CB-PSA.4 Anti Total PSA

Antigen Used for Immunization

Clarified seminal plasma from healthy males was used forimmunization at a concentration of 100 mg of total protein/dose.

Method of Immunization

BALB/c mice were immunized subcutaneously every 15 dayswith semen for a total of eight immunizations, followed bytwo doses of 50 mg of pure PSA. The first immunization usedcomplete Freund’s adjuvant and the rest were prepared inincomplete Freund’s adjuvant.

Parental Cell Line Used for Fusion

Mouse spleen cells were fused with myeloma cells (P3/X63.Ag8.653 cell line) using the method of Kearney and asso-ciates.(1) The splenocyte-myeloma ratio was 10:1, and thefused cell mixture was distributed in conventional 96-wellculture plates at a final concentration of 1�105 cells/well.

Selection and Cloning Procedure

Selection of positive clones was carried out using ELISAassay. Polystyrene strips were coated with 1mg/mL of thepurified PSA in phosphate buffered saline (PBS; 0.13 mol/LNaCl; 0.27 mol/L KCl; 0.0015 mol/L KH2PO4; 0.0065 mol/LNa2HPO4) pH 7.2, 100 mL/well, at 378C for 3 h. Plates werethen blocked with PBS-1% skimmed milk (200 mL/well).Hybridoma supernatant fluids were diluted 1:2 in PBS andincubated in the wells for 1 h at 378C. After washing withPBS-0.05% Tween-20, the strips were incubated with ananti-mouse IgG polyclonal sheep antibodies horseradishperoxidase conjugate for 1 h at 378C. The reaction was de-veloped with citrate-phosphate buffer (pH 5.5) 0.014%H2O2, and 0.25% OPD (ortho-phenylenediamine) for10 min, and stopped with 2.5 mol/L sulfuric acid (50 mL/well). The absorbance was measured at 492 nm using aspectrophotometer. An unrelated MAb was used as nega-tive control.

Heavy and Light Chains of Immunoglobulin

MAb isotype determination was made with concentratedculture supernatant and specific anti-mouse IgG isotypereagents from Sigma (CA) using radial double diffusion.Ascitic fluid was obtained by the intraperitoneal inocula-tion of hybridoma anti-PSA cells at 1�106 cells/mice(BALB/c). Purification of the immunoglobulins from theascitic fluid was carried out by the Protein A method,using a pH gradient. Briefly, Sepharose Protein A wasequilibrated in 3 mol/L NaCl, 1.5 mol/L glycine buffer(pH 8.9). Ascitic fluid samples were put into the columnand the non-bound materials were discarded. Mouse IgGs

were eluted using 0.1 mol/L citric acid in a pH gradientof pH 6�3.

Specificity

To identify the PSA epitope profile recognized by our MAb, acompetition ELISA was developed. Polystyrene plates werecoated with 1mg/mL of different PSA MAbs in PBS for 3 h, andblocked with PBS-1% bovine serum albumin for 1 h at 378C.Purified MAbs were preincubated at 378C, with different con-centrations of PSA conjugated with NHS-biotin. One-hundredmicroliters of the pre-incubated samples were added to theplates followed by incubation for 1 h at 378C. A Streptavidin-HRP conjugate was then added to the wells at a 1:2500 dilutionand the plates incubated for 30 min. The other steps weresimilar to the ones described previously.(2) Assays were per-formed in duplicate, and the experiment was repeated at leasttwice. Percent inhibition was calculated using the formula:

% inhibition¼ [100� (absorbance sample=

absorbance at 0 mg=mL PSA)] · 100

Competition was considered to have occurred when the sig-nal decreased> 50% in comparison with the sample withoutanti-PSA MAbs.

Specific Antigen Identified

Association and dissociation rate constants for the MAb toPSA were determined by surface plasmon resonance on aBiacore instrument. Briefly, the sensor chip was activated forimmobilization according to methods outlined by Pharmacia(Uppsala, Sweden). Polyclonal rabbit anti-mouse immuno-globulin antibodies were coupled to the surface. Mouse anti-PSA MAbs at 100mg/mL in PBS-0.1%Tween-20/3.4 mmol/LEDTA were injected onto the sensor chip. Binding to theantigen was studied by injection of pure PSA at 20mg/L inthe same diluent. Association and dissociation rate constantswere calculated using Biacore kinetics evaluation software(Pharmacia).PSA epitope recognition by Western blot technique was de-veloped to characterize the epitope recognition pattern forevery MAb. PSA was run in 12% denaturing (2 mercap-toethanol) or non-denaturing sodium-dodecyl-sulfate-polyacrylamide gels (SDS-PAGE) and transferred to anitrocellulose membrane using the semi-dry procedure. Themembranes were blocked with PBS-1% BSA and incubatedfor 1 h with 750mL of each MAb. After washing with PBS-T,the membranes were incubated for 1 h at 378C with a com-mercial sheep anti mouse-IgG-HRP conjugate according tothe manufacturer’s instructions. The reaction was developedwith PBS, 0.25% 3.3 diaminobenzidine for 15 min, and stop-ped by washing with water.

HYBRIDOMAVolume 30, Number 3, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/hyb.2011.0008.MAb

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Availability

Tissue culture supernatant Yes No �Ascitic fluid Yes � NoHybridoma cells Yes � No

References

1. Kearney JF, Radbruch A, Liesegang B, and Rajewsky K:A new mouse myeloma cell line that has lost immunoglob-ulin expression but permits the construction of antibody-secreting hybrid cell line. J Immun 1979;123(4):1548–1550.

2. Acevedo B, Perera Y, Ruiz M, Rojas G, Benıtez J, Ayala M,and Gavilondo J: Development and validation of a quantita-

tive ELISA for the measurement of PSA concentration. ClinChim Acta 2002;317:55–63.

Address correspondence to:

Dr. Lilian Perez LopezProtein PurificationCenter for Immunoassay25 Ave. and 146 St.PlayaHavana 10600CubaTelephone: 537-208-2929/ext. 312E-mail: iqlilian@cie.sld.cu

320 MONOCLONAL ANTIBODIES