Monoclonal antibodies against rabies virus have been widely used in the diagnosis and immunological analysis of rabies [7, 13]. Human monoclonal antibodies to rabies G protein are also expected to be used as a replacement for rabies immune globulin(RIG) [4, 10]in the post-exposure treatment of rabies. Two techniques are recommended by WHO and used world wide. These are the gold standard fluorescent antibody (FA) technique, a rapid and sensitive method for diagnosing rabies infection in animals; and the rapid fluorescent focus inhibition test (RFFIT) for the evaluation of neutralizing antibodies of rabies virus (RV). Both these techniques need fluorescent-labeled high potency and specific antibodies to RV antigens. However, this detection reagent is not yet approved for commercial manufacture in China, while the imported products are expensive and therefore are not readily available. This mitigates against the use of these techniques as standards. Thus, the preparation of potent and specific monoclonal antibodies against rabies virus will contribute significantly to develop ment of techniques for laboratory diagnosis of rabies. Since these will have local independent property rights it will facilitate rabies surveillance in China.
In 1978, Wiktor reported the preparation of rabies virus monoclonal antibodies. Since then, rabies virus monoclonal antibody (McAb) technology has been more and more widely used in basic research, and the testing and diagnosis of rabies. In this study, anti-RV monoclonal antibodies were prepared using a human RV vaccine strain and a baculovirus-expressed RV nucleoprotein as antigens.
Mouse SP2/0 myeloma cell strains were provided by the Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention; BSR cells were kept in our laboratory. Animals used for immunization were 6-8 weeks old female BABL/C mice.
Concentrated solutions of human rabies virus vaccine PV strain were provided by the ChengDa Bio Co., Ltd. Rabies virus nucleoproteins (CVS-11 strain) were expressed and purified by a baculovirus eukaryotic expression system  in our laboratory.
PEG4000 (Sigma Corporation), FITC labeled goat anti-mouse IgG and murine monoclonal antibodies subtype identification kit ISO. 2 (Sigma, USA); HT and HAT medium (Invitrogen, USA); nProtein A Sepharose 4 Fast Flow (Pharmacia Corporation, USA); anti-RV nucleoprotein fluorescent antibody (Millipore Corporation); Freund's complete and incomplete adjuvant (Sigma, USA).
Antigens used for immunization were rabies virus nucleoproteins (CVS-11 strain) and concentrated solution of human rabies virus vaccine PV strain. The concentrations of two antigen suspensions were in the range of 1-1.5 mg/mL after measurement by UV-spectrophotometry, they were then diluted with PBS to the required concentrations (Table 1).
Table 1. Immunization process of rabies virus (CVS-11 strain) nucleoprotein and PV strain antigens in mice
Ten 6-8 week-old female BABL/C mice were divided into two groups. Immunizations were conducted on d 1, 15, 29 and 43 respectively according to methods described elsewhere , four times in total. The first three injections were on both sides of the subcutaneous tissue of the neck, back and into the peritoneal cavity. The final immunization was into the peritoneal cavity, the other details are shown in Table 1. After immunization, blood was collected from the caudal vein and specific anti-RV antibody levels in serum were detected using indirect immunofluorescence to evaluate immune efficacy.
Preparation of myeloma cells: SP2/0 myeloma cells were cultured in RMPI1640 medium containing 10% serum at 37℃ and 5% CO2, and passaged once every two days. SP2/0 cells from the logarithmic phase were selected for the fusion with spleen cells.
Preparation of feeder cells: One healthy Balb/c mouse was killed by cervical dislocation the day before cell fusion, 5 mL 1640 medium (containing 20% serum) was injected into the peritoneal cavity of the mouse, then the medium was pumped out after gentle massage on the mouse peritoneal cavity and transferred into flasks. The sufficient medium was added and mixed with the feeder cells, 200 μL/well was distributed into 96-well plates and incubated at 37℃, 5% CO2overnight.
A booster immunization was conducted on the immunized mice. Three days later, spleen cells of the mice which showed better immune efficacy were selected for the fusion according to the methods described [1, 2, 8], the spleen cells were mixed with SP2/0 cells at a 5-10:1 ratio. After centrifugation, 1 mL 50% PEG4000 was added into the mixed cells drop by drop over 1 min and after 90 sec serum-free RMPI-1640 medium was added to stop the reaction. After centrifugation, the fusion cells were resuspended using RMPI-1640 selective medium containing 20% serum (FBS) and 1% HAT, and 100 μL/well of it was added into 96-well plates already loaded with the feeder cells. The plates were then incubated at 37℃, 5% CO2.
Preparation of CVS-11 RV antigen plate: BSR cells were infected with rabies virus CVS-11 strain then paved in a monolayer in 96-well plates; after 24 h of incubation, each infected cell plate was fixed with acetone and then used for screening of the hybridoma.
Screening of the hybridoma: The fusion result was checked every day. On day 8, cell supernatant of the wells in 96-well plates was detected using CVS-11 RV antigen plate by indirect fluorescent antibody (IFA) technique:50µL supernatant of the selected hybridoma was added into the wells of CVS-11 RV antigen plate and incubated at 37℃ for 30 min, then FITC-goat anti-mouse IgG was added. After 30 min at 37℃, 60% glycerol was added, observation was conducted through the fluorescence microscope. The positive results showed as specific yellow-green fluorescent granules against the red colored cell background.
After the screening of positive hybridoma, Double dilution could be conducted directly for single cell cloning. The positive cloned wells were selected and cloning continued, this was repeated for the second cloning, after which the positive cells were used for the construction of cell strains.
Antibody secretion of cell strains was detected every other generation after the construction of cellstrains. Cell strains which showed four consecutive positive detection results were selected, the hybridoma was gradually expanded from 48-well plates to 24-well plates and then to 6-well plates and finally a flask.
After being expanded in the flask, the monoclonal cell strains then expanded in vitro using mouse intraperitoneal amplification. 0.5 mL cell suspension containing 1.0×106 monoclonal aitibody cells was injected intraperitoneally into multiparous BABL/C mice; each monoclonal cell strain was injected into 5 mice. Ascitic fluid was collected after 7-10 days. Ascitic fluid obtained was purified using SPA affinity chromatography; concentration of purified monoclonal antibodies was detected using UV spectrophotometry. The purification result was checked by SDS-PAGE.
Ascitic fluid titration: Using IFA technique, the ascitic fluid obtained was taken as the primary antibody source for double dilution and 10-fold serial dilution respectively starting from 1:1000. The final concentrations for the two dilutions were 10000 and 1010respectively. Observation and analysis were conducted by inverted fluorescence microscope.
Using the RFFIT method , the viruses obtained with the optimum infection level and monoclonal antibodies supernatant were neutralized. The greatest dilution of monoclonal antibody supernatant able to neutralize 50% of the virus was calculated and antibody neutralizing titer of monoclonal antibody supernatant was compared with standard serum with known titer, thus demonstrating the ability of the obtained monoclonal antibodies to neutralize rabies virus.
ELISA plates were coated with different subclasses of murine monoclonal antibodies. Purified ascitic fluid was taken for primary antibodies, the types of monoclonal antibodies being detected using ELISA capture according to the ISO.2 (Sigma, USA) manual.
Cells and animals for immunization
Antigens for immunization
Reagents and instruments
Processing of antigens for immunization
Preparation of cells
Fusion of spleen cells with SP2/0 cells
Screening of positive hybridoma
Cloning and cell strain construction of the positive hybridoma
Preparation of anti-rabies virus monoclonal antibody ascitic fluid and titration
Anti-rabies virus neutralizing monoclonal antibodies titer
Identification of anti-rabies virus monoclonal antibody sub class
Blood from the tails of immunized mice was used for the immunization assessment and the serum antibody titer was detected using IFA technique. The results showed that the highest antibody titers of 10 immunized mice reached 1:6000, indicating effective immunization.
The fusion rate was higher than 80%. Clone screening was carried out twice. Results showed screening reduced the number of positive clone wells, because positive hybridoma can lose the ability to secrete antibodies due to factors such as damage during the passage process. Eventually, positive hybridoma that showed high-titer and specific secretions of anti-rabies virus were selected for construction of cell strains. Details are shown in Table 2.
Table 2. Fusion and screening results of mice immunized with rabies virus nucleoprotein and concentrated vaccine for human use
The passage number after the construction of cell strains was taken as the first generation. The stability of the first eleven generations of cells was detected at every other generation, as shown in Table 3. The antibody titer of the supernatant of monoclonal cell strains was maintained at 1:800-900, showing a good stability and high specificity. The indirect FA results of the monoclonal antibody supernatant are shown in Fig. 1. The presence of specific yellow-green fluorescent granules indicates viral antigens are in the monoclonal antibody supernatant. This demonstrates that the monoclonal antibodies obtained showed good specificity, sensitivity and stability. These granules do not appear in the negative control.
Table 3. Detection of antibody-secretion of hybridoma cell strains after construction of cell strains
Figure 1. IFA results of the monoclonal antibody cell supernatant under 200× inverted fluorescence microscope. A, 3B12 strain monoclonal antibody; B, 3B12 blank cell control; C, 4A12 strain monoclonal antibody; D, anti-RV nucleoprotein monoclonal antibody from the Millipore Corporation; E, 4A12 blank cell control; F, blank negative control.
The two monoclonal antibody cell strains, 3B12 and 4A12 were detected for the neutralization titer through RFFIT, the results are shown in Table 4.
Table 4. Neutralization titer of 3B12 and 4A12 detected by RFFIT.
The titers of obtained ascitic fluid for the two strains, determined by indirect immunofluorescence, were 1:10000 and 1:8000 respectively. UV spectro photometric analysis showed the concentration of monoclonal antibodies of purified ascitic fluid were 1.06 mg/mL and 0.80 mg/mL respectively. SDS-PAGE analysis after purification by electrophoresis (Fig. 2) shows that, compared to the vague band of unpurified samples, the purified monoclonal antibodies from the ascitic fluid present two clear bands near 25 KD and 50 KD, consistent with the expected molecular size of the IgG light chain and heavy chain.
Sub-class identification was conducted using ELISA capture with a monoclonal antibodies subclass identification kit (Sigma), and both monoclonal antibodies obtained were determined as IgG2a monoclonal antibodies.