1. Abstract:
The purpose of this research is to create a prostate
specific antigen (PSA) that is complexed to alpha 1-
antichymotrypsin (ACT), resulting in a PSA-ACT product for
the use as a calibrator in immunoassays. PSA-ACT is used
to detect early prostate cancer (PCa), to monitor the
progress of the disease, and to determine therapeutic
response. In order to create PSA-ACT, the PSA must first
be complexed with the ACT. The resulting mixture is then
purified. The purified product is confirmed with SDS PAGE
and the final PSA-ACT product is evaluated by
manufacturers for the use as a calibrator in immunoassays.
Background and Introduction:
Prostate Specific Antigen is a major protein in human
seminal fluid with a molecular weight of 28kDa. PSA is
produced both naturally by prostate epithelial cells and by
prostate cancer cells. PSA’s function is to cleave
seminogelin, the main component of semen coagulum. PSA
is a serine regulated protease that is secreted into the
prostatic ducts as an inactive 244 amino acid proenzyme
(proPSA). ProPSA is activated by the cleavage of seven n-
terminal amino acids. PSA that enters circulation intact is
rapidly bound by protease inhibitors, primarily alpha-1-
antichymotripsin.
Free PSA levels can rise because of other medical
conditions, not just in the case of PCa. It is essential to
determine an alternative to testing only for free PSA, which is
where PSA-ACT becomes an invaluable asset to this
process. To get a better understanding of whether or not a
patient has PCa, free PSA can be measured against total
PSA, which includes the PSA-ACT complex. As explained by
the Mayo Clinic, “higher total PSA levels and lower
percentages of free PSA are associated with higher risks of
prostate cancer”. Depending on the results, a doctor can
better determine if a patient may have PCa.
The Creation, Purification, and Analysis of the Prostate Specific Antigen and Alpha-1-
Antichymotripsin Complex
Authors: Jessie Schreiber and Meghan Bish
Methods:
Mix and Incubate PSA
with ACT at 37ºC
Centrifugal
Concentration of
PSA-ACT
Gel Filtration
Chromatography
with S-100 resin
Buffer Exchange
PSA-ACT
Ion-exchange with
CM-Cellulose
Column
Results:
Results Continued:
Figure 1.0
An example of an immunoassay that the PSA-ACT would be
used for as a calibrator.
Figure 2.0
The chromatogram from the S-100 Column showing two peaks. The first
peak contains both the PSA-ACT complex and the free ACT. The second
peak contains free PSA.
Conclusions:
• Successfully complexed PSA and ACT
• Low purity ( >45%)
• Unknown contaminant observed on SDS PAGE (possibly albumin)
• Low resolution between PSA-ACT/free ACT and free PSA
• Incomplete separation of free ACT
• Synthesized PSA-ACT comparable to commercially available PSA/ACT
protein complex.
Centrifugal
Concentration of
PSA-ACT
Chart 1.0
This is a graph that shows the CM-cellulose column’s fractions versus the
fraction’s optical density measured at 280nm that contained the protein of
interest.
-0.01
0
0.01
0.02
0.03
0.04
0.05
50 55 60 65 70 75 80 85 90 95 100
Absorbanceat280nm
Fraction Number
Optical Density of CM-Cellulose Fractions
Lane Sample
1 N/A
2 Protein Ladder
3 PSA
4 ACT
5 PSA/ACT post-complexing
6 Flow-thru from conc.
7 Protein Ladder
8 Fraction 12
9 Fraction 14
10 Fraction 15
11 Fraction 16
12 Fraction 17
13 Fraction 21
Figure 3.0
SDS-PAGE of S-100 column fractions and originl
PSA and ACT samples. Fractions 12,14,15,16,17,
and 21 were of interest in order to determine which
fractions to pool.
Lane Number: 1 2 3 4 5 6 7 8 9 10 11 12 13
Figure 4.0
SDS-PAGE of CM-Cellulose fractions and original PSA and ACT samples.
Lane Number: 1 2 3 4 5 6 7 8 9 10 11 12 13
Lane Sample
1 N/A
2 Protein Ladder
3 N/A
4 PSA (starting material)
5 ACT (starting material)
6 Pre-S-100 Complex
7 Post S-100 Complex
8 Protein Ladder
9 N/A
10 "ACT" (CM cellulose wash)
11 Post CM-Cellulose Complex
12
Commercially Available
PSA/ACT
13
Commercially Available
PSA/ACT (2X conc)
References:
Prostate-Specific Antigen (PSA), Total and Free, Serum." PSAFT. Mayo Clinic, n.d. Web. 03 Aug. 2016.