key: cord-005397-i7x50taa
authors: Wang, Wen-juan; Li, Xiong; Wang, Li-hua; Shan, Hu; Cao, Lei; Yu, Peng-cheng; Tang, Qing; Liang, Guo-dong
title: Preparation and identification of anti-rabies virus monoclonal antibodies
date: 2012-06-09
journal: Virol Sin
DOI: 10.1007/s12250-012-3242-0
sha: 
doc_id: 5397
cord_uid: i7x50taa

To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6–8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents.

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 development 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 [11] 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 [3] in our laboratory. 

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) .

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 [9] , 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 

Preparation of myeloma cells: SP2/0 myeloma cells were cultured in RMPI1640 medium containing 10% serum at 37℃ and 5% CO 2 , 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% CO 2 overnight.

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% CO 2 . 

Using the RFFIT method [12] , the viruses obtained 

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 Table 2 . Number of positive cells after second screening 6 7 Number of positive cells after third screening 1 1

Positive proportion of single-well cloning after the first cloning 40.6% 43.8%

Positive proportion of single-well cloning after the second cloning 100% 100% 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 two monoclonal antibody cell strains, 3B12

and 4A12 were detected for the neutralization titer through RFFIT, the results are shown in Table 4 .

The titers of obtained ascitic fluid for the two strains, determined by indirect immunofluorescence, were 1:10000 and 1:8000 respectively. UV spectrophotometric 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. instability of fusion cells [6] . That is why the number of positive hybridoma decreased with time, as shown in the result. Timely cloning is a more important consideration for efficient generation of monoclonal antibodies because in the same well, the growth ability of cells which do not secrete antibodies will be greater than those secreting antibodies [4] .

The titer of the neutralizing antibodies obtained was detected using the RFFIT and the results showed that the generated monoclonal antibodies had the ability to neutralize rabies virus. The neutralizing activity needed to be further validated and the details are shown in Table 4 .

There are many ways to purify monoclonal antibodies [14] : including ammonium sulfate precipitation, octanoic acid precipitation and affinity chromatography.

Of these, ammonium sulfate precipitation shows a higher purified rate of ascitic fluid but a lower recovery rate, and it has a significant impact on the immune activity of IgG [5] , while octanoic acid precipitation achieves the opposite result. Therefore, in this experiment, using affinity chromatography, our experimental results verified that this purification 

Development and characterization of neutralizing monoclonal antibody to the SARS-coronavirus

Novel monoclonal antibodies that bind to wild and fixed rabies virus strains

Identification and baculovirus expression of Rabies virus N gene

A human monoclonal antibodies cocktail as a novel component of rabies postexposure prophylaxis

Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory

Continuous cultures of fused cells secreting antibody of predefined specificity

Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis

Purification and Application of the Rabies Viral Monoclonal Antibody

Laboratory techniques in rabies. Geneva: World Health Organization

Use of neutralizing murine monoclonal antibodies to rabies glycoprotein in passive immunotherapy against rabies

Monoclonal antibodies against rabies virus produced by somatic cell hybridization: Detection of antigenic variants

The establishment of a rapid fluorescent focus inhibition test for testing rabies virus neutralizing antibody

Competitive ELISA using a rabies glycoprotein-transformed cell line to semi-quantify rabies neutralizing-related antibodies in dogs

The study of purified methods of mice ascitic fluid IgG McAb


