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A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts A. Dupont-Deshorgue a,b,1, J.B. Oudart a,b,c,1, B. Brassart a,b, G. Deslee d, J.M. Perotin d, M.D. Diebold a,b,e, J.C. Monboisse a,b,c, L. Ramont a,b,c, S. Brassart-Pasco a,b,⇑ a
Centre National de la Recherche Scientifique (CNRS), Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), 51095 Reims, France Université de Reims Champagne–Ardenne, 51097 Reims, France Laboratoire Central de Biochimie, Centre Hospitalier Universitaire (CHU) de Reims, 51092 Reims, France d Service des Maladies Respiratoires, Centre Hospitalier Universitaire (CHU) de Reims, 51092 Reims, France e Service d’Anatomie Pathologique, Centre Hospitalier Universitaire (CHU) de Reims, 51092 Reims, France b c
a r t i c l e
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Article history: Received 17 November 2014 Received in revised form 25 March 2015 Accepted 23 April 2015 Available online xxxx Keywords: Tetrastatin Collagen IV Competitive ELISA Pathologies
a b s t r a c t Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression. Ó 2015 Elsevier Inc. All rights reserved.
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Basement membranes are thin and amorphous specialized extracellular matrices that play roles in various biological events, including embryonic development. They are mainly composed of laminins, nidogens, heparan sulfate proteoglycans (perlecan and/or agrin), and type IV collagen. More recently, associations between basement membrane and minor collagens such as types XVIII, XV, and XIX have been reported and named basement membrane zone [1]. Basement membranes were long considered simple structural supports to the tissue as the constitutive proteins form an organized scaffold. However, they were reported to exert important biological functions, and their roles in many physiological and pathological situations are now well established [2]. Basement membrane components were suggested as diagnostic or prognostication markers in many diseases [3], and some of them were proposed as cancer biomarkers [4]. For example, type XVIII collagen, a basement membrane-associated collagen, was proposed as a cancer biomarker [5,6]. ⇑ Corresponding author at: Université de Reims Champagne–Ardenne, 51097 Reims, France. Fax: +33 326918055. E-mail address: [email protected] (S. Brassart-Pasco). 1 These two authors contributed equally to the study.
Collagen IV is the major component of basement membranes. It is formed by the association of three a(IV) chains among six possible, a1(IV) to a6(IV), each encoded by a different gene. Tissue distribution of a(IV) chains is variable. Whereas a1(IV) and a2(IV) chains are ubiquitous, the other four chains are expressed in specialized basement membranes. The a3(IV) and a4(IV) chains occur only in [a3(IV); a4(IV); a5(IV)] heterotrimers in the aorta, the pulmonary alveoli, the glomerulus, the cochlea, or the lens capsule. From the N terminus to the C terminus, each a(IV) chain comprises (i) an N-terminal domain comprising the 7S domain, (ii) a long central triple-helical domain with several interruptions, and (iii) a C-terminal NC1 domain of approximately 230 residues [7]. Different a(IV) NC1 domains were reported to exert antitumor or antiangiogenic activities [8]. The NC1 alpha 4(IV) domain, also named tetrastatin, induced inhibition of in vivo tumor growth in a mouse melanoma xenograft model and inhibition of in vitro tumor cell proliferation and invasion [9]. Peptides reproducing several tetrastatin sequences and named tetrastatin-1, -2, and -3 strongly inhibited endothelial cell migration without any significant effect on their proliferation [10,11].
http://dx.doi.org/10.1016/j.ab.2015.04.023 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: A. Dupont-Deshorgue et al., A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.023
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Tumstatin, the NC1 alpha 3(IV) domain, was also reported to exert antiangiogenic and antitumor properties [12–14]. Circulating tumstatin was first detected in mouse serum [15]. It was confirmed in human serum by Luo and coworkers, who also studied tumstatin amount in lung tissue extract by enzyme-linked immunosorbent assay (ELISA)2 [16]. No studies on circulating tetrastatin have been reported to date. The aim of the current work was to develop and validate a rapid and convenient method allowing tetrastatin detection and its quantification in human fluids and tissue extracts. A competitive ELISA was developed to achieve this goal. Unselected patients were used to detect tetrastatin in body fluids (serum, bronchial aspiration fluids, and bronchoalveolar lavage fluids). We also tried to detect tetrastatin in tissue extracts of patients with lung cancer.
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Materials and methods
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Reagents
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Biochemical reagents were purchased from Sigma (St. Quentin– Fallavier, France). Rabbit anti-human tetrastatin antibody was produced by Covalab (Villeurbanne, France) by immunizing the TLKESQAQRQKISRC sequence in rabbit. The His-Tag monoclonal antibody was obtained from GenScript (Piscataway, NJ, USA). pQE-31 vector and Ni-NTA resin were obtained from Qiagen (Courtaboeuf, France).
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Patients and specimens
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Blood samples from 53 unselected patients (22 female and 31 male, age range = 10–89 years, mean = 55 years) were obtained from the Central Laboratory of Biochemistry at Reims University Hospital (CHU de Reims). Sera were then separated and stored at 80 °C. Bronchoalveolar lavage fluids from 20 patients (8 female and 12 male, age range = 24–81 years) and bronchial aspiration fluids from 22 patients (11 female and 11 male, age range = 27–81 years) (mean = 59 years), were obtained from the Department of Respiratory Diseases at Reims University Hospital and centrifuged at 2000g for 10 min at 4 °C, and supernatants were stored at 80 °C. Tissue extracts from 12 patients (cancer tissue and healthy tissue taken at a distance from the tumor and checked as normal by a collaborating pathologist) were obtained from the Department of Pathology at Reims University Hospital and stored at 80 °C. Collection and use of human specimens were approved by the institutional review board of Reims University Hospital and conducted according to the Declaration of Helsinki principles. Written informed consent was obtained from all subjects.
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Protein extraction from tissues
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Tissues were cut into small pieces, sonicated on ice in a Tris buffer (50 mM Tris [pH 7.6], 0.5 M NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.02% NaN3, 1 mM phenylmethanesulfonyl fluoride [PMSF], 5 mM ethylenediaminetetraacetic acid [EDTA], and 1 mM iodoacetamide), homogenized overnight at 4 °C, and centrifuged at 10,000g for 10 min at 4 °C. Supernatants were kept frozen until use.
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Abbreviations used: ELISA, enzyme-linked immunosorbent assay; SDS, sodium dodecyl sulfate; rh tetrastatin, recombinant human tetrastatin; IPTG, isopropyl-b-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; TBS-T, Tween 20 in Tris–HCl buffer and NaCl; BSA, bovine serum albumin; TMB, tetramethylbenzidine; CV, coefficient of variation; HUVEC, human umbilical vein endothelial cell.
Expression and purification of recombinant human tetrastatin
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The sequence encoding the complete human tetrastatin (NC1 [a4(IV)1–229]) was amplified by reverse transcription–polymerase chain reaction from kidney messenger RNA (mRNA) using the following sets of primers: 50 -TTTGGCCCTGGATACCTCGGT-30 and 50 -CGCATTCTCTAGCTATACTTC-30 . The resulting complementary DNA (cDNA) fragments were cloned into a pQE-31 vector. The orientation and complete sequence of the insert were checked by sequencing. Recombinant human tetrastatin (rh tetrastatin) was expressed in Escherichia coli JM109, DE3 strain (Promega, Charbonnière-les-bains, France). Transformed bacteria were grown at 37 °C in 100 ml of Luria–Bertani medium containing 50 lg/ml ampicillin until the absorbance at 600 nm was approximately 0.6. Protein expression was induced by the addition of 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG). After 4 h of culture at 37 °C under agitation, bacterial suspension was harvested by centrifugation at 4000g for 15 min at 4 °C. The pellets were resuspended in 5 ml of 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole (pH 8.0). The cells were disrupted by thaw/freeze cycle and sonication. After centrifugation at 10,000g for 30 min, supernatant (4 ml) was incubated for 1 h with 1 ml of Ni-NTA Superflow resin. The resin was then poured into a chromatography column and washed with 4 ml of 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH 8.0). Finally, rh tetrastatin was eluted with 3 0.5 ml of elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, pH 8.0). After dialysis against distilled water, its purity was assessed by SDS–PAGE (polyacrylamide gel electrophoresis) and by Western blotting using an anti-tetrastatin polyclonal antibody or an anti-6 His-Tag monoclonal antibody.
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Western blot
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For Western blot analysis, samples were reduced by 10 mM dithiothreitol and subjected to SDS–PAGE (0.1% SDS and 10% polyacrylamide gel) (100 lg total protein/lane) and then transferred onto Immobilon-P membranes (Millipore, St. Quentin en Yvelines, France). Membranes were blocked by incubation with 5% nonfat dry milk and 0.1% Tween 20 in 50 mM Tris–HCl buffer and 150 mM NaCl (pH 7.5) (TBS-T) for 2 h at room temperature. They were incubated overnight with a 1/5000 diluted rabbit anti-human tetrastatin polyclonal antibody or with a 1/5000 anti-6 His-Tag monoclonal antibody and then for 1 h with the 1/10,000 diluted corresponding peroxidase-conjugated secondary antibody at room temperature. Immune complexes were visualized using the ECL Prime Chemiluminescence Detection Kit (GE Healthcare, Orsay, France).
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Competitive ELISA procedure
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Direct ELISA development Serial dilutions were performed from a single stock solution to obtain the different dilutions of antibody or rh tetrastatin concentrations. rh tetrastatin (0–10 lg/well) was adsorbed to the 96-well microtiter plate in carbonate buffer (0.2 M sodium carbonate and 0.2 M sodium bicarbonate, pH 9.6) overnight at 4 °C. After washing with TBS-T, the plate was blocked with TBS-T supplemented with 1% bovine serum albumin (BSA) for 1 h at 37 °C and washed with TBS-T. Then, 100 ll of the primary antibody solution (rabbit anti-human tetrastatin polyclonal antibody) at different dilutions (1/100 to 1/3200) in TBS-T containing 1% BSA was added to each well and incubated for 1 h at room temperature. After three washes with TBS-T, 100 ll of the secondary antibody solution (anti-rabbit IgG coupled to peroxidase) diluted 1/10,000 was added to each well and incubated for 1 h at room temperature. After three washes, 100 ll per well of tetramethylbenzidine (TMB), a
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peroxidase substrate, was added to the wells and incubated for 30 min in the dark. The enzymatic reaction was stopped by the addition of 50 ll per well of 0.5 M H2SO4. The intensity of the yellow coloration was measured at 450 nm with an LB940 spectrophotometer (Berthold Technologies, Yvelines, France). Competitive ELISA development Step 1 (coating). Recombinant human tetrastatin (500 ng/well) was adsorbed to the 96-well microtiter plate in carbonate buffer (0.2 M sodium carbonate and 0.2 M sodium bicarbonate, pH 9.6) overnight at 4 °C. After washing with TBS-T, the plate was blocked with TBS-T supplemented with 1% BSA for 1 h at 37 °C. Step 2 (competition). Next, 60 ll of each sample or calibrator was incubated with 60 ll of primary antibody (rabbit anti-human tetrastatin polyclonal antibody diluted 1/10,000e in TBS-T–1% BSA (final dilution 1/20,000e) for 1 h at room temperature. Step 3. After that, 100 ll of each competitive mixture from step 2 was added into each well coated with rh tetrastatin in step 1. The plate was incubated for 1 h at room temperature. Step 4. After three washes with TBS-T, 100 ll of secondary antibody solution (anti-rabbit IgG coupled to peroxidase) diluted 1/10,000e was added to each well and incubated for 1 h at room temperature. Step 5. After washing with TBS-T, 100 ll per well of a peroxidase substrate, TMB, was added and incubated in the dark for 30 min.
Fig.1. Recombinant human tetrastatin production. Recombinant human tetrastatin domain was expressed in E. coli JM109, DE3 strain. (A) SDS–PAGE analysis. Lane LMW: low-molecular-weight marker; lane 1: crude lysate (4-h IPTG induction); lane 2: rh tetrastatin domain purified by chromatography on Ni-NTA Superflow resin. (B) Western blot using an anti-tetrastatin antibody. Lane 1: crude lysate (4-h IPTG induction); lane 2: rh tetrastatin domain purified by chromatography on NiNTA Superflow resin. (C) Western blot using an anti-His-Tag antibody. Lane 1: crude lysate (4-h IPTG induction); lane 2: rh tetrastatin domain purified by chromatography on Ni-NTA Superflow resin.
primary antibody (1/5000, 1/10,000, 1/20,000, and 1/40,000) and different amounts of coated rh tetrastatin (0–2500 ng/well). The calibration curve was performed using serial dilutions of a single stock rh tetrastatin solution. The different amounts were 0, 2.44, 4.88, 9.76, 19.5, 39, 78, 156, 312.5, 625, 1250, and 2500 ng/well. We selected the conditions giving an absorbance of 1 unit: a dilution of 1/10,000 of the primary antibody and a coating of 500 ng/well of rh tetrastatin (Fig. 2). The selected combination permitted obtaining an absorbance of 1 unit using the lowest amount of rh tetrastatin and the highest dilution of antibody.
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Validation of competitive ELISA
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Step 6. The enzymatic reaction was stopped by adding 50 ll per well of 0.5 M H2SO4. The intensity of the yellow coloration was measured at 450 nm with an LB940 spectrophotometer (Berthold Technologies).
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Assay performance and validation
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To evaluate intra-assay variations, three different serum samples were assayed in eight replicates at the same time. For inter-assay variations, three different serum samples were assayed in triplicate for 3 days consecutively. Recovery was assessed by adding known concentrations of rh tetrastatin.
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Statistical analysis
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Paired Student’s t test was performed to compare tetrastatin content in tumor with healthy tissue from the same patient.
The calibration curve was determined with increasing concentrations of rh tetrastatin from 0 to 2500 ng/ml (Fig. 3A). To determine the tetrastatin concentration in the samples, we performed a logarithmic transformation of the curve, as is usually done in the literature (Fig. 3B). The obtained calibration curve was linear (y = 27.537x + 104.71 and R2 = 0.9983). To validate the accuracy of our competitive ELISA, we measured several serum samples supplemented with known rh tetrastatin. The measured concentrations were well correlated with the expected concentration (Table 1). The recovery ranged from 88.9 to 97.7%. The precision of the ELISA was estimated using clinically available serum of different tetrastatin concentrations. Intra- and inter-assay coefficients of variation (CVs) were 1.06 and 6.7% at 5 lg/ml, 2.6 and 7.8% at 0.3125 lg/ml, and 2.8 and 9.5% at 0.0198 lg/ml, respectively (n = 8 and n = 3, respectively).
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Results
Quantification of tetrastatin in blood
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Production of rh tetrastatin
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Recombinant human tetrastatin was expressed in E. coli JM109, DE3 strain, and purified by Ni-NTA resin Superflow affinity column via its 6 His-Tag. The column-purified recombinant protein appeared as a single band in SDS–PAGE at 29 kDa (Fig. 1A). The identity of the protein was further confirmed by Western blot using an anti-tetrastatin antibody (Fig. 1B) and an anti-6 His-Tag antibody (Fig. 1C).
The presence of circulating tetrastatin has not been reported until now. An attempt to detect tetrastatin in serum from patients was performed. The optimized validated competitive ELISA was used to determine tetrastatin levels in the serum of 53 different donors. Tetrastatin concentrations were measured in triplicate and varied from 18 to 300 ng/ml, depending on the patient. Fig. 4A shows a simple plot for each patient that corresponds to the mean of triplicates for each patient. Quantification of tetrastatin in bronchial aspiration fluids
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Determination of ELISA conditions For competitive ELISA, we determined the optimal primary antibody concentration to use and the optimal amount of rh tetrastatin for the plate coating using different concentrations of the
The competitive ELISA was used to determine tetrastatin levels in bronchial aspiration fluids of 22 patients. Tetrastatin concentrations were measured in triplicate and varied from 7.3 to 210.7 ng/ml, depending on the patient (Fig. 4B).
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Fig.2. Direct ELISA. Intensity of signal was obtained at 450 nm with different primary antibody concentrations and different amounts of rh tetrastatin coated. Antibody dilutions: , 1/40,000; j, 1/20,000; N, 1/10,000; , 1/5000.
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Quantification of tetrastatin in bronchoalveolar lavage fluids
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The competitive ELISA was also used to determine tetrastatin levels in bronchoalveolar lavage fluids of 20 patients. Tetrastatin concentrations were measured in triplicate and varied from 2.5 to 16.3 ng/ml, depending on the patient (Fig. 4C).
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Tetrastatin level increases in pulmonary tumor extracts
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For this experiment, tetrastatin content was determined in cancer tissue and compared with healthy tissue from the same patient; healthy tissue was taken at a distance from the tumor
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and checked as normal by a collaborating pathologist. Tetrastatin concentrations were measured in triplicate in 12 patients. In healthy tissue, tetrastatin concentration varied from 11.6 to 40.7 ng/mg of total protein. In cancer tissue, tetrastatin concentration varied from 20.7 to 80.8 ng/mg of total protein. The tetrastatin level was significantly higher in cancer tissue than in healthy tissue (paired Student’s t test, P < 0.002) (Fig. 5).
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Discussion
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Extracellular matrix components, especially those of basement membrane, are involved in many physiopathological situations such as inflammation, cell migration, tumor invasion, angiogenesis, and embryonic development [1,2]. The diagnosis of diseases is partly based on the measurement of circulating proteins. Such proteins or their proteolytic fragments can be measured in biological fluids, such as blood and urine, and used for diagnosis, prognostication, or treatment monitoring. Collagen IV is involved in various diseases such as Alport’s syndrome, characterized by different COL4A3-5 gene mutations [17], and Goodpasture’s syndrome, characterized by the presence of autoantibodies directed against the a3(IV) collagen chain [18]. Several basement membrane components were proposed as diagnostic and prognostic markers in many diseases [3]. The serum endostatin level is associated with poor outcome in patients with non-Hodgkin lymphoma [6] and was suggested to predict tumor vascularity [19,20]. Measurement of collagen IV in serum using a sandwich enzyme immunoassay (EIA) kit employing two monoclonal antibodies to the 7S domain and some non-7S and
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Fig.3. ELISA calibration. (A) The calibration curve was determined with increasing concentrations of rh tetrastatin (0–2500 ng/ml). (B) The logarithmic transformation of the calibration curve was linear (y = 27.537x + 104.71 and R2 = 0.9983).
Please cite this article in press as: A. Dupont-Deshorgue et al., A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.023
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Sample absorbance
Inhibition [(A/A0) 100]
Expected concentration (lg/ml)
Measured concentration (lg/ml)
% Error
0.205 0.379 0.552 0.727 0.902
19.2 35.5 51.7 68.0 84.5
5 1.25 0.3125 0.0780 0.0198
5.132 1.307 0.337 0.085 0.022
2.6 4.5 7.8 8.9 11.1
non-NCl domains suggests that the urinary level of type IV collagen is useful for detection of early diabetic nephropathy and for the differential diagnosis of diabetic nephropathy and chronic glomerulonephritis [21,22]. In pancreatic cancer tissue, a high level of type IV collagen was found close to cancer cells in the tumor stroma [23]. Luo and coworkers developed an ELISA to measure tumstatin in serum and tissue extracts. Tumstatin levels in poorly differentiated lung tumor tissues were significantly lower than those in nontumor tissues and well-differentiated tumor tissues (P < 0.001) [16]. The collagen IV NC1 alpha 4(IV) domain, also named tetrastatin, appeared to be devoid of biological activity on human umbilical vein endothelial cell (HUVEC) adhesion and migration and on basic fibroblast growth factor (bFGF)-induced angiogenesis and hamster melanoma growth in the chick CAM model [24]. By contrast, several synthetic peptides derived from the type IV collagen a4(IV) chain, named tetrastatin-1, -2, and -3 and corresponding to NC1[a4(IV)54–74], NC1[a4(IV)64–84], and NC1[a4(IV)168–187], respectively, were reported to inhibit HUVEC migration in vitro in modified Boyden chambers [9,10]. More recently, we showed that tetrastatin induces inhibition of tumor growth in vivo in a mouse melanoma model and inhibition of in vitro tumor cell proliferation and invasion. The results are different from those observed by Petitclerc and coworkers [24], probably due to the model used to study tumor growth (mouse xenograft model with human melanoma cells vs. chick CAM model with hamster melanoma cells). To date, no studies have quantified tetrastatin level in body fluids and extracts. Therefore, in our study we focused on the development of a competitive ELISA, a rapid and convenient method, to measure tetrastatin. We first performed a direct ELISA to select the appropriate antibody dilution and coating amount, a dilution of 1/10,000 of the primary antibody and a coating of 500 ng/well of rh tetrastatin permitted to obtain an absorbance
Fig.4. ELISA measurement of tetrastatin in body fluids. (A) Serum samples from 53 different patients were assessed for their tetrastatin content. A simple plot for each patient corresponds to the mean of triplicates. (B) Bronchial aspiration fluids from 22 different patients were assessed for their tetrastatin content. A simple plot for each patient corresponds to the mean of triplicates. (C) Bronchoalveolar lavage fluids from 20 different patients were assessed for their tetrastatin content. A simple plot for each patient corresponds to the mean of triplicates.
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of 1 unit, and we selected these two parameters for the competitive ELISA development. Intra-CVs ranged from 1.06% at 5 lg/ml to 2.8% at 0.0198 lg/ml. Inter-CVs ranged from 6.7% at 5 lg/ml to 9.5% at 0.0198 lg/ml. Inter-CVs are better for higher concentrations than for lower concentrations but are widely acceptable if we refer to commercially available competitive ELISA. No circulating tetrastatin has been reported to date. We tried to detect tetrastatin in serum of patients because it was easy to obtain and implement. This assay was sensitive and had a linear range from 2 to 2500 ng/ml (R2 = 0.9983). We detected amounts ranging from 18 to 300 ng/ml in serum. Tumstatin was also detected in human serum by quantitative ELISA (12.6– 147.2 ng/ml) [16] and was reported to be cleaved by matrix metallopeptidase 9 (MMP-9) [15]. Because the a4(IV) collagen chain is present as [a3(IV); a4(IV); a5(IV)] heterotrimers, we can assume that tetrastatin is also cleaved in vivo by such proteases. The aim of this study was to detect circulating tetrastatin. For that purpose, unselected patients with very different pathologies were tested. The small number of patients for each pathology tested did not allow performing statistical analysis. Determining the tetrastatin level in serum from patients with lung carcinomas, for instance, could be the aim of another study with large cohorts of patients. It could also be interesting to detect tetrastatin in other body fluids such as bronchial aspiration and bronchoalveolar lavage fluids. Our competitive ELISA was also used to determine tetrastatin level in bronchial aspirations of 22 patients. Tetrastatin concentrations varied from 7.3 to 210.7 ng/ml. The small number of patients included in this study did not permit finding significant differences according to pathologies. Type IV collagen fragments were reported as markers of liver and pulmonary fibrosis [25,26]. We used our competitive ELISA to determine tetrastatin levels in bronchoalveolar lavage fluids of 20 patients. Such an approach was previously used to study collagen-like glycoprotein (surfactant protein A and surfactant protein D) modulation with aging and emphysema development [27]. Tetrastatin concentrations varied from 2.5 to 16.3 ng/ml, depending on the patient. A clinical study is currently under investigation to determine tetrastatin amounts in bronchial aspiration and bronchoalveolar lavage fluids from patients with different infectious, inflammatory, or malignant bronchoalveolar diseases. Because the expression of other NC1 domains from basement membrane collagens was previously reported to be altered in cancer progression [28–30], we used the competitive ELISA to detect tetrastatin in healthy and tumor tissue extracts. The tetrastatin level was studied in pulmonary tissue extracts from 12 patients because this tissue contains large amounts of [a3(IV); a4(IV); a5(IV)] heterotrimer. In healthy tissue, tetrastatin concentrations ranged from 11.6 to 40.7 ng/mg of total protein. In cancer tissue, tetrastatin concentrations ranged from 20.7 to 80.8 ng/mg of total
Fig.5. ELISA measurement of tetrastatin in lung tissue extracts. Lung tissue extracts from 12 different patients were assessed for their tetrastatin content. For each patient, cancer tissue was compared with healthy lung tissue. A simple plot for each patient corresponds to the mean of triplicates. ***P < 0.02 according to paired Student’s t test.
Please cite this article in press as: A. Dupont-Deshorgue et al., A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.023
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protein. The tetrastatin level was significantly higher in cancer tissue than in healthy tissue (paired Student’s t test, P < 0.002) (Fig. 5). The small number of patients included in the study did not allow performing statistical analysis depending on the grade of the tumor. Larger cohorts of patients would be needed to eventually propose tetrastatin as a diagnostic cancer biomarker. Nevertheless, this study allowed us to demonstrate the ability of the ELISA to point out differences in the level of tetrastatin in healthy and cancer lung tissues. This could be extended to other cancer types. In conclusion, the competitive ELISA we have developed is a rapid and convenient method to detect tetrastatin in body fluids and tissue extracts from patients whose basement membrane metabolism may be altered.
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Acknowledgments
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This work was supported by grants from the Université de Reims Champagne–Ardenne, the Centre National de la Recherche Scientifique (CNRS), the Ligue Nationale Contre le Cancer, the Region Champagne–Ardenne, and the GIRCI est (AAP Jeune Chercheur Broncoll). The authors thank François-Xavier Maquart, Lukasz Szpak, Vincent Baudouin, and Saviz Nasri for their contributions to the work. The authors also thank Sylvette Gobert (Université de Reims Champagne–Ardenne) for improving the English language of the manuscript.
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Please cite this article in press as: A. Dupont-Deshorgue et al., A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.023
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A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts A. Dupont-Deshorgue a,b,1, J.B. Oudart a,b,c,1, B. Brassart a,b, G. Deslee d, J.M. Perotin d, M.D. Diebold a,b,e, J.C. Monboisse a,b,c, L. Ramont a,b,c, S. Brassart-Pasco a,b,⇑ a
Centre National de la Recherche Scientifique (CNRS), Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), 51095 Reims, France Université de Reims Champagne–Ardenne, 51097 Reims, France Laboratoire Central de Biochimie, Centre Hospitalier Universitaire (CHU) de Reims, 51092 Reims, France d Service des Maladies Respiratoires, Centre Hospitalier Universitaire (CHU) de Reims, 51092 Reims, France e Service d’Anatomie Pathologique, Centre Hospitalier Universitaire (CHU) de Reims, 51092 Reims, France b c
a r t i c l e
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Article history: Received 17 November 2014 Received in revised form 25 March 2015 Accepted 23 April 2015 Available online xxxx Keywords: Tetrastatin Collagen IV Competitive ELISA Pathologies
a b s t r a c t Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression. Ó 2015 Elsevier Inc. All rights reserved.
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Basement membranes are thin and amorphous specialized extracellular matrices that play roles in various biological events, including embryonic development. They are mainly composed of laminins, nidogens, heparan sulfate proteoglycans (perlecan and/or agrin), and type IV collagen. More recently, associations between basement membrane and minor collagens such as types XVIII, XV, and XIX have been reported and named basement membrane zone [1]. Basement membranes were long considered simple structural supports to the tissue as the constitutive proteins form an organized scaffold. However, they were reported to exert important biological functions, and their roles in many physiological and pathological situations are now well established [2]. Basement membrane components were suggested as diagnostic or prognostication markers in many diseases [3], and some of them were proposed as cancer biomarkers [4]. For example, type XVIII collagen, a basement membrane-associated collagen, was proposed as a cancer biomarker [5,6]. ⇑ Corresponding author at: Université de Reims Champagne–Ardenne, 51097 Reims, France. Fax: +33 326918055. E-mail address: [email protected] (S. Brassart-Pasco). 1 These two authors contributed equally to the study.
Collagen IV is the major component of basement membranes. It is formed by the association of three a(IV) chains among six possible, a1(IV) to a6(IV), each encoded by a different gene. Tissue distribution of a(IV) chains is variable. Whereas a1(IV) and a2(IV) chains are ubiquitous, the other four chains are expressed in specialized basement membranes. The a3(IV) and a4(IV) chains occur only in [a3(IV); a4(IV); a5(IV)] heterotrimers in the aorta, the pulmonary alveoli, the glomerulus, the cochlea, or the lens capsule. From the N terminus to the C terminus, each a(IV) chain comprises (i) an N-terminal domain comprising the 7S domain, (ii) a long central triple-helical domain with several interruptions, and (iii) a C-terminal NC1 domain of approximately 230 residues [7]. Different a(IV) NC1 domains were reported to exert antitumor or antiangiogenic activities [8]. The NC1 alpha 4(IV) domain, also named tetrastatin, induced inhibition of in vivo tumor growth in a mouse melanoma xenograft model and inhibition of in vitro tumor cell proliferation and invasion [9]. Peptides reproducing several tetrastatin sequences and named tetrastatin-1, -2, and -3 strongly inhibited endothelial cell migration without any significant effect on their proliferation [10,11].
http://dx.doi.org/10.1016/j.ab.2015.04.023 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: A. Dupont-Deshorgue et al., A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.023
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Tumstatin, the NC1 alpha 3(IV) domain, was also reported to exert antiangiogenic and antitumor properties [12–14]. Circulating tumstatin was first detected in mouse serum [15]. It was confirmed in human serum by Luo and coworkers, who also studied tumstatin amount in lung tissue extract by enzyme-linked immunosorbent assay (ELISA)2 [16]. No studies on circulating tetrastatin have been reported to date. The aim of the current work was to develop and validate a rapid and convenient method allowing tetrastatin detection and its quantification in human fluids and tissue extracts. A competitive ELISA was developed to achieve this goal. Unselected patients were used to detect tetrastatin in body fluids (serum, bronchial aspiration fluids, and bronchoalveolar lavage fluids). We also tried to detect tetrastatin in tissue extracts of patients with lung cancer.
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Materials and methods
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Reagents
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Biochemical reagents were purchased from Sigma (St. Quentin– Fallavier, France). Rabbit anti-human tetrastatin antibody was produced by Covalab (Villeurbanne, France) by immunizing the TLKESQAQRQKISRC sequence in rabbit. The His-Tag monoclonal antibody was obtained from GenScript (Piscataway, NJ, USA). pQE-31 vector and Ni-NTA resin were obtained from Qiagen (Courtaboeuf, France).
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Patients and specimens
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Blood samples from 53 unselected patients (22 female and 31 male, age range = 10–89 years, mean = 55 years) were obtained from the Central Laboratory of Biochemistry at Reims University Hospital (CHU de Reims). Sera were then separated and stored at 80 °C. Bronchoalveolar lavage fluids from 20 patients (8 female and 12 male, age range = 24–81 years) and bronchial aspiration fluids from 22 patients (11 female and 11 male, age range = 27–81 years) (mean = 59 years), were obtained from the Department of Respiratory Diseases at Reims University Hospital and centrifuged at 2000g for 10 min at 4 °C, and supernatants were stored at 80 °C. Tissue extracts from 12 patients (cancer tissue and healthy tissue taken at a distance from the tumor and checked as normal by a collaborating pathologist) were obtained from the Department of Pathology at Reims University Hospital and stored at 80 °C. Collection and use of human specimens were approved by the institutional review board of Reims University Hospital and conducted according to the Declaration of Helsinki principles. Written informed consent was obtained from all subjects.
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Protein extraction from tissues
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Tissues were cut into small pieces, sonicated on ice in a Tris buffer (50 mM Tris [pH 7.6], 0.5 M NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.02% NaN3, 1 mM phenylmethanesulfonyl fluoride [PMSF], 5 mM ethylenediaminetetraacetic acid [EDTA], and 1 mM iodoacetamide), homogenized overnight at 4 °C, and centrifuged at 10,000g for 10 min at 4 °C. Supernatants were kept frozen until use.
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Abbreviations used: ELISA, enzyme-linked immunosorbent assay; SDS, sodium dodecyl sulfate; rh tetrastatin, recombinant human tetrastatin; IPTG, isopropyl-b-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; TBS-T, Tween 20 in Tris–HCl buffer and NaCl; BSA, bovine serum albumin; TMB, tetramethylbenzidine; CV, coefficient of variation; HUVEC, human umbilical vein endothelial cell.
Expression and purification of recombinant human tetrastatin
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The sequence encoding the complete human tetrastatin (NC1 [a4(IV)1–229]) was amplified by reverse transcription–polymerase chain reaction from kidney messenger RNA (mRNA) using the following sets of primers: 50 -TTTGGCCCTGGATACCTCGGT-30 and 50 -CGCATTCTCTAGCTATACTTC-30 . The resulting complementary DNA (cDNA) fragments were cloned into a pQE-31 vector. The orientation and complete sequence of the insert were checked by sequencing. Recombinant human tetrastatin (rh tetrastatin) was expressed in Escherichia coli JM109, DE3 strain (Promega, Charbonnière-les-bains, France). Transformed bacteria were grown at 37 °C in 100 ml of Luria–Bertani medium containing 50 lg/ml ampicillin until the absorbance at 600 nm was approximately 0.6. Protein expression was induced by the addition of 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG). After 4 h of culture at 37 °C under agitation, bacterial suspension was harvested by centrifugation at 4000g for 15 min at 4 °C. The pellets were resuspended in 5 ml of 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole (pH 8.0). The cells were disrupted by thaw/freeze cycle and sonication. After centrifugation at 10,000g for 30 min, supernatant (4 ml) was incubated for 1 h with 1 ml of Ni-NTA Superflow resin. The resin was then poured into a chromatography column and washed with 4 ml of 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH 8.0). Finally, rh tetrastatin was eluted with 3 0.5 ml of elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, pH 8.0). After dialysis against distilled water, its purity was assessed by SDS–PAGE (polyacrylamide gel electrophoresis) and by Western blotting using an anti-tetrastatin polyclonal antibody or an anti-6 His-Tag monoclonal antibody.
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Western blot
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For Western blot analysis, samples were reduced by 10 mM dithiothreitol and subjected to SDS–PAGE (0.1% SDS and 10% polyacrylamide gel) (100 lg total protein/lane) and then transferred onto Immobilon-P membranes (Millipore, St. Quentin en Yvelines, France). Membranes were blocked by incubation with 5% nonfat dry milk and 0.1% Tween 20 in 50 mM Tris–HCl buffer and 150 mM NaCl (pH 7.5) (TBS-T) for 2 h at room temperature. They were incubated overnight with a 1/5000 diluted rabbit anti-human tetrastatin polyclonal antibody or with a 1/5000 anti-6 His-Tag monoclonal antibody and then for 1 h with the 1/10,000 diluted corresponding peroxidase-conjugated secondary antibody at room temperature. Immune complexes were visualized using the ECL Prime Chemiluminescence Detection Kit (GE Healthcare, Orsay, France).
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Competitive ELISA procedure
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Direct ELISA development Serial dilutions were performed from a single stock solution to obtain the different dilutions of antibody or rh tetrastatin concentrations. rh tetrastatin (0–10 lg/well) was adsorbed to the 96-well microtiter plate in carbonate buffer (0.2 M sodium carbonate and 0.2 M sodium bicarbonate, pH 9.6) overnight at 4 °C. After washing with TBS-T, the plate was blocked with TBS-T supplemented with 1% bovine serum albumin (BSA) for 1 h at 37 °C and washed with TBS-T. Then, 100 ll of the primary antibody solution (rabbit anti-human tetrastatin polyclonal antibody) at different dilutions (1/100 to 1/3200) in TBS-T containing 1% BSA was added to each well and incubated for 1 h at room temperature. After three washes with TBS-T, 100 ll of the secondary antibody solution (anti-rabbit IgG coupled to peroxidase) diluted 1/10,000 was added to each well and incubated for 1 h at room temperature. After three washes, 100 ll per well of tetramethylbenzidine (TMB), a
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peroxidase substrate, was added to the wells and incubated for 30 min in the dark. The enzymatic reaction was stopped by the addition of 50 ll per well of 0.5 M H2SO4. The intensity of the yellow coloration was measured at 450 nm with an LB940 spectrophotometer (Berthold Technologies, Yvelines, France). Competitive ELISA development Step 1 (coating). Recombinant human tetrastatin (500 ng/well) was adsorbed to the 96-well microtiter plate in carbonate buffer (0.2 M sodium carbonate and 0.2 M sodium bicarbonate, pH 9.6) overnight at 4 °C. After washing with TBS-T, the plate was blocked with TBS-T supplemented with 1% BSA for 1 h at 37 °C. Step 2 (competition). Next, 60 ll of each sample or calibrator was incubated with 60 ll of primary antibody (rabbit anti-human tetrastatin polyclonal antibody diluted 1/10,000e in TBS-T–1% BSA (final dilution 1/20,000e) for 1 h at room temperature. Step 3. After that, 100 ll of each competitive mixture from step 2 was added into each well coated with rh tetrastatin in step 1. The plate was incubated for 1 h at room temperature. Step 4. After three washes with TBS-T, 100 ll of secondary antibody solution (anti-rabbit IgG coupled to peroxidase) diluted 1/10,000e was added to each well and incubated for 1 h at room temperature. Step 5. After washing with TBS-T, 100 ll per well of a peroxidase substrate, TMB, was added and incubated in the dark for 30 min.
Fig.1. Recombinant human tetrastatin production. Recombinant human tetrastatin domain was expressed in E. coli JM109, DE3 strain. (A) SDS–PAGE analysis. Lane LMW: low-molecular-weight marker; lane 1: crude lysate (4-h IPTG induction); lane 2: rh tetrastatin domain purified by chromatography on Ni-NTA Superflow resin. (B) Western blot using an anti-tetrastatin antibody. Lane 1: crude lysate (4-h IPTG induction); lane 2: rh tetrastatin domain purified by chromatography on NiNTA Superflow resin. (C) Western blot using an anti-His-Tag antibody. Lane 1: crude lysate (4-h IPTG induction); lane 2: rh tetrastatin domain purified by chromatography on Ni-NTA Superflow resin.
primary antibody (1/5000, 1/10,000, 1/20,000, and 1/40,000) and different amounts of coated rh tetrastatin (0–2500 ng/well). The calibration curve was performed using serial dilutions of a single stock rh tetrastatin solution. The different amounts were 0, 2.44, 4.88, 9.76, 19.5, 39, 78, 156, 312.5, 625, 1250, and 2500 ng/well. We selected the conditions giving an absorbance of 1 unit: a dilution of 1/10,000 of the primary antibody and a coating of 500 ng/well of rh tetrastatin (Fig. 2). The selected combination permitted obtaining an absorbance of 1 unit using the lowest amount of rh tetrastatin and the highest dilution of antibody.
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Validation of competitive ELISA
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Step 6. The enzymatic reaction was stopped by adding 50 ll per well of 0.5 M H2SO4. The intensity of the yellow coloration was measured at 450 nm with an LB940 spectrophotometer (Berthold Technologies).
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Assay performance and validation
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To evaluate intra-assay variations, three different serum samples were assayed in eight replicates at the same time. For inter-assay variations, three different serum samples were assayed in triplicate for 3 days consecutively. Recovery was assessed by adding known concentrations of rh tetrastatin.
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Statistical analysis
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Paired Student’s t test was performed to compare tetrastatin content in tumor with healthy tissue from the same patient.
The calibration curve was determined with increasing concentrations of rh tetrastatin from 0 to 2500 ng/ml (Fig. 3A). To determine the tetrastatin concentration in the samples, we performed a logarithmic transformation of the curve, as is usually done in the literature (Fig. 3B). The obtained calibration curve was linear (y = 27.537x + 104.71 and R2 = 0.9983). To validate the accuracy of our competitive ELISA, we measured several serum samples supplemented with known rh tetrastatin. The measured concentrations were well correlated with the expected concentration (Table 1). The recovery ranged from 88.9 to 97.7%. The precision of the ELISA was estimated using clinically available serum of different tetrastatin concentrations. Intra- and inter-assay coefficients of variation (CVs) were 1.06 and 6.7% at 5 lg/ml, 2.6 and 7.8% at 0.3125 lg/ml, and 2.8 and 9.5% at 0.0198 lg/ml, respectively (n = 8 and n = 3, respectively).
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Quantification of tetrastatin in blood
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Production of rh tetrastatin
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Recombinant human tetrastatin was expressed in E. coli JM109, DE3 strain, and purified by Ni-NTA resin Superflow affinity column via its 6 His-Tag. The column-purified recombinant protein appeared as a single band in SDS–PAGE at 29 kDa (Fig. 1A). The identity of the protein was further confirmed by Western blot using an anti-tetrastatin antibody (Fig. 1B) and an anti-6 His-Tag antibody (Fig. 1C).
The presence of circulating tetrastatin has not been reported until now. An attempt to detect tetrastatin in serum from patients was performed. The optimized validated competitive ELISA was used to determine tetrastatin levels in the serum of 53 different donors. Tetrastatin concentrations were measured in triplicate and varied from 18 to 300 ng/ml, depending on the patient. Fig. 4A shows a simple plot for each patient that corresponds to the mean of triplicates for each patient. Quantification of tetrastatin in bronchial aspiration fluids
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Determination of ELISA conditions For competitive ELISA, we determined the optimal primary antibody concentration to use and the optimal amount of rh tetrastatin for the plate coating using different concentrations of the
The competitive ELISA was used to determine tetrastatin levels in bronchial aspiration fluids of 22 patients. Tetrastatin concentrations were measured in triplicate and varied from 7.3 to 210.7 ng/ml, depending on the patient (Fig. 4B).
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Fig.2. Direct ELISA. Intensity of signal was obtained at 450 nm with different primary antibody concentrations and different amounts of rh tetrastatin coated. Antibody dilutions: , 1/40,000; j, 1/20,000; N, 1/10,000; , 1/5000.
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Quantification of tetrastatin in bronchoalveolar lavage fluids
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The competitive ELISA was also used to determine tetrastatin levels in bronchoalveolar lavage fluids of 20 patients. Tetrastatin concentrations were measured in triplicate and varied from 2.5 to 16.3 ng/ml, depending on the patient (Fig. 4C).
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Tetrastatin level increases in pulmonary tumor extracts
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For this experiment, tetrastatin content was determined in cancer tissue and compared with healthy tissue from the same patient; healthy tissue was taken at a distance from the tumor
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and checked as normal by a collaborating pathologist. Tetrastatin concentrations were measured in triplicate in 12 patients. In healthy tissue, tetrastatin concentration varied from 11.6 to 40.7 ng/mg of total protein. In cancer tissue, tetrastatin concentration varied from 20.7 to 80.8 ng/mg of total protein. The tetrastatin level was significantly higher in cancer tissue than in healthy tissue (paired Student’s t test, P < 0.002) (Fig. 5).
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Discussion
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Extracellular matrix components, especially those of basement membrane, are involved in many physiopathological situations such as inflammation, cell migration, tumor invasion, angiogenesis, and embryonic development [1,2]. The diagnosis of diseases is partly based on the measurement of circulating proteins. Such proteins or their proteolytic fragments can be measured in biological fluids, such as blood and urine, and used for diagnosis, prognostication, or treatment monitoring. Collagen IV is involved in various diseases such as Alport’s syndrome, characterized by different COL4A3-5 gene mutations [17], and Goodpasture’s syndrome, characterized by the presence of autoantibodies directed against the a3(IV) collagen chain [18]. Several basement membrane components were proposed as diagnostic and prognostic markers in many diseases [3]. The serum endostatin level is associated with poor outcome in patients with non-Hodgkin lymphoma [6] and was suggested to predict tumor vascularity [19,20]. Measurement of collagen IV in serum using a sandwich enzyme immunoassay (EIA) kit employing two monoclonal antibodies to the 7S domain and some non-7S and
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Fig.3. ELISA calibration. (A) The calibration curve was determined with increasing concentrations of rh tetrastatin (0–2500 ng/ml). (B) The logarithmic transformation of the calibration curve was linear (y = 27.537x + 104.71 and R2 = 0.9983).
Please cite this article in press as: A. Dupont-Deshorgue et al., A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.023
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Sample absorbance
Inhibition [(A/A0) 100]
Expected concentration (lg/ml)
Measured concentration (lg/ml)
% Error
0.205 0.379 0.552 0.727 0.902
19.2 35.5 51.7 68.0 84.5
5 1.25 0.3125 0.0780 0.0198
5.132 1.307 0.337 0.085 0.022
2.6 4.5 7.8 8.9 11.1
non-NCl domains suggests that the urinary level of type IV collagen is useful for detection of early diabetic nephropathy and for the differential diagnosis of diabetic nephropathy and chronic glomerulonephritis [21,22]. In pancreatic cancer tissue, a high level of type IV collagen was found close to cancer cells in the tumor stroma [23]. Luo and coworkers developed an ELISA to measure tumstatin in serum and tissue extracts. Tumstatin levels in poorly differentiated lung tumor tissues were significantly lower than those in nontumor tissues and well-differentiated tumor tissues (P < 0.001) [16]. The collagen IV NC1 alpha 4(IV) domain, also named tetrastatin, appeared to be devoid of biological activity on human umbilical vein endothelial cell (HUVEC) adhesion and migration and on basic fibroblast growth factor (bFGF)-induced angiogenesis and hamster melanoma growth in the chick CAM model [24]. By contrast, several synthetic peptides derived from the type IV collagen a4(IV) chain, named tetrastatin-1, -2, and -3 and corresponding to NC1[a4(IV)54–74], NC1[a4(IV)64–84], and NC1[a4(IV)168–187], respectively, were reported to inhibit HUVEC migration in vitro in modified Boyden chambers [9,10]. More recently, we showed that tetrastatin induces inhibition of tumor growth in vivo in a mouse melanoma model and inhibition of in vitro tumor cell proliferation and invasion. The results are different from those observed by Petitclerc and coworkers [24], probably due to the model used to study tumor growth (mouse xenograft model with human melanoma cells vs. chick CAM model with hamster melanoma cells). To date, no studies have quantified tetrastatin level in body fluids and extracts. Therefore, in our study we focused on the development of a competitive ELISA, a rapid and convenient method, to measure tetrastatin. We first performed a direct ELISA to select the appropriate antibody dilution and coating amount, a dilution of 1/10,000 of the primary antibody and a coating of 500 ng/well of rh tetrastatin permitted to obtain an absorbance
Fig.4. ELISA measurement of tetrastatin in body fluids. (A) Serum samples from 53 different patients were assessed for their tetrastatin content. A simple plot for each patient corresponds to the mean of triplicates. (B) Bronchial aspiration fluids from 22 different patients were assessed for their tetrastatin content. A simple plot for each patient corresponds to the mean of triplicates. (C) Bronchoalveolar lavage fluids from 20 different patients were assessed for their tetrastatin content. A simple plot for each patient corresponds to the mean of triplicates.
5
of 1 unit, and we selected these two parameters for the competitive ELISA development. Intra-CVs ranged from 1.06% at 5 lg/ml to 2.8% at 0.0198 lg/ml. Inter-CVs ranged from 6.7% at 5 lg/ml to 9.5% at 0.0198 lg/ml. Inter-CVs are better for higher concentrations than for lower concentrations but are widely acceptable if we refer to commercially available competitive ELISA. No circulating tetrastatin has been reported to date. We tried to detect tetrastatin in serum of patients because it was easy to obtain and implement. This assay was sensitive and had a linear range from 2 to 2500 ng/ml (R2 = 0.9983). We detected amounts ranging from 18 to 300 ng/ml in serum. Tumstatin was also detected in human serum by quantitative ELISA (12.6– 147.2 ng/ml) [16] and was reported to be cleaved by matrix metallopeptidase 9 (MMP-9) [15]. Because the a4(IV) collagen chain is present as [a3(IV); a4(IV); a5(IV)] heterotrimers, we can assume that tetrastatin is also cleaved in vivo by such proteases. The aim of this study was to detect circulating tetrastatin. For that purpose, unselected patients with very different pathologies were tested. The small number of patients for each pathology tested did not allow performing statistical analysis. Determining the tetrastatin level in serum from patients with lung carcinomas, for instance, could be the aim of another study with large cohorts of patients. It could also be interesting to detect tetrastatin in other body fluids such as bronchial aspiration and bronchoalveolar lavage fluids. Our competitive ELISA was also used to determine tetrastatin level in bronchial aspirations of 22 patients. Tetrastatin concentrations varied from 7.3 to 210.7 ng/ml. The small number of patients included in this study did not permit finding significant differences according to pathologies. Type IV collagen fragments were reported as markers of liver and pulmonary fibrosis [25,26]. We used our competitive ELISA to determine tetrastatin levels in bronchoalveolar lavage fluids of 20 patients. Such an approach was previously used to study collagen-like glycoprotein (surfactant protein A and surfactant protein D) modulation with aging and emphysema development [27]. Tetrastatin concentrations varied from 2.5 to 16.3 ng/ml, depending on the patient. A clinical study is currently under investigation to determine tetrastatin amounts in bronchial aspiration and bronchoalveolar lavage fluids from patients with different infectious, inflammatory, or malignant bronchoalveolar diseases. Because the expression of other NC1 domains from basement membrane collagens was previously reported to be altered in cancer progression [28–30], we used the competitive ELISA to detect tetrastatin in healthy and tumor tissue extracts. The tetrastatin level was studied in pulmonary tissue extracts from 12 patients because this tissue contains large amounts of [a3(IV); a4(IV); a5(IV)] heterotrimer. In healthy tissue, tetrastatin concentrations ranged from 11.6 to 40.7 ng/mg of total protein. In cancer tissue, tetrastatin concentrations ranged from 20.7 to 80.8 ng/mg of total
Fig.5. ELISA measurement of tetrastatin in lung tissue extracts. Lung tissue extracts from 12 different patients were assessed for their tetrastatin content. For each patient, cancer tissue was compared with healthy lung tissue. A simple plot for each patient corresponds to the mean of triplicates. ***P < 0.02 according to paired Student’s t test.
Please cite this article in press as: A. Dupont-Deshorgue et al., A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.023
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protein. The tetrastatin level was significantly higher in cancer tissue than in healthy tissue (paired Student’s t test, P < 0.002) (Fig. 5). The small number of patients included in the study did not allow performing statistical analysis depending on the grade of the tumor. Larger cohorts of patients would be needed to eventually propose tetrastatin as a diagnostic cancer biomarker. Nevertheless, this study allowed us to demonstrate the ability of the ELISA to point out differences in the level of tetrastatin in healthy and cancer lung tissues. This could be extended to other cancer types. In conclusion, the competitive ELISA we have developed is a rapid and convenient method to detect tetrastatin in body fluids and tissue extracts from patients whose basement membrane metabolism may be altered.
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Acknowledgments
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This work was supported by grants from the Université de Reims Champagne–Ardenne, the Centre National de la Recherche Scientifique (CNRS), the Ligue Nationale Contre le Cancer, the Region Champagne–Ardenne, and the GIRCI est (AAP Jeune Chercheur Broncoll). The authors thank François-Xavier Maquart, Lukasz Szpak, Vincent Baudouin, and Saviz Nasri for their contributions to the work. The authors also thank Sylvette Gobert (Université de Reims Champagne–Ardenne) for improving the English language of the manuscript.
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Please cite this article in press as: A. Dupont-Deshorgue et al., A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.04.023
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