Article pubs.acs.org/ac
Specific Probe Selection from Landscape Phage Display Library and Its Application in Enzyme-Linked Immunosorbent Assay of Free Prostate-Specific Antigen Qiaolin Lang,†,⊥ Fei Wang,†,‡,⊥ Long Yin,†,‡ Mingjun Liu,§ Valery A. Petrenko,∥ and Aihua Liu*,†,‡ †
Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China ‡ University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China § Department of Clinical Laboratory, The Affiliated Hospital of Medical College, Qingdao University, 16 Jiangsu Road, Qingdao 266003, China ∥ Department of Pathobiology, Auburn University, 269 Greene Hall, Auburn, Alabama 36849-5519, United States S Supporting Information *
ABSTRACT: Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825−165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.
P
and PSA−ACT are known as the total prostate-specific antigen (t-PSA) on behalf of the level of PSA in serum.6−8 In the 1980s, PSA was introduced into clinical medicine by the U.S. Food and Drug Administration as the tumor marker used in prediction and early diagnosis of prostate cancer, monitoring of surgery treatment response, and detection of disease recurrence.9−11 For healthy people, t-PSA concentration in serum is usually under 4 ng/mL, and therefore, the value of 4 ng/mL is admittedly regarded as the clinical testing indicator. PCa testing based on PSA level is simple and convenient and can effectively reduce the number of biopsies and lessen the pain of PCa patients. In addition, the ratio of f-PSA/t-PSA is an available index to improve the diagnostic certainty.12−15 Thus, the detection of t-PSA and f-PSA plays an important role in the early diagnosis of prostate cancer. PSA is detected by immunoassay,16 radioimmunoassay,17 enzyme-linked immuno-
rostate cancer (PCa) is the second leading cause of cancerrelated death in men in the United States. PCa accounts for about 29% of male cancers, and the deaths induced by PCa accounted for 9% of male cancer deaths in the United States in 2012.1 Early diagnosis of PCa is often based on the level of prostate-specific antigen (PSA) in serum. PSA, first identified in the 1960s,2 is a 33−34 kDa single-chain glycoprotein secreted from the prostate gland bubble and ductal epithelial cells. PSA is a serine protease that belongs to the kallilkrein family and is composed of 93% amino acid protein and N-linked oligosaccharide. PSA is especially present in the prostate, and its physiological functions include assisting in semen liquefaction and improving sperm motility.3−5 When the lesions occur in the prostate, such as benign prostatic hyperplasia and prostate cancer, PSA could be secreted substantially into the blood. PSA exists in several forms in serum, including free (f-PSA) and in complexes with α1-antichymotrypsin (PSA−ACT) and α2-macroglobulin (PSA−α2M). Of these, PSA−α2M has no immune activity, because its epitopes are completely blocked. Therefore, f-PSA © 2014 American Chemical Society
Received: December 24, 2013 Accepted: February 6, 2014 Published: February 18, 2014 2767
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Figure 1. (A) Schematic illustration of biopanning for f-PSA. f-PSA was immobilized on the surface of a dish. After wells were blocked with BSA, the f8/8 landscape library was added to interact with the coated f-PSA. Unbound phages were washed away, and bound phages were eluted with mild acids. Then the eluted phages were amplified to use as sublibrary for the next round of biopanning. After three rounds, the monoclonal phages were picked and their nucleotides were sequenced to obtain the peptide sequence. (B) Phage recovery during biopanning.
sorbent assay (ELISA),18 electrochemiluminescence immunoassay (ECLIA),19−22 time-resolved immunofluorometric assay with nanoparticle labeling,23 nanoparticle-based biobar code,24 multitumor markers protein chip,25 and surface plasmon resonance26,27 due to their high sensitivity, specificity, and reproducibility. However, a simple, stable, and inexpensive approach for rapid f-PSA detection is highly requested. Phage display presented by Smith in 1985,28 is the methodology to insert the DNA of foreign proteins or polypeptide into the specific position of genes encoding the coat proteins to display the protein or polypeptide on the surface of the phage along with the assembly of phage particle. Meanwhile, the recombinant phages can keep independent spatial structure and biological activity, without losing their ability to infect the host bacterium. Phage display has been widely used in different areas of bioscience and biotechnology, including epitope screening, cancer diagnosis, targeted drug development,29−34 and biosensing.35−46 Filamentous phages (M13, f1, and fd) (Supporting Information, Figure S1A), belonging to the class of nonlytic linear viruses, are widely used to construct phage display libraries. In 1996, some of us constructed the first landscape phage library f8/8 by splicing oligonucleotides of random octapeptide into gene VIII of fd-tet phage.47 The oligonucleotides replaced the nucleic acid
sequences encoding the N-terminal Glu-Gly-Glu (E2-G3-E4) of protein pVIII (Supporting Information, Figure S1B). As a result, foreign random octapeptides were displayed on the phage surface as the N-terminal portion of the major coat protein pVIII.47 This landscape phage library contained about 2.0 × 109 individual clones, each displaying a unique octapeptide fused to 4000 copies of pVIII proteins.47 Landscape phage libraries are a rich source of many specific phage ligands, which can bind to given targets such as avidin,48 enzymes,49 bacteria,50,51 and cancer cells.30,31 Our previous work investigated the dose-dependent binding of β-galactosidase to the landscape phage immobilized to an ELISA plate and found that the dissociation constant was 30 nM, which indicated that the selected phage exhibited good affinity for β-galactosidase.48 The landscape phages, selected from the libraries, bind the given targets specifically with high sensitivity and could serve as novel biomolecular probes in detection methods with several advantages. First, landscape phages display almost 4000 copies of octapeptide on the surface and show a multivalent interaction with a given target.50 Second, the production of the phage is very simple and low-cost, and once the specific phage monoclones were screened, they could be amplified infinitely by culture. Third, the filamentous phage has a symmetrical three-dimensional structure, so the active 2768
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remove nonbound phage. Aliquots (100 μL) of elution buffer (0.2 M glycine hydrochloride, pH 2.2, containing 1 mg/mL BSA) was then added to each well and incubated for 10 min at room temperature to elute the bound phage. The eluate was neutralized with 19 μL of 1 M Tris-HCl (pH 9.1). The eluted phages were propagated and purified for use in the next round. In the next round of biopanning, the phage clones selected and amplified in the previous round were used as the input library. All other procedures remained as the same as before except for washing 10 times. After three rounds of selection, individual phage clones were picked randomly and amplified, and their genes were sequenced to determine the amino acid sequences of the phages. Specificity Array of Selected Phage Clones. The specificity testing procedure is schematically shown in Figure S2 (Supporting Information). Briefly, the wells of a NuncImmuno MicroWell 96-well plate were coated with f-PSA, PSA−ACT, hK-2, AFP, CA125, CA15-3, CA19-9, or CEA (each 0.5 μg in 50 μL of PBS) separately and incubated overnight at 4 °C with gentle shaking on an oscillator. Then wells were covered with blocking buffer (10 mg/mL BSA) for 1 h at room temperature and washed 10 times with TBST. Candidate phages (∼108 phage virions in 50 μL of TBS) were added to wells and incubated overnight at 4 °C with gentle shaking on an oscillator. Wells were then washed 10 times with TBST to remove nonbound phage. Elution buffer was then added to each well and incubated for 10 min at room temperature to elute the bound phage. Subsequently, the elution was neutralized with 19 μL of 1 M Tris-HCl (pH 9.1). Eluted phages were titered as described previously. Phage recovery was calculated as a ratio of eluted phages to input phages to compare the captured phages by different targets. Phage ELISA for f-PSA. The phage ELISA procedure is schematically shown in Figure S3 (Supporting Information). The phage was loaded into wells of a 96-well microtiter plate, and the plate was incubated at 4 °C for 16 h. The wells were washed four times with PBST (phosphate-buffered saline containing 0.5% Tween 20). f-PSA solution (containing 0.5% Tween 20) was added to the wells, and the plate was incubated at 37 °C. Then the wells were washed six times with PBST, anti-PSA mAb (containing 0.5% Tween 20) was added, and the plate was incubated for 1 h at room temperature. The plate was washed again, and then 50 μL of IgG-HRP (containing 0.5% Tween 20) was added and the plate was incubated for 1 h at room temperature. To develop the plate color, 100 μL of 0.04% o-phenylenediamine (substrate) was added. After 15 min, the reaction was stopped by adding 50 μL of 2 M H2SO4. The OD492 value was recorded on a microplate reader. Optimization of Phage ELISA Conditions. Effect of Coating Buffer pH. Various coating buffers including 0.1 M TBS (pH 7.0), 0.1 M TBS (pH 7.5), 0.1 M NaHCO3 (pH 8.6), and 0.1 M Na2CO3 (pH 9.6) were tested. The aliquot of selected phage monoclones (40 μL, 5 × 1011 virions/mL) (as input) was separately dissolved in the above buffers. Then the prepared phage solutions were loaded into wells of a 96-well microtiter plate, and the plate was incubated at 4 °C for 16 h. After the wells were washed with PBST four times, phages immobilized on the plate were eluted by elution buffer and titered as output. The output/input ratio was used to measure the coating efficiency. Effect of Phage Concentration. To evaluate the effect of selected phage concentrations on the response of phage ELISA, 40 μL of phage solution with gradually decreasing phage
sites (random octapeptides) could avoid being blocked during immobilization. And finally, our previous work also showed that phage can withstand harsh environments, such as acidic and alkaline pH, nucleases,52 heat,53 and nonaqueous solvents.54 In view of the attractive features of landscape phage, it is promising to use them as novel probes in new fields. In this study, we screened phage probes from the f8/8 landscape library for f-PSA-binding clones. The specific landscape phages were identified by specificity array. Then the phage with better specificity and affinity was used as captured probe to establish a sandwich ELISA of “phage/fPSA/anti-PSA mAb”, combining with anti-PSA mAb. The optimization of phage ELISA conditions including coating buffer pH, blocking agent, phage concentration, incubation time, and anti-PSA mAb dilution was carried out. Under the established optimal conditions without blocking, the absorbance at 492 nm (OD492) was linear with f-PSA concentration within 0.825−165 ng/mL. The limit of detection of the array was 0.16 ng/mL f-PSA.
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EXPERIMENTAL SECTION Materials and Reagents. f-PSA, PSA−ACT, α-fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), and anti-human total PSA monoclonal antibody (anti-PSA mAb) were purchased from Meridian Life Science (Memphis, TN). Carcinoembryonic antigen (CEA) was kindly gifted from the Antibody Center of Biocell Biotechnology Co., Ltd. (Zhengzhou, China). Human glandular kallikrein 2 (hK-2) was generously provided by Novoprotein Co. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Solarbio (Beijing, China). Goat anti-mouse immunoglobulin Gconjugated HRP (IgG-HRP) was from Tiangen Biotech Co., Ltd. (Beijing, China). o-Phenylenediamine was from Aladdin (Shanghai, China). Tween 20, tris(hydroxymethyl)aminomethane (Tris), potassium phosphate monobasic (KH 2 PO 4 ), and disodium phosphate dodecahydrate (Na2HPO4·12H2O) were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The Nunc-Immuno MicroWell 96-well plate used in biopanning was from Nunc (Roskilde, Denmark). The 96-well microtiter plate (Costar EIA/RIA plate) used in ELISA was from Corning (New York). Phage Display Library. The f8/8 landscape phage library, constructed by Petrenko et al.,47 contains ∼2 × 109 different clones. The phage from this library display random octapeptides by replacing amino acids E2, G3, and D4 on every copy of pVIII coat protein. All general methods dealing with the phage, including phage cultivation, amplification, purification, titration, PCR of phage DNA, and production of the carving cells, were recommended previously.30,55 Selection of Target-Binding Phage. The biopanning procedure is schematically shown in Figure 1A. The wells of a Nunc-Immuno MicroWell 96-well plate were coated with 50 μL of f-PSA (10 μg/mL in phosphate-buffered saline, PBS) and incubated overnight at 4 °C with gentle shaking on an oscillator. Then wells containing f-PSA were blocked with blocking buffer (10 mg/mL BSA) for 1 h at room temperature and washed six times with TBST (Tris-buffered saline, TBS, containing 0.5% Tween 20). In the first round of biopanning, ∼1011 phage virions from f8/8 phage library in 50 μL library diluents (TBST containing 1 mg/mL BSA) were added to each well and incubated overnight at 4 °C with gentle shaking on an oscillator. Wells were then washed six times with TBST to 2769
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concentrations (from 40 × 1011 to 0.625 × 1011 virions/mL) were immobilized in ELISA array as described above. The OD492 values for the resultant arrays were recorded for comparison. Effect of Antigen Incubation Time. Under the optimal selected phage concentration (2.0 × 1012 virions/mL) with 0.1 M TBS (pH 7.5) as coating buffer, f-PSA was incubated with the immobilized phages at 37 °C for different durations. Then the OD492 values for the arrays were recorded. Effect of Anti-PSA mAb Dilution. Under the above optimal phage ELISA conditions, anti-PSA mAb with different dilutions was applied to construct the phage ELISA array. The OD492 values were measured for the wells with f-PSA (as positive value) and the wells without f-PSA (as negative value), and the ratio of positive/negative (P/N) was calculated for comparison. Detection of f-PSA with ECLIA. f-PSA content in the serum samples was also detected by use of a Cobas e601 analyzer (Roche Diagnostics, Basel, Switzerland). Details of the assay, based on the sandwich principle, can be found in the experimental protocol of the instrument provided by Roche. Briefly, 20 μL of sample, a biotinylated monoclonal PSAspecific antibody, and a monoclonal PSA-specific antibody labeling tris(2,2′-bipyridyl)ruthenium(II) complex are mixed together to form a sandwich complex. Then streptavidin-coated microparticles are added to bind the resultant complex to the solid phase. Subsequently, the above reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are removed with ProCell. A voltage to the electrode is applied to induce chemiluminescent emission, which is recorded by a photomultiplier. The f-PSA amount in the sample is determined via the instrument-specifically generated two-point calibration.
Table 1. Amino Acid Sequences of Selected Phages phage
peptide sequence
P1 P2 P3 P4
ERNSVSPS DRNPPLPS EPFQVGDQ VHNTTSSS
serum. Therefore, PSA−ACT and some tumor markers were chosen as controls to study the specificity of the selected phage. In particular, human glandular kallikrein-2 (hK2) is a protein localized to the prostate, which is 80% homologous to fPSA.21,56 In this array, f-PSA, PSA−ACT, AFP, CA125, CA153, CA19-9, CEA, and hK-2 were incubated separately in the wells of the Nunc-Immuno MicroWell 96-well plate, and the plastic plate was used as blank control. After blocking and washing of the wells, candidate phages were individually added and incubated with the biomarkers at 4 °C overnight. Wells were washed again to remove unbound phages. Then bound phages were eluted and tittered, and the phage recovery was calculated to compare the captured phages by different targets (Figure 2). The phage clones P1−P4 showed good specificity for f-PSA compared with other tumor markers in the phage capture assay (Figure 2). In addition, the phages captured by f-PSA were obviously higher than those captured by PSA−ACT (Figure 2). Among these four phages, P1 and P2 showed similar specificity, which probably originated from their belonging to the same family, because both displayed the octapeptide sequence (D/ E)RNXXXPS. They might bind to f-PSA by the same acting site. In addition, compared with the other three phages, the recovery of phage P1 was 6.90%, higher than others, which indicated that P1 had better binding to f-PSA. Optimization of Conditions for Phage ELISA. The usual ELISA method to detect antigen is based on a double-antibody sandwich, by which one antibody is used to capture the antigen, followed by a second antibody that is used to recognize the captured antigen and produce signal amplification for detection (Supporting Information, Figure S4). In this phage ELISA array, phage P1 was used because it showed the best affinity as the capture probe immobilized on the surface of the ELISA plate to capture f-PSA. Then an anti-PSA monoclonal antibody is used to recognize the captured antigen to form the complex phage/f-PSA/anti-PSA mAb. An HRP-conjugated antibody and o-phenylenediamine were successively added to develop plate color. To improve the detection, optimization of ELISA conditions was carried out. In ELISA arrays, nonspecific background is a common problem. If the background is high, the detection sensitivity will be lowered. To reduce the nonspecific background, blocking is a usual strategy adopted in ELISA, and Tween 20 is also used to reduce the nonspecific background under nonblocking conditions because of its surfactant function. In the optimization of phage ELISA conditions, the phage concentration, incubation time, and anti-PSA mAb dilution were carried out under nonblocking conditions and with blocking with 1% skim milk (Supporting Information, Figure S4). Effect of Coating Buffer pH. Phage was immobilized onto a 96-well microtiter plate as the probe to capture the target molecules. For different proteins or antibodies, the coating buffer pH might have different effects on immobilization. Buffers such as Tris-HCl and carbonate are frequently used for protein or antibody coating. To examine the effect of buffer pH
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RESULTS AND DISCUSSION Selection of PSA-Binding Phage. In this work, we selected phage clones binding f-PSA through a biopanning procedure (Figure 1A). Phages from f8/8 landscape library were added to wells of the Nunc-Immuno MicroWell 96-well plate with immobilized f-PSA. After washing to remove the unbound phages, the PSA binding phages were eluted and amplified to be used as the input (instead of the primary library) in the next round of selection. This procedure was repeated in three rounds. It should be mentioned here that the conditions of elution are more stringent in the second and third round of the biopanning procedure. The numbers of phage particles present in the input and output of each round were determined by titration. The phage recovery (output/input) in each round (Figure 1B) and the increase of phage recovery after each round indicated that the targeted phages capable of binding to PSA were enriched effectively. After three rounds of selection, 10 randomly picked clones were isolated, and the DNA sequence was obtained by PCR.47 Then the PCR products were sequenced and four unique peptide sequences were found (Table 1). The octapeptides P1 and P2 could be assigned to the same family because they showed some similar structural features. Both octapeptides contained the consensus sequence (D/E)RNXXXPS with no similarity to other octapeptides. Specificity of Phage Binding to f-PSA. The specificity (or selectivity) of the selected phage is the ability to distinguish f-PSA from other cancer biomarkers. PSA is a cancer biomarker, and there are other forms and many other tumor markers in the 2770
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Figure 2. Specificity of different phage monoclones (P1−P4) binding to different targets.
Figure 3. Optimization of conditions for phage ELISA. (A) Effect of coating buffer pH; (B) effect of phage concentration; (C) effect of incubation time; (D) effect of anti-PSA mAb dilution.
incubation overnight, unbound phage particles were eluted and titration was measured as output. The efficiency of the phage coating was measured by calculating output/input ratio (Figure 3A). The output/input ratio for phage P1 in TBS (pH 7.5) was
on phage coating, several buffers including TBS (pH 7.0), TBS (pH 7.5), 0.1 M NaHCO3 (pH 8.6), and 0.1 M Na2CO3 (pH 9.6) were tested. The same amount of phage P1 in each buffer as input was loaded in a 96-well microtiter plate. After 2771
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obviously better than the other pH values tested, suggesting that TBS (pH 7.5) was an efficient coating buffer. Therefore, TBS buffer (pH 7.5) was preferred as the coating buffer for phage ELISA. Effect of Phage Concentration. As capture probe, the phage concentration will have different effects on the coating on the plate. To determine an appropriate amount of phage for coating, plates were coated with phage P1 that was gradientdiluted in TBS buffer. After reaction with f-PSA and treatment with PSA antibody, the HRP-conjugated secondary antibody was added to catalyze the chromogenic substrate. The OD492 value increased with phage concentration, and a plateau was reached when the phage concentration was higher than 2.0 × 1012 virions/mL (Figure 3B). For better use of phage, a concentration of 2.0 × 1012 virions/mL was adopted in the following experiments. Effect of Antigen Incubation Time. In this array, the core step is the specific binding between immobilized phages and fPSA. The usual process includes incubation of f-PSA with the immobilized phages at 37 °C for different durations. The OD492 values for incubation for 1 or 2 h were relatively lower; however, the value increased as incubation time extended from 1 to 3 h (Figure 3C). Further, the OD492 value reached a plateau after 3 h (Figure 3C). Given that the detection should be simple and fast, reaction at 37 °C for 3 h was used for the following experiments. Effect of Anti-PSA mAb Dilution. In this array, the anti-PSA mAb was used to recognize the f-PSA captured by phage P1. Several dilutions of anti-PSA mAb (1:500, 1:1000, 1:2000, 1:4000, and 1:8000) were tested. The P/N ratios varying with mAb dilutions were summarized, and the dilutions of 1:500 and 1:1000 were obviously higher than the values for other dilutions (Figure 3D). For effective use of reagents, an anti-PSA mAb dilution of 1:1000 was applied for the final array. Standard Curve for f-PSA Detection. Under the optimal phage ELISA conditions established above, 40 μL of 2.0 × 1012 virions/mL phage in TBS was loaded into wells of a 96-well microtiter plate. After coating of the wells overnight, f-PSA in different concentrations (containing 0.5% Tween 20) was added, and the plate was incubated at 37 °C for 3 h, followed by the mAb (1:1000), IgG-HRP, and substrate added successively. Finally, the OD492 values were measured to make the standard curve (Figure 4). The OD492 value was linear with f-PSA concentration from 0.825 to 165 ng/mL (R = 0.998), which covered the “diagnostic gray zone” of 4−10 ng/ mL for diagnosing patients.57 The limit of detection (LOD) was calculated to be 0.16 ng/mL in this ELISA array (signal-tonoise ratio S/N = 3). The relative standard deviation of 10 tested arrays was within 6%, suggesting good reproducibility of the method. The standard curve for f-PSA detection after blocking with 1% skim milk was also checked, and the LOD could reach 0.66 ng/mL f-PSA (Supporting Information, Figure S5). The detection sensitivity of phage ELISA without blocking was obviously better than that with blocking. As the octapeptide is of small size compared with blocking agent, some active sites of the probe phage could probably be blocked by the blocking agents. It is reasonable that there are more exposed active sites to capture f-PSA without blocking, and therefore, more target molecules could be captured by the phages to result in better sensitivity. For context, the detection sensitivity for f-PSA by conventional methods has achieved subnanogram levels, for example, the detection limits for f-PSA are 0.1 ng/mL for magnetic-
Figure 4. Standard curve for f-PSA. (Inset) Enlarged portion of standard curve for f-PSA at low concentrations.
particle-based chemiluminescence enzyme immunoassay,21 0.2 ng/mL for multisequential surface plasmon resonance analysis on the basis of haptoglobin−lectin complex,26 1 ng/mL for the protein chip system,25 and 0.74 ng/mL for fluorescence resonance energy transfer-based array.58 The LOD for our phage ELISA array (0.16 ng/mL) is lower than or comparable with other conventional methods. However, phage probe has many merits such as multivalency, high stability against harsh environments,52,53 simplicity, and low cost in preparation, which could not be met for antibody. Therefore, it is prospective that the landscape phage can be widely used as a novel probe in bioassays. Moreover, a further biopanning can be carried out to get more phage clones that show better affinity binding to PSA to improve the sensitivity of the phage ELISA. Additionally, the phages bound to PSA by different epitopes could be screened to serve as probes with different binding sites. Thus, f8/8 phages can be used as both capturing and detecting probes in ELISA, by which a double-phage sandwich array could be explored. Analysis of Real Serum Samples. The as-established phage ELISA was used to determine f-PSA in real serum samples. ECLIA was carried out for comparison. The results are shown in Table 2. Clearly, the results by phage ELISA were in Table 2. Detection of f-PSA Content in Real Serum Samples f-PSA conca (ng/mL) serum sample 1 2 3 4
this method 3.16 2.61 9.76 0.87
± ± ± ±
0.24 0.20 0.40 0.06
ECLIA
relative error (%)
± ± ± ±
+2.9 −7.8 +5.0 +8.7
3.07 2.83 9.30 0.80
0.20 0.21 0.43 0.08
a
All values were obtained as the average of three repetitive measurements plus-minus standard deviation.
good agreement with those achieved by ECLIA, which demonstrated that this phage ELISA was capable of precise detection of f-PSA in real serum samples. Compared with ECLIA and other traditional methods, our strategy is simple in both sample treatments and instrumentation. 2772
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(10) Montie, J. E.; Meyers, S. E. Urol. Clin. North Am. 1997, 24, 247− 252. (11) Lilja, H.; Ulmert, D.; Vickers, A. J. Nat. Rev. Cancer 2008, 8, 268−278. (12) Pannek, J.; Subong, E. N. P.; Jones, K. A.; Marschke, P. L. S.; Epstein, J. I.; Chan, D. W.; Carter, H. B.; Luderer, A. A.; Partin, A. W. Urology 1996, 48, 51−54. (13) Jung, K.; Elgeti, U.; Lein, M.; Brux, B.; Sinha, P.; Rudolph, B.; Hauptmann, S.; Schnorr, D.; Loening, S. A. Clin. Chem. 2000, 46, 55− 62. (14) Grossklaus, D. J.; Shappell, S. B.; Gautam, S.; Smith, J. A.; Cookson, M. S. J. Urol. 2001, 165, 455−458. (15) Filella, X.; Truan, D.; Alcover, J.; Quinto, L.; Molina, R.; Luque, P.; Coca, F.; Ballesta, A. M. Urology 2004, 63, 1100−1103. (16) Catalona, W. J.; Bartsch, G.; Rittenhouse, H. G.; Evans, C. L.; Linton, H. J.; Amirkhan, A.; Horninger, W.; Klocker, H.; Mikolajczyk, S. D. J. Urol. 2003, 170, 2181−2185. (17) Johnson, E. D.; Kotowski, T. M. J. Forensic Sci. 1993, 38, 250− 258. (18) Asafudullah, S. M.; Salam, M. A.; Badruddoza, S. M.; Rahman, M. K. TAJ: J. Teach. Assoc. 2009, 22, 30−35. (19) Zhang, X.; Liu, Y.; Jia, J.; Xu, W.; Li, Z.; Chen, Y.; Han, S. At. Energy Sci. Technol. 2008, 42, 112−116. (20) Adachi, T.; Moriya, K.; Esaki, K. Acta Urol. Jpn. 1998, 44, 469− 476. (21) Liu, R.; Wang, C.; Jiang, Q.; Zhang, W.; Yue, Z.; Liu, G. Anal. Chim. Acta 2013, 801, 91−96. (22) Xie, C.; Wang, G. J. Clin. Lab. Anal. 2011, 25, 37−42. (23) Soukka, T.; Paukkunen, J.; Härmä, H.; Lönnberg, S.; Lindroos, H.; Lövgren, T. Clin. Chem. 2001, 47, 1269−1278. (24) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884−1886. (25) Sun, Z. H.; Fu, X. L.; Zang, L.; Yang, X. L.; Liu, F. Z.; Hu, G. X. Anticancer Res. 2004, 24, 1159−1165. (26) Kazuno, S.; Fujimura, T.; Arai, T.; Ueno, T.; Nagao, K.; Fujime, M.; Murayama, K. Anal. Biochem. 2011, 419, 241−249. (27) Su, L.-C.; Chen, R.-C.; Li, Y.-C.; Chang, Y.-F.; Lee, Y.-J.; Lee, C.-C.; Chou, C. Anal. Chem. 2010, 82, 3714−3718. (28) Smith, G. P. Science 1985, 228, 1315−1317. (29) Peng, W.-P.; Hou, Q.; Xia, Z.-H.; Chen, D.; Li, N.; Sun, Y.; Qiu, H.-J. Virus Res. 2008, 135, 267−272. (30) Abbineni, G.; Modali, S.; Safiejko-Mroczka, B.; Petrenko, V. A.; Mao, C. Mol. Pharmaceutics 2010, 7, 1629−1642. (31) Jayanna, P. K.; Bedi, D.; Deinnocentes, P.; Bird, R. C.; Petrenko, V. A. Protein Eng., Des. Sel. 2010, 23, 423−430. (32) Zang, L.; Shi, L.; Guo, J.; Pan, Q.; Wu, W.; Pan, X.; Wang, J. Cancer Lett. 2009, 281, 64−70. (33) Molek, P.; Strukelj, B.; Bratkovic, T. Molecules 2011, 16, 857− 887. (34) Qi, H.; Lu, H.; Qiu, H.-J.; Petrenko, V.; Liu, A. J. Mol. Biol. 2012, 417, 129−143. (35) Liu, A. H.; Abbineni, G.; Mao, C. B. Adv. Mater. 2009, 21, 1001−1005. (36) Mao, C. B.; Liu, A. H.; Cao, B. R. Angew. Chem., Int. Ed. 2009, 48, 6790−6810. (37) Olsen, E. V.; Sorokulova, I. B.; Petrenko, V. A.; Chen, I. H.; Barbaree, J. M.; Vodyanoy, V. J. Biosens. Bioelectron. 2006, 21, 1434− 1442. (38) Nanduri, V.; Sorokulova, I. B.; Samoylov, A. M.; Simonian, A. L.; Petrenko, V. A.; Vodyanoy, V. Biosens. Bioelectron. 2007, 22, 986− 992. (39) Horikawa, S.; Bedi, D.; Li, S.; Shen, W.; Huang, S.; Chen, I. H.; Chai, Y.; Auad, M. L.; Bozack, M. J.; Barbaree, J. M.; Petrenko, V. A.; Chin, B. A. Biosens. Bioelectron. 2011, 26, 2361−2367. (40) Yang, L.-M. C.; Tam, P. Y.; Murray, B. J.; McIntire, T. M.; Overstreet, C. M.; Weiss, G. A.; Penner, R. M. Anal. Chem. 2006, 78, 3265−3270. (41) Donavan, K. C.; Arter, J. A.; Pilolli, R.; Cioffi, N.; Weiss, G. A.; Penner, R. M. Anal. Chem. 2011, 83, 2420−2424.
CONCLUSIONS In summary, phage probes against f-PSA were screened from the f8/8 landscape phage library and many targeted phages were enriched effectively. Four phage clones were obtained and characterized by phage capture array. Compared with other tumor markers, all four phages exhibited good specificity for fPSA. The phage clone P1 displaying ERNSVSPS, with the best affinity for f-PSA, was selected as the capture probe to establish a sandwich ELISA array with anti-PSA monoclonal antibody. The proposed phage ELISA had a dynamic linear range of 0.825−165 ng/mL f-PSA, which covered the “diagnostic gray zone” of 4−10 ng/mL for diagnosing patients. In consideration of the excellent performance of the landscape phages, the phages can be screened to detect PSA−ACT, which is underway in this laboratory. Taken together, the data can be applicable for clinical diagnosis and early detection of PCa. Furthermore, specific phage probes for other tumor markers could be selected from the f8/8 landscape library and phage microarrays could be constructed to achieve simultaneous detection of multiple tumor markers. Finally, the phages could be used in phage-based immunoassays, substituting for antibodies.
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ASSOCIATED CONTENT
S Supporting Information *
Five figures showing structure of phage fd, schematic illustrations of phage capture array and phage ELISA for fPSA detection, effect of blocking wells, and standard curve for fPSA with phage ELISA after blocking. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail [email protected]. Author Contributions ⊥
Q.L. and F.W. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (91227116, 21275152) and the HundredTalent-Project (KSCX2-YW-BR-7), Chinese Academy of Sciences (to A.L.).
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REFERENCES
(1) Siegel, R.; Naishadham, D.; Jemal, A. Ca-Cancer J. Clin. 2012, 62, 10−29. (2) Rao, A. R.; Motiwala, H. G.; Karim, O. M. A. BJU Int. 2008, 101, 5−10. (3) Seregni, E.; Botti, C.; Ballabio, G.; Bombardieri, E. Tumori 1996, 82, 72−77. (4) Sokoll, L. J.; Chan, D. W. Urol. Clin. North Am. 1997, 24, 253− 259. (5) Armbruster, D. A. Clin. Chem. 1993, 39, 181−195. (6) McCormack, R. T.; Rittenhouse, H. G.; Finlay, J. A.; Sokoloff, R. L.; Wang, T. J.; Wolfert, R. L.; Lilja, H.; Oesterling, J. E. Urology 1995, 45, 729−744. (7) Leinonen, J.; Lovgren, T.; Vornanen, T.; Stenman, U. H. Clin. Chem. 1993, 39, 2098−2103. (8) Zhou, A. M.; Tewari, P. C.; Bluestein, B. I.; Caldwell, G. W.; Larsen, F. L. Clin. Chem. 1993, 39, 2483−2491. (9) Pienta, K. J. Urology 2009, 73, 11−20. 2773
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Specific Probe Selection from Landscape Phage Display Library and Its Application in Enzyme-Linked Immunosorbent Assay of Free Prostate-Specific Antigen Qiaolin Lang,†,⊥ Fei Wang,†,‡,⊥ Long Yin,†,‡ Mingjun Liu,§ Valery A. Petrenko,∥ and Aihua Liu*,†,‡ †
Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China ‡ University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China § Department of Clinical Laboratory, The Affiliated Hospital of Medical College, Qingdao University, 16 Jiangsu Road, Qingdao 266003, China ∥ Department of Pathobiology, Auburn University, 269 Greene Hall, Auburn, Alabama 36849-5519, United States S Supporting Information *
ABSTRACT: Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825−165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.
P
and PSA−ACT are known as the total prostate-specific antigen (t-PSA) on behalf of the level of PSA in serum.6−8 In the 1980s, PSA was introduced into clinical medicine by the U.S. Food and Drug Administration as the tumor marker used in prediction and early diagnosis of prostate cancer, monitoring of surgery treatment response, and detection of disease recurrence.9−11 For healthy people, t-PSA concentration in serum is usually under 4 ng/mL, and therefore, the value of 4 ng/mL is admittedly regarded as the clinical testing indicator. PCa testing based on PSA level is simple and convenient and can effectively reduce the number of biopsies and lessen the pain of PCa patients. In addition, the ratio of f-PSA/t-PSA is an available index to improve the diagnostic certainty.12−15 Thus, the detection of t-PSA and f-PSA plays an important role in the early diagnosis of prostate cancer. PSA is detected by immunoassay,16 radioimmunoassay,17 enzyme-linked immuno-
rostate cancer (PCa) is the second leading cause of cancerrelated death in men in the United States. PCa accounts for about 29% of male cancers, and the deaths induced by PCa accounted for 9% of male cancer deaths in the United States in 2012.1 Early diagnosis of PCa is often based on the level of prostate-specific antigen (PSA) in serum. PSA, first identified in the 1960s,2 is a 33−34 kDa single-chain glycoprotein secreted from the prostate gland bubble and ductal epithelial cells. PSA is a serine protease that belongs to the kallilkrein family and is composed of 93% amino acid protein and N-linked oligosaccharide. PSA is especially present in the prostate, and its physiological functions include assisting in semen liquefaction and improving sperm motility.3−5 When the lesions occur in the prostate, such as benign prostatic hyperplasia and prostate cancer, PSA could be secreted substantially into the blood. PSA exists in several forms in serum, including free (f-PSA) and in complexes with α1-antichymotrypsin (PSA−ACT) and α2-macroglobulin (PSA−α2M). Of these, PSA−α2M has no immune activity, because its epitopes are completely blocked. Therefore, f-PSA © 2014 American Chemical Society
Received: December 24, 2013 Accepted: February 6, 2014 Published: February 18, 2014 2767
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Figure 1. (A) Schematic illustration of biopanning for f-PSA. f-PSA was immobilized on the surface of a dish. After wells were blocked with BSA, the f8/8 landscape library was added to interact with the coated f-PSA. Unbound phages were washed away, and bound phages were eluted with mild acids. Then the eluted phages were amplified to use as sublibrary for the next round of biopanning. After three rounds, the monoclonal phages were picked and their nucleotides were sequenced to obtain the peptide sequence. (B) Phage recovery during biopanning.
sorbent assay (ELISA),18 electrochemiluminescence immunoassay (ECLIA),19−22 time-resolved immunofluorometric assay with nanoparticle labeling,23 nanoparticle-based biobar code,24 multitumor markers protein chip,25 and surface plasmon resonance26,27 due to their high sensitivity, specificity, and reproducibility. However, a simple, stable, and inexpensive approach for rapid f-PSA detection is highly requested. Phage display presented by Smith in 1985,28 is the methodology to insert the DNA of foreign proteins or polypeptide into the specific position of genes encoding the coat proteins to display the protein or polypeptide on the surface of the phage along with the assembly of phage particle. Meanwhile, the recombinant phages can keep independent spatial structure and biological activity, without losing their ability to infect the host bacterium. Phage display has been widely used in different areas of bioscience and biotechnology, including epitope screening, cancer diagnosis, targeted drug development,29−34 and biosensing.35−46 Filamentous phages (M13, f1, and fd) (Supporting Information, Figure S1A), belonging to the class of nonlytic linear viruses, are widely used to construct phage display libraries. In 1996, some of us constructed the first landscape phage library f8/8 by splicing oligonucleotides of random octapeptide into gene VIII of fd-tet phage.47 The oligonucleotides replaced the nucleic acid
sequences encoding the N-terminal Glu-Gly-Glu (E2-G3-E4) of protein pVIII (Supporting Information, Figure S1B). As a result, foreign random octapeptides were displayed on the phage surface as the N-terminal portion of the major coat protein pVIII.47 This landscape phage library contained about 2.0 × 109 individual clones, each displaying a unique octapeptide fused to 4000 copies of pVIII proteins.47 Landscape phage libraries are a rich source of many specific phage ligands, which can bind to given targets such as avidin,48 enzymes,49 bacteria,50,51 and cancer cells.30,31 Our previous work investigated the dose-dependent binding of β-galactosidase to the landscape phage immobilized to an ELISA plate and found that the dissociation constant was 30 nM, which indicated that the selected phage exhibited good affinity for β-galactosidase.48 The landscape phages, selected from the libraries, bind the given targets specifically with high sensitivity and could serve as novel biomolecular probes in detection methods with several advantages. First, landscape phages display almost 4000 copies of octapeptide on the surface and show a multivalent interaction with a given target.50 Second, the production of the phage is very simple and low-cost, and once the specific phage monoclones were screened, they could be amplified infinitely by culture. Third, the filamentous phage has a symmetrical three-dimensional structure, so the active 2768
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remove nonbound phage. Aliquots (100 μL) of elution buffer (0.2 M glycine hydrochloride, pH 2.2, containing 1 mg/mL BSA) was then added to each well and incubated for 10 min at room temperature to elute the bound phage. The eluate was neutralized with 19 μL of 1 M Tris-HCl (pH 9.1). The eluted phages were propagated and purified for use in the next round. In the next round of biopanning, the phage clones selected and amplified in the previous round were used as the input library. All other procedures remained as the same as before except for washing 10 times. After three rounds of selection, individual phage clones were picked randomly and amplified, and their genes were sequenced to determine the amino acid sequences of the phages. Specificity Array of Selected Phage Clones. The specificity testing procedure is schematically shown in Figure S2 (Supporting Information). Briefly, the wells of a NuncImmuno MicroWell 96-well plate were coated with f-PSA, PSA−ACT, hK-2, AFP, CA125, CA15-3, CA19-9, or CEA (each 0.5 μg in 50 μL of PBS) separately and incubated overnight at 4 °C with gentle shaking on an oscillator. Then wells were covered with blocking buffer (10 mg/mL BSA) for 1 h at room temperature and washed 10 times with TBST. Candidate phages (∼108 phage virions in 50 μL of TBS) were added to wells and incubated overnight at 4 °C with gentle shaking on an oscillator. Wells were then washed 10 times with TBST to remove nonbound phage. Elution buffer was then added to each well and incubated for 10 min at room temperature to elute the bound phage. Subsequently, the elution was neutralized with 19 μL of 1 M Tris-HCl (pH 9.1). Eluted phages were titered as described previously. Phage recovery was calculated as a ratio of eluted phages to input phages to compare the captured phages by different targets. Phage ELISA for f-PSA. The phage ELISA procedure is schematically shown in Figure S3 (Supporting Information). The phage was loaded into wells of a 96-well microtiter plate, and the plate was incubated at 4 °C for 16 h. The wells were washed four times with PBST (phosphate-buffered saline containing 0.5% Tween 20). f-PSA solution (containing 0.5% Tween 20) was added to the wells, and the plate was incubated at 37 °C. Then the wells were washed six times with PBST, anti-PSA mAb (containing 0.5% Tween 20) was added, and the plate was incubated for 1 h at room temperature. The plate was washed again, and then 50 μL of IgG-HRP (containing 0.5% Tween 20) was added and the plate was incubated for 1 h at room temperature. To develop the plate color, 100 μL of 0.04% o-phenylenediamine (substrate) was added. After 15 min, the reaction was stopped by adding 50 μL of 2 M H2SO4. The OD492 value was recorded on a microplate reader. Optimization of Phage ELISA Conditions. Effect of Coating Buffer pH. Various coating buffers including 0.1 M TBS (pH 7.0), 0.1 M TBS (pH 7.5), 0.1 M NaHCO3 (pH 8.6), and 0.1 M Na2CO3 (pH 9.6) were tested. The aliquot of selected phage monoclones (40 μL, 5 × 1011 virions/mL) (as input) was separately dissolved in the above buffers. Then the prepared phage solutions were loaded into wells of a 96-well microtiter plate, and the plate was incubated at 4 °C for 16 h. After the wells were washed with PBST four times, phages immobilized on the plate were eluted by elution buffer and titered as output. The output/input ratio was used to measure the coating efficiency. Effect of Phage Concentration. To evaluate the effect of selected phage concentrations on the response of phage ELISA, 40 μL of phage solution with gradually decreasing phage
sites (random octapeptides) could avoid being blocked during immobilization. And finally, our previous work also showed that phage can withstand harsh environments, such as acidic and alkaline pH, nucleases,52 heat,53 and nonaqueous solvents.54 In view of the attractive features of landscape phage, it is promising to use them as novel probes in new fields. In this study, we screened phage probes from the f8/8 landscape library for f-PSA-binding clones. The specific landscape phages were identified by specificity array. Then the phage with better specificity and affinity was used as captured probe to establish a sandwich ELISA of “phage/fPSA/anti-PSA mAb”, combining with anti-PSA mAb. The optimization of phage ELISA conditions including coating buffer pH, blocking agent, phage concentration, incubation time, and anti-PSA mAb dilution was carried out. Under the established optimal conditions without blocking, the absorbance at 492 nm (OD492) was linear with f-PSA concentration within 0.825−165 ng/mL. The limit of detection of the array was 0.16 ng/mL f-PSA.
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EXPERIMENTAL SECTION Materials and Reagents. f-PSA, PSA−ACT, α-fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), and anti-human total PSA monoclonal antibody (anti-PSA mAb) were purchased from Meridian Life Science (Memphis, TN). Carcinoembryonic antigen (CEA) was kindly gifted from the Antibody Center of Biocell Biotechnology Co., Ltd. (Zhengzhou, China). Human glandular kallikrein 2 (hK-2) was generously provided by Novoprotein Co. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Solarbio (Beijing, China). Goat anti-mouse immunoglobulin Gconjugated HRP (IgG-HRP) was from Tiangen Biotech Co., Ltd. (Beijing, China). o-Phenylenediamine was from Aladdin (Shanghai, China). Tween 20, tris(hydroxymethyl)aminomethane (Tris), potassium phosphate monobasic (KH 2 PO 4 ), and disodium phosphate dodecahydrate (Na2HPO4·12H2O) were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The Nunc-Immuno MicroWell 96-well plate used in biopanning was from Nunc (Roskilde, Denmark). The 96-well microtiter plate (Costar EIA/RIA plate) used in ELISA was from Corning (New York). Phage Display Library. The f8/8 landscape phage library, constructed by Petrenko et al.,47 contains ∼2 × 109 different clones. The phage from this library display random octapeptides by replacing amino acids E2, G3, and D4 on every copy of pVIII coat protein. All general methods dealing with the phage, including phage cultivation, amplification, purification, titration, PCR of phage DNA, and production of the carving cells, were recommended previously.30,55 Selection of Target-Binding Phage. The biopanning procedure is schematically shown in Figure 1A. The wells of a Nunc-Immuno MicroWell 96-well plate were coated with 50 μL of f-PSA (10 μg/mL in phosphate-buffered saline, PBS) and incubated overnight at 4 °C with gentle shaking on an oscillator. Then wells containing f-PSA were blocked with blocking buffer (10 mg/mL BSA) for 1 h at room temperature and washed six times with TBST (Tris-buffered saline, TBS, containing 0.5% Tween 20). In the first round of biopanning, ∼1011 phage virions from f8/8 phage library in 50 μL library diluents (TBST containing 1 mg/mL BSA) were added to each well and incubated overnight at 4 °C with gentle shaking on an oscillator. Wells were then washed six times with TBST to 2769
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concentrations (from 40 × 1011 to 0.625 × 1011 virions/mL) were immobilized in ELISA array as described above. The OD492 values for the resultant arrays were recorded for comparison. Effect of Antigen Incubation Time. Under the optimal selected phage concentration (2.0 × 1012 virions/mL) with 0.1 M TBS (pH 7.5) as coating buffer, f-PSA was incubated with the immobilized phages at 37 °C for different durations. Then the OD492 values for the arrays were recorded. Effect of Anti-PSA mAb Dilution. Under the above optimal phage ELISA conditions, anti-PSA mAb with different dilutions was applied to construct the phage ELISA array. The OD492 values were measured for the wells with f-PSA (as positive value) and the wells without f-PSA (as negative value), and the ratio of positive/negative (P/N) was calculated for comparison. Detection of f-PSA with ECLIA. f-PSA content in the serum samples was also detected by use of a Cobas e601 analyzer (Roche Diagnostics, Basel, Switzerland). Details of the assay, based on the sandwich principle, can be found in the experimental protocol of the instrument provided by Roche. Briefly, 20 μL of sample, a biotinylated monoclonal PSAspecific antibody, and a monoclonal PSA-specific antibody labeling tris(2,2′-bipyridyl)ruthenium(II) complex are mixed together to form a sandwich complex. Then streptavidin-coated microparticles are added to bind the resultant complex to the solid phase. Subsequently, the above reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are removed with ProCell. A voltage to the electrode is applied to induce chemiluminescent emission, which is recorded by a photomultiplier. The f-PSA amount in the sample is determined via the instrument-specifically generated two-point calibration.
Table 1. Amino Acid Sequences of Selected Phages phage
peptide sequence
P1 P2 P3 P4
ERNSVSPS DRNPPLPS EPFQVGDQ VHNTTSSS
serum. Therefore, PSA−ACT and some tumor markers were chosen as controls to study the specificity of the selected phage. In particular, human glandular kallikrein-2 (hK2) is a protein localized to the prostate, which is 80% homologous to fPSA.21,56 In this array, f-PSA, PSA−ACT, AFP, CA125, CA153, CA19-9, CEA, and hK-2 were incubated separately in the wells of the Nunc-Immuno MicroWell 96-well plate, and the plastic plate was used as blank control. After blocking and washing of the wells, candidate phages were individually added and incubated with the biomarkers at 4 °C overnight. Wells were washed again to remove unbound phages. Then bound phages were eluted and tittered, and the phage recovery was calculated to compare the captured phages by different targets (Figure 2). The phage clones P1−P4 showed good specificity for f-PSA compared with other tumor markers in the phage capture assay (Figure 2). In addition, the phages captured by f-PSA were obviously higher than those captured by PSA−ACT (Figure 2). Among these four phages, P1 and P2 showed similar specificity, which probably originated from their belonging to the same family, because both displayed the octapeptide sequence (D/ E)RNXXXPS. They might bind to f-PSA by the same acting site. In addition, compared with the other three phages, the recovery of phage P1 was 6.90%, higher than others, which indicated that P1 had better binding to f-PSA. Optimization of Conditions for Phage ELISA. The usual ELISA method to detect antigen is based on a double-antibody sandwich, by which one antibody is used to capture the antigen, followed by a second antibody that is used to recognize the captured antigen and produce signal amplification for detection (Supporting Information, Figure S4). In this phage ELISA array, phage P1 was used because it showed the best affinity as the capture probe immobilized on the surface of the ELISA plate to capture f-PSA. Then an anti-PSA monoclonal antibody is used to recognize the captured antigen to form the complex phage/f-PSA/anti-PSA mAb. An HRP-conjugated antibody and o-phenylenediamine were successively added to develop plate color. To improve the detection, optimization of ELISA conditions was carried out. In ELISA arrays, nonspecific background is a common problem. If the background is high, the detection sensitivity will be lowered. To reduce the nonspecific background, blocking is a usual strategy adopted in ELISA, and Tween 20 is also used to reduce the nonspecific background under nonblocking conditions because of its surfactant function. In the optimization of phage ELISA conditions, the phage concentration, incubation time, and anti-PSA mAb dilution were carried out under nonblocking conditions and with blocking with 1% skim milk (Supporting Information, Figure S4). Effect of Coating Buffer pH. Phage was immobilized onto a 96-well microtiter plate as the probe to capture the target molecules. For different proteins or antibodies, the coating buffer pH might have different effects on immobilization. Buffers such as Tris-HCl and carbonate are frequently used for protein or antibody coating. To examine the effect of buffer pH
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RESULTS AND DISCUSSION Selection of PSA-Binding Phage. In this work, we selected phage clones binding f-PSA through a biopanning procedure (Figure 1A). Phages from f8/8 landscape library were added to wells of the Nunc-Immuno MicroWell 96-well plate with immobilized f-PSA. After washing to remove the unbound phages, the PSA binding phages were eluted and amplified to be used as the input (instead of the primary library) in the next round of selection. This procedure was repeated in three rounds. It should be mentioned here that the conditions of elution are more stringent in the second and third round of the biopanning procedure. The numbers of phage particles present in the input and output of each round were determined by titration. The phage recovery (output/input) in each round (Figure 1B) and the increase of phage recovery after each round indicated that the targeted phages capable of binding to PSA were enriched effectively. After three rounds of selection, 10 randomly picked clones were isolated, and the DNA sequence was obtained by PCR.47 Then the PCR products were sequenced and four unique peptide sequences were found (Table 1). The octapeptides P1 and P2 could be assigned to the same family because they showed some similar structural features. Both octapeptides contained the consensus sequence (D/E)RNXXXPS with no similarity to other octapeptides. Specificity of Phage Binding to f-PSA. The specificity (or selectivity) of the selected phage is the ability to distinguish f-PSA from other cancer biomarkers. PSA is a cancer biomarker, and there are other forms and many other tumor markers in the 2770
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Figure 2. Specificity of different phage monoclones (P1−P4) binding to different targets.
Figure 3. Optimization of conditions for phage ELISA. (A) Effect of coating buffer pH; (B) effect of phage concentration; (C) effect of incubation time; (D) effect of anti-PSA mAb dilution.
incubation overnight, unbound phage particles were eluted and titration was measured as output. The efficiency of the phage coating was measured by calculating output/input ratio (Figure 3A). The output/input ratio for phage P1 in TBS (pH 7.5) was
on phage coating, several buffers including TBS (pH 7.0), TBS (pH 7.5), 0.1 M NaHCO3 (pH 8.6), and 0.1 M Na2CO3 (pH 9.6) were tested. The same amount of phage P1 in each buffer as input was loaded in a 96-well microtiter plate. After 2771
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obviously better than the other pH values tested, suggesting that TBS (pH 7.5) was an efficient coating buffer. Therefore, TBS buffer (pH 7.5) was preferred as the coating buffer for phage ELISA. Effect of Phage Concentration. As capture probe, the phage concentration will have different effects on the coating on the plate. To determine an appropriate amount of phage for coating, plates were coated with phage P1 that was gradientdiluted in TBS buffer. After reaction with f-PSA and treatment with PSA antibody, the HRP-conjugated secondary antibody was added to catalyze the chromogenic substrate. The OD492 value increased with phage concentration, and a plateau was reached when the phage concentration was higher than 2.0 × 1012 virions/mL (Figure 3B). For better use of phage, a concentration of 2.0 × 1012 virions/mL was adopted in the following experiments. Effect of Antigen Incubation Time. In this array, the core step is the specific binding between immobilized phages and fPSA. The usual process includes incubation of f-PSA with the immobilized phages at 37 °C for different durations. The OD492 values for incubation for 1 or 2 h were relatively lower; however, the value increased as incubation time extended from 1 to 3 h (Figure 3C). Further, the OD492 value reached a plateau after 3 h (Figure 3C). Given that the detection should be simple and fast, reaction at 37 °C for 3 h was used for the following experiments. Effect of Anti-PSA mAb Dilution. In this array, the anti-PSA mAb was used to recognize the f-PSA captured by phage P1. Several dilutions of anti-PSA mAb (1:500, 1:1000, 1:2000, 1:4000, and 1:8000) were tested. The P/N ratios varying with mAb dilutions were summarized, and the dilutions of 1:500 and 1:1000 were obviously higher than the values for other dilutions (Figure 3D). For effective use of reagents, an anti-PSA mAb dilution of 1:1000 was applied for the final array. Standard Curve for f-PSA Detection. Under the optimal phage ELISA conditions established above, 40 μL of 2.0 × 1012 virions/mL phage in TBS was loaded into wells of a 96-well microtiter plate. After coating of the wells overnight, f-PSA in different concentrations (containing 0.5% Tween 20) was added, and the plate was incubated at 37 °C for 3 h, followed by the mAb (1:1000), IgG-HRP, and substrate added successively. Finally, the OD492 values were measured to make the standard curve (Figure 4). The OD492 value was linear with f-PSA concentration from 0.825 to 165 ng/mL (R = 0.998), which covered the “diagnostic gray zone” of 4−10 ng/ mL for diagnosing patients.57 The limit of detection (LOD) was calculated to be 0.16 ng/mL in this ELISA array (signal-tonoise ratio S/N = 3). The relative standard deviation of 10 tested arrays was within 6%, suggesting good reproducibility of the method. The standard curve for f-PSA detection after blocking with 1% skim milk was also checked, and the LOD could reach 0.66 ng/mL f-PSA (Supporting Information, Figure S5). The detection sensitivity of phage ELISA without blocking was obviously better than that with blocking. As the octapeptide is of small size compared with blocking agent, some active sites of the probe phage could probably be blocked by the blocking agents. It is reasonable that there are more exposed active sites to capture f-PSA without blocking, and therefore, more target molecules could be captured by the phages to result in better sensitivity. For context, the detection sensitivity for f-PSA by conventional methods has achieved subnanogram levels, for example, the detection limits for f-PSA are 0.1 ng/mL for magnetic-
Figure 4. Standard curve for f-PSA. (Inset) Enlarged portion of standard curve for f-PSA at low concentrations.
particle-based chemiluminescence enzyme immunoassay,21 0.2 ng/mL for multisequential surface plasmon resonance analysis on the basis of haptoglobin−lectin complex,26 1 ng/mL for the protein chip system,25 and 0.74 ng/mL for fluorescence resonance energy transfer-based array.58 The LOD for our phage ELISA array (0.16 ng/mL) is lower than or comparable with other conventional methods. However, phage probe has many merits such as multivalency, high stability against harsh environments,52,53 simplicity, and low cost in preparation, which could not be met for antibody. Therefore, it is prospective that the landscape phage can be widely used as a novel probe in bioassays. Moreover, a further biopanning can be carried out to get more phage clones that show better affinity binding to PSA to improve the sensitivity of the phage ELISA. Additionally, the phages bound to PSA by different epitopes could be screened to serve as probes with different binding sites. Thus, f8/8 phages can be used as both capturing and detecting probes in ELISA, by which a double-phage sandwich array could be explored. Analysis of Real Serum Samples. The as-established phage ELISA was used to determine f-PSA in real serum samples. ECLIA was carried out for comparison. The results are shown in Table 2. Clearly, the results by phage ELISA were in Table 2. Detection of f-PSA Content in Real Serum Samples f-PSA conca (ng/mL) serum sample 1 2 3 4
this method 3.16 2.61 9.76 0.87
± ± ± ±
0.24 0.20 0.40 0.06
ECLIA
relative error (%)
± ± ± ±
+2.9 −7.8 +5.0 +8.7
3.07 2.83 9.30 0.80
0.20 0.21 0.43 0.08
a
All values were obtained as the average of three repetitive measurements plus-minus standard deviation.
good agreement with those achieved by ECLIA, which demonstrated that this phage ELISA was capable of precise detection of f-PSA in real serum samples. Compared with ECLIA and other traditional methods, our strategy is simple in both sample treatments and instrumentation. 2772
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(10) Montie, J. E.; Meyers, S. E. Urol. Clin. North Am. 1997, 24, 247− 252. (11) Lilja, H.; Ulmert, D.; Vickers, A. J. Nat. Rev. Cancer 2008, 8, 268−278. (12) Pannek, J.; Subong, E. N. P.; Jones, K. A.; Marschke, P. L. S.; Epstein, J. I.; Chan, D. W.; Carter, H. B.; Luderer, A. A.; Partin, A. W. Urology 1996, 48, 51−54. (13) Jung, K.; Elgeti, U.; Lein, M.; Brux, B.; Sinha, P.; Rudolph, B.; Hauptmann, S.; Schnorr, D.; Loening, S. A. Clin. Chem. 2000, 46, 55− 62. (14) Grossklaus, D. J.; Shappell, S. B.; Gautam, S.; Smith, J. A.; Cookson, M. S. J. Urol. 2001, 165, 455−458. (15) Filella, X.; Truan, D.; Alcover, J.; Quinto, L.; Molina, R.; Luque, P.; Coca, F.; Ballesta, A. M. Urology 2004, 63, 1100−1103. (16) Catalona, W. J.; Bartsch, G.; Rittenhouse, H. G.; Evans, C. L.; Linton, H. J.; Amirkhan, A.; Horninger, W.; Klocker, H.; Mikolajczyk, S. D. J. Urol. 2003, 170, 2181−2185. (17) Johnson, E. D.; Kotowski, T. M. J. Forensic Sci. 1993, 38, 250− 258. (18) Asafudullah, S. M.; Salam, M. A.; Badruddoza, S. M.; Rahman, M. K. TAJ: J. Teach. Assoc. 2009, 22, 30−35. (19) Zhang, X.; Liu, Y.; Jia, J.; Xu, W.; Li, Z.; Chen, Y.; Han, S. At. Energy Sci. Technol. 2008, 42, 112−116. (20) Adachi, T.; Moriya, K.; Esaki, K. Acta Urol. Jpn. 1998, 44, 469− 476. (21) Liu, R.; Wang, C.; Jiang, Q.; Zhang, W.; Yue, Z.; Liu, G. Anal. Chim. Acta 2013, 801, 91−96. (22) Xie, C.; Wang, G. J. Clin. Lab. Anal. 2011, 25, 37−42. (23) Soukka, T.; Paukkunen, J.; Härmä, H.; Lönnberg, S.; Lindroos, H.; Lövgren, T. Clin. Chem. 2001, 47, 1269−1278. (24) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884−1886. (25) Sun, Z. H.; Fu, X. L.; Zang, L.; Yang, X. L.; Liu, F. Z.; Hu, G. X. Anticancer Res. 2004, 24, 1159−1165. (26) Kazuno, S.; Fujimura, T.; Arai, T.; Ueno, T.; Nagao, K.; Fujime, M.; Murayama, K. Anal. Biochem. 2011, 419, 241−249. (27) Su, L.-C.; Chen, R.-C.; Li, Y.-C.; Chang, Y.-F.; Lee, Y.-J.; Lee, C.-C.; Chou, C. Anal. Chem. 2010, 82, 3714−3718. (28) Smith, G. P. Science 1985, 228, 1315−1317. (29) Peng, W.-P.; Hou, Q.; Xia, Z.-H.; Chen, D.; Li, N.; Sun, Y.; Qiu, H.-J. Virus Res. 2008, 135, 267−272. (30) Abbineni, G.; Modali, S.; Safiejko-Mroczka, B.; Petrenko, V. A.; Mao, C. Mol. Pharmaceutics 2010, 7, 1629−1642. (31) Jayanna, P. K.; Bedi, D.; Deinnocentes, P.; Bird, R. C.; Petrenko, V. A. Protein Eng., Des. Sel. 2010, 23, 423−430. (32) Zang, L.; Shi, L.; Guo, J.; Pan, Q.; Wu, W.; Pan, X.; Wang, J. Cancer Lett. 2009, 281, 64−70. (33) Molek, P.; Strukelj, B.; Bratkovic, T. Molecules 2011, 16, 857− 887. (34) Qi, H.; Lu, H.; Qiu, H.-J.; Petrenko, V.; Liu, A. J. Mol. Biol. 2012, 417, 129−143. (35) Liu, A. H.; Abbineni, G.; Mao, C. B. Adv. Mater. 2009, 21, 1001−1005. (36) Mao, C. B.; Liu, A. H.; Cao, B. R. Angew. Chem., Int. Ed. 2009, 48, 6790−6810. (37) Olsen, E. V.; Sorokulova, I. B.; Petrenko, V. A.; Chen, I. H.; Barbaree, J. M.; Vodyanoy, V. J. Biosens. Bioelectron. 2006, 21, 1434− 1442. (38) Nanduri, V.; Sorokulova, I. B.; Samoylov, A. M.; Simonian, A. L.; Petrenko, V. A.; Vodyanoy, V. Biosens. Bioelectron. 2007, 22, 986− 992. (39) Horikawa, S.; Bedi, D.; Li, S.; Shen, W.; Huang, S.; Chen, I. H.; Chai, Y.; Auad, M. L.; Bozack, M. J.; Barbaree, J. M.; Petrenko, V. A.; Chin, B. A. Biosens. Bioelectron. 2011, 26, 2361−2367. (40) Yang, L.-M. C.; Tam, P. Y.; Murray, B. J.; McIntire, T. M.; Overstreet, C. M.; Weiss, G. A.; Penner, R. M. Anal. Chem. 2006, 78, 3265−3270. (41) Donavan, K. C.; Arter, J. A.; Pilolli, R.; Cioffi, N.; Weiss, G. A.; Penner, R. M. Anal. Chem. 2011, 83, 2420−2424.
CONCLUSIONS In summary, phage probes against f-PSA were screened from the f8/8 landscape phage library and many targeted phages were enriched effectively. Four phage clones were obtained and characterized by phage capture array. Compared with other tumor markers, all four phages exhibited good specificity for fPSA. The phage clone P1 displaying ERNSVSPS, with the best affinity for f-PSA, was selected as the capture probe to establish a sandwich ELISA array with anti-PSA monoclonal antibody. The proposed phage ELISA had a dynamic linear range of 0.825−165 ng/mL f-PSA, which covered the “diagnostic gray zone” of 4−10 ng/mL for diagnosing patients. In consideration of the excellent performance of the landscape phages, the phages can be screened to detect PSA−ACT, which is underway in this laboratory. Taken together, the data can be applicable for clinical diagnosis and early detection of PCa. Furthermore, specific phage probes for other tumor markers could be selected from the f8/8 landscape library and phage microarrays could be constructed to achieve simultaneous detection of multiple tumor markers. Finally, the phages could be used in phage-based immunoassays, substituting for antibodies.
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ASSOCIATED CONTENT
S Supporting Information *
Five figures showing structure of phage fd, schematic illustrations of phage capture array and phage ELISA for fPSA detection, effect of blocking wells, and standard curve for fPSA with phage ELISA after blocking. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail [email protected]. Author Contributions ⊥
Q.L. and F.W. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (91227116, 21275152) and the HundredTalent-Project (KSCX2-YW-BR-7), Chinese Academy of Sciences (to A.L.).
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