title: Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.
abstract: OBJECTIVE
  Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility.
  
  
  METHODS
  We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels.
  
  
  RESULTS
  The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech).
  
  
  CONCLUSIONS
  Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.
authors.affiliation: Hybritech Incorporated, San Diego, California 92196-9006, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Finlay
authors.forename: C L
authors.initials: CL
authors.lastname: Evans
authors.forename: J R
authors.initials: JR
authors.lastname: Day
authors.forename: J K
authors.initials: JK
authors.lastname: Payne
authors.forename: S D
authors.initials: SD
authors.lastname: Mikolajczyk
authors.forename: L S
authors.initials: LS
authors.lastname: Millar
authors.forename: K
authors.initials: K
authors.lastname: Kuus-Reichel
authors.forename: R L
authors.initials: RL
authors.lastname: Wolfert
authors.forename: H G
authors.initials: HG
authors.lastname: Rittenhouse
mesh_terms: D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000032:analysis / Q000097:blood; D014408:Biomarkers, Tumor / Q000032:analysis / Q000097:blood; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007610:Kallikreins / Q000032:analysis* / Q000276:immunology; D008297:Male; D017430:Prostate-Specific Antigen / Q000276:immunology; D011469:Prostatic Diseases / Q000097:blood; D011470:Prostatic Hyperplasia / Q000097:blood; D011471:Prostatic Neoplasms / Q000097:blood; D012661:Semen / Q000737:chemistry; D000514:alpha 1-Antichymotrypsin / Q000097:blood
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D014408:Biomarkers, Tumor; D000514:alpha 1-Antichymotrypsin; D007610:Kallikreins; D017430:Prostate-Specific Antigen