Abstract
Antigen Used for Immunization
Clarified seminal plasma from healthy males was used for immunization at a concentration of 100 mg of total protein/dose.
Method of Immunization
BALB/c mice were immunized subcutaneously every 15 days with semen for a total of eight immunizations, followed by two doses of 50 μg of pure PSA. The first immunization used complete Freund's adjuvant and the rest were prepared in incomplete Freund's adjuvant.
Parental Cell Line Used for Fusion
Mouse spleen cells were fused with myeloma cells (P3/X 63.Ag8.653 cell line) using the method of Kearney and associates.(1) The splenocyte-myeloma ratio was 10:1, and the fused cell mixture was distributed in conventional 96-well culture plates at a final concentration of 1 × 105 cells/well.
Selection and Cloning Procedure
Selection of positive clones was carried out using ELISA assay. Polystyrene strips were coated with 1 μg/mL of the purified PSA in phosphate buffered saline (PBS; 0.13 mol/L NaCl; 0.27 mol/L KCl; 0.0015 mol/L KH2PO4; 0.0065 mol/L Na2HPO4) pH 7.2, 100 μL/well, at 37°C for 3 h. Plates were then blocked with PBS-1% skimmed milk (200 μL/well). Hybridoma supernatant fluids were diluted 1:2 in PBS and incubated in the wells for 1 h at 37°C. After washing with PBS-0.05% Tween-20, the strips were incubated with an anti-mouse IgG polyclonal sheep antibodies horseradish peroxidase conjugate for 1 h at 37°C. The reaction was developed with citrate-phosphate buffer (pH 5.5) 0.014% H2O2, and 0.25% OPD (ortho-phenylenediamine) for 10 min, and stopped with 2.5 mol/L sulfuric acid (50 μL/well). The absorbance was measured at 492 nm using a spectrophotometer. An unrelated MAb was used as negative control.
Heavy and Light Chains of Immunoglobulin
MAb isotype determination was made with concentrated culture supernatant and specific anti-mouse IgG isotype reagents from Sigma (CA) using radial double diffusion. Ascitic fluid was obtained by the intraperitoneal inoculation of hybridoma anti-PSA cells at 1 × 106 cells/mice (BALB/c). Purification of the immunoglobulins from the ascitic fluid was carried out by the Protein A method, using a pH gradient. Briefly, Sepharose Protein A was equilibrated in 3 mol/L NaCl, 1.5 mol/L glycine buffer (pH 8.9). Ascitic fluid samples were put into the column and the non-bound materials were discarded. Mouse IgGs were eluted using 0.1 mol/L citric acid in a pH gradient of pH 6−3.
Specificity
To identify the PSA epitope profile recognized by our MAb, a competition ELISA was developed. Polystyrene plates were coated with 1μg/mL of different PSA MAbs in PBS for 3 h, and blocked with PBS-1% bovine serum albumin for 1 h at 37°C. Purified MAbs were preincubated at 37°C, with different concentrations of PSA conjugated with NHS-biotin. One-hundred microliters of the pre-incubated samples were added to the plates followed by incubation for 1 h at 37°C. A Streptavidin-HRP conjugate was then added to the wells at a 1:2500 dilution and the plates incubated for 30 min. The other steps were similar to the ones described previously.(2) Assays were performed in duplicate, and the experiment was repeated at least twice. Percent inhibition was calculated using the formula:
Competition was considered to have occurred when the signal decreased > 50% in comparison with the sample without anti-PSA MAbs.
Specific Antigen Identified
Association and dissociation rate constants for the MAb to PSA were determined by surface plasmon resonance on a Biacore instrument. Briefly, the sensor chip was activated for immobilization according to methods outlined by Pharmacia (Uppsala, Sweden). Polyclonal rabbit anti-mouse immunoglobulin antibodies were coupled to the surface. Mouse anti-PSA MAbs at 100 μg/mL in PBS-0.1%Tween-20/3.4 mmol/L EDTA were injected onto the sensor chip. Binding to the antigen was studied by injection of pure PSA at 20 μg/L in the same diluent. Association and dissociation rate constants were calculated using Biacore kinetics evaluation software (Pharmacia).
PSA epitope recognition by Western blot technique was developed to characterize the epitope recognition pattern for every MAb. PSA was run in 12% denaturing (2 mercaptoethanol) or non-denaturing sodium-dodecyl-sulfate-polyacrylamide gels (SDS-PAGE) and transferred to a nitrocellulose membrane using the semi-dry procedure. The membranes were blocked with PBS-1% BSA and incubated for 1 h with 750 μL of each MAb. After washing with PBS-T, the membranes were incubated for 1 h at 37°C with a commercial sheep anti mouse-IgG-HRP conjugate according to the manufacturer's instructions. The reaction was developed with PBS, 0.25% 3.3 diaminobenzidine for 15 min, and stopped by washing with water.