Journal of Medical Virology 86:1256–1266 (2014)

Respiratory Syncytial Virus Detection in Cells and Clinical Samples by Using Three New Monoclonal Antibodies  mez,1,2 Jorge E. Mora,1 Claudia M. Corte´s,3 Claudia A. Riedel,3 Marcela Ferre´s,4 Roberto S. Go Susan M. Bueno,1,2 and Alexis M. Kalergis1,2,5* 1

Millennium Institute on Immunology and Immunotherapy, Departamento de Gene´tica Molecular y Microbiologı´a, Facultad de Ciencias Biol ogicas, Pontificia Universidad Cat olica de Chile, Santiago, Chile 2 INSERM U1064, Nantes, France 3 Millenium Institute on Immunology and Immunotherapy, Departamento de Ciencias Biol ogicas, Facultad de Ciencias Biol ogicas y Facultad de Medicina, Universidad Andre´s Bello, Santiago, Chile 4 Centro de Investigaciones Medicas, Facultad de Medicina Pontificia Universidad Cat olica de Chile, Santiago, Chile 5 Departamento de Inmunologı´a Clı´nica y Reumatologı´a, Pontificia Universidad Cat olica de Chile, Santiago, Chile

Acute respiratory infections caused by the respiratory syncytial virus (RSV) are important health burdens that affect infants worldwide. RSV is also an important cause of morbidity and disease in adults, which causes enormous economic losses. At the present time, RSV infection is diagnosed by immunofluorescence, test pack and/or PCR, obtaining better results with PCR than with any other technique. The production of new monoclonal antibodies (mAbs) capable of detecting RSV in clinical samples is necessary to generate better and faster diagnosis tools for RSV. In this study, three new mAbs, directed against the RSV N and M2-1 proteins, were evaluated for the detection of RSV in clinical samples. Nasopharyngeal swabs were obtained from: 27 RSVpositive patients; 15 human metapneumovirus (hMPV)-positive patients; and 6 healthy controls. To evaluate RSV presence in these samples, clinical samples and RSV-infected cells were tested by Enzyme-Linked ImmunoSorbent Assay (ELISA), flow cytometry, immunofluorescence, and dot-blot assays. Specificity and sensitivity were determined for each mAb by using purified RSV antigens and antigens from different viruses. Infected cells and clinical samples tested with the three new mAbs resulted positive by immunofluorescence, ELISA, flow cytometry, and dot blot. No false positives were obtained in samples infected with other respiratory virus (hMPV) or from healthy controls. These results suggest that these new antiRSV mAbs can be considered for the rapid and reliable detection of RSV on infected cells and clinical specimens by multiple immunological approaches. J. Med. Virol. 86:1256–1266, 2014. # 2013 Wiley Periodicals, Inc. C 2013 WILEY PERIODICALS, INC. 

KEY WORDS:

RSV; monoclonal antibodies; clinical samples; diagnosis; ELISA

INTRODUCTION The respiratory syncytial virus (RSV) is an enveloped, negative, single-stranded RNA virus belonging to the Mononegavirales order, Paramyxoviridae family, Pneumovirus genus, and Respiratory Syncytial virus species, with a genome that encodes 11 proteins [Hacking and Hull, 2002]. This virus is the major causative agent of acute respiratory tract infections in infants worldwide, leading to severe outbreaks during the winter season. According to the World Abbreviations: RSV, respiratory syncytial virus; hMPV, human metapneumovirus; ADV, adenovirus; ELISA, EnzymeLinked ImmunoSorbent Assay; PBS, phosphate-buffered saline; PBS/1% BSA, phosphate-buffered saline containing 1% of bovine serum albumin; E. coli, Escherichia coli; mAbs, monoclonal antibodies; RT, room temperature; RT-PCR, reverse transcriptase polymerase chain reaction Grant sponsor: FONDECYT (Fondo Nacional de Desarrollo Cientı´fico y Tecnol ogico); Grant numbers: 1110397; 1110518; 1100926; Grant sponsor: Biomedical Research ConsortiumChileand Millennium Nucleus on Immunology and Immunotherapy; Grant number: P04/030-F. Competing interests: A patent has been filed for some of the antibodies described in this work.
Correspondence to: Alexis M. Kalergis, Millennium Institute on Immunology and Immunotherapy, Departamento de Gene´tica Molecular y Microbiologı´a, Facultad de Ciencias Biol ogicas, Pontificia Universidad Cat olica de Chile. Santiago 8331010, Chile. E-mail: [email protected], [email protected] Accepted 3 September 2013 DOI 10.1002/jmv.23807 Published online 22 October 2013 in Wiley Online Library (wileyonlinelibrary.com).

RSV Detection With Three New Antibodies

Health Organization, RSV infects 64 million people annually, of which 160,000 die [WHO, 2009]. RSV infection causes a wide range of medical conditions, from a mild rhinitis to more severe respiratory diseases, such as pneumonia or bronchiolitis [Domachowske and Rosenberg, 1999; Openshaw and Tregoning, 2005]. Furthermore, RSV infection is significantly more severe in infants, premature babies, children with congenital heart disease and immunocompromised individuals [Weisman, 2003; Cabalka, 2004; Carpenter and Stenmark, 2004]. RSV infection fails to induce protective immunological memory [Bont et al., 2002]. Therefore, reinfections are common, reducing their severity as the age of the patient increases. An accurate diagnosis is essential due to the severity and sequels of the disease caused by RSV. In public health services, current diagnostic methods include a test based on the detection of viral antigens by direct immunofluorescence of nasopharyngeal swab (NS), reverse transcriptase polymerase chain reaction (RT-PCR), and immunochromatographic tests (quick test) [Falsey et al., 2002]. The sensitivity of these tests to detect RSV are: about 95% by immunofluorescence; near to 100% by RT-PCR; and 85% by fast immunochromatographic tests [Henrickson, 2004]. Mahony [2008] has described that fast immunochromatographic tests and immunofluorescence assays have been replaced by even more efficient laboratory techniques for viral detection, such as new nucleic acid amplification technology, particularly multiplex PCR [Mahony, 2008]. Along these lines, molecular assays are important for the development of new and better techniques to detect different viral infections [Henrickson and Hall, 2007]. On the other hand, the generation of new monoclonal antibodies (mAbs) with high specificity and sensitivity is important to both research laboratories and public health services, because this kind of antibodies show high efficacy and specificity at detecting viral antigens. MAbs are proteins secreted by one B cell clone that show high specificity for a unique antigen. Expansion of B cell clones in culture could allow the production and purification of large amount of mAbs [Nelson et al., 2000]. MAbs can be used in several different applications for the treatment of diseases, such as cancer [Kelley et al., 1997; Esteva, 2004; Goodin, 2008], autoimmunity [Bennett et al., 2005; Robak and Robak, 2009], and viral infections [Mejias et al., 2004]. These molecules also can be used in various laboratory techniques, such as flow cytometry, immunofluorescence, Western blot, and EnzymeLinked ImmunoSorbent Assay (ELISA). mAbs have been important for understanding the molecular mechanisms of pathogenicity of various organisms by blocking specific molecular receptors [Kauffman et al., 1983; Shepley and Racaniello, 1994]. Likewise, mAbs can be used efficiently for direct detection of different viruses [Manoha et al., 2008], especially

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those that are the cause of major health burdens, such as RSV [Borek et al., 2006]. MAbs can be considered as a rapid, effective, and accurate for detecting RSV infection, providing an early diagnosis method that could greatly aid the physician to prescribe an appropriate treatment for the disease. In this report, we have evaluated the properties of three new mAbs for the detection of RSV antigens in samples from patients infected with this virus and in RSV-infected cells in vitro, using different immunological techniques. MATERIALS AND METHODS Virus Preparation Monolayers of 70% confluence of the human laryngeal carcinoma cell line HEp-2 cells (American Type Culture Collection) were infected with 3  107 pfu of RSV serogroup A, strain 12018-8 (clinical isolate obtained from the Instituto de Salud Pu´blica de Chile), in OptiMEM (GIBCO, Life Technologies, Carlsbad, CA) at 37˚C and 5% CO2. After 12 hr of incubation, culture media was replaced with fresh OptiMEM and incubated for another 48 hr. Next, small aliquots of supernatant from infected HEp-2 cells were frozen at 80˚C. Virus was tittered over HEp-2 cells in 96-well plates and screened for syncytia formation after crystal violet staining, as previously described [Gonzalez et al., 2008]. Generation of Purified RSV Antigens The use of expression vectors pET15b-N and pET15b-M2-1 with His-tag has been reported [Bueno et al., 2008]. These plasmids were used to produce N and M2-1 proteins, respectively, through individual electroporation into the Escherichia coli BL21 strain. Each isolated colony, selected by ampicillin resistance, was sub-cultured into 8–10 L of Luria Bertani broth supplemented with IPTG (0.5 mM). Cultures were incubated for 3 hr at 37˚C and then centrifuged at 4,600g for 5 min to pellet bacteria expressing recombinant proteins. The bacterial mass was lysed using lysis buffer (20 mM sodium phosphate, pH 7.5; 500 mM NaCl, 20 mM Imidazole) plus lysozyme (1 mg/ ml final concentration), supplemented with proteases inhibitors and incubated for 30 min at 4˚C. The lysed bacterial were sonicated for 10 cycles of 1 min at maximum power on ice. Lysates were centrifuged for 30 min at 20,000g and the filtered supernatants were incubated for 45 min in stirring at 4˚C, in a Ni-NTA column (Invitrogen, Life Technologies, Carlsbad, CA), for recombinant protein purification. The recombinant proteins were purified by Imidazole elution and collected in a 1 ml aliquot. The clear protein content was measured at 280-nm absorbance. Finally, the protein preparation was analyzed by Western blot using a commercial HRP-conjugated anti-RSV polyclonal antibody (US Biological, MA). J. Med. Virol. DOI 10.1002/jmv

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Generation of Monoclonal and Polyclonal Antibodies Against RSV N and M2-1 Proteins mAbs were generated by GrupoBios S.A. (Santiago, Chile) after immunizing mice with the purified proteins described above, according to standard protocols [Kohler and Milstein, 1975; Becker et al., 1994, 1996]. The concentration of antibodies was determined by ELISA, in which 96-well plates (NUNC, Thermo Scientific, Rochester, NY) were incubated for 1 hr at 37˚C with each of the three mAbs at different dilutions, using an anti-Melan A mouse mAb (Santa Cruz Biotechnology, Dallas, TX) to prepare the standard curve. Next, plates were blocked with 1% gelatin from cold water fish skin (Sigma-Aldrich, St. Louis, MO) for 2 hr at room temperature (RT), washed with phosphate-buffered saline (PBS)/Tween-20 (0.02%) and incubated for 1 hr at RT with anti-mouse IgGHRP (1 mg/ml) (Invitrogen, Life Technologies, Carlsbad, CA) at 1:1,500 dilution (final concentration 0.66 mg/ml) in blocking solution. Finally, the plate was washed with PBS/Tween-20 (0.02%), and 3–30 -5–50 tetramethyl-benzidine (100 mg/ml, Sigma-Aldrich) was used as a colorimetric substrate. The enzymatic reaction was stopped with 2 M H2SO4, and optical densities (OD) were measured at 450 nm. Based on this method, concentrations determined were: for the antiN clone 1E9/D1, 488 mg/ml, for anti-N clone 8E4/A7, 125 mg/ml and for anti-M21 clone 8A4/G9, 425 mg/ml. Further, the purified anti-M21 antibody clone 8A4/G9 was also generated through Grupobios S.A. (Santiago, Chile) and this antibody was used in a dot-blot assay. Polyclonal antibodies against N and M2-1 proteins were generated in New Zeeland rabbits, which were immunized with 200 mg of purified N and M2-1 proteins in alum (Pierce, Thermo Scientific, Rochester, NY). At days 14 and 28, rabbits were boosted with the same dose of proteins and sera were collected at day 35. Next, sera were adsorbed on lysates of HEp-2 cells and BL-21 E. coli. Briefly 1 ml of culture of BL-21 or HEp-2 cells were centrifuged for 10 min at 850g, then pellets were dissolved in extraction buffer (50 mM Tris–Cl pH 7.5 þ 5 mM EDTAþ 0.6% SDS) with proteases inhibitors, sonicated 10 times for 1 min at maximum power on ice, and centrifuged for 5 min at 850g. Next, supernatants were diluted in 50 ml of 0.1% PBS 1/Tween-20, and added onto a nitrocellulose membrane for 2 hr at RT with agitation. After this time, the membrane was washed three times with 0.1% PBS 1/Tween-20 and blocked with 0.1% PBS 1/Tween-20 and 5% milk for 3 hr. The membrane was washed for two times with 0.1% PBS 1/ Tween-20 for 10 min. Finally the membrane was incubated with sera diluted 1/10 in 0.1% PBS 1/ Tween-20 and 2% milk for 1 hr at RT, then 8 hr at 4˚ C, after this time the adsorbed sera were collected. Clinical Samples Clinical samples were obtained from infants with respiratory diseases in the Laboratorio de Infectologı´a J. Med. Virol. DOI 10.1002/jmv

and Hospital Clı´nico of the Pontificia Universidad Catolica de Chile. NSs samples from patients with respiratory tract infections (27), from patients with episodes of human metapneumovirus (hMPV) (15) and from healthy controls (6), were first diagnosed by immunofluorescence to determine whether they were positive for RSV or other virus with D3 UltraTM 8 DFA Kit (Diagnostics Hybrids, Athens, OH). Then, a fraction of each sample was stored at 20˚C. Once the experiments started, samples were thawed and assessed by ELISA. ELISA For the detection of RSV N and M2-1 proteins, direct ELISA was performed using Nunc 96-well plates. N and M2-1 proteins were obtained from recombinant bacteria and placed in wells, with quantities ranging from 500 ng to 25 ng/well. To activate ELISA plates with RSV, 2 ml vials of icepacked virus preparations, containing 1  107 PFU/ ml, were first exposed for 30 min to UV radiation (302 nm), using a 15-W lamp transiluminator, and then boiled for 5 min at 65˚C. Seventy-five microliters per well of the inactivated RSV sample were placed in the 96-well plates and incubated overnight (ON) at 4˚C. Next, plates were blocked by the addition of 3% PBS/BSA (Sigma-Aldrich) for 2 hr at RT, and then washed once with 0.02% PBS/Tween-20 and twice with PBS for 5 min each wash. Anti-N 1E9/D1 and 8E4/A7 clones, anti-M2-1 8A4/G9 clone, anti-F RSV antibody (Millipore, Billerica, MA; MAB8599 131-2A clone), or the antibody against RSV from D3 UltraTM 8 DFA Kit (Diagnostics Hybrids, Athens, OH), were added in 1% PBS/BSA at different dilutions. Finally, plates were washed and incubated with anti-mouse IgG-HRP (1 mg/ml) (Invitrogen, Life Technologies) at 1:1,500 dilutions in 1% PBS/BSA for 1 hr at RT. Next, plates were washed again, and 100 mg/ml 3–30 5–50 -tetramethyl-benzidine (Sigma-Aldrich) was used as a colorimetric substrate. The enzymatic reaction was stopped with 2 M H2SO4, and optical density was measured at 450 nm. Clinical samples were evaluated by sandwich ELISA, in which plates were first incubated with 1:350 dilution of each anti-N 1E9/D1 clone, anti-N 8E4/A7 clone or anti-M2-1 8A4/G9 clone in PBS for 1 hr at 37˚C. Final concentrations for: anti-N 1E9/D1 clone was 1.40 mg/ml; for anti-N 8E4/A7 clone was 357 ng/ml; and for anti-M2-1 8A4/G9 clone was 1.21 mg/ml. Then plates were blocked with 1% gelatin from cold-water fish skin (Sigma-Aldrich) in PBS for 3 hr. Later, plates were washed once with 0.02% PBS/Tween-20 and twice with 1 PBS. Plates were then incubated with the samples ON at 4˚C (50 ml per well). Next day plates were washed and incubated with rabbit polyclonal antibodies against RSV N or M2-1 proteins in 1:1,000 dilutions, in blocking solution for 2 hr at RT. Finally, plates were washed and incubated with 1:1,500 dilution of goat anti-rabbit

RSV Detection With Three New Antibodies

(H þ L) IgG-HRP (0.9 mg/ml, Bio-Rad, Hercules, CA) in blocking solution (final concentration 0.6 mg/ml) for 1 hr at RT. Then, plates were washed, and 100 mg/ml 3–30 -5–50 -tetramethyl-benzidine, at a final concentration of 100 mg/ml (Sigma-Aldrich), was used as a colorimetric substrate. Enzymatic reaction was stopped with 2 M H2SO4, and optical density at 450 nm was determined. Statistical analysis was performed in GraphPad Prism v5.0 software. Flow Cytometry HEp-2 cells were infected with RSV for 48 hr, and these cells were centrifuged for 6 min at 300g. The cells obtained were fixed with 2% paraformaldehyde for 10 min at 4˚C. Then, cells were permeabilized with 0.2% saponin in PBS for 30 min at RT. Next, cells were stained with 1:3,500 and 1:7,000 dilutions of each anti-N 1E9/D1 clone (139 ng/ml, 69 ng/ml), anti-N 8E4/A7 clone (35 ng/ml, 17 ng/ml), anti-M2-1 8A4/G9 clone (121 ng/ml, 60 ng/ml) or 1:3,500 dilution of anti-F RSV 348 mAbs in 1% PBS/BSA for 1 hr at 4˚ C. Cells were then washed with 1 PBS, centrifuged for 6 min at 300g and then incubated with IgG/IgM FITC-conjugated anti-mouse antibody (0.5 mg/ml, BD Pharmingen, San Jose, CA) at a dilution 1:1,000 for 1 hr at 4˚C, and washed with 1 PBS. Data acquisition was performed on a FACSCANTO II cytometer (BD Biosciences, San Jose, CA). Data were analyzed with FCS Express V3 software. Immunofluorescence Assay RSV infected HEp-2 cells were examined by immunofluorescence, as described previously [Bueno et al., 2008]. Briefly, 5  104 cells were cultured in 24well plates with 12-mm cover, and infected with 1  105 PFU of RSV. After 2 days, cells were fixed with 4% paraformaldehyde in 0.1 M PBS for 10 min at RT. The cells were permeabilized with 0.2% saponin in 0.1 M PBS–2% BSA for 30 min at RT. Later, cells were incubated ON at 4˚C with the antiN 1E9/D1 clone at final concentration of 4.88 mg/ml; anti-N 8E4/A7 clone at final concentration of 1.25 mg/ ml; anti-M2-1 8A4/G9 clone at final concentration of 4.25 mg/ml; or anti-F RSV 348 mAbs at a 1:100 dilution in 0.1 M PBS–2% BSA. The fixed cells were incubated with IgG/IgM FITC-conjugated anti-mouse antibody (0.5 mg/ml) (BD Pharmingen) at a 1:200 dilution (final concentration 2.5 mg/ml) for 1 hr at RT and then washed with PBS 1. Nuclei were stained with Hoescht 33342 (5 mg/ml) for 30 min. Coverslips were mounted and examined using an Olympus IX70 inverted microscope upright microscope Olympus BX51 (Center Valley, PA) and the pictures were analyzed in INFINITY Software V5.00. Dot-Blot Assays Lysates of RSV-infected HEp-2 cells or non-infected cells, BSA, and different amounts of purified viral

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proteins were spotted onto a nitrocellulose membrane (Thermo Scientific). Next, loaded membranes were air-dried for 15 min. The dot containing BSA was used as a negative control. Nitrocellulose membranes were subsequently blocked with 0.1 M PBS, 0.1% Tween-20, 2% non-fat milk, for 1 hr at RT. Then, membranes were incubated ON at 4˚C with the mAbs for RSV, in blocking solution at different concentrations: anti-N 1E9/D1 clone, 4.88 mg/ml to 48.8 ng/ml; anti-N 8E4/A7 clone, 1.25 mg/ml to 12.5 ng/ml; and anti-M2-1 8A4/G9 clone, 4.15 mg/ml to 41.5 ng/ml. A 1:10,000 Peroxidase-AffiniPure Goat Anti-Mouse IgG (H þ L) (Jackson ImmunoResearch, West Grove, PA, #115-035-003) in PBS–0.1% Tween-20 (final concentration 80 ng/ml) was used as secondary antibody and incubated for 1 hr at RT. Then, membranes were washed in PBS–0.1% Tween-20 and then incubated with the enhanced chemiluminescence Western blot detection system (ECL, Amersham, Uppsala, Sweden), to visualize the proteins. The antibody–antigen reaction was visualized and summarized in Table I. RESULTS Detection of Purified RSV Antigens by ELISA To evaluate the specificity of the mAbs developed in this study, ELISA was performed using purified recombinant RSV antigens obtained as described in the Materials and Methods Section. To determine the specificity of these antibodies, different amounts of antigens were used, ranging from 500 to 25 ng. In each case, samples without antigen were included as controls. As shown in Figure 1, all the mAbs were able to recognize their respective antigens with significant higher sensitivity as compared to the nonantigen control. For the case of anti-N 1E9/D1 clone, we observed significant higher detection in all the tested antigencoated compared to the control (without antigens) samples. Similar data were obtained for the anti-M21 8A4/G9 clone (Fig. 1A,C). However, for the anti-N 8E4/A7 clone, significant detection was observed only up to 50 ng of the RSV antigen (Fig. 1B). Thus, antiN and anti-M2-1 clones were capable of detecting their cognate antigens with significant specificity on purified RSV antigen-coated ELISA plates. In order to study the capacity of these mAbs to recognize purified antigens, and to determine the minimal dilution able to detect RSV proteins, a direct ELISA was performed. In this assay, plates were coated with 100 ng of the purified antigen (or 500 ng, data not shown) or uncoated, and different concentrations of each of the three mAbs was added (anti-N 1E9/D1 clone, ranged from 4.88 mg/ml to 9.76 ng/ml; anti-N 8E4/A7 clone, from 2.5 mg/ml to 6.25 ng/ml; and anti-M2-1 8A4/G9 clone, from 4.25 mg/ml to 6.5 ng/ml). Interestingly, in the case of anti-N 1E9/D1 clone, RSV proteins were detected in all the dilutions (Fig. 2A). Similar results were obtained with the J. Med. Virol. DOI 10.1002/jmv

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Gomez et al. TABLE I. Summary of the Sensitivity and Specificity of the Monoclonal Antibodies in the Dot-Blot Assays

A:

Monoclonal antibody anti-N clone 1E9/D1

RSV infected HEp-2 cells 1 mg

48.8 ng/ml 488 ng/ml 1 mg/ml 4.88 mg/ml Monoclonal antibody anti-N clone 1E9/D1

B:

48.8 ng/ml 488 ng/ml 1 mg/ml 4.88 mg/ml Monoclonal antibody anti-N clone 8E4/A7 12.5 ng/ml 125 ng/ml 0.25 mg/ml 1.25 mg/ml Monoclonal antibody anti-N clone 8E4/A7

C:

12.5 ng/ml 125 ng/ml 0.25 mg/ml 1.25 mg/ml Monoclonal antibody anti-M2-1 clone 8A4/G9 42.5 ng/ml 425 ng/ml 0.85 mg/ml 4.25 mg/ml Monoclonal antibody anti-M2-1 clone 8A4/G9

42.5 ng/ml 425 ng/ml 0.85 mg/ml 4.25 mg/ml

20 mg

 þ  þþþ  þþþ  þþþ His-tag His-tag RSV M2 RSV P 1 mg 1 mg         RSV infected HEp-2 cells 1 mg 20 mg    þþþ  þþþ  þþþ His-tag His-tag RSV M2 RSV P 1 mg 1 mg         RSV infected HEp-2 cells 1 mg 20 mg þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ His-tag His-tag RSV N RSV P 1 mg 1 mg        

Uninfected HEp-2 cells 1 mg

20 mg

        Control Control () () BSA P3 ADV         Uninfected HEp-2 cells 1 mg 20 mg         Control Control () () BSA P3 ADV         Uninfected HEp-2 cells 1 mg 20 mg         Control Control () () BSA P3 ADV        

Purified proteins 1 mg

0.5 mg

25 ng

þþþ þþþ þþþ þþþ

þþþ þþþ þþþ þþþ

þþþ þþþ þþþ þþþ

Purified proteins 1 mg 0.5 mg 25 ng þþ þþ þþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ

Purified proteins 1 mg 0.5 mg 25ng þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ þþþ

Data shown are the concentrations of the mAbs. (A) anti-N 1E9/D1 clone, (B) anti-N 8E4/A7 clone, and (C) anti-M2-1 8A4/G9 clone from 1:10,000 to 1:100. It also includes samples, which were incubated with the mAb. HEp-2 cells infected with RSV, uninfected cells, purified protein of interest, purified viral protein (indicated as His-tag, as the same His-tag of the protein of interest, in each case) and negative controls (BSA and protein of ADV). The highest sensitivity is indicated as þþþ, medium as þþ, low as þ, and null is .

Fig. 1. Direct ELISA for the detection of purified RSV antigens by different mAbs. Plates were coated with different amounts of purified RSV antigens: 500–25 ng of protein per well, per duplicate. Data shown in graphs are optical density results obtained with (A) anti-N 1E9/D1 clone at 4.88 mg/ml, (B) anti-N 8EA/A7 clone at 1.25 mg/ml, and (C) anti-M2-1 8A4/G9

J. Med. Virol. DOI 10.1002/jmv

clone at 4.25 mg/ml. Wells with no antigen (controls) were included in all the experiments. Data are means (SEM) of at least two independent experiments.
P < 0.05;

P < 0.01;


P < 0.0001; ns: no significant difference, treatment versus control by one-way ANOVA and posteriori Dunnett’s multiple comparisons test.

RSV Detection With Three New Antibodies

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Fig. 2. Limiting-dilution assays for detection of purified RSV antigens. Wells were coated with 100 ng of purified antigens, N protein in (A) and (B), and M2-1 protein in (C). Graphs show optical density at 450 nm. A: For anti-N 1E9/D1 clone, concentration ranged from 4.88 mg/ml to 9.76 ng/ml. B: For anti-N 8E4/ A7 clone, concentration ranged from 2.5 mg/ml to 6.25 ng/ml. C:

For anti-M2-1 8A4/G9 clone, concentration ranged from 4.25 mg/ ml to 8.5 ng/ml. As controls, we included wells with no antigen. Data are means (SEM) of at least two independent experiments.
P < 0.05;

P < 0.01;


P < 0.0001; ns: no significant difference, proteins versus control by one-way ANOVA and posteriori Dunnett’s multiple comparisons test.

anti-M2-1 mAb (Fig. 2C). However, in the case with the anti-N 8E4/A7 clone, the limit of detection was 25 ng/ml (Fig. 2B). These results suggest that this ELISA was highly sensitive, using decreasing concentrations of these three mAbs, for detecting purified RSV antigens. Importantly, the anti-N 1E9/D1 clone and anti-M2-1 8A4/G9 clone were able to detect RSV antigens, at all the tested dilutions.

agent of respiratory infections in patients attending healthcare services. To determine the ability of the mAbs to recognize RSV antigens on infected cells, mAbs were tested by immunofluorescence microscopy. HEp-2 cells were infected with RSV for two days and evaluated for cytopathic effect. Antibodies previously tested for RSV detection were used, as positive controls, to demonstrate cell infection by RSV (see the Materials and Methods Section). Permeabilized cells were stained with each of the three new mAbs, anti-N 1E9/D1 and 8E4/A7 clones and anti-M2-1 8A4/ G9 clone. Data show that all the tested antibodies were able of detecting RSV-infected cells and a similar cytoplasmatic staining was observed for the three mAbs (Fig. 4). These findings suggest that these antibodies can be used as new diagnostic tool to detect RSV-infected cells from clinical samples.

Detection of RSV Infected Cells by Flow Cytometry With New Monoclonal Antibodies In order to evaluate different uses for the mAbs generated in this study, RSV infected cells were analyzed by flow cytometry using the new mAbs and compared with a previously reported monoclonal anti-F antibody. HEp-2 cells were infected with RSV for 2 days and evaluated for virus-induced cytopathic effects by microscopy. Syncytia production in these cells has been described as a parameter of infection with RSV [Shigeta et al., 1968]. In addition, cells were stained with anti-F RSV 348 antibody, which has been previously shown to recognize F protein on RSV infected cells [Bourgeois et al., 1991; Gonzalez et al., 2008] and analyzed by flow cytometry (Fig. 3). Later, fixed samples were permeabilized to detect the presence of N and M2-1 proteins in the cytoplasm of RSV infected cells and stained with the three different mAbs. For each of the three mAbs evaluated, we obtained similar results: approximately 70% of cells showed increased fluorescence for both N and M2-1 proteins (Fig. 3). These data suggest that these three mAbs are efficient at detecting RSV infected cells by flow cytometry. Detection of RSV Proteins in Infected Cells by Immunofluorescence Immunofluorescence has been used as the standard method, to determine whether RSV is the etiologic

Detection of RSV Antigens by Dot Blot Dot-blot assays were carried out by adding purified RSV proteins or lysated from RSV infected cells onto a nitrocellulose membrane to be detected by each of the respective mAbs. Testing the three mAbs by this technique allows the selection of the best antibody combination that could be considered for developing an immunochromatographic diagnostic kit. It was observed that a concentration equal to 488 ng/ml of the anti-N 1E9/D1 clone was able to recognize specifically RSV antigens on the dot-blot assays (Fig. 5B). Furthermore, this clone was able to recognize as low as 25 ng of purified RSV N protein. Moreover, by using this technique, it was also possible to detect the presence of RSV proteins in infected cells (20 mg of proteins from infected cells). These results suggest that this clone is highly specific, because it does not recognize negative controls, such as uninfected cells or other His-tagged proteins that were produced in the same manner as N-RSV, such J. Med. Virol. DOI 10.1002/jmv

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Fig. 3. RSV can be detected in infected HEp-2 cells by flow cytometry. HEp-2 cells were infected with RSV (1  105 PFU) for 48 hr and analyzed by flow cytometry using different antibodies. A: Histograms are representative of RSV-infected HEp-2 cells for all the three monoclonal antibodies, showing results obtained with the anti-N 1E9/D1 clone (upper left), anti-N 8E4/A7 clone (upper right), anti-M2-1 8A4/G9 clone (bottom left) and positive control with the anti-F RSV 348 antibody (bottom right). The marker on histograms was defined according to control samples (red line). Graphs in (B) and (C) show the percentage of RSV infected HEp-2 by flow cytometry, white bars are uninfected HEp-2 cells and black

bars are RSV-infected HEp-2 cells. B: Data shown were obtained with 1 in 3,500 dilution (for the anti-N clones 1E9D1 and 8E4/A7, were 140 and 35.7 ng/ml, respectively; and for the anti-M2-18A4/G9 clone was 121 ng/ml). C: Data shown were obtained with 1 in 7,000 dilution (for the anti-N clones 1E9D1 and 8E4/A7, were 69.7 and 17.8 ng/ml, respectively; and for the anti-M2-1 8A4/G9 clone was 60.7 ng/ml). As a control, cells were stained with the secondary antibody only. Data are means (SEM) of three independent experiments.

P < 0.01;


P < 0.001; ns: no significant difference, infected versus uninfected by two-way ANOVA and posteriori Bonferroni posttest.

as P-RSV and M2-1-RSV proteins. Table IA summarizes the results obtained by dot-blot assays for the anti-N 1E9/D1 clone antibody. In addition, the anti-N 8E4/A7 clone antibody was able to detect RSV antigens in infected cells (Fig. 5C). Specifically, using this mAb at a concentration equal to 125 ng/ml it is possible to detect 20 mg of protein from RSV infected cells. However, 1 mg of RSV proteins was not detected in infected HEp-2 cells at all the concentrations tested. Nevertheless, this clone was able to detect purified RSV N protein as low as 25 ng, at a concentration of the antibody equal to 12.5 ng/ml. These results suggest that the antibody is specific, because it does not recognize negative controls, such as uninfected cells nor other Histagged RSV proteins, such as M2-1 and P. Table IB

Fig. 4. Detection of RSV antigens in infected HEp-2 cells by immunofluorescence. HEp-2 cells were infected with RSV for 48 hr and analyzed by immunofluorescence. Images show in green the fluorescence derived from (A) anti-N 1E9/D1 clone (4.8 mg/ml), (B) anti-N 8E4/A7 clone (1.25 mg/ml), and (C) antiM2-1 8A4/G9 clone (4.25 mg/ml). Images were taken at 40 magnification and were representative for two independent experiments. We included nuclear staining by using Hoescht 33342.

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RSV Detection With Three New Antibodies

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because it does not show signal for negative controls, such as uninfected cells or proteins from another virus (ADV). Table IC summarizes the results obtained by dot-blot assays performed with the antiM2-1 8A4/G9 clone. Detection of RSV in Clinical Samples From Patients Diagnosed With RSV Using the Three New mAbs by Sandwich ELISA

Fig. 5. Dot-blot assays confirm monoclonal antibody specificity for RSV antigens. A: Schematic representation of the membrane used to load samples and the protein concentrations used. The exposed film is shown below. B: Dot blots for the anti-N 1E9/D1 clone. C: Dot blots for the anti-N 8E4/A7 clone. D: Schematic representation for the dot-blot assays with the anti-M2-1 8A4/G9 clone. E: Dot blots for the clone against RSV M2-1 protein. The effective recognition of antigens by each antibody is reflected by the spot intensity, which appears with variability according to the different antibody concentrations tested. The film on the right corresponds to the dot blot of infected or uninfected cells. In all the experiments, controls were included. These consisted of other viral proteins, such as P3 from ADV, His-tagged P from RSV or a non-related protein, such as BSA.

summarizes the results obtained by dot-blot assays for the anti-N 8E4/A7 clone. As described above, nitrocellulose membranes with purified viral proteins or cells infected with RSV were incubated with the mAb anti-M2-1 8A4/G9 clone. In this assay we used purified antibody. Figure 5E shows that a dilution of 42.5 ng/ml of antiM2-1 8A4/G9 clone antibody could span a wide range of RSV M2-1 protein detection (25 ng to 1 mg). Moreover, this antibody did not recognize the proteins: the adenovirus (ADV) P3; RSV P; RSV N or BSA, at any of the dilutions tested. Furthermore, this mAb can detect efficiently RSV in infected cells (1 and 20 mg of protein from whole cell lysate of RSV infected cells, using an antibody concentration equal to 42.5 ng/ml). At this concentration, the antibody is highly specific,

Sandwich ELISAs were performed to verify the ability of mAbs to diagnose patients with RSV on clinical samples (NSs). Samples were obtained from the Laboratorio de Infectologı´a, Hospital Clı´nico, Facultad de Medicina, Pontificia Universidad Catolica de Chile. Patients were previously diagnosed with RSV by the standard Immunofluorescence technique. Sandwich ELISAs were performed, using the mAbs anti-N 1E9/D1 and 8E4/A7 clones, and anti-M2-1 8A4/G9 clone as capture antibodies. Rabbit polyclonal antibodies were used against the same RSV proteins. Table II describes the groups of patients that were tested by the sandwich ELISA approach. Twentyseven different RSV patients were tested in total (Table II and Fig. 6). In addition, 15 hMPV positive patients and 6 healthy controls were included in these experiments as controls. As a result, no detection was observed in samples from all the 21 controls determined by sandwich ELISA (Table II and Fig. 6). Particularly for the anti-N 1E9/D1 clone, 18 patients diagnosed with RSV were analyzed and 17 resulted positive (mean  SD; 0.616  0.163), equivalent to a ratio of 0.944 positive to total. For the case of anti-N 8E4/A7 clone, 18 patients diagnosed with RSV were analyzed and 17 resulted positive (0.588  0.215), with a ratio equal to 0.944. Using the anti-M2-1 8A4/G9 clone in 16 patients diagnosed with RSV, 15 resulted positive (0.641  0.186), with a ratio equal to 0.938 (Table II). These results suggest that the new mAbs can efficiently discriminate controls from patients infected with RSV and can be used for designing new diagnostic strategies.

DISCUSSION In this study, the use of three new mAbs against two RSV antigens, the N and M2-1 proteins, was evaluated. These mAbs were used to detect RSV by five different approaches: direct ELISA; sandwich ELISA; flow cytometry; immunofluorescence; and dotblot. It is essential to develop new tools for detection of RSV infection, which can allow fast, sensitive, efficient, and affordable diagnostic kits. Thus, the mAbs described above were shown by several different techniques that can efficiently detect RSV antigens, which suggest that they could be considered as useful tools for the diagnosis of RSV infection. Dot-blot assays, along with the other techniques described in this article, provide useful information about the sensitivity of each particular mAb for viral J. Med. Virol. DOI 10.1002/jmv

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Gomez et al. TABLE II. Clinical Samples and Their Analyses With the Three Anti-RSV Monoclonal Antibodies RSV patients

Anti-N RSV 18 (0.616  0.163) clone 1E9/D1 Anti-N RSV 18 (0.588  0.215) clone 8E4/A7 Anti-M2-1 RSV 16 (0.641  0.186) clone 8A4/G9

Positive Negative analysis analysis

hMPV patients

Positive Negative analysis analysis

Healthy patients

Positive Negative Ratio analysis analysis P/T

17

1

15 (0.161  0.054)

0

15

6 (0.140  0.023)

0

6

0.944

17

1

15 (0.104  0.064)

0

15

6 (0.123  0.030)

0

6

0.944

15

1

15 (0.083  0.044)

0

15

6 (0.095  0.031)

0

6

0.938

RSV patients: patients diagnosed positive for RSV. Positive analysis: patients diagnosed positive for RSV and were positive by ELISA analyses. Negative analysis: patients diagnosed positive for RSV and were negative by ELISA analyses. hMPV patients: patients diagnosed positive for hMPV. Healthy patients: healthy patients. P/T ratio: ratio between positive analysis and total RSV patients analyzed for each antibody. In parenthesis mean  SD.

antigens detection. As reported previously [Bhunia et al., 1991; Tsurumi et al., 2003], the dot-blot technique can be considered as a valid, useful and affordable method to qualitatively study the antigen– antibody binding reaction. These studies showed that these mAbs are specific for the detection of the respective RSV protein tested. Importantly, these mAbs neither recognize the His-tag domain from their cognate recombinant proteins nor other unrelated viral proteins. A specificity assay was included, in which all the three mAbs were tested with RSV, hMPV, and ADV-infected cells. In these assays, positive staining was only observed in samples derived from RSV-infected cells and not in samples from hMPV- or ADV-infected cells, suggesting that these mAbs are specific for RSV detection. There is a need for new and affordable approaches to detect RSV infection in clinical samples [Henrickson, 2004]. Currently, determination of RSV infection has been performed by standard Immunofluorescence (Diagnostic Hybrids) and PCR assays, but always persists the idea to generate more affordable and quickly tests for diagnosis of viral infections in affected patients. New tests that can be accessible, rapid, sensitive, and specific could greatly help the public health system in outbreaks caused by RSV. For the same reason, mAb production has significantly advanced in the last years, producing more sensitive and specific antibodies [Falsey et al., 2002; Mahony, 2008]. Along these lines, the results obtained by immunofluorescence assays shown in the Figure 4, suggest that the use of the new mAbs described in this work, could be useful to generate new diagnosis tests. Detection of N and M2-1 antigens, in RSV-infected HEp-2 cells by immunofluorescence is the first approach to obtain further reliable results with clinical samples. However, it is important to consider that measurement of cytopathy due to virus infection in cell culture after immunofluorescence might be required to verify uncertain Immunofluorescence results [Bromberg and Tannis, 1991]. For instance, in Chile cell culture is still the “gold standard” method. The three new mAbs presented in this study could be an important contribution, because they have an J. Med. Virol. DOI 10.1002/jmv

equivalent capacity to detect RSV in clinical samples as compared to the commercial kits currently used by the Chilean healthcare system to diagnose infection by this virus (D3Ultra 8TM DFA—Diagnostic Hybrids). Consistent with this notion is the observation that by sandwich ELISA these mAbs failed to detect only 1 out of 27 samples characterized previously as RSV-positive by immunofluorescence RSV. Comparison was carried out by ELISAs of the commercial anti-F-RSV protein antibody (MAB8599 clone 131-2A, Millipore, Billerica, MA) and the antibody used to detect this virus by means of the Diagnostics Hybrids kits with anti-M2-1 8A4/G9 clone mAb. As a result, improved detection was achieved with the anti-M2-1 8A4/G9 clone as compared with the both commercial antibodies (Supplementary Fig. 1). These data further support the notion that these three mAbs can be considered as useful tools to detecting RSV infection in clinical and research samples. While as part of this study was not possible to determine the RSV serogroup in the clinical samples tested, a report from the Instituto de Salud Publica de Chile-informed that the predominant serotype during 2011 was B (76.7%) over A (23.3%). Therefore, it is very likely that a higher proportion of the samples tested in this study were of the B serotype [ISP, 2012]. However, because the sequences of N and M2-1 proteins are conserved in both A and B serogroups, it is likely that these three mAbs are able to detect both RSV serogroups. In support of this notion, positive results were obtained in HEp-2 cells infected with RSV serotype A. Furthermore, the positive results obtained by flow cytometry lead to the possibility of developing new techniques to detecting RSV in clinical samples from patients, cell cultures or in vivo experiments, such as cytometric bead arrays or luminex. These techniques have been used to detect cytokines [Tarnok et al., 2003; Wong et al., 2006; Frey et al., 2008], antibodies [Ferbas et al., 2007], and intracellular signaling molecules [Morgan et al., 2004; Zartman et al., 2004]. Therefore, these mAbs may represent new tools to generate improved detection systems for RSV antigens and expand the range of techniques available to detecting the infection by this virus.

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Fig. 6. ELISA sandwich for detection of RSV in clinical samples. Samples obtained from the Laboratorio de Infectologı´a of the Pontificia Universidad Cat olica de Chile that were RSV positive by the standard immunofluorescence diagnostic assay, were analyzed by ELISA sandwich technique. In the graph are shown optical densities obtained for samples from 18 patients infected with RSV using the anti-N 1E9/D1 and 8E4/A7 clones; 16 with the anti-M2-1

8A4/G9 clone; 15 from patients infected with hMPV; and 6 from healthy patients tested with all the clones. Capture monoclonal antibodies were used in 1:350 dilution, and detection polyclonal antibodies in 1:1,000 dilutions. Data are means  SD of at least three independent experiments.


P < 0.001; ns: no significant difference, RSV or hMPV patients versus healthy patients by twoway ANOVA and Bonferroni posttest.

The mAbs characterized in this study could also be considered for further evaluation as an immune therapy to treat or prevent RSV infection. There is a need for new efficient antibodies to treat RSV infection that could complement the only currently clinical approved, but limited and high cost, humanized anti-F mAb (Palivizumab) [Johnson et al., 1997; The IMpact-RSV Study Group, 1998]. These mAbs can become excellent candidates for immune therapy for RSV, because they recognize proteins that are essential for the viral infectious process. In fact, the N and M2-1 proteins are the required for the formation of the nucleocapsid and transcription of RSV genome, respectively. Further, the ability of these new mAbs to recognize these antigens could aid to neutralize RSV infection and eventually translate into new passive immunization approaches. However, these potential approaches must be demonstrated by extensive additional research, which is out of the scope of the present study. In conclusion, three new mAbs are described and characterized in this study, which are specific for RSV N and M2-1 proteins. These three antibodies can be used as new tools to detect RSV infection in both, cells and in clinical samples. These reagents can become very useful, in view of the broad spectrum of techniques in which they can be applied. mAbs have also the potential to be used as protective therapies against RSV infection, leading to new research and clinical opportunities to prevent infection by this virus.

CONCLUSIONS Three mAbs against RSV were presented here, which are perfectly effective in detecting this virus in infected cells or purified RSV antigens by techniques such as: flow cytometry, with a percentage of detection of approximately 70% of infected cells; or by immunofluorescene, which is the currently standard technique to determine RSV infection in patients; ELISA, obtaining a high ratio of positive samples, which were previously confirmed for RSV infection, with the three mAbs, especially with the anti-N 1E9/ D1 clone. For this reason, the three new antibodies generated in this study, can be considered as important tools for RSV detection in clinical samples and thus for an accurate diagnosis for RSV infection. ACKNOWLEDGMENTS We are grateful to Ana Marı´a Contreras for assistance with the clinical samples, to Drs. Marı´a Olga Bargsted and Margarita Lay for critically proofreading of the manuscript and to Dr. Pierre Pothier for providing the anti-F 348 RSV antibody. REFERENCES Becker MI, Juica F, Jamett A, Tzichinovsky S, Barros S, Aguayo J, De Ioannes AE. 1994. Development of anti-human B blood group monoclonal antibodies suitable as a blood typing reagent. Hybridoma 13:303–310. Becker MI, Aguayo JE, Jamett A, Juica F, Yudelevich A, Foradori A, De Ioannes AE. 1996. An alternative ELISA for T4

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