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Two-dimensional gel electrophoresis detects prostate-specific antigen--[[alpha].sub.1]-antic hymotrypsin complex in serum but not in prostatic fluid.

Prostate-specific antigen (PSA) is a 34-kDa secretory protein of the human prostate [1, 2] that mixes with secretions of other male sex accessory glands during ejaculation as a part of the seminal plasma [3, 4]. (3) The concentration of PSA in prostatic fluid and seminal plasma is very high, ranging from 0.5 to 2 g/L [5-7]. Being a serine protease, PSA in the seminal plasma is responsible for the liquefaction of semen immediately upon ejaculation [8,91. By mechanisms yet unknown, a small amount of PSA is released from the prostate into the circulation [10]. The amount of PSA present in serum varies, depending on the individual's health status. In the presence of prostatic malignancy, the concentration of serum PSA increases; therefore, serum PSA is an important marker for the diagnosis and management of prostatic cancer [11,12].

Although produced predominantly in the liver [13,14], ACT is also synthesized in other organs, including the prostate [15,16]. ACT is a glycoprotein with mass ranging between 55 and 68 kDa; the variation in size is attributed to a variation in glycosylation [17]. ACT is one of the major antiproteases in the human serum, present in concentrations ranging from 5 to 10 [micro]mol/L. It inhibits the proteolytic activity of PSA by covalent binding to form PSA-ACT complex [18]. This PSA-ACT complex is the major form of PSA in the serum and can be detected by commercially available immunoassays [19-24].

Recognition of the formation of the complex between PSA and ACT has prompted the development of diagnostic systems that compare the ratio of the total and free PSA in serum, in hopes of improving the sensitivity of the test over that of conventional assays that detect total PSA only [19,25,26]. Given that the majority of the mass of immunologically detectable complexes of PSA is PSA-ACT [19,20,25], an understanding of the biology of the inter' action between PSA and ACT would enhance our ability to improve diagnostic tools to aid the detection and management of patients with prostatic cancer. Serum contains high concentrations of ACT [17]; prostatic fluid, the main source for serum PSA, also contains ACT but at much lower concentrations [16]. It remains unclear as to what extent an interaction between PSA and ACT in the prostate affects serum concentrations of PSA-ACT complex. In the present study, we investigated the formation of PSA-ACT complex in serum and in prostatic fluid.

Materials and Methods

REAGENTS

PSA was purified from seminal plasma according to the method of Sensabaugh and Grim [2]. The polyclonal antibody against PSA was purchased from Dako (Carpinteria, CA). Monoclonal antibodies against different epitope sites of the PSA molecule were developed by Hybritech (San Diego, CA). Purified ACT was purchased from Biodesign (Kennebunkport, ME), and the polyclonal antibody against ACT was obtained from Calbiochem (La Jolla, CA).

Polyacrylamide, Streptavidin-AP (streptavidin-alkaline phosphatase) conjugate, 4-nitroblue tetrazolium (NBT), and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were purchased from Boehringer Mannheim (Indianapolis, IN). Sodium dodecyl sulfate (SDS) and Servalyte (pH 3-10) were purchased from Serva (Heidelberg, Germany); Pharmalyte and Ampholine (pH 5-7) were obtained from LKB-Pharmacia (Uppsala, Sweden). Urea, 2-mercaptoethanol, dithiothreitol, N,N,N',N'-tetramethylethylenediamine, bisacrylamide, ammonium persulfate, Coomassie Brilliant Blue 8250, nitrocellulose membrane, goat anti-rabbit IgG, and biotinylated goat anti-rabbit IgG were obtained from Bio-Rad (Hercules, CA). Sigma Chemical Co. (St. Louis, MO) was the source for 4-chloro-l-naphthol, glycine, phosphate-buffered saline (PBS), bromphenol blue, Nonidet P-40, Tris ("Trisma"), bovine serum albumin (BSA), and sulfosalicylic acid. Ammonium hydroxide, sodium hydroxide, glacial acetic acid, 300-350 mL/L hydrogen peroxide, 30 mL/L formamide, Tween 20 surfactant, sodium chloride, phosphoric acid, magnesium chloride, and zinc chloride were purchased from Fisher Scientific Co. (Itasca, IL). Silver nitrate was purchased from Mallinckrodt (Paris, KY) and nonfat milk powder from Carnation (Glendale, CA).

CLINICAL SPECIMENS

Prostatic fluid specimens were collected from men of ages [less than or equal to] 40 years who were seen as outpatients in the Urology Clinic at Northwestern Memorial Hospital (Chicago, IL). Prostatic fluid was obtained by rectal digital massage 10 to 30 min after voiding and was collected on a glass slide. The fluid was transferred to a small plastic or glass container and stored at 4[degrees]C. A cover slip was placed on the residual fluid on the slide, and the cells and particles on multiple fields were promptly evaluated microscopically. The sample was transported to the laboratory within 2 h of collection for storage at -80[degrees]C. At the time of analysis, samples collected from multiple patients were thawed and pooled.

Seminal plasma specimens were collected by masturbation from men who visited the Fertility Clinic in the Department of Urology for fertility evaluation. Those specimens that showed normal ranges of sperm count and morphology were studied. Upon collection, samples were placed in a rack at room temperature for 30 min to allow complete liquefaction. They were then centrifuged at 3000g for 30 min to remove spermatozoa. The supernatant fraction was stored at -80[degrees]C until analysis.

Blood specimens were collected by venipuncture from patients who were diagnosed with advanced stages of prostate cancer. At each collection, 10 mL of blood was drawn into a Vacutainer Tube (Becton Dickinson, Rutherford, NJ). The blood was left at room temperature to allow clot formation, and the serum was obtained by centrifugation at 3000g for 30 min. Serum specimens were analyzed for PSA with a commercial kit (Tandem-E PSA; Hybritech). Samples in the present study were derived from pooled sera from patients whose PSA concentrations were 980, 2500, and 3000 [micro]g/L. The samples were stored at -80[degrees]C until analysis. Before electrophoresis and Western blot analysis, some serum specimens were subjected to immunoaffinity chromatography to enrich the PSA-containing fraction. Some specimens of seminal plasma and prostatic fluid were also subjected to affinity chromatography before electrophoresis for comparison. The present study was approved by the Institutional Review Board, and informed consent was obtained before specimen collection.

PROCEDURES

Immunoaffinity chromatography. Five different monoclonal antibodies against different epitope sites of the PSA molecule were mixed in equal amounts and covalently coupled to 3 M Emphaze AB1 resin (Pierce Chemical Co., Rockford, IL). A 1.0-mL aliquot of the affinity resin (equivalent to 3.5 mg of monoclonal antibodies per milliliter) was mixed with either 10 mL of serum or 0.5 mL of prostatic fluid or seminal plasma, the latter two having been diluted to 5.0 mL with PBS. The mixture was kept at 4[degrees]C for 4 h with constant shaking, washed three times each with 10.0 mL of PBS, and centrifuged at 3000g for 10 min. The washed resin was treated with 0.5 mL of Urea Mix (9 mol/L urea, 40 mL/L Nonidet P-40, 2% Ampholyte pH 9-11, and 20 mL/L mercaptoethanol, in distilled water) for 2 h at room temperature with constant shaking. The supernatant fraction obtained after centrifugation of the Urea Mix-treated affinity resin was stored at -80[degrees]C until analysis. The protein content of the preparation was estimated according to a modification of Bradford's procedure [27].

In vitro formation of PSA-ACT complex. Purified PSA was incubated with purified ACT to study the formation of PSA-ACT complex. Aliquots of prostatic fluid were also incubated with the ACT or PSA to determine the biological activity of endogenous PSA or ACT. Unless otherwise specified, incubations were carried out in a small conical microcentrifuge tube under the following conditions: 5.0 Ag of PSA in 5.0 [micro]L of 100 mmol/L ammonium bicarbonate buffer containing 1 g/L sodium azide, or 5.0 [micro]L of prostatic fluid or seminal plasma, was mixed with 35 [micro]g of ACT in 7 [micro]L of 20 mmol/L Tris-HCI buffer, pH 7.4, containing 200 mmol/L NaCl. After incubation at 37'C for various intervals ([less than or equal to] 3 h), the samples were denatured: For all one-dimensional (1-D) electrophoresis procedures, this involved mixing with 12 [micro]L of SDS Mix (20 mL/L SDS, 100 mmol/L dithiothreitol, 62.5 mmol/L Tris base, 100 mL/L glycerol) and then boiling for 5 min; samples prepared for two-dimensional (2-D) electrophoresis were mixed with 12 [micro]L of Urea Mix. Specimens were subjected to gel electrophoresis immediately or stored at -80[degrees]C for future analysis.

Incubation of PSA and ACT with zinc chloride. Purified PSA and ACT were incubated with various concentrations of zinc chloride for 30 min at 37[degrees]C. After incubation, the samples were mixed with Urea Mix for 2-D electrophoresis analysis, or with SDS Mix for 1-D analysis. Western blot analysis for PSA or ACT was carried out on 2-D gels to identify the migration pattern of these proteins. In some experiments, we replaced the zinc chloride with magnesium chloride, at the same concentrations, for comparison.

1-D and 2-D electrophoresis. 1-D gel electrophoresis was carried out on 10% polyacrylamide gel according to the method of Laemmli [28]. Rainbow high-molecular-weight markers (Amersham, Arlington Heights, IL) were applied to one lane as a molecular mass reference. The ISO-DALT system described by Anderson and Anderson [29, 30] was used for the 2-D gel electrophoresis. The first dimension, isoelectric focusing, separated individual proteins by their intrinsic charges, ranging from pH 4 to 9. The second dimension was SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separated individual proteins by their relative molecular masses. A 9-18% acrylamide gradient gel system was used, yielding molecular masses ranging from 10 to 200 kDa [31]. At the completion of electrophoresis, gels either were directly stained with silver nitrate or Coomassie Blue or were prepared for transfer blotting to nitrocellulose membrane for Western blot analysis.

Western blot analysis. Western blot analyses were performed according to the method of Towbin et al. [32]. A semidry blotting apparatus (Hoefer-Pharmacia, San Francisco, CA) was used to transfer the protein spots from the acrylamide gel onto 0.2-[micro]m (pore size) nitrocellulose membrane (Bio-Rad). The transferred membrane was blocked overnight in a solution of 30 g/L BSA in TweenTris-buffered saline (TTBS; 20 mmol/L Tris-HCI, 500 mmol/L NaCl, 5 mL/L Tween 20, pH 7.2).

To detect ACT, we applied polyclonal rabbit anti-ACT antibody (Biodesign) at 1:1000 dilution to the membrane and incubated this overnight at 4[degrees]C. The membranes were washed with TTBS and incubated with biotinylated goat anti-rabbit IgG (Bio-Rad) diluted 1:40 000. After 3 h, the membranes were washed with TTBS and further incubated for 1 h with Streptavidin-AP conjugate diluted 1:10 000. After further washes with TTBS, the proteins were visualized by incubating the membrane in substrate solution containing NBT and BCIP in 100 mmol/L TrisHCI, pH 9.5, 100 mmol/L NaCl, and 5 mmol/L Mg[Cl.sub.2].

To detect PSA, we incubated the membrane overnight with a polyclonal rabbit anti-PSA antibody (Dako) diluted 1:1500 in blocking solution (50 g/L nonfat milk in PBS containing 0.01 g/L sodium azide). We then washed the membrane with blocking solution and incubated it with horseradish peroxidase conjugated to goat anti-rabbit IgG (Bio-Rad) for 4 h. Immunoreactive proteins were detected by incubating the membrane in enough substrate solution to cover the membrane. We used substrate solution prepared as 10 mL of 3 g/L 4-chloro-l-naphthol in methanol plus 30 [micro]L of hydrogen peroxide in 50 mL of TTBS.

Results

2-D ELECTROPHORETIC PROFILES OF PSA, ACT, AND PSA-ACT

In serum. An investigation of 2-D electrophoretic profiles for PSA, ACT, and PSA-ACT provided information regarding molecular forms of these substances at the time of sample collection. Fig. 1A shows a representative silverstained 2-D protein profile of human serum from a prostatic cancer patient. Based on the result of Western blot analysis for ACT (Fig. 1B), the location of ACT was identified as a series of protein spots with molecular masses ranging from 68 to 72 kDa and pI from 4.5 to 5.8. Because ACT is a known glycoprotein, the variation in these results probably results from variation in glycosylation of the ACT protein moiety [17]. The data in Fig. 1B suggest that variation in glycosylation is accompanied by apparently higher MT, possibly through loss of sialic acid residues, resulting in a less acidic form of ACT. Attempts to perform Western blot analysis for PSA on serum specimens not subjected to immunoaffinity column chromatography were not successful (data not shown).

[FIGURE 1 OMITTED]

To investigate the 2-D electrophoretic profile of PSA and PSA-ACT in serum, we subjected specimens to PSA immunoaffinity column chromatography before 2-D gel electrophoresis and Western blot analysis. Fig. 2A shows a representative silver-stained 2-D protein profile of a serum specimen from a prostatic cancer patient (after affinity chromatography to enrich the PSA-containing fraction). Locations for PSA and PSA-ACT in the profile could be identified by Western blot analysis for PSA (Fig. 2B). PSA occupied the familiar location at molecular mass of 34 kDa and pI 8.0-8.8 [4,9]. PSA-ACT was located by its molecular mass of 96 kDa and pI 6.5-7.0. Fig. 2C is a Western blot profile of the same sample probed for ACT, which shows the location of PSA-ACT complex. The 2-D Western blot profile in Fig. 2C did not detect free ACT, presumably because the ACT did not bind during the PSA affinity chromatography before 2-D PAGE analysis.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

In prostatic fluid. Fig. 3A is a representative silver-stained 2-D protein profile of a prostatic fluid specimen. Locations of PSA and ACT were identified by Western blot analyses for PSA and ACT, respectively (Fig. 3B and C). In addition to the 34-kDa PSA detected in serum specimens, prostatic fluid specimens contained characteristic immunoreactive PSA spots with higher (36 kDa) and lower (14-30 kDa) masses and a wide spread of pI values [4]. Multiple forms of PSA in human seminal fluid have also been described [33]. Immunoreactive ACT in prostatic fluid occupied the same location as serum ACT in the 2-D electrophoretic profile. However, unlike that in serum, which had a more defined outline, ACT in prostatic fluid showed a diffused boundary with a wide and more acidic spread of pl. Of interest was an apparent absence of PSA-ACT complexes from the 2-D Western blot profiles probed for either PSA (Fig. 3B) or ACT (Fig. 3C). The protein profile of PSA in seminal plasma was similar to that for prostatic fluid (data not shown).

IN VITRO REACTION OF PROSTATIC FLUID WITH EXOGENOUS PSA OR ACT

Results of the above studies indicated that both serum and prostatic fluid specimens contained free PSA as well as free ACT. However, at least at the level of Western blot analysis, PSA-ACT complex was detected only in serum, not in prostatic fluid. In an attempt to further investigate this difference, we performed the following experiments. Given that prostatic fluid contains materials secreted from prostatic epithelial cells, we though it possible that prostatic fluid contained an inhibitor of the formation of PSA-ACT complexes. To test whether PSA in prostatic fluid was biologically active, we incubated aliquots of undiluted prostatic fluid specimens with various amounts of purified human ACT at 37[degrees]C for 30 min. The incubation mixture was subjected to 1-D gel electrophoresis and Western blot analysis with an anti-PSA antibody. Because uncomplexed PSA in the prostatic fluid contains immunoreactive bands of 14-36 kDa, detection of PSA bands with a higher mass (-96 kDa) is indicative of PSA-ACT complex. Fig. 4 shows that the PSA-ACT complex was detectable but the amount of the complex depended on the amount of exogenous ACT added to the incubation mixture, which suggests that prostatic fluid contained biologically active PSA. In contrast, incubation of aliquots of prostatic fluid with purified PSA under the same conditions failed to form the PSA-ACT complex (data not shown). These findings suggested that the ACT in prostatic fluid was unlikely to be biologically active, for it failed to form the complex with exogenously added purified PSA that was biologically active (see below).

[FIGURE 4 OMITTED]

EFFECT OF ZINC ON FORMATION OF PSA-ACT COMPLEX

Because prostatic fluid and seminal plasma are rich in zinc, 390-450 mg/L (6 mmol/L) and 150-200 mg/L (2 mmol/L), respectively [34], and PSA is known to be inhibited by zinc [35], zinc could play a role in preventing the formation of the PSA-ACT complex. To determine if zinc has any effect on PSA interaction with ACT, we incubated purified PSA with purified ACT in PBS at 37[degrees]C for 30 min in the presence of various amounts of Zn[Cl.sub.2]. The resulting mixtures were subjected to 1-D PAGE and silver-staining. Fig. 5 illustrates that purified PSA and ACT were able to form the PSA-ACT complex under these in vitro conditions, but Zn[Cl.sub.2], at 100 mmol/L or more, prevented the formation of PSA-ACT complex. Mg[Cl.sub.2] at the same concentrations was ineffective in blocking the formation of the complex (data not shown).

Discussion

Results of the present study demonstrate that, although both serum and prostatic fluid contain PSA and ACT, a significant amount of PSA-ACT complex is detected in serum but not in prostatic fluid. Initially we thought that the high concentration of zinc in prostatic fluid prevented the formation of PSA-ACT complex. However, zinc inhibition cannot be the sole explanation because PSA-ACT complex would form in prostatic fluid if excess endogenous ACT was added. This suggested that exogenous ACT, not PSA in the prostatic fluid, had been inactivated.

2-D gel electrophoresis allows the separation of a mixture of proteins by intrinsic charges in the first dimension and by relative molecular masses in the second dimension [29, 30]. The characteristic 2-D electrophoretic migration pattern of each individual protein offers a powerful tool for protein separation and protein identification [31]. Any deviation in the electrophoretic migration pattern for a specific protein would suggest structural and, perhaps, functional alterations. In a previous study, 2-D electrophoresis was used to demonstrate the functional role of PSA in semen liquefaction [9]. In the present study, with the aid of Western blot analysis, locations of PSA, ACT, and PSA-ACT were identified in the 2-D electrophoretic profile. An alteration in the pattern of migration for ACT from prostatic fluid was associated with the absence of PSA-ACT complex in these specimens. However, one should exercise caution in interpreting the above findings, due to the intrinsic limitations of the technique of 2-D electrophoresis. Despite its ability to separate a large number of proteins in body fluids and in cell lysates, this technique is not considered as the method of choice for quantitative assessment of proteins [31]. Therefore, inability of this technique to detect any PSAACT complex in prostatic fluid specimens should not be taken as proof of a complete absence of this complex. Indeed, using a highly sensitive ELISA technique, Espana and associates [7,36] were able to detect PSA-ACT complex in the prostatic fluid and seminal plasma. Nonetheless, the present data suggest that the interaction between PSA and ACT is, at least, not as predominant in prostatic fluid as in serum. We were also unable to detect any PSA complexed with Protein C inhibitor (PCI) in any of the body fluids we tested, although others have detected it [7,37]. Our inability to detect the PSA:PCI complex could be a result of the low concentrations of this complex in prostate fluid. Although Espana et al. were able to detect the PSA:PCI complex by ELISA, the amount was only 0.2% of the total PSA in prostatic fluid [7]. Therefore, it is not surprising that, using the less sensitive technique of 2-D PAGE and Western blotting, we could not detect it.

[FIGURE 5 OMITTED]

That PSA and ACT did not form a significant amount of the complex in prostatic fluid may be physiologically important. Because PSA-ACT complex is formed through a covalent bond, it follows that PSA might be secreted from the prostatic epithelial cells as an active, uncomplexed form (free PSA), despite the understanding that prostatic epithelial cells are able to synthesize both PSA and ACT [16]. Hence, the formation of the PSA-ACT complex found in serum may not take place inside the prostatic epithelial cells or in prostate fluid but may take place outside these compartments.

The lack of significant amount of complex formation between PSA and ACT in the prostatic fluid was possibly attributable, in part, to the 6 mmol/L concentration of zinc in this fluid [34]. This was consistent with our findings that incubation of zinc chloride between 6 and 100 mmol/L with purified PSA and ACT inhibited complex formation in vitro. Previous studies have shown that zinc is able to inhibit the enzymatic activity of PSA [35]. However, the present results also indicated that at least some of the PSA molecules in the prostatic fluid remain biologically active, for the addition of purified ACT to prostatic fluid led to the formation of PSA-ACT complex. Indeed, the ACT found in the prostatic fluid may not be biologically active, given that the addition of purified PSA to the prostatic fluid did not result in formation of the complex. The mechanism for this inability of prostatic ACT to complex with PSA is at present unclear. Proteases are known to bind, cleave, and release inactivated protease inhibitors before finally forming stable, covalent protease-serpin complexes [38]. Our explanation of our data and the work of prior workers is that serum contains relatively high concentrations of ACT, 500 mg/L [39], and relatively low concentrations of PSA, <4 [micro]g/L [25]. The few PSA molecules found in serum will have many ACT molecules to react with. All the PSA will react and inactivate several ACT molecules before almost exclusively forming PSA-ACT complex. A small number of the inactive ACT molecules will be undetectable among the large number of active ACT molecules left over. In prostatic fluid, however, the concentrations of ACT and PSA are much more similar, 300 and 1800 mg/L, respectively [7], such that there will be many PSA molecules for every ACT molecule. A large portion of the finite amount of ACT present will react with PSA, be cleaved, and be released as inactivated ACT. Christensson et al. showed that PSA cleaved ACT at Len 358 [18]. We suspect that the inactivated ACT was similarly clipped at Len 358 by PSA, which should remove -6 kDa of the C-terminus of ACT. The removal of 6 kDa from the cleaved ACT molecule apparently has no effect on the molecular mass, as both cleaved and uncleaved show the same molecular mass on SDS-PAGE [38]; removal of a section of the C-terminus of ACT, however, could change the pl of the protein. Our findings are consistent with these results: We found ACT at the same molecular mass in both serum and prostatic fluid, but pl values for ACT differed between serum and prostatic fluid. A small amount of PSA-ACT would be expected to be formed, which could explain why the complex can be detected with a sensitive immunofluorometric technique but not with the less-sensitive 2-D electrophoresis and Western blot analysis.

Interestingly, only intact (30-kDa) PSA was found in serum (Fig. 2B). Because, as shown in Fig. 3B, our detection system was capable of detecting fragmented PSA in prostatic fluid, apparently an intact (30-kDa) form of PSA is present in human serum that is incapable of reacting with ACT. Given that most PSA is inactivated by cleavage [33], this unique, 30-kDa inactive form of PSA in serum must be inactive for some other reason.

In summary, results of the present study demonstrate that PSA-ACT complex is readily detected in serum but not in prostatic fluid or in seminal plasma. Our findings suggest that the high concentration of zinc, the presence of other inhibitors, the proportion of PSA to ACT, or the inactivation of prostatic ACT may be the mechanism for a lack of PSA-ACT complex in prostatic fluid. These advances in understanding the biology of PSA and ACT interaction in two body compartments provide further insights into the physiological relations between PSA and ACT.

This work was supported in part by grants from the National Institutes of Health (DK39250, CA69851). A.J.S. was the recipient of a Summer Student Research Fellowship from the Department of Urology, Northwestern University Medical School.

Received August 16, 1996; revised October 23, 1996; accepted October 30, 1996.

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YI QIAN, (1) JULIA A. SENSIBAR, (1) DAVID J. ZELNER, (1) ANTHONY J. SCHAEFFER, (1) JUDITH A. FINLAY, (2) HARRY G. RITTENHOUSE, (2) and CHUNG LEE (1) *

(1) Department of Urology, Northwestern University Medical School, Chicago, IL 60611.

(2) Cancer Research Program, Flybritech, San Diego, CA 92196.

(3) Nonstandard abbreviations: PSA, prostate-specific antigen; ACT, [[alpha].sub.1]-antichymotrypsin; 1-D, one-dimensional; 2-D, two-dimensional; PAGE, polyacrylamide gel electrophoresis; NBT, 4-nitroblue tetrazolium; BCIP, 5-bromo4-chloro-3-indolyl-phosphate; SDS, sodium dodecyl sulfate; PBS, phosphatebuffered saline; BSA, bovine serum albumin; TTBS, Tween-Tris-buffered saline (see text); and PCI, Protein C inhibitor.

* Author for correspondence. Fax 312-908-7275; e-mail c-lee7@nwu.edu.
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Title Annotation:Enzynes and Protein Markers
Author:Qian, Yi; Sensibar, Julia A.; Zelner, David J.; Schaeffer, Anthony J.; Finlay, Judith A.; Rittenhous
Publication:Clinical Chemistry
Date:Feb 1, 1997
Words:5354
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