Development of Neutralizing Monoclonal Antibodies Against VP4 of Rotavirus CC0812-1

Cultivation of RV CC0812-1

MA104 cells were routinely cultured in DMEM (Gibco, Life Technologies, Carlsbad, CA) supplemented with 10% fetal calf serum (Gibco) and 1% L-glutamine (Amresco, Cleveland, OH) in a 5% CO₂ incubator at 37°C. The cultivation procedure of RV CC0812-1 was carried out as described by Urasawa and colleagues.⁽¹⁰⁾ Briefly, 1 mL of RV CC0812-1 (1.0×10³ TCID₅₀) was treated with 15 μg/mL trypsin for 30 min at 37°C and inoculated to MA104 cells cultured in a 25 cm² flask. Following adsorption to the MA10 cells for 1 h at 37°C, the virus inoculums were pipetted off. After being washed three times with Dulbecco's phosphate-buffered saline (DPBS), the cells were maintained in DMEM containing 2 μg/mL trypsin. The culture supernatant of RV-infected MA104 cells was harvested at 96 h post-infection.

Purification of RV CC0812-1

The harvested culture supernatant of RV-infected MA104 cells were frozen and thawed three times and clarified by centrifugation at 7000 rpm for 30 min. The polyethylene glycol (PEG) 6000 (Merck, Darmstadt, Germany) was added to the supernatant to a final concentration of 5%. The supernatant was then kept in a 4°C refrigerator overnight to allow virus precipitation. The precipitated RVs were collected by centrifugation at 15,000 rpm for 30 min and were resuspended in DPBS. The clarified suspension was subjected to Sepharose CL-4B gel for filtration chromatography and eluted with DPBS. The elutions were inspected by ultraviolet spectrophotometer at the wavelength of 280 nm. The RV fractions were collected and combined; the concentration of RVs was determined by the ultraviolet spectrophotometer method. The purified RV CC0812-1 was negative stained by phosphotungstenic acid and was observed by electron microscopy (Hitachi H-7000FA).

Mice immunization

Six-week-old female BALB/c mice were purchased from the Medical Laboratory Animal Center of Hubei Province and were maintained and bred in the laboratory animal house of Huazhong Normal University (Wuhan, China) under specific pathogen-free conditions and 22±1°C standard with a regular 12 h light/12 h dark cycle and allowed free access to standard laboratory food and water. Animal studies were approved by the Science and Technology Department of Hubei Province. Female BALB/c mice were injected intraperitoneally four times with 100 μg purified RV CC0812-1 at 1-week intervals. Complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO) was used in the first immunization and incomplete Freund's adjuvant was used in the subsequent three booster shots. A final immunization with 100 μg purified RV CC0812-1 only was given intravenously 3 days before cell fusion.

Mice bearing myeloma

The sp2/0 cells were maintained in RPMI-1640 with 10% fetal calf serum (Gibco) and 1% L-glutamine (Amresco, Solon, OH) in a 5% CO₂ incubator at 37°C. After being washed three times with DPBS, 5–10×10⁶ sp2/0 cells were intramuscularly implanted into the back of BALB/c mice to form a solid tumor of mice myeloma 10–14 days before cell fusion.

Cell fusion and cloning

Cell fusion was performed using the modified methods described by Campbell.⁽¹¹⁾ Spleens were dissected from BALB/c mice immunized with purified RV CC0812-1, and splenocytes were prepared in serum-free RPMI-1640 medium (Invitrogen, Carlsbad, CA). The myeloma sp2/0 cells were isolated from the sp2/0 solid tumor of BALB/c mice-borne myeloma. For hybridoma preparation, the splenocytes were mixed with mouse myeloma sp2/0 cells at a ratio of 5–10:1 in serum-free RPMI-1640 medium at room temperature and were centrifuged at 1000 rpm for 10 min. The mixture pellet of splenocytes and myeloma cells was resuspended in 1 mL 50% PEG 1450 (Sigma-Aldrich) over 1 min, followed by the serial addition of 3 mL RPMI-1640 over 3 min and 10 mL RPMI-1640 over 1 min, with gentle stirring. Fused cells were centrifuged at 1000 rpm for 5 min and resuspended in RPMI-1640 medium with 20% fetal calf serum and HAT medium (hypoxanthine, aminopterin, and thymidine, Sigma-Aldrich) at 2.5×10⁶ splenocytes/mL. The fused cells were then dispensed at 100 μL/well into 96-well cell culture microplates (Greiner Bio-one, Frickenhausen, Germany) previously plated with BALB/c mice macrophage feeder cells of 10⁴ cells/well. Cells were incubated at 37°C with 5% CO₂. The medium was half-replaced with fresh selection medium 7 days later. The culture supernatants were pipetted off for antibody screening by ELISA. Antibody-producing hybridoma clones were cloned three times in RPMI-1640 medium supplemented with 20% fetal calf serum and HT medium (Gibco) by limiting dilution method.

Screening ELISA

For screening antibody-producing hybridoma clones, 96-well microplates were coated with 100 μL purified RV CC0812-1/well at 8 μg/mL diluted in carbonic acid buffer (coating buffer, 33.7 mM NaHCO₃, 15 mM Na₂CO₃ [pH 9.6]) and incubated at 4°C overnight. The microplates were blocked by incubation with 10 mg/mL bovine serum albumin (BSA) in DPBS containing 0.05% Tween-20 (DPBS-T) for 2 h at 37°C and washed three times with DPBS-T. Then 100 μL culture supernatant was added and incubated for 1–2 h at 37°C. After being washed three times with DPBS-T, 100 μL of rabbit anti-mouse IgG HRP conjugate (1:5000 diluted in DPBS-T with 0.5 mg/mL BSA) were added and incubated for 1–2 h at 37°C. After being washed three times with DPBS-T, ELISA analyses were developed using 100 μL of 0.04 mg/mL of freshly prepared OPD (o-phenylenediamine) substrate, stopped with 50 μL of 2 M H₂SO₄, and read at 492 nm on the microplate reader (SpectraMax M5/M5e, Molecular Devices LLC, Sunnyvale, CA).

Isotyping of MAbs

The immunoglobulin class and subclass of MAbs were determined with an indirect ELISA assay using a mouse immunoglobulin isotyping kit (Proteintech Group, Chicago, IL) according to the manufacturer's instructions.

Western blot analysis

The specificity of the MAbs was evaluated by Western blot analysis using purified RV CC0812-1. Western blot analysis of MAbs was carried out as described by Towbin.^(13a) Purified RV CC0812-1 were dissolved in protein sample buffer, immediately boiled for 10 min, and then separated with 10% SDS-PAGE. After electrophoresis, RV proteins were transferred to a nitrocellulose membrane (Immobilon-P, Millipore, Billerica, MA). The membrane was blocked by immersion in Tris-buffered saline (TBS) containing 5% nonfat milk at 37°C for 1 h, and washed three times with TBS containing 0.05% Tween-20 (TBS-T). The membrane was then incubated with MAbs of RV CC0812-1 at 37°C for 1 h; rabbit anti-mouse IgG HRP conjugate (1:500 diluted in DPBS-T with 0.5 mg/mL BSA) was added after washing three times with TBST and incubated at 37°C for 1 h. Finally the membranes washed three times with TBST were developed using an enhanced DAB reagent.

Neutralization test

For the neutralization test, MA104 cells were plated in 96-well plates. The MAbs serially two-fold diluted with PBS were added to an equal volume of 100 TCID₅₀ trypsin-treated RV CC0812-1 and incubated for 1 h at 37°C. The serial reaction resultants of the MAb-RV were regularly inoculated to MA104 cells in 96-well plates. The infected MA104 cells were maintained in DMEM containing 2 μg/mL trypsin. The CPE on MA104 in the 96-well plates was checked daily and the neutralization titer was calculated using the Reed-Muench method.

Morphology of purified RV CC0812-1

Calculated according to an optical density value of 260 and 280 nm, the concentration of RV CC0812-1 purified by PEG precipitation combined with Sepharose CL-6B gel filtration chromatography was up to 600 μg protein/mL. Under transmitted electron microscopy, most of the purified RV CC0812-1 were distinctive, intact, and triple-layered particles and exhibited a typical morphologic appearance of rotavirus virion resembling a wheel with short spokes and a well-defined, smooth outer rim (Fig. 1).

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FIG. 1.

Morphology of purified RV CC0812-1 (10,000×).

Efficiency of cell fusion

Seventy-two hours after cell fusion, hybridoma cells were observed emerging in 133 wells of two 96-well plates and the efficiency of the cell fusion was up to 69%. Screening the supernatant of these 133 clones for RV antibodies, 57 clones were positive by indirect screening ELISA. After cloning three times by the limiting dilution method, four hybridoma cell lines, designated as 1B1, 1B8, 1F11, and 1G10, respectively, which stably secret antibodies against RV CC0812-1, were selected and established from these 57 positive clones.

Characterization of MAbs

The isotypes of four MAbs were determined by indirect ELISA. The indirect ELISA analyses revealed that MAbs 1B1, 1B8, and 1G10 belonged to the IgM class, and MAb 1F11 belonged to the IgG1 subclass.

Specificity of the MAbs was further analyzed by Western blot. The Western blot analysis demonstrated that brown bands at approximately 88 kDa, consistent with the expected molecular weight of VP4 of RV CC0812-1, were all developed on nitrocellulose membranes for MAbs 1B1, 1B8, 1F11, and 1G10 (Fig. 2). These four MAbs were all directed against the structural protein VP4 of RV CC0812-1.

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FIG. 2.

Western blot analysis of MAbs secreted by four hybridoma cell lines. M, molecular weight marker of proteins; lane 1, 1-B1; lane 2, 1-B8; lane 3, 1-F11; lane 4, 1-G10.

The neutralizing activity of the MAbs was further investigated by neutralization test. The neutralization test showed that all four MAbs had the capacity of neutralizing RV CC0812-1; they were all neutralizing MAbs against VP4 of RV CC0812-1 although their neutralization titers were different. The neutralization titers of MAbs 1B1, 1B8, 1F11, and 1G10 in BALB/c mice ascetic fluids were 1:2048, 1:1024, 1:512, and 1:512 for RV CC0812-1, respectively.