A dual-monoclonal sandwich assay for prostate-specific membrane antigen: Levels in tissues, seminal fluid and urine

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a 750-residue integral membrane glycoprotein and the target of an in-vivo imaging agent for metastatic prostate carcinoma (PCa). PSMA expression in normal and diseased prostatic tissues has previously been demonstrated by immunohistochemical techniques. In order to quantify PSMA levels in tissue homogenates and physiological fluids, we have developed a dual monoclonal antibody (mAb) sandwich assay which detects the antigen at a sensitivity <1 ng/mL and which is linear across the working range 0-50 ng/mL. METHODS: The assay involves capture of the PSMA by a biotinylated mAb (7E11) immobilized onto a streptavidin-coated microtiter plate; this mAb binds to the N-terminus of the antigen. The captured PSMA is detected by an Eu-labelled mAb (PEQ226) which binds in the region corresponding to Residues 134-437 of the antigen. PSMA was purified from LNCaP cells by immunoaffinity chromatography, and used as a calibrator, based on its concentration by the bicinchoninic acid (BCA) protein assay. RESULTS: The assay was applied to a panel of normal and tumor tissues. Levels were highest in the prostate tissues (292-4254 ng/mg protein). Low levels (21-51 ng/mL) were observed in membranes from ovary and breast, and neglible levels (1-10 ng/mg) in membranes from skin, liver, intestine, and kidney. Levels in the corresponding cytosol fractions were 20-to 50-fold lower. The average PSMA level in seminal fluid from 21 donors was 9, 012 ng/mL. On average, levels in normal-male urine (3.47 ng/mL) were ten-fold higher than in normal-female urine (0.3 ng/mL). CONCLUSIONS: This report is the first to describe absolute quantitation of PSMA in tissues and fluids. Congruent with earlier tissue studies based on immunohistochemical staining and Western-blot analysis, prostate tissue membranes expressed the highest levels of PSMA.