Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays.
We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.
Chen
Z
Z
Department of Urology S287, Stanford University School of Medicine, Stanford University Medical Center, CA 94305-5118, USA.
Prestigiacomo
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A
Stamey
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TA
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay / Q000592:standards*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017430:Prostate-Specific Antigen / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D012015:Reference Standards; D012661:Semen / Q000737:chemistry; D000514:alpha 1-Antichymotrypsin / Q000378:metabolism*
D000514:alpha 1-Antichymotrypsin; D017430:Prostate-Specific Antigen