Viruses and sera:
NDV strain chicken/EGY/NR730/2016 (NR730; GenBank Acc. no. MH899939; genotype VII.1.1) was isolated from an ND vaccinated layer flock in Egypt suffering respiratory distress. The virus was characterized as velogenic having an intracerebral pathogenicity index (ICPI) of 1.8 and belonged to genotype 2.VIIb . Pigeon type paramyxovirus-1 (pigeon/Germany/R75/1998), genotype 2.VI, was derived from the repository of the ND reference laboratory (Acc. No. KJ736742) and NDV/clone 30, genotype 2.II, was derived from a commercial vaccine (MSD, New Jersey, USA). As a source of polyclonal reference antibodies, the watery egg yolk preparation from eggs was used of specific pathogen free (SPF) chickens, immunized repeatedly with a commercial inactivated ND-vaccine containing NDV clone 30. A monospecific rabbit α-NDV-HN serum and α-NDV-F serum  was used for specific detection of NDV-HN protein by western blot (WB) analysis. Immunizations were carried out in accordance with the legally approved protocol (MV-LALLF- 7221.3-2.5-010/10).
Virus propagation and purification.
Virus was propagated in embryonated SPF chicken eggs (ECE) as described  (VALO BioMedia GmbH, Osterholz-Scharmbeck , Lower Saxony, Germany). Amino-allantioc fluid (AAF) was harvested on day 3 post infection (dpi) and purified by sucrose gradient ultra-centrifugation. Briefly, debris was cleared from AAF by low speed centrifugation (30 min at 10,976 x g; 10,000 rpm Rotor JA-10; Beckman Coulter, Brea, California, USA). Then virus was spun down by ultra-centrifugation (1.5 hour at 96,281 x g; 28,000 rpm, 32Ti Rotor, Beckman Coulter). Virus pellets from 6 tubes were re-suspended in a total of 45 mL of phosphate buffered saline (PBS, pH 7.2) containing 1 M KCl (KCl-PBS) before adding 15 mL of the virus suspension on top of a discontinuous sucrose gradient (30-60%, in KCl-PBS). Visible bands forming after ultra-centrifugation overnight (96,281 x g; 28,000 rpm, 32Ti Rotor; Beckman Coulter) were collected and diluted 1:5 in KCl-PBS before pelleting the virus by ultra-centrifugation for 1.5 h (96,281 x g; 28,000 rpm, 32Ti Rotor; Beckman Coulter). The final virus pellets, representing a total of 228 mL AAF were collected and resuspended in 1.5 ml KCl-PBS. The protein concentration of the obtained virus suspension was determined according to Bradford using the Roti®-Quant protein quantitation assay (Carl Roth GmbH, Karlsruhe, Baden-Württemberg, Germany) following the producer’s instructions. Haemagglutination activity was determined using the HA test according to standard procedures . Purification for pigeon type paramyxovirus (PPMV-1) R75/98 and vaccine type APMV-1 clone 30 was done accordingly, but virus was resuspended in PBS.
Enrichment of HN-protein
Separation of NDV spike proteins was done as described by . Briefly, protein concentration of purified virus was adjusted to 1.5 mg/mL in KCl-PBS before 0.1 mL Triton X-100 in PBS (20% (v/v)) was added, gently mixed and kept at room temperature (RT) for 20 min. The suspension was centrifuged (20 min at 10,000 x g, 9,703 rpm, A-4-81-11 Rotor, (Eppendorf, Hamburg, Germany)) and the obtained pellet (p1) was resuspended in 1 mL PBS (0.01 M, ph 7.2) and kept for analysis whereas the supernatant was further cleared by ultra-centrifugation at high speed (1 h at 200,000 x g, 55,000 rpm Rotor TSL 55 (Beckman Coulter, Brea, California, USA)) for 1 h. Again the pellet (p2) was kept for analysis after resuspension in 0.2 mL PBS. The supernatant was collected and dialyzed against 0.01 M phosphate buffer in order to remove the potassium chloride from the buffer used during purification. Any precipitate that formed during dialysis was sedimented by centrifugation (20 min at 10,000 x g, 9,703 rpm, A-4-81-11 Rotor (Eppendorf, Hamburg, Germany)) and the pellet was resuspended in 0.1 mL PBS (p3). The supernatant of the dialyzed material (s3) was the fraction that was subsequently used as antigen for immunization.
SDS-PAGE and Western blot
Proteins were separated under denaturing conditions in 10% sodium dodecyl sulphate (SDS) polyacrylamide gels using a minigel system (Biorad, Hercules, California, USA) according to standard guides (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf). Shortly, samples were diluted in sample buffer (Roti load® (Carl Roth GmbH, Karlsruhe, Baden-Württemberg, Germany)) heated at 100°C for five minutes before adding 16 µL per lane into the gel pocket. Protein separation was conducted applying constant voltage setting (200 V) and the gel was either stained by Coomassie blue (Biorad, Hercules, California, USA) or proteins were blotted on a nitrocellulose membrane (Amersham™ Protran, Cytiva, Marlborough, MA, USA) applying constant voltage setting (15 V) for 1.5 hours. For western blot analysis the membrane was blocked for one hour with 1% skimmed milk powder in 0.025% Tween 20 in PBS (PBS-Tween) and subsequently incubated with target specific antibodies over night at 4°C. After washing three times with PBS-Tween, blots were incubated with peroxidase (POD) labeled species-specific anti-immune globulin G (IgG) or immune globulin Y (IgY) conjugates (Sigma-Aldrich, St. Louis, Missouri, USA) for 1 h at RT. After washing three times, peroxidase activity was visualized by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific™. Waltham, Massachusetts, USA) and the Chemi Doc XRS+ imaging system (Bio-Rad, Hercules, California, USA).
Mouse inoculation and monoclonal production
Two female BALB/c mice were immunized five times intraperitoneally with 20 µg of purified protein fraction (S3) mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik, Heidelberg, Germany) over a period of 26, at weeks 4, 7, 11 after the first immunization, and the last boost was administrated four days before extraction of the spleen. Blood samples were taken on days 35, 49, and 84 after first immunization (dpi) from the submandibular vein and at 183 dpi, at the end of the experiment. Four days after the final boost mice were euthanized, their spleen removed under aseptic conditions and splenocytes harvested into serum-free RPMI-1640 medium (Invitrogen, Carlsbad, California, USA/Thermo Scientific™. Waltham, Massachusetts, USA) by using a cell strainer (BD Biosciences, Franklin Lakes, New Jersey, USA). In presence of polyethylene glycol 1500 (Roche Applied Science, Penzberg, Germany) the isolated splenocytes were fused with murine myeloma SP2/0 cells following a slightly modified standard protocol  by using a cell-to-cell ratio of 1:4. Fused spleen cells were seeded in three different cell densities (30,000, 15,000, and 7,500 spleen cells per well, two plates per density) in 96well flat-bottomed plates (Greiner bio-one, Kremsmünster, Austria) and incubated for 10 days (37 °C, 90% RH, and 5% CO2) by using complete RPMI-1640 culture medium 10% FCS (Fischer Scientific, Hampton, New Hampshire, USA ), 1x MEM non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate (Invitrogen, Carlsbad, California, USA/Thermo Scientific™. Waltham, Massachusetts, USA) supplemented with 1x BM Condimed H1 (Hybridoma Cloning Supplement, Sigma-Aldrich). For selection of growing hybridoma clones the complete medium was additionally supplemented with 1x HAT Media Supplement (50×) Hybri-Max™ (Sigma-Aldrich). Growing cultures were screened for specific antibodies by Con-A ELISA, IF and HI. The haemaglutination inhibition test (HI) was used to determine the haemagglutination inhibition activity according to standard procedures . For generating MAb producing cell clones, cells from positive cultures were cloned at least twice by limiting dilution (0.1 cells per well) in complete RPMI-1640 medium supplemented with 1x HT Media Supplement (50×) Hybri-Max™ (Sigma-Aldrich). Final clones were adapted to complete RPMI-1640 medium without any further supplements.
The ELISA procedure was done according to a previously published protocol [40, 41]. Briefly, ELISA plates (Immunolon II, Thermo Scientific™. Waltham, Massachusetts, USA) were pretreated with Concanavalin A (ConA) (Carl Roth GmbH) by adding 50 µl of Con-A (50 µg/mL in PBS) to each well. After incubation for 1 h at RT plates were washed three times using PBS-Tween and coated with 50 µL (20 µg/mL) pre-treated antigen. For preparation of the antigen, gradient purified virus stock at a concentration of 200 µg/mL was incubated with TritonX100 (1% v/v) for 45 min at RT and subsequently diluted 1:10 with PBS for coating the plates for 1 h. Thereafter, plates were washed three times with PBS-Tween and blocked with 1% FCS in PBS for 30 min at RT. Test sera (50 µL/well) at indicated dilutions were incubated for 30 min at RT and after three washings with PBS-Tween, plates were incubated with POD labeled species specific anti-IgG or IgY conjugates (50 µL/well) for 30 min at RT. After washing the plates three times with PBS-Tween, bound antibodies were visualized by incubation with o-Phenylenediamine dihydrochloride (Sigma-Aldrich). (50 µL/(100 µL/well, 1 mg/mL in 0.05 M phosphate-citrate buffer) for 20 min. Colour reaction was stopped with 2.5 M H2SO4 well density was measured at 492 nm using Sunrise™, Tecan`s microplate readers (Männedor) and the optical f, Zürich, Switzerland).
Indirect immunofluorescence (IF)
Immunofluorescence test was done with NDV/NR730/16 infected LMH cells (ATCC CRL-2117™) cultivated in 96 well plates (Corning, Corning, New York, USA) grown to 70-80% confluency before infection. Infected plates were fixed with 3.7% formaldehyde after incubation for 24 hours and stored at 4 °C for up to four weeks. For use, plates were emptied and treated with TritonX-100 (1% v/v) in PBS (100 µL/well) for 30 minutes at RT and after flicking off the supernatant, blocked with 1% fetal calf serum in PBS for 30 minutes at RT. Subsequently, cells were incubated with MAb or indicated sera (50 µL/well) for 1 h at RT. After washing the plates three times with PBS-Tween, wells were incubated with FITC-conjugated anti-species specific IgG or IgY conjugate (Sigma-Aldrich, country) for 1 h at RT. After final washing for three times with PBS-Tween, wells were mounted with glycerol in water 1:5 and inspected under the microscope (Eclipse TS100, Nikon, Minato, Tokyo, Japan ) with the appropriate filter (excitation 495 nm, emission 525 nm).
Serum neutralization test (SNT)
Serial dilutions (log2) of MAb´s, starting with concentrated supernatants, were mixed with equal volume of NDV/NR730/16 (100 µL) containing 400 KID50. For each dilution row, the last well was kept without serum to serve as virus control. For each test a heat inactivated (56 °C, 30 min) NDV reference serum was included as positive neutralization control. After incubation for 30 min at 37 °C, 50 µL of antibody / virus mixture was transferred in triplicate to 100 µL LMH cells that were cultivated without fetal calf serum but with TPCK treated Trypsin (2 µg/mL) (Sigma-Aldrich) in 96well plates (Corning ) at a density 106 cells/cm2. The plates were incubated at 37 °C with 5% CO2 atmosphere for four days and SNT-titer was determined by determining the last dilution without presence of a cytopathic effect (cpe) and calculated according to Reed and Muench .
Haemagglutination inhibition test (HI)
Haemagglutination inhibition test was done according standard procedures, applying four haemagglutinating units and HI-titer are given as last serum dilution (log2) that inhibit agglutination . For analyzing antigenic differences by polyclonal sera, the test were done in triplicate and repeated in three independent experiments. Results were analyzed by Sigma Plot 11 (Systat Software, INC) applying Kruskal-Wallis One Way Analysis of Variance on Ranks.