Immunohistochemical characterization of novel murine monoclonal antibodies against human placenta‐specific 1

Abstract

Human PLAC1 (placenta-specific 1) is a new member of cancer-testis antigens with 212 amino acids, and its expression is restricted to placenta and at much lower levels to testis. Recently, ectopic expression of the PLAC1 transcript has been demonstrated in a wide range of human tumors and cancer cell lines with a proposed function in tumor cell growth. No monoclonal anti-PLAC1 antibody applicable to immunohis-tochemical staining is available so far. To better understand the PLAC1 expression and localization, we aimed to produce monoclonal antibodies (mAbs) against the extracellular region of PLAC1. Mice were immunized with a synthetic peptide corresponding to the C-terminal 11 amino acids of PLAC1 conjugated with a carrier protein. Hybridomas were produced by standard protocol and screened for positive reactivity by enzyme-linked immunosorbent assay. Reactivity of final two clones was then assessed by Western blotting (WB), immunohistochemistry (IHC), and immunocytochemistry (ICC). Both clones showed a specific immunostaining pattern in human term placenta as the positive control. Reactivity was mostly localized to the cytoplasm of syncytiotrophoblasts. One of the clones showed an excellent staining signal in breast, ovary, and prostate cancer cell lines. Importantly, no reactivity was observed with human lymph node cells or prostate. None of the mAbs were able to detect PLAC1 in Western blot. Based on the present results, these mAbs can be used for detection of PLAC1 in IHC and ICC techniques.

Vaccination with human amniotic epithelial cells confer effective protection in a murine model of Colon adenocarcinoma

“…Placenta is a unique tissue in the body that exists for a very limited period of pregnancy and provides an exceptional microenvironment capable of controlling invasion of trophoblast cells. Notably, as we and others reported recently, vital parameters of cancer cells are modulated by placental factors and microenvironment and that placenta expresses such novel markers as PLAC1 (placenta‐specific 1) shared by many cancer cell types . It also hosts a collection of cells with stem cell properties .…”

“…Notably, as we and others reported recently, vital parameters of cancer cells are modulated by placental factors and microenvironment 21 and that placenta expresses such novel markers as PLAC1 (placenta-specific 1) shared by many cancer cell types. 22,23 It also hosts a collection of cells with stem cell properties. 24 More importantly, placental-derived cells and proteins could effectively hinder the outgrowth of cancer cells in murine models.…”

Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1)

“…Two PLAC1-specific Rabbit polyclonal antibodies [4,39], were produced. According to the published amino acid sequence, two synthetic peptides; PLAC1 166-177 (CVFSEEEHTQVP) [11,39] and PLAC1 117-127 (APQKSPWLTKP) [4] corresponding to extracellular segment of human PLAC1 were conjugated separately with Imject Maleimide-activated mcKeyhole Limpet Hemocyanin (Thermo Scientific, Rockford, IL, USA) and injected to New Zealand White Rabbits. Antibody purification was performed using peptide-coupled affinity column after five immunizations.…”

“…After separation on SDS-PAGE gel, proteins were transferred to nitrocellulose membranes antibody at a concentration of (50 ng ml -1 ) [4,11,39]. In the latter case, membranes were washed and incubated for 1.5 h at room temperature with the corresponding secondary antibodies, HRP-conjugated sheep anti-rabbit (Sina Biotech, Tehran, Iran) at a 1:3000 dilution.…”

Detection Systems in Immunohistochemistry

“…To overcome this limitation, a polymer-enhanced two-step IHC detection system (EnVision, EV) was introduced in 1995. EV system contains secondary antimouse and antirabbit Ig antibodies and could be applied to localize tissue-bound primary antibodies of mouse and rabbit origins (Figure 6b) [83][84][85]. The EnVision complex is composed of up to 20 secondary antibodies and nearly 100 molecules of peroxidase molecules, which all are directly attached to an activated dextran polymer backbone [86].…”