Preparation and Application of two Monoclonal Antibodies against Canine Parvovirus Vaccine and Field Strains

Canine parvovirus disease is an acute and highly infectious disease caused by canine parvovirus type 2 (CPV-2). The outbreak of canine parvovirus disease could be a seriously threat to the health of dogs, and also caused huge economic losses to the general economic animal breeding.CPV-2 was fi rst recognized in the late 1970s, and then widely spread via genetic mutations and evolution with affecting different ages of dogs all over the world with infection rate from 50% to 100% and mortality of at least 50% [1-2]. Until the mid1980s, CPV variants including CPV-2a and CPV-2b emerged in some countries with different pathogenicity [3-6]. In the past 30 years, CPV-2a and CPV-2b have mainly spread in different proportions among dog populations throughout the world, and then CPV new variants (CPV-2c) were detected in Europe, Asia, and North and South America [7-9]. Up to now, except a product containing modifi ed-live CPV-2b strain (Galaxy DA2PPv; Schering-Plough Animal Health), CPV strain subtype of the other commercial vaccines was CPV-2a such as Nobivac®DHPPi, Progard®-CPV, Vanguard®Plus 5, Recombitek® Canine Parvo and “Rabies, Canine Distemper, Parainfl uenza, Adenovirus and Parvovirus Vaccine for Dogs, Live” Vaccine. Therefore, monoclonal antibodies that can differentiate the vaccine strains from fi eld strains of CPV will be valuable.


Introduction
Canine parvovirus disease is an acute and highly infectious disease caused by canine parvovirus type 2 (CPV-2). The outbreak of canine parvovirus disease could be a seriously threat to the health of dogs, and also caused huge economic losses to the general economic animal breeding.CPV-2 was fi rst recognized in the late 1970s, and then widely spread via genetic mutations and evolution with affecting different ages of dogs all over the world with infection rate from 50% to 100% and mortality of at least 50% [1][2]. Until the mid-1980s, CPV variants including CPV-2a and CPV-2b emerged in some countries with different pathogenicity [3][4][5][6]. In the past 30 years, CPV-2a and CPV-2b have mainly spread in different proportions among dog populations throughout the world, and then CPV new variants (CPV-2c) were detected in Europe, Asia, and North and South America [7][8][9]. Up to now, except a product containing modifi ed-live CPV-2b strain (Galaxy DA2PPv; Schering-Plough Animal Health), CPV strain subtype of the other commercial vaccines was CPV-2a such as Nobivac ® DHPPi, Progard ® -CPV, Vanguard ® Plus 5, Recombitek ® Canine Parvo and "Rabies, Canine Distemper, Parainfl uenza, Adenovirus and Parvovirus Vaccine for Dogs, Live" Vaccine. Therefore, monoclonal antibodies that can differentiate the vaccine strains from fi eld strains of CPV will be valuable.
MAbs work as a useful tool to study the CPV pathogenic mechanisms and to develop corresponding diagnostic reagents. The principal objective of present study is to screen CPV MAbs by using hybridoma techniques and generate MAbs with good reactivity of CPV vaccine and/or fi eld strains.

Preparation of anti-canine parvovirus MAbs
In order to prepare M Abs, S P2/0 myeloma cells were in advance cultured with RPMI1640 medium (

Purifi cation of MAbs
Two MAbs were respectively purifi ed from ascetics by affi nity binding with protein G resin. In brief, 5 ml a scite plus 5ml binding/wash buffer (containing of 20mM Na 2 HPO 4 and 0.15 M NaCl, pH8.0) was mixed and applied for equilibrating 5 ml column of protein G. One ascetic fl uid was then needed to the column for binding. After washing the column with 30ml binding/wash buffer, the purifi ed MAb was eluted by appropriate volume of elute buffer (0.1M glycine, pH2.5) and collected the Elute and immediately neutralize to pH7.4 with Neutralization Buffer (1M Tris-HCl, pH8.5). The purity of MAbs was measured by SDS-PAGE ( B io-Rad Laboratories, Inc.) and t he concentration of MAbs was measured by B CA Protein Assay Kit (Beyotime Biotechnology, China).

Determination of relative affi nity of MAbs
The relative affi nity of MAbs was determined by indirect ELISA using thiocyanate.
In brief, ascites of 10H4, 10B11 and SP2/0 cells were diluted with different concentrations of NaSCN and OD 450 was measured to evaluate the reactivity between antigen and antibody. The concentration of NaSCN was considered as the relative affi nity Ka of 10H4 and 10B11 when the OD 450 value declined by 50% as much as that when it was not eluted. The unit of relative affi nity is expressed by mol/L.

H emagglutination inhibition (HI) assay
H I assay was followed as described previously [10].

M E V and FPV (abbreviation for Mink enteritis and Feline
Parvovirus virus, stored in our laboratory) were simultaneously detected by HI tests for verifying t w o MAbs' specifi city.

Indirect immunofl uorescence (IFA)
IFA was used to detect the reactivity of two MAbs to CPV.

Virus neutralization (VN) tests
VN tests were performed as described previously [11] . In brief, 50μl 2-fold serial dilutions of ascetic fl uid from 1:100, and viruses (100TCID 50 /50μl, with 4 replicates of Strains S2, S0425, S0304 or S0425-vaccine) were respectively mixed and incubated at 37°C for 1h. After that, add F81 cells (10 4 cells/100μl) into this mixture. The cells were examined for 5 days, and the VN titers were expressed as the reciprocal of the highest dilution which inhibited the viral cytopathic effect in at least 2/4 wells with the particular dilution.

Specifi city and reactivity of two MAbs
In order to detect the reactivity and HI titer of two MAbs, HI assay was performed. As shown in fi gure 1, both of two

Neutralizing capacity of two MAbs
The VN titers of 10H4 and 10B11 were summarized in

Discussion
Monoclonal antibodies to different genotypes CPV were previously reported. In Masato Nakamura's study, MAbs 2G5 and 20G4 recognized CPV-2a, CPV-2b, and CPV-2c, MAb 21C3 only reacted with CPV-2b and CPV-2c, and MAb 19D7 recognized all types of the FPV subgroup viruses [12]. However, there were no reports about any MAbs which could differentiate CPV vaccine from fi eld viruses.
In this study, we are the fi rst group to report that the MAb 10B11 could only react with CPV fi eld virus by the results of HI, IFA, and VN tests. By contrast, another MAb 10H4 reacted with    The present widely-used techniques to discriminate between vaccine and fi eld viruses were PCR and real-time PCR, bu t these methods are expensive and time-consuming [13,14]. The two MAbs in this study provide good biological reagents to develop high throughput and sensitive method such as ELISA to differentiate the vaccine from fi eld viruses.
10H4 and 10B11 were generated by immunization of mice with CVCC AV298, a vaccine strain of CPV. The proteins that these two antibodies recognized need to be explored in the future study. Since both MAbs showed very high neutralizing antibodies to different genotypes of fi eld CPVs, they may also work as therapeutic MAbs to treat CPV infection.

Conclusions
To conclude, two CPV MAbs were characterized in this study which MAb 10H4 reacted with both fi eld and vaccine strains of CPV, Whereas, MAb 10B11 only reacted with fi eld strain but not vaccine strain of CPV. These CPV MAbs may provide valuable biological reagents to study the CPV pathogenic mechanisms as well as to work as therapeutic antibodies.