Monoclonal Antibodies as Ligands for Purificaion of Rabies virus Proteins from the Brain Tissues of Infected Dogs and Mice (ANTIBODI MONOCLONAL SEBAGAI LIGAND UNTUK PURIFIKASI PROTEIN VIRUS RABIES ASAL JARINGAN OTAK ANJING DAN MENCIT TERINFEKSI)

Immunoaffinity chromatography using monoclonal antibodies (mAbs) as ligands has been used to purify rabies virus (RV) individual proteins. In this method, mAbs against RV were firstly purified, coupled to CnBr-agarose resin and used for purification RV individual proteins. Brain tissue homogenates derived from infected and uninfected dogs and mice were mixed with mAbs-CnBr agarose resin and washed extensivelly phosphate buffered salin (PBS). Following elution and neutralization, purified proteins were detected by enzyme-linked immunosirbent assay (ELISA) and Western blotting assay. Of the three mAbs (BB5, AE11 and AF6) as ligands, mAb AE11-CnBr agarose resin yielded highest protein levels as compared to those of mAb BB5-CnBr agarose and mAb AF6-CnBr agarose resins. In Western Blotting assay, the purified protein appeared to be 65 Kda (glycoprotein) and 38 kDa proteins. In ELISA test, the purified proteins reacted with both mAbs and policlonal antibodies (pAbs). Kata kunci: CnBr-agarose, rabies virus, protein, purificaion, chromaography Jurnal Veteriner Desember 2016 Vol. 17 No. 4 : 556-563 pISSN: 1411-8327; eISSN: 2477-5665 DOI: 10.19087/jveteriner.2016.17.4.556 Terakreditasi Nasional, Dirjen Penguatan Riset dan Pengembangan, online pada http://ojs.unud.ac.id/php.index/jvet Kemenristek Dikti RI S.K. No. 36a/E/KPT/2016


INTRODUCTION
Rabies is a zoonotic viral disease and is still endemic in many conntries arround the world including Indonesia (WHO, 2010).In Indonesia, disease is transmitted by rabid animals such dogs, cats and monkeys.However, dog bites play a major role in rabies transmission among animals and also from rabid animals to humans (OIE, 2016).One effort in preventing and eradicating the disease from the infected areas is mass vaccination of susceptible animals especially dogs using the appropriate vaccines.The succes of such vaccination, however, depends on the protective immune response induced by the vaccine and the coverage of vaccination.The vaccination coverage to induce herd immunity in susceptible dog population has been suggested to be higher than 70% (Conan et al, 2015).To determine the immune response induced by vaccination, the availability of a good serological method such indirect enzyme-linked immunosorbent assay (ELISA) is important.The use purified antigen is required in the development of serological tests with high level of accuracy for rabies virus antibody.
Rabies virus (RV) belongs to lyssavirus of the family Rhabdoviridae (Balaul dan Lafon, 2003).The virion consist of single-stranded RNA genome (Consales and Bolzan, 2007) which codes for viral proteins such as N (nucleocapsid), P (phosphoprotein) dan M (membrane) proteins, G (envelope glycoprotein) and L (replicase) (Bradame and Tordo, 2001).All of those proteins play roles in the infection of virus into target cells.However, glycoprotein (G) is the most important protein for use in the development of both vaccine for preventing rabies and serological test for examining the immune response induced by rabies vaccines.G protein is the virus attachment protein which initiates the binding of the virion with its receptors on the surface of target cells (Kuzmina et al, 2013, Mori dan Marimoto, 2014).The antibody against G protein induced by vaccines in hosts will prevent the infection by blocking the viral entry into cells.The use of purified RV protein such as G protein in serological test such as ELISA will ,therefore, increase the sensitivity and specificity of the test.
Many methods can used in the purification of protein used as antigen for the development of ELISA test such as density gradient centrifugation and affinity chromatography (Launa et al, 2012;Feng et al. 2016).The use of affinity chromatography is particularly interesting as it is simple and can be used to purify a protein from a mixture composed of many proteins (Launa et al, 2012).In recent years, one affinity chromatography technique that has been widely used for protein purification is immunoaffinity chromatography using antibody against a protein as ligands (Abdolalizadeh et al, 2013).In this method, purified antibody against a protein is coupled into CnBr-activated agarose gel bead and the bead is then used as ligand for protein purification (Kavran and Leahy, 2014).Both polyclonal and monoclonal antibodies can be used as ligands in this purification method.However, monoclonal antibodies (mAbs) appear to be better ligand compared to polyclonal antibodies (pAbs) as mAbs only bind with a single epitope in a protein molecule (Moser and Hage, 2010 ) and is therefore likely to obtain a purer individual protein.Currently, mAbs against rabies virus have been produced and some of which reacted specifically with rabies virus antigen (Astawa et al, 2015).A study was therefore conducted to examine the potential use of such mAbs as ligands for purification of rabies virus individual proteins.

Production and Purification of Monoclonal antibodies
In this study, three (BB5, AC11 and AF6)) of eight mAbs against rabies virus produced in the previous study (Astawa et al, 2015) were used.The stocks of high titer mAbs were produced by growing hybridomas in culture until they reached a dying stage when most cells were at the stage of dying.The supernatant was then clarified by centrifugation at 1000 x g for 10 minutes.The mAbs were firstly tested and titrated by ELISA as described by Astawa et al, 2015.Fifty ml stock of each mAb was used for purification.The mAbs were then purified using rapid antibody purification kit (Cell Biolab, USA) according to the procedures as described by the manufacturer.Brieftly, mAbs stock was diluted 1:2 in binding buffer and was passed three times through resin coupled with protein A/G to maximase the binding of IgG to protein A/G.The resin was then washed with 30 ml binding buffer until all unbound components of hybridoma media was washed away from the resin.Bound mAb was then eluted by elution buffer and the pH of mAb elutes were normalized to 7.4 by adding neutralization buffer.Five elutes of 1 ml for each mAbs collected and tested by ELISA according to the procedures as described below.

Coupling of mAbs to CnBr-Activated Agarose Bead
Coupling of mAbs to CnBr-activated agarose was conducted according to procedures as described by Kavran and Leahy, 2014 with modification.The purified mAbs were dialyzed for six hours at roo temperatures in two changes of 500 ml coupling buffer (100 mM NaHCO3 pH 8.6, 500 mM NaCl).One gram of CnBr-activated agarose resin was then suspended in activation buffer (15 ml 0.1 N HCl) and left at 4 o C for two Mantik Astawa, et al Jurnal Veteriner The tissue homogenate was then subjected for three times freezing-thawing to break the cells in the brain tissue homogenate.The homogenates were treated with 0.01% âpropiolacton for 24 hours at 4 o C to inactivate the virus, followed by incubation at 37 o C for another 24 hours to inactivate the â-propiolacton.Sample of â-propiolacton-treated tissue homogenate was inoculated intrcerebrally into suckling mice.When no rabies clinical sign was observed after three week post-inoculation and immunoflouresecne test, the brain tissue homogenate was used for RV protein purification.Brain tissue homogenates from uninfected dogs and mice were also prepared using the same procedures as described for those from rabid dogs or mice.

Purification of Rabies Virus Proteins
Tissue homogenate prepared as above was firstly centrifuged at 10000 x g for 15 minutes at 4 o C. The supernatant was then collected and mixed with mAbs-CnBr-activated agarose resin and left for one hour at room temperature with a constant gentle shaking.The resin was then poured into 15 ml column and washed with 50 ml PBS until all unbound proteins were washed away from the column.The rabies virus protein bound to resin via mAbligands was the eluted by elution buffer (200 mM glycine pH 2.8 ) and the pH of the elutes was then normalized by adding neutralization buffer (3 M TRIS-HCl, pH 8.8.).Five elutes from each mAb-CnBr agarose resin were collected and tested by ELISA using mixed mAbs (BB5, AC11 and AF6).

Enzyme-Linked Immunosorbent Assay for Detection of Purified Proteins
Protein samples from each elute was firstly diluted 1/20 in carbonate-bicarbonate coating buffer (50 mM NaHCO2, 50mM Na2CO3, pH.9.6) and was coated into 96 well ELISA plate (100 µl per well) for overnight at 4 o C .Wells of the ELISA plate were washed twice with PBS containing 0.1% Tween-20 (PBST), and blocked with 5% skim milk in PBS for 1 hour at 37 o C. Following twice washes as above, 100 µl monoclonal antibody against rabies protein diluted 1:10 was added into each well and incubated for 1 hour at 37 o C. Wells on ELISA plate was washed three times with PBS and 100 µl anti-mouse IgG-horseradish peroixidase was added and incubated for another 1 hour as above.After three times washes as above, 100 µl TMB substrate was added and left for 20 minutes at room temperature in dark enviromnemt and hours.The activated resin was washed three times with coupling buffer by centrifugation at 500 x g for five minutes.After the last wash, the supernatant was discarded and two ml dialysed mAbs diluted in 2 ml of coupling buffer was mixed with the CnBr activated resin.The coupling of mAbs to CnBr activated resin was conducted by constant shaking of the mAbs and CnBr agarose resin mixture at room temperature for two hours.The mAbs-coupled resin was the washed three times with coupling buffer as above.Five ml blocking buffer (100 mM ethanolamine in coupling buffer) was the added to the resin and left at room temperature for another one hour with a constant shaking as above.The resin was washed for four times with alternating high pH buffer (100 mM Tris-HCl pH.9.2, 500 mM NaHCl) and low pH buffer (NaOAc pH 4.0, 500 mM NaCl).Finally the resin was washed twice phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,2 mM KH2PO4,, pH.7.4) and kept at 4 o C in PBS containing 0.1% NaN3 (sodium azide).

Preparation of Brain Tissue Homogenate of Rabid Dogs and Mice
Brain tissues of infected dogs were obtained from field rabid dogs necropsied in the Pathology section of Animal Disease Investigation Centre Denpasar, Bali Indonesia.The rabies virusinfected mice were obtained by inoculation of suckling mice with the brain homogenate derived from rabid dogs which have previously been confirmed by direct immunoflourescence and/or by mouse inoculation test.The presence of rabies virus in the brain tissue samples of dog or mice was detected direct immunoflourescene assay.Fisrtly, touch smear of infected brain tissues was prepared on mciroscope slides treated with poly-L-lysine and fixed with cold acetone for 10 minutes.Fifty ml antiabies mAb-FITC conjugate (Bio-Rad) was added to the smear of brain tissues.The smears were left at 37 o C for 30 minutes and washed five times with PBS, mounted in 90% buffered glycerol and examined under Flourescene microscope.Brain tissues of infeced dogs and mouse with high intensity of RV antigen were then used for purification procedures.
The brain samples of infected mice or dogs were firstly homogenized by grinding in a mortar using piston and passed through 10 ml syringe with 18 G needle.The homogenate was divided into two tubes; one treated with 1% Triton X-100 in PBS and one tube treated with PBS only.

Rabies Virus Antigen Detected by Direct Immunoflourescen in Brain Tissues
Before they were used as the sources of RV protein purification, brain tissues of infected and uninfected dogs and mice were tested by direct immunoflourescence.Rabies virus antigen was detected in smears of infected dogs and mice and mouse brain tissues, but not in uninfected brain tissues.(Figure 1).

Profiles of Purified RV proteins in ELISA Test
In this study, purified RV proteins eluted from mAbs-CnBr agarose resin were tested by ELISA.Using the resin, purified proteins were detected in elutes 1-2 using mAb AF6-CnBr agarose-resin, in elutes 1-3 using mAbs BB5-CnBr agarose resin, and in elutes 1-5 using mAb AC11-CnBr agarose resin.The use of brain tissue homogente treated with triton-X100 resulted in lower titer of protein.No diference was observed proteins titer obtained from the bain of infected dogs as compared to those obtained from the brain of infcted mice (Figure 2).

Estimated Molecular Weight of Purified RV proteins
Following purification by mAbs-CnBr agarose resins, two bands of purified proteins were detected in elutes of homogenates derived from infected dogs.The molecular weights of the proteins were 65 kDa and and 38 kDa.No protein band was detected in elutes of brain tissue homagenate derived from uninfected dogs were used (Figure 3).stopped with 1 N H 2 SO 4 .The optical density (OD) of the substrate was then read by ELISA reader.

Titers of mAbs before and after Purification
The titers of mAbs before purification were as follows mAb BB5 (2 8 ), mAbs AC11 (2 6 ) and AF6 (2 6 ).After pufication, mAb titers increased  Three mAbs (BB5, AC11 and AF6) against rabies virus were used as ligands of immunoaffinity chromatography for purification of rabies virus proteins from brain tissues of infected dogs and mice.Purification of mAbs is required to enable coupling of mAbs to CnBractivated agarose resin (Read et al, 2009;Abi-Ghanem and Berghman, 2012).Purification of antibodies including mAbs is generally achieved by affinity chromatography using agarose matrix coupled with protein A or G which can bind to all mammalian IgGs with different level of affinities (Page and Torp, 2002).A volume of 50 ml hybridoma culture supernatant was used for this purification method.As the concentration of mAbs in hybridoma culture supernatant was reported to be around 25 ug per ml (10-100 ug per ml) (Goding, 1996), the use of 50 ml hybridoma culture supernatant was expected to yield around 1 mg mAb which was sufficient for coupling the mAbs to CnBr activated agarose resin.
By using direct immuoflourescence, the rabies virus antigen was detected in the brain smears of infected but not in uninfected dogs and mice (Figure 1), but not in the brain smear of uninfected dogs and mice.Such detection is inportant to determine the intensity and severity of rabies infection in the brain tissues.Direct immunofluorescence assay has been considered as confirmatory standard for detection of rabies virus in tissues of infected animals or human (Jackson, 2013).By this technique, it appears that rabies virus infection in brain tissues of infected dogs was more intense than those of infected mice.Other test which is also used to detect rabies virus in brain tissues is mouse inoculation test (MIT).Compoared to the direct immunoflourescence test, MIT is higher in sensitivity and is lower in spesificity (Kadam et al, 2011).MT has been used as both confirmatory and gold standard of rabies diagnosis.Combination of both mouse inoculation and direct flourescence assay are often required for accurate detection of rabies virus infection in the infected tissues.Rabid mice used in this study also the result of MIT as further confirmatory test for the detection of rabies virus in the brain of infected dogs.
When the brain tissue homogenates of infected dogs or mice were used as sources of rabies virus proteins for purification, the RV proteins bind to their homogolog mAbs in mAb-CnBr agarose resin.The rabies virus individual protein is therefore trapped in the resin.Following several times washes to remove the unbound proteins, only proteins bound to mAbs retained in the resin and elution by low pH buffer can yield rabies virus protein with high level of purity.In this study, both untreated and triton X100-treated homogenates were used as the sources of rabies proteins.It appeared that the ELISA optical density of proteins purified from homogenates of rabid dog brain tissues treated with triton-x 100 was slightly lower than those untreated homogenates.Triton X is a detergent widely used in lysing cells used for protein extraction.Triton X-100 disrupts the cellular integrity by interfering with polar head of lipid bilayers in cell membrane (Koley and Bard, 2010).By lysing the infected cells using triton X, it was expected that the level of viral proteins obtained was higher than those in untreated homogenate.Similarly, OD of rabies virus proteins obtained from homogenates of rabid mouse brain homogenates was similar to those of rabid dogs.No immunoreactive protein was detected in the brain homogenate of uninfected dogs and mice which indicated that mAbs-CnBr agarose resin only binds and therefore trapped the rabies virus proteins but not normal cellular proteins.
The purified rabies virus proteins detected by ELISA were further confirmed by Western blotting assay.However, two bands of purified rabies virus proteins were detected by Western blotting assay from elutes of brain homogenates of rabid dogs and none was detected from non rabid dogs.The result confirmed that the purified proteins were rabies virus proteins with minimum contaminant of cellular proteins.The molecular weights of proteins detected by Western blotting assay were around 65 kDa and 38 kDa.The two proteins appeared to be glycoprotein and phosphoprotein respectively as it has been reported that the molecular weight of rabies virus glycoprotein and phosphoprotein were 64-68 kDa and 37-40 kDa respectively (Jackson, 2010).However, the two proteins bands detected by Western blotting assay need further confirmation using polyclonal antibody which is not available at this stage.
Purification of rabies virus proteins by immunoaffinity chromatography using mAbs as ligands is expected to yield purer proteins as compared to those using other purification methods.This immunoaffinity chromatography technique has been used to purify cytokines such as TNF á (Abdolalizadeh et al, 2013), enzymes (Karimi et al, 2012) and other biological markers (Mousavi and Nasiri. 2015).For rabies virus, affinity chromatography using DEAE cellulose resin has been used to purify the virus for use in vaccines or serological tests (Frazatti-Gallina et al, 2004).The availability of immunoaffinity chromatography using mAbs as ligands for purification of rabies virus proteins provides an alternative method of antigen preparation which can be performed when individual proteins are required.In addition, the individual proteins obtained from these purification methods are expected to be purer and free from contaminating cellular proteins.

Figure 1 .
Figure 1.Rabies virus antigen detected by direct immunoflourescent in brain tissues touch smears of infected dog and mice.A: infected dog, B: infected mice C: uninfected dog, D: uninfected mice.Note that rabies virus was detected in the infected but not in uninfected dog or mice

Table 1 .
Titers of mAbs before and after purification using protein A/G-sepharose resin