN. One BALB/c mouse was immunized with CHO/bPDPN cells (1 × 10 8 ) intraperitoneally (i.p.)…”