PSA was purified from human seminal fluid by PSA affinity chromatography method. In this regard, anti-PSA mAb was coupled to CNBr-activated Sepharose 4B (GE Healthcare, Uppsala, Sweden). The seminal fluid was diluted with PBS in 1:10 ratio, centrifuged at 1200 g for 10 min and then filtered through 0.45 µm filters (Orange Scientific, Braine-1’ Alleud, Belgium). The cleared seminal fluid was loaded on column. Captured PSA proteins were eluted by glycine-HCl (0.1 M, pH = 2.7) and then immediately dialyzed against PBS (pH = 7.5) at 4°C overnight. The purity of purified PSA was analyzed by SDS-PAGE.

Female Balb/c mice aged 6 to 8 weeks (Pasture Institute, Tehran, Iran) were immunized intraperitoneally with 50 µg of highly-purified PSA emulsified with complete Freund's adjuvant (Sigma-Aldrich, Wisconsin, USA) followed by four booster injections of PSA emulsified with incomplete Freund's adjuvant (Sigma-Aldrich). One week after the last immunization, blood was collected from the tail vein for determination of anti-PSA antibody titers by enzyme-linked immuno-sorbent assay (ELISA). Three days before the cell fusion, 20 µg of PSA (without any adjuvant) were injected intravenously ^{(15, 16)}. The use of animals were approved by the ethical committee of Avicenna Research Institute.

Anti-PSA monoclonal antibodies (mAbs) were generated as described elsewhere ⁽¹⁵⁾. Briefly, murine myeloma cell line, Sp2/0, was cultured in RPMI-1640 medium (Gibco, Gran Island, NY, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco) and used as the fusion partner. Splenocytes from the immunized mouse were mixed with Sp2/0 cells at a ratio of 1:5. The cell mixture was washed twice with pre-warmed RPMI-1640 (37°C). Fusion was then performed using pre-warmed 50% polyethylene glycol (PEG) 1500 (Sigma-Aldrich). Selective HAT medium (Sigma-Aldrich) was then added to the wells for selection of hybridoma cells. The reactivities of hybridoma supernatants were checked by ELISA method as described below. Finally, positive hybridoma cells were cloned four times by limiting dilution to select the stable hybridomas.

For titration of ant-PSA in the mice sera and screening of anti-PSA producing hybridoma cells, purified PSA and human seminal plasma were separately coated at 10 µg/ml and 1:2000 dilution in 96-well ELISA plates, respectively (Nunc, Roskilde, Denmark). The plates were incubated at 37°C for 1 hr followed by incubation at 4°C overnight. The wells were washed 3 times with PBS containing 0.05% Tween 20, (PBS-T) for 3 min and then blocked with 2.5% skim milk in PBS-T at 37°C for 1 hr. Serial dilutions of mouse sera and hybridoma supernatants were added to the wells and subsequently incubated for 1 hr. After washing, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig (1:1000) (Avicenna Research Institute, Tehran, Iran) was added to the wells and incubated for 1 hr at 37°C. Then, 50 µl of ortho-phenylenediamine (OPD) or tetramethylbenzidine (TMB) substrates (Sigma-Aldrich) were added to each well and the plates were incubated at room temperature in the dark. After 15 min, the reaction was stopped by adding 15 µl stopping solution (20% H2SO4) to each well. The optical density (OD) was measured at appropriate wavelength by an ELISA reader (BioTek, Winooski, VT, USA). The explained procedure was applied for isotype determination of produced anti-PSA mAbs, except that goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM and IgA (1/1000 dilution) (Sigma-Aldrich) were coated in the 96-well plates ⁽¹⁵⁾. In addition, the captured anti-PSA mAbs were detected by HRP-conjugated rabbit anti-mouse Ig (1:1000).

Briefly, Balb/c mice were peritoneally injected with 0.5 ml 2,6,10,14-Tetramethyl-pentadecane (Pristane) (Sigma-Aldrich). After one week, 5×10⁶ hybridoma cells were inoculated into peritoneal cavity of the mice. The ascitic fluids were harvested after 7-10 days ⁽¹⁷⁾.

Anti-PSA mAbs were purified by Hi-Trap protein G column (GE Healthcare) according to the manufacturer's instructions. Briefly, the ascitic fluids were diluted 1:5 with PBS and filtered through 0.45 µm filters (Orange Scientific). The diluted ascetic fluids were passed through the column and unbound proteins washed out with PBS. The captured antibodies were eluted by glycine-HCl (0.1 M, pH = 2.7) and then dialyzed against PBS (pH = 7.5) at 4°C overnight ⁽¹⁵⁾. The concentrations of purified mAbs were calculated by optical densitometry method at 280 nm.

Human seminal plasma samples were run on a 12% SDS-PAGE gel. After electrophoresis, resolved proteins were transferred onto PVDF membranes (Millipore corporation, Billerica, Massachusetts, USA). The membranes were blocked with 5% skim milk in PBS-T at 4°C overnight. After gentle washing with PBS-T, all purified mAbs were added to the membrane and incubated for 1.5 hr at room temperature. Purified normal mouse Ig at the same concentration of anti-PSA mAbs was used as a negative control in the experiments. After extensive washing with PBS-T, the membrane was incubated with HRP-conjugated rabbit anti-mouse Ig (Avicenna Research Institute) (1:2500) for 1 hr at room temperature followed by washing with ECL chemiluminescence detection system (GE Healthcare).

Formalin-fixed paraffin-embedded human PCa and BPH tissues (Department of pathology, Khatam al anbia Hospital, Tehran, Iran) were checked for PSA expression by IHC method. For assessing anti-PSA mAbs specificity, brain cancer sections were served as PSA-negative tissue in our experiments ⁽¹⁸⁾. In the next step, 4 µm of thick tissue sections were deparaffinized with xylene, then rehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed in citrate buffer at 94°C for 30 min in water bath. After washing with Tris-buffered saline (TBS), endogenous peroxidase activity was quenched by addition of hydrogen peroxidase (3%) in TBS for 10 min. Nonspecific binding was blocked with 5% goat serum in TBS-Tween 0.05% for 20 min and slides were then tilted and incubated with anti-PSA mAbs (10 µg/ml) in TBS containing 1% BSA for 60 min. Furthermore, irrelevant isotype-matching antibody (an anti-idiotype mAb, clone 2F9-G5 (IgG1/κ)) was included in the experiments. The sections were then washed with TBS containing 1% BSA and incubated with EnVision detection system (Dako, Copenhagen, Denmark) for 20 min. Color was developed using 3, 3’-diaminobenzidine (DAB) substrate solution (Sigma-Aldrich) and the slides were counterstained with Mayer's hematoxylin ⁽¹⁹⁾.

The PSA was successfully purified form seminal plasma. Detection of purified PSA in SDS-PAGE demonstrated a single specific band at ∼33 kDa (Figure 1). The PSA was highly purified with no contamination with other proteins of seminal fluid.

After immunization of Balb/c mice, the titers of anti-PSA antibodies in the mice sera were detected by indirect ELISA. Mouse 1 with higher titer of anti-PSA antibody was selected for hybridoma production (Figure 2). After cell fusion, supernatants of growing hybridoma cells were screened based on their reactivity with the purified PSA by ELISA. After four times of cloning, five hybridoma cells named 2G3-E2, 2F9-F4, 2G2-B2, 2D6-E8 and 2C8-E9 were produced. All five anti-PSA mAbs were produced in ascitic fluids and purified.

First of all, the isotypes of all specific mAbs were determined by home-made ELISA as described in methods section. Our results showed that the isotypes of 2G2-B2, 2F9-F4 and 2D6-E8 were IgG1/k and those of 2G3-E2 and 2C8-E9 were IgG2a/k (Tables 1 and 2). In the next step, reactivities of the purified mAbs were assessed by an indirect ELISA on purified PSA (Figure 3A) and also seminal fluids (Figure 3B). Our findings showed that all produced mAbs specifically recognized PSA in both purified PSA and crude mixture of seminal fluids. The highest reaction was observed for clone 2C8-E9 which recognized the target at lowest concentration of the mAb (9.7 ng/ml). Besides, reactivities of the mAbs were checked in western blot on human semen samples. In this regard, all anti-PSA mAbs detected a 33 kDa protein band in human semen corresponding to free PSA. Two distinct bands were also observed at 28 and 90 kDa (Figure 4). For more characterization, the mAbs were applied for staining of PSA expressing tissues, PCa and BPH, in IHC experiments. Although all clones except 2F9-F4 strongly reacted with PCa and BPH tissues, no reactivity was observed in brain cancer sections as PSA-negative control tissue (Figure 5).