Anti-Human MUC16 Recombinant Antibody (OC125) (CAT#: TAB-1401CL)

Figure 1 Comparison staining of high-grade serous ovarian carcinomas using OC125 (left panel) and 4H11 (right panel)

Rao, T. D., Park, K. J., Smith-Jones, P., Iasonos, A., Linkov, I., Soslow, R. A., & Spriggs, D. R. (2010). Novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16. Applied immunohistochemistry & molecular morphology: AIMM/official publication of the Society for Applied Immunohistochemistry, 18(5), 462.

Figure 2 Immunohistochemical scoring of OC125 and 4H11 on tissue microarrays of high-grade ovarian serous carcinoma. Only membranous and/or cytoplasmic staining was considered positive.

Score 0: No staining; Score 1: <5% strong or weak; Score 2: 5–50% strong or weak; Score 3: 51–75% strong or 51–100% weak; Score 4: 76–99% strong; Score 5: 100% strong. Figure A: OC125 (Score 0); Figure B: OC125 (Score 1); Figure C: OC125 (Score 2); Figure D: OC125 (Score 3); Figure E: OC125 (Score 4); Figure F: OC125 (Score 5); Figure G: 4H11 (Score 0); Figure H: 4H11 (Score 1); Figure I: 4H11 (Score 2); Figure J: 4H11 (Score 3); Figure K: 4H11 (Score 4); Figure L: 4H11 (Score 5)

Rao, T. D., Park, K. J., Smith-Jones, P., Iasonos, A., Linkov, I., Soslow, R. A., & Spriggs, D. R. (2010). Novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16. Applied immunohistochemistry & molecular morphology: AIMM/official publication of the Society for Applied Immunohistochemistry, 18(5), 462.

Figure 3 Internalization of radio-labelled 4H11 and OC125 monoclonal antibodies on SKOV3-phrGFP-ΔMUC16c334 stable transfected cells

Rao, T. D., Park, K. J., Smith-Jones, P., Iasonos, A., Linkov, I., Soslow, R. A., & Spriggs, D. R. (2010). Novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16. Applied immunohistochemistry & molecular morphology: AIMM/official publication of the Society for Applied Immunohistochemistry, 18(5), 462.

Figure 4 Immunofluorescence micrographs demonstrating the presence of the membrane-associated mucin MUC16 in corneal and conjunctival epithelial cells.

Binding of the monoclonal antibody OC125 (A) or VK-8 (B) or the polyclonal antibody R16 (C) raised against the MUC16 mucin was observed in apical cells of the corneal epithelium. There was no background binding associated with the secondary antibodies, anti-mouse IgG (D), and anti-rabbit IgG (E). Binding of the OC125 antibody to the suprabasal cells of the conjunctival epithelium is demonstrated in (F). Bottom inset: Intracellular binding of the OC125 antibody was observed in some goblet cells. Top inset: Phase-contrast image of the section in the bottom inset demonstrating that the cell shown in the bottom inset is a goblet cell. Binding of the OC125 antibody to scattered islands of cells in HCLE cultures is shown in (G). Propidium iodide was included in the mounting medium to localize the position of the nuclei of cells in culture (G, arrows). Dashed lines: basement membrane region of the epithelia. Scale bar, 25 μm.

Argüeso, P., Spurr-Michaud, S., Russo, C. L., Tisdale, A., & Gipson, I. K. (2003). MUC16 mucin is expressed by the human ocular surface epithelia and carries the H185 carbohydrate epitope. Investigative ophthalmology & visual science, 44(6), 2487-2495.

Figure 5 Western blot analysis of immunoprecipitated mucin demonstrating that the H185 epitope is on MUC16.

(A) One-step immunoprecipitation of HCLE protein extracts using the MUC16-specific antibody OC125, followed by detection with OC125 (OC125/OC125 [capture Ab/detection Ab]) (lane 1), OC125/H185 (lane 2), H185/H185 (lane 3) and H185/OC125 (lane 4). Two-step immunoprecipitation using H185-OC125/OC125 (first capture Ab-second capture Ab/detection Ab) (lane 5). Demonstration of nonspecific binding of mucins in the sample to agarose beads used in two-step immunoprecipitation (lane 6). (B) Immunodepletion experiments demonstrating that removal of H185 epitope from corneal cell protein extracts resulted in absence of H185 (control) and OC125 antibody binding (lanes 1 and 2). Removal of MUC16 mucin results in absence of OC125 antibody binding (control) and weak binding of the H185 antibody (lanes 3 and 4). The low molecular weight bands (∼75 kDa) observed in (A) correspond to the primary antibodies used in the mucin immunoprecipitation assay and to secondary antibodies removed from agarose beads by the Laemmli buffer. Arrowheads: molecular weight of the mucins analyzed.

Argüeso, P., Spurr-Michaud, S., Russo, C. L., Tisdale, A., & Gipson, I. K. (2003). MUC16 mucin is expressed by the human ocular surface epithelia and carries the H185 carbohydrate epitope. Investigative ophthalmology & visual science, 44(6), 2487-2495.

Figure 6 MUC16 is cleaved and a fraction of cleaved fragments remain non-covalently associated in the cells.

Lysates of ovarian cancer cell lines (SKOV3, OVCAR-5 and OVCAR-3) were resolved on 12% SDS-PAGE (A) or 0.1% SDS-2% agarose gels (B), transferred to PVDF membranes and probed with the indicated antibodies. (C) OVCAR-3 culture supernatant was resolved on a gradient (4–12%) SDS-PAGE, transferred to PVDF membranes and probed with the indicated antibodies. (D, E and F) OVCAR-3 cells were lysed and precipitated with MUC16 CT antiserum (LUM16-4) or CA125 mAbs (M11 and OC125). The precipitates were resolved by SDS-PAGE under reducing (D and E) and non-reducing conditions (F) followed by transfer to PVDF membrane and probed with the indicated antibodies. Mouse IgG1 and non-specific rabbit serum (NRS) were used as negative controls. Full length MUC16 (FL MUC16) was detected as a high molecular weight smear recognized by both CA125 and 5E6 mAbs. MAb 5E6 recognized the cleaved C-terminal fragment of MUC16 (MUC16 CT). Intact non-reduced immunoglobulin (IgG) and reduced heavy and light chains (IgG HC and IgG LC respectively) are indicated.

Aithal, A., Junker, W. M., Kshirsagar, P., Das, S., Kaur, S., Orzechowski, C.,... & Ponnusamy, M. P. (2018). Development and characterization of carboxy-terminus specific monoclonal antibodies for understanding MUC16 cleavage in human ovarian cancer. PloS one, 13(4), e0193907.

Figure 7 OC125 weakly binds to cells that are generally considered negative for MUC16.

SKOV-3 cells (identity confirmed by STR analysis) were stained with the primary (1°) antibodies OC125 or VK8 followed by incubation with allophycocyanine (APC)-labeled donkey anti-mouse (DAM) secondary antibody. Binding of the antibodies to SKOV-3 cells was determined on a LSR-II flow cytometer. RT-PCR using MUC16 primers previously reported, was used to determine expression of MUC16 in OVCAR-3, ECC-1 and SKOV-3 cells.

Felder, M., Kapur, A., Gonzalez-Bosquet, J., Horibata, S., Heintz, J., Albrecht, R.,... & Whelan, R. J. (2014). MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress. Molecular cancer, 13(1), 129.

Figure 8 Cell surface MUC16 is sensitive to proteolysis.

A, Binding of OC125 and VK8 to OVCAR-3 cells that were harvested with trypsin or EDTA containing media was determined by flow cytometry. A significant decrease in VK8 binding to cells harvested after trypsin treatment was observed whereas under these conditions, OC125 binding was less affected.

Felder, M., Kapur, A., Gonzalez-Bosquet, J., Horibata, S., Heintz, J., Albrecht, R.,... & Whelan, R. J. (2014). MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress. Molecular cancer, 13(1), 129.