Thiophilic interaction chromatography facilitates detection of various molecular complexes of prostate-specific antigen in biological fluids.
BACKGROUND
It is known that total PSA values are often greater than the sum total of free-PSA (f-PSA) and PSA-ACT complex. It is primarily because other PSA complexes are present in the patient serum and currently cannot be quantified. Our studies were aimed in developing methodology that will permit recovery of all molecular forms of PSA from various biological fluids.


METHODS
Thiophilic interaction (3S,T-gel) chromatography procedure was used to isolate various PSA molecular forms present in biological fluids. f-PSA and PSA complexes were shown to have strong affinity for T-gel. PSA forms eluted from the column were analyzed by SDS-PAGE/Western blot and were identified by immunostaining with antibody-specific to PSA and to various protease inhibitors. The identity of PSA complexes was also confirmed by Enzyme-linked Immunosorbent-assay (ELISA).


RESULTS
One step affinity chromatography procedure (3S,T-gel) was used to isolate different molecular forms of PSA in seminal plasma, patient sera, and in prostate cancer cell culture medium. Seminal plasma was shown to contain f-PSA, PSA-ACT, PSA-A2M, and PSA-PCI. Sera from prostate cancer patients at different stages of the disease contained f-PSA, PSA-ACT, and PSA-A2M. Besides these PSA forms, some patients at late stage of the disease (T3 and T4) also contained two additional PSA-complexes that have not been identified as of yet. Conditioned medium from LNCaP cells was shown to contain f-PSA, PSA-ACT, and PSA-A2M complexes.


CONCLUSIONS
Thiophilic gel has strong affinity for all known molecular forms of PSA present in any biological material. Visualization of PSA molecular forms in Western-blot analysis was feasible only after patient serum was processed through T-gel. Our procedure can be used to screen hybridoma and antibodies specific to the individual PSA complex. The PSA molecular forms isolated from patient serum after T-gel procedure may provide useful internal calibrators, and thus may significantly enhance the reliability of PSA measurements.
Kawinski
Elzbieta
E
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Levine
Ellis
E
Chadha
Kailash
K
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002850:Chromatography, Gel / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D015394:Molecular Structure; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D011471:Prostatic Neoplasms / Q000473:pathology; D012661:Semen / Q000737:chemistry*; D012680:Sensitivity and Specificity
D017430:Prostate-Specific Antigen