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Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20

Production and Characterization of Monoclonal Antibodies against Horse Immunoglobulins Useful for the Diagnosis of Equine Diseases a

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Tiziana Di Febo , Mirella Luciani , Antonella Ciarelli , Grazia a

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Bortone , Chiara Di Pancrazio , Diamante Rodomonti , Liana Teodori a

& Manuela Tittarelli a

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Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, via Campo Boario, Teramo, Italy Accepted author version posted online: 06 Jun 2014.Published online: 25 Sep 2014.

To cite this article: Tiziana Di Febo, Mirella Luciani, Antonella Ciarelli, Grazia Bortone, Chiara Di Pancrazio, Diamante Rodomonti, Liana Teodori & Manuela Tittarelli (2015) Production and Characterization of Monoclonal Antibodies against Horse Immunoglobulins Useful for the Diagnosis of Equine Diseases, Journal of Immunoassay and Immunochemistry, 36:3, 253-264, DOI: 10.1080/15321819.2014.928780 To link to this article: http://dx.doi.org/10.1080/15321819.2014.928780

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Journal of Immunoassay and Immunochemistry, 36:253–264, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2014.928780

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PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST HORSE IMMUNOGLOBULINS USEFUL FOR THE DIAGNOSIS OF EQUINE DISEASES

Tiziana Di Febo, Mirella Luciani, Antonella Ciarelli, Grazia Bortone, Chiara Di Pancrazio, Diamante Rodomonti, Liana Teodori, and Manuela Tittarelli Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, via Campo Boario, Teramo, Italy



Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELISA versus purified immunoglobulins from donkey, cow, buffalo, sheep, pig, and chicken. Three MAbs (1B10B6C9, 1B10B6C10, 1B10B6E9) reacted only with horse and donkey IgG and IgM and, in western blotting, were specific for the Fc fragment of equine IgG. MAb 1B10B6E9 was used in chemiluminescent immunoblotting assay for the diagnosis of dourine and in indirect immunofluorescence assay (IFA) for the diagnosis of African horse sickness and dourine. Keywords horse immunoglobulins, immunoblotting assay, immunofluorescence assay, monoclonal antibodies, equine diseases

INTRODUCTION Many tests for the diagnosis of the equine diseases were developed and are presently in use, based on polyclonal antibodies as secondary antibodies.[1−4] The secondary polyclonal antibodies versus immunoglobulins of common animal species can easily be found in the market; moreover, their production in laboratory animals is simple and a large quantity of antibodies can be produced in a relatively short time. However, polyclonal antibodies have a lack of homogeneity between lots, due to the variability of the animal immune response; they may also react in a non specific way in immunological tests, with a consequent increase in the background signal. The elimination or the reduction of the non specific IgG component in the antisera requires tedious processes of immunoabsorption Address correspondence to Tiziana Di Febo, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, via Campo Boario, 64100 Teramo, Italy. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/ljii.

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that lengthen the time of preparation and increase the cost. In a different way, the production of mononoclonal antibodies requires highly competent staff and appropriate laboratories, but provides specific reagents easily purified and with batch to batch homogeneity. They can be used not only for the development of diagnostic tests, but also for the study of the human and animal immune system and for the purification and characterization of immunoglobulins. The use of anti-immunoglobulins monoclonal antibodies for the research and for diagnostic purposes is well described in the literature.[5−10] The aim of the present study was the production and characterization of anti-horse IgG monoclonal antibodies for use in diagnosis of equine diseases. One of the produced monoclonal antibodies (MAb 1B10B6E9) was tested both in immunoblotting and in indirect immunofluorescence for the diagnosis of dourine, a contagious disease of horses caused by protozoan parasite Trypanosoma equiperdum, and in indirect immunofluorescence for the diagnosis of African horse sickness, an infectious disease caused by an Orbivirus (Reoviridae). EXPERIMENTAL Animal Experimentation Animal experimentation was carried out in compliance with Italian national law (Legislative Decree 116/92[11] ) implementing Directive 86/609/EEC of the Council of the European Communities on the protection of animals used for experimental and other scientific purposes.[12] Horse IgG Purification Horse IgG were purified from 50 mL of horse serum, previously filtered through 0.22 μm filters (Millipore, Billerica, MA, USA), by affinity chromatography using a column prepacked with recombinant Protein A (HiTrap rProtein A FF, 5 mL, GE Healthcare, Uppsala, Sweden) according to the manufacturers’ instructions. Purified IgG were concentrated with a 100 kDa molecular weight cut-off centrifugal filters (Millipore) and resuspended in sterile 0.01 M phosphate buffered saline, pH 7.2 (PBS). Protein concentration of the purified IgG was determined spectrophotometrically using the ratio A280nm /IgG molar extinction coefficient. Purification of Immunoglobulins for MAbs Characterization Immunoglobulins (IgG and IgM) to be used for MAbs characterization were purified by affinity chromatography: donkey, sheep, and pig IgG were purified using a column HiTrap rProtein A FF, 5 mL (GE Healthcare); bovine and buffalo IgG were purified using a column HiTrap Protein G

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HP, 5 mL (GE Healthcare); purified horse, donkey, sheep, buffalo, and pig IgM were obtained using a column HiTrap IgM Purification HP, 1 mL (GE Healthcare). Purified bovine IgM and chicken IgG were purchased from Sigma (Sigma, St. Louis, MO, USA).

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Immunization of Mice and MAbs Production Four female Balb/c mice were inoculated intraperitoneally with 50 μg/mL (500 μL/mouse) of purified horse IgG diluted in complete Freund’s adjuvant (Sigma). Two weeks after the first immunization, mice were inoculated with 50 μg/mL (500 μL/mouse) of purified IgG diluted in incomplete Freund’s adjuvant (Sigma); a third inoculation was carried out on the 28th day with 50 μg/mL (500 μL/mouse) of horse IgG diluted in sterile PBS. On the 46th day a “booster” was carried out intraperitoneally with 50 μg/mL (500 μL/mouse) of IgG diluted in sterile PBS. After euthanasia of mice, splenocytes were collected and fused with murine Sp2/O-Ag-14 myeloma cell line (American Type Culture Collection, Manassas, VA, USA). Hybridomas obtained were cultured in Dulbecco’s Modified Eagle’s Medium containing 20% fetal bovine serum (Sigma) and HAT Media supplement 50X (HybriMax® , Sigma) and after two weeks they were screened by indirect ELISA. Ninety-six-well microplates (PolySorp, Nunc Brand Products, Roskilde, DK) were coated with purified horse IgG, diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6) to a final concentration of 10 μg/mL, and incubated overnight at 4◦ C. After blocking with 1% yeast extract in PBS containing 0.05% Tween 20 (PBST), hybridoma supernatants were added into the wells and the plates incubated for 1 hr at 37◦ C. After washing with PBST, a HRP-conjugated goat anti-mouse IgG (GE Healthcare) was added and the plates were incubated for 30 min at room temperature (RT). After further washings, 100 μL/well of the chromogen substrate 3,3 , 5,5 tetramethylbenzidine (TMB) (Sigma) was added into each well for 30 min, the reaction stopped with 0.5 N sulphuric acid and the optical density (OD) values were measured at 450 nm. Hybridomas with OD450 nm values ≥ 0.300 were considered positive. Hybridomas secreting antibodies against horse IgG were cloned according to the method of limiting dilutions.[13−15] Monoclonal Antibodies Characterization MAbs isotype was determined using a commercial kit (Mouse-Typer® Isotyping Panel, Bio-Rad, Hercules, CA, USA) according to the manufacturers’ instructions. Crossreactivity of MAbs against IgG and IgM of different species (donkey, cow, buffalo, sheep, pig, and chicken) were determined in indirect ELISA, as described above, using microplates coated with 10 μg/mL of purified IgG and IgM.

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Monoclonal Antibodies Purification Supernatants produced on a large scale were purified in the same manner of horse IgG used for the immunization of mice; after the elution step, the buffer was replaced with PBS using 100 kDa molecular weight cut-off centrifugal filters (Millipore). The concentration of purified MAb IgG was determined spectrophotometrically (280 nm).

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MAb Labeling with HRP and FITC An aliquot of the purified MAbs was conjugated with horseradish peroxidase (Sigma) as described in the literature.[16] Another aliquot was conjugated with fluorescein-5-isothiocyanate (FITC): MAb was dialyzed in 0.5 M carbonate buffer, pH 9.5; 320 μg of FITC (Invitrogen, Carlsbad, CA, USA) solubilized in 32 μL of DMSO (Sigma) were added to 1 mg of IgG and incubated for 1 hr at RT. The conjugate was then dialyzed in PBS. Papain Digestion of Horse IgG Horse IgG purified by affinity chromatography were digested with papain. Papain from papaya latex (2x crystallized, Sigma), was solubilized in PBS 0.01 M pH 7.5, 0.02 M EDTA, 0.02 M L-Cysteine at a concentration of 0.1 mg/mL and then was added to an equal volume of horse IgG, suspended in PBS at a concentration of 2 mg/mL. The mixture was incubated for 20 hr at 37◦ C in a water bath. The enzymatic reaction was stopped by addition of 0.3 M iodoacetamide solubilized in PBS (100 μL per mL of reaction mixture). After incubation at RT for 30 min, the digested IgG were used for the characterization of MAbs anti-horse IgG in SDS-PAGE. SDS-PAGE and Western Blotting MAbs anti-horse IgG were further characterized by SDS-PAGE and western blotting. 10 μg of papain digested and undigested horse IgG were loaded into a NuPAGE® 10% Bis-Tris gel (Invitrogen) and separated by electrophoresis at constant voltage of 200V. At the end of the run, gel was stained with Simply Blue Safe Stain (Invitrogen) overnight at RT and destained in distilled water to remove the background. The electrophoretic separation of horse IgG was repeated as described above and proteins were blotted on iBlot® transfer stack mini nitrocellulose membranes (Invitrogen) using iBlot® Dry Blotting System (Invitrogen). At the end of the blotting, membranes were blocked with PBST containing 5% skimmed milk for 2 hr at RT and, after washing with PBST for 5 min, incubated overnight at 4◦ C with

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MAb supernatants. After 3 washes with PBST, membranes were incubated for 1 hr at RT with a secondary antibody anti-mouse IgG HRP-conjugated (GE Healthcare) diluted 1:8000 in PBST containing 2.5% skim milk. After 3 washes with PBST and one with PBS, membranes were incubated with ECL SelectTM Western Blotting Detection Reagent (GE Healthcare). Image acquisition of stained gels and membranes was carried out with Chemidoc MP (Bio-Rad) and analyses performed using Image Lab Software version 4.0.1 (Bio-Rad).

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Immunoblotting Test for the Diagnosis of Dourine One of the selected anti-horse IgG MAbs (MAb 1B10B6E9) was employed to develop a chemiluminescent immunoblotting test (cIB) for the study of the antigenic patterns recognized by antibodies present in the sera of horses naturally and experimentally infected with Trypanosoma equiperdum.[17] Briefly, purified T. equiperdum OVI antigen (Onderstepoort Veterinary Institute, Onderstepoort, South Africa), used at a concentration of 1 × 108 Tryp/mL, was separated by SDS-PAGE using NuPAGE® 12% Bis-Tris gels (Invitrogen) at 200 V. Proteins were blotted onto nitrocellulose using an iBlot® Dry Blotting System (Invitrogen); membranes were then blocked with PBST containing 5% skimmed milk, cut into strips and then incubated overnight at 4◦ C with equine sera diluted 1:10 in PBST containing 2.5% skimmed milk. Strips were washed and incubated with MAb 1B10B6E9 HRP-conjugated diluted 1:100000 in PBST containing 2.5% skimmed milk and, after further washing, chemiluminescent substrate ECL SelectTM Western Blotting Detection Reagent (GE Healthcare) was added; detection of protein bands was carried out using the Chemidoc MP (BioRad). Molecular weight of immunocomplexes was estimated using Image Lab Software version 4.0.1 (Bio-Rad). Indirect Immunofluorescence Assay for the Diagnosis of Dourine The indirect immunofluorescence assay (IFA) was performed according to OIE Manual of Diagnostic Tests and Vaccines.[1] 10 μL of positive (IFA titer 1:320) and negative horse sera, diluted 1:80 in PBS, were, respectively, applied on the wells of slides coated with T. equiperdum OVI antigen and incubated in a humid chamber at 37◦ C for 30 min. Slides were then washed three times in PBS, air-dried, added with 10 μL of MAb 1B10B6E9 FITCconjugated diluted 1:320 in PBS containing 1% Evans Blue and incubated at 37◦ C for 30 min in a humid chamber. After further washes, slides were air-dried, mounted in glycerol/PBS (50/50) and examined by a fluorescence microscope (40× magnification; Axio Lab. A1, Zeiss, Oberkochen,

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D). The results obtained with the MAb 1B10B6E9 were compared with those obtained using an anti-horse IgG polyclonal antibody FITC-conjugated (Sigma) diluted 1:64.

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Indirect Immunofluorescence Assay for the Diagnosis of African Horse Sickness Vero cells cultures were prepared and infected with virus AHS serotype 9 (107,01 TCID50/mL). Glass slides were coated with infected Vero cells, fixed with cold acetone for 20 min and air dried. 10 μL of AHSV9 naturally infected horse positive serum (SN titre >1280) and negative serum, both diluted 1:20 in PBS, were applied on the antigen coated slides and incubated in a humid chamber at 37◦ C for 30 min. Slides were washed two times in PBS, rinsed in distilled water and air dried. Ten μL of MAb 1B10B6E9 FITCconjugated, diluted 1:320 in PBS containing 1% Evans Blue, were added to slides and incubated at 37◦ C for 30 min in a humid chamber. Slides were washed in PBS and distilled water as described above. After being washed and air-dried, the slides were mounted with glycerol buffer and cover slips and evaluated by fluorescence microscopy (40× magnification; Axio Lab. A1, Zeiss). RESULTS AND DISCUSSION Monoclonal Antibody Production and Characterization Two weeks after the cell fusion, about 200 hybridomas were produced; supernatants were tested in indirect ELISA to assess the production of antihorse IgG antibodies. Twenty-one clones with an OD450 nm greater than or equal to 2.000 were selected and further tested towards donkey, cow, buffalo, sheep, and pig immunoglobulins. Eighteen of the 21 selected MAbs crossreacted with IgG and IgM of species different from equids (cow, buffalo, sheep, and pig), while 3 clones (1B10B6C9, 1B10B6C10, and 1B10B6E9) reacted only with horse and donkey IgG and IgM. These last 3 MAbs showed isotype IgG2a , κ light chain. Fractionation of Horse IgG To determine which region of IgG is recognized by MAbs, horse IgG were digested with papain. Analysis performed by SDS-PAGE showed that the band of molecular weight between 50 and 60 kDa (heavy chain) disappeared almost completely, and a band of approximately 30 kDa (Fc fragment) and 3 closely spaced bands of approximately 25 kDa (Fab fragment) appeared; a 95 kDa band (probably IgG aggregates) also disappeared (Figure 1). Similar

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FIGURE 1 Monoclonal antibodies characterization. Comassie stain of undigested horse IgG (lane 1A) and IgG digested with 0.1 mg/mL papain for 20 hr (lane 1B). Western blot of undigested and papain digested horse IgG with MAb 1B10B6C9 (lanes 2A-2B), MAb 1B10B6C10 (lanes 3A-3B), and MAb 1B10B6E9 (lanes 4A-4B).

results were obtained by shortening the digestion time to 16 hr and 18 hr. The lysis of the heavy chain was rather less efficient using a lower concentration of papain (0.02 mg/mL) with incubation time of 16 hr, 18 hr, and 20 hr (data not shown). SDS-PAGE and Western Blotting MAbs that reacted only with horse and donkey IgG (1B10B6C9, 1B10B6C10, and 1B10B6E9) were characterized by western blotting using digested and undigested horse IgG. These 3 MAbs reacted with the heavy chain of horse IgG (50 kDa) and with the 95 kDa band of undigested IgG, while they reacted with the Fc fragment of the heavy chain (30 kDa) in the case of IgG digested with papain (Figure 1). Immunoblotting Test for the Diagnosis of Dourine MAb 1B10B6E9, HRP-conjugated, was used to develop a chemiluminescent immunoblotting test in order to characterize the antigenic pattern recognized by antibodies present in the sera of naturally and experimentally horses infected by Trypanosoma equiperdum and to compare the antigenic pattern with the corresponding pattern obtained from healthy horses. Data obtained showed that the cIB allowed to discriminate between healthy and infected animals. In fact, as in naturally as in experimentally infected horses, bands with molecular weight between 16 and 35 kDa were detected, while

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FIGURE 2 Immunoblotting test for the diagnosis of dourine. Analysis of positive sera (lanes 1–5) and negative sera (lanes 6–10).

they are not present in healthy horses (Figure 2). The cIB also allowed to study the kinetics of the humoral response in horses after infection with T. equiperdum (Figure 3, Table 1).[17] Indirect Immunofluorescence Assay for the Diagnosis of Dourine MAb 1B10B6E9, FITC-conjugated, was used in IFA for the diagnosis of dourine in horses, in substitution of a commercial polyclonal anti-horse-IgGFITC currently used. The MAb-FITC allowed an excellent discrimination between positive serum and negative serum and there were no unspecific reactions with the antigen T. equiperdum bound to the slides (no background); in the case of commercial polyclonal, there was instead a weak reaction with the antigen (Figure 4). Indirect Immunofluorescence Assay for the Diagnosis of African Horse Sickness MAb 1B10B6E9, FITC-conjugated, was able to discriminate the positive serum from negative serum. AHSV9-infected Vero cells, incubated with the positive serum, showed in the cytoplasm a granular green fluorescence, as “dust-like” small particles. No fluorescence in the infected cells incubated with the negative serum (Figure 5) and no unspecific reactions of MAb with not infected Vero cells (data not shown) were seen.

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FIGURE 3 Immunoblotting test for the diagnosis of dourine: analysis of sera collected from an horse experimentally infected by transfusion of Trypanosoma equiperdum infected horse blood (DBI: days before infection; DPI: days post infection). TABLE 1 Results of complement fixation test (CFT), immunofluorescence assay (IFA) and RT-PCR analysis of sera collected from an horse experimentally infected by transfusion of Trypanosoma equiperdum infected horse blood Time (days)a 84 DBI 3 DPI 10 DPI 14 DPI 17 DPI 70 DPI 125 DPI a DBI:

CFTb

IFAc

RT-PCR (blood)

Negative Negative Negative 1:5 1:5 1:160 1:2560

Negative Negative Negative Negative 1:80 1:320 ≥1:640

Negative Positive Positive Positive Positive Positive Positive

days before infection; DPI: days post infection; b Negative CFT: < 1:5; c Negative IFA: < 1:80.

Discussion Some serological diagnosis of human and animal diseases by indirect methods require the use of anti-immunoglobulins secondary antibodies. Currently, polyclonal antibodies constitute the majority of the commercially secondary antibodies, because they are easy and cheap to produce, even

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FIGURE 4 Indirect immunofluorescence assay for the diagnosis of dourine using MAb 1B10B6E9 and a commercial anti-horse IgG (polyclonal) FITC-conjugated as secondary antibodies (40× magnification). Positive serum: IFA titre 1:320.

FIGURE 5 Indirect immunofluorescence assay for the diagnosis of African horse sickness using MAb 1B10B6E9 as secondary antibody (40× magnification). Positive serum: IFA titre 1:320.

if often cross-reactive with immunoglobulins of other species or with different antigens (e.g., blocking agents used in the tests). The use of anti-immunoglobulins monoclonal antibodies could be a valid alternative to improve the diagnostic tests. In this paper we describe the production and characterization of anti-horse IgG monoclonal antibodies to be used in

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the serological diagnosis of equine diseases. Three (1B10B6C9, 1B10B6C10, and 1B10B6E9; isotype IgG2a , κ light chain) out of the 21 MAbs obtained, reacted only with horse and donkey IgG and IgM and are specific to the Fc fragment of the immunoglobulins. One (MAb 1B10B6E9) of these 3 MAbs, which have similar characteristics, was tested in immunoblotting and in indirect immunofluorescence assay for the diagnosis of dourine in equids. The immunoblotting test, developed with MAb 1B10B6E9 and a highly purified antigen, allowed to show differences in antigenic patterns recognized by antibodies present in the sera from horses naturally and experimentally infected with Trypanosoma equiperdum and by antibodies present in the sera of non infected horses. Therefore, the immunoblotting test developed with the MAb 1B10B6E9 could be used as confirmatory test, in parallel with complement fixation and indirect immunofluorescence, for the diagnosis of dourine in equids. In addition, FITC-conjugated MAb 1B10B6E9, tested in indirect immunofluorescence assay (IFA-dourine and IFA-AHSV), was able to discriminate in a clear manner positive sera from negative sera and did not cross-react with the antigens (T. equiperdum and Vero cells) attached to the slides; in addition, in the case of the IFA-dourine, the MAb 1B10B6E9 was found to be more specific than the commercial polyclonal, which showed a weak cross-reaction with T. equiperdum; so MAb could be used in the test in place of the polyclonal secondary antibody after suitable validation of the method.[18] CONCLUSIONS In conclusion, the results obtained by testing MAb 1B10B6E9 in immunoblotting and in immunofluorescence indicate that it can be used to develop and improve diagnostic methods that involve the use of antihorse IgG polyclonal antibodies. The MAb can also be used in the study of the immunitary response of equids following natural or experimental infection with different types of pathogens. Moreover, the other two MAbs (1B10B6C9 and 1B10B6C10), having similar characteristics to MAb 1B10B6E9, could be used as an alternative for the diagnosis of the equids diseases or in research. REFERENCES 1. World Organisation of Animal Health (Office International des Épizooties, OIE). Dourine. In Manual of Diagnostic Tests and Vaccines for Terrestrial Animals; OIE: Paris, 2013; Ch 2.5.3, 1–10. 2. World Organisation of Animal Health (Office International des Épizooties, OIE). African Horse Sickness. In Manual of Diagnostic Tests and Vaccines for Terrestrial Animals; OIE: Paris, 2012; Ch. 2.5.1, 1–12. 3. Clausen, P.H.; Chuluun, S.; Sodnomdarjaa, R.; Greiner, M.; Noeckler, K.; Staak, C.; Zessin, K.H.; Schein, E. A Field Study to Estimate the Prevalence of Trypanosoma equiperdum in Mongolian Horses. Vet. Parasitol. 2003, 115, 9–18.

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4. Toledo Piza, A.S.; Pereira, A.R.; Terreran, M.T.; Mozzer, O.; Tanuri, A.; Brandão, P.E.; Richtzenhain, L.J. Serodiagnosis of Equine Infectious Anemia by Agar Gel Immunodiffusion and ELISA using a Recombinant p26 Viral Protein Expressed in Escherichia coli as Antigen. Prev. Vet. Med. 2007, 78, 239–245. 5. Arce, C.; Moreno, A.; Millán, Y.; Martín de las Mulas, J.; Llanes, D. Production and Characterization of Monoclonal Antibodies against Dog Immunoglobulin Isotypes. Vet. Immunol. Immunopathol. 2002, 88, 31–41. 6. Beh, K.J. Production and Characterization of Monoclonal Antibodies Specific for Sheep IgG Subclasses IgG1 or IgG2 . Vet. Immunol. Immunopathol. 1987, 14, 187–196. 7. Estes, D.M.; Templeton, J.W.; Hunter, D.M.; Adams, L.G. Production and Use of Murine Monoclonal Antibodies Reactive with Bovine IgM Isotype and IgG Subisotypes (IgG1 , IgG2a and IgG2b ) in Assessing Immunoglobulin Levels in Serum of Cattle. Vet. Immunol. Immunopathol. 1990, 25, 61–72. 8. Penha, T.R.; Krüger, E.R.; Thomaz-Soccol, V.; Bacila Agottani, J.V.; Itano, F.H.; Della Coletta Troiano, L.; Brodzinski, J. Production and Characterization of Monoclonal Antibodies Anti Fragment Fc of Bovine IgG. Braz. Arch. Biol. Technol. 2010, 53(1), 105–114. 9. Sugiura, T.; Kondo, T.; Imagawa, H.; Kamada, M. Production of Monoclonal Antibodies to Six Isotypes of Horse Immunoglobulin. Vet. Immunol. Immunopathol. 1998, 62, 145–151. 10. Wagner, B.; Radbruch, A.; Rohwer, J.; Leibold, W. Monoclonal Anti-Equine IgE Antibodies with Specificity for Different Epitopes on the Immunoglobulin Heavy Chain of Native IgE. Vet. Immunol. Immunopathol. 2003, 92, 45–60. 11. Decreto legislativo 27 Gennaio 1992, n. 116. Attuazione della direttiva n. 86/609/CEE in Materia di Protezione Degli Animali Utilizzati a Fini Sperimentali o ad altri fini Scientifici (Gazzetta Ufficiale n. 40 del 18/02/1992, Supplemento ordinario n. 33). 12. European Commission. Council Directive of 24 November 1986 on the Approximation of Laws, Regulations and Administrative Provisions of the Member States Regarding the Protection of Animals used for Experimental and Other Scientific Purposes (86/609/EEC). 13. Campbell, A.M. Monoclonal Antibody Technology. In Laboratory Techniques in Biochemistry and Molecular Biology; Burdon, R.H., van Knippernberg, P.H., Eds.; Elsevier Science BV: Amserdam, 1987; 1–265. 14. Goding, J.W. Monoclonal Antibodies: Principles and Practice, 3rd ed. Academic Press Limited: London, 1996; pp 154–156. 15. Luciani, M.; Armillotta, G.; Magliulo, M.; Portanti, O.; Di Febo, T.; Di Giannatale, E.; Roda, A.; Lelli, R. Production and Characterisation of Monoclonal Antibodies Specific for Escherichia coli O157:H7. Vet. Ital. 2006, 42(3),173–182. 16. Nakane, P.K.; Kawaoi, A. Peroxidase-Labeled Antibody. A New Method of Conjugation. J. Histochem. Cytochem. 1974, 22, 1084–1091. 17. Luciani, M.; Di Pancrazio, C.; Di Febo, T.; Tittarelli, M.; Podaliri Vulpiani, M.; Puglielli, M.O.; Naessens, J.; Sacchini, F. IgG Antibodies from Dourine Infected Horses Identify a Distinctive Trypanosoma equiperdum Antigenic Pattern of Low Molecular Weight Molecules. Vet. Immunol. Immunopathol. 2013, 151, 140–146. 18. World Organisation of Animal Health (Office International des Épizooties, OIE). Principles and methods of validation of diagnostic assays for infectious diseases. In Manual of Diagnostic Tests and Vaccines for Terrestrial Animals; OIE: Paris, 2013; Ch. 1.1.5, 1–16.

Production and characterization of monoclonal antibodies against horse immunoglobulins useful for the diagnosis of equine diseases.

Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELIS...
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