A Novel Monoclonal Antibody Against the Constant Region of Goose Immunoglobulin Light Chain

Animals

BALB/c mice (5∼6 weeks old) were purchased from the veterinary institute in Harbin and maintained under standard conditions with free access to laboratory food and water.

Purification of goose Ig

Ig was purified roughly by anhydrous sodium sulfate from goose serum and further purified using protein A affinity chromatography.⁽⁹⁾ The purified Ig mixed with sodium dodecyl sulfate (SDS)-loading buffer was subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Protein expression and purification

A pair of specific primers F/λIgCL (5′-TAGGATCCGTCCTGGGCCAGCCCAAGG-3′, BamH I site underlined) and R/λIgCL (5′-CCGAAGCTTTTACGAGCACTCGGATTTGTTCA-3′, Hind III site underlined) that were used to amplify the GoIgCL gene (309bp) were designed according to the sequence of GoIgL (GenBank ID: HQ852946).⁽⁷⁾ The PCR products were cloned into pET30a (+) and the positive recombinant plasmid was transformed into Escherichia coli strain Rosetta(DE3) Lys cells; then rGoCL was induced with isopropyl β-D-thiogalactoside (IPTG) (Sigma-Aldrich, St. Louis, MO) and the expression product was analyzed by SDS-PAGE.⁽¹⁰⁾ The supernatant was purified by a nickel-charged column (GenScript, Nanjing, China), according to the manufacturer's protocol, and rGoCL was dialyzed as screening antigen, which was used to detect the MAb against GoIgCL.

Cell fusion

BALB/c (6∼8 weeks old) mice were immunized with subcutaneous (s.c.) injections of 50 μg/mouse purified goose Ig emulsions in Freund's complete adjuvant (Sigma-Aldrich) and boosted with an additional 50 mg/mouse of goose Ig intraperitoneally (i.p.) without adjuvant on day 21. After 3 days, the serum was separated to detect the indirect enzyme-linked immunosorbent assay (I-ELISA) method for selecting the positive hybridoma cells, and the spleen was removed for the fusion process. The spleen cells were separated from the spleen, removed according to conventional methods, and then fused with SP2/0 cells at a ratio of 9:1 in serum-free medium using PEG3500 (Sigma-Aldrich). The resulting hybridoma cells were plated onto 96-well plates (Costar, Corning, NY) and cultured in selection medium 1640 (Gibco, Carlsbad, CA) with hypoxanthine, aminopterin, and thymidine (HAT, Sigma-Aldrich). Five days post-fusion, half medium was changed into selection medium 1640 with hypoxanthine and thymidine (HT, Sigma-Aldrich). After 10–14 days, the hybridoma supernatants were screened using the I-ELISA detected to select hybridomas secreting specific antibodies against GoIgCL. After 14 days post-fusion, the selected hybridoma clones were subcloned by limiting dilution and rescreened by I-ELISA three to four times until all the subclones secreting antibodies reacted with rGoCL. The selected hybridomas were expanded for storage in liquid nitrogen after being checked for sterility.⁽¹¹⁾

Establishment of screening method

An I-ELISA method used for selection of hybridoma-secreted antibodies against GoIgCL was developed. 96-well polystyrene plates (Jet, Toronto, Canada) were coated with 0.5 μg/mL rGoCL diluted in a binding solution (0.01 M NaOH buffer [pH 12.0]) with a 100 μL volume and incubated at 4°C overnight. The plate was then washed with PBST (0.01 M PBS [pH 7.4], 0.05% (v/v) Tween-20) four times. The coated wells were blocked by 300 μL blocking buffer (PBST containing 5% skimmed milk) at 37°C for 2 h. After four washes with PBST, the serum separated above was diluted by multiproportion with PBST in a 100 μL volume per well at 37°C for 1 h. Unbound serum was removed by washing four times with PBST, and volumes (100 μL) of HRP-conjugated goat anti-mouse (ZSGB, Beijing, China) were diluted at 1:10,000 and dispensed into all wells. After 1 h incubation at 37°C and subsequent washing, the color was developed using the chromogen/substrate mixture TMB/H₂O₂ (TMB, 3,3′,5,5′-tetramethyl-benzidine; Sigma-Aldrich). The reaction was stopped after 15 min by adding 1 M H₂SO₄ and the OD450 nm of each well was read using a microplate reader (Molecular Devices, Sunnyvale, CA).⁽¹²⁾

Basic characterization of MAb

The isotype of the selected MAb was identified using a mouse monoclonal antibody isotyping kit (Sigma-Aldrich) according to the manufacturer's instructions. The chromosome number of hybridoma cells was analyzed by the conventional method using colchicines (Sigma-Aldrich) with the control of SP2/0 cells.^(13,14) The stability of passage and cryopreservation was also measured by conventional techniques.^(13,15) The ascites of MAb against GoIgCL was prepared using standard procedures as described previously⁽¹⁶⁾ and purified by affinity chromatography using Protein G (Genscript). The titer of ascites produced here was tested by I-ELISA.

Analysis of MAb by Western blot, ELISA, and flow cytometry

Western blot was performed to analyze the immunoreactivity of MAb with goose Ig under standard conditions. The goose serum, goose bile (our lab conserved), and fusion protein rGoCL were separated on 12% gels and then electro-transferred onto a nitrocellulose membrane (NC membrane) in transfer buffer. After blocking with 5% skimmed milk in PBST overnight at 4°C, the NC membrane was incubated with hybridoma supernatant for 2 h at 37°C. Then the membrane was washed with PBST four times and incubated with HRP-conjugated goat anti-mouse (1:1000) for 1 h at 37°C. The HRP substrate, 4-chloro-I-naphthol (4CN, Sigma-Aldrich) was used for color development and visualization of reacted bands.

The reactivity of MAb with other animal Ig was tested by the I-ELISA as described above. The plates were coated with serum (four animals per species) diluted 1:100 in 0.01 M NaOH buffer (pH 12.0), and each serum was repeated three times. At the same time, the purified rGoCL and serum dilution were used as control. Finally, the results were analyzed by conventional statistical methods. The sera that were examined for immunoreactivity analysis were from Gallus gallus, Gallus domesticus, turkey, duck, goose, cow, sheep, pig, rabbit, Anas platyrhynchos, and Grus japonensis.

The reactivity of MAb with native Ig expressed on the surface of B lymphocytes was analyzed by flow cytometry. First, the lymphocytes were isolated from goose peripheral blood by lymphocyte separation medium (TBD, Tianjin, China) according to the manufacturer's instructions. Two×10⁶ cells were added to the centrifuge tube respectively; then the MAb and SP2/0 diluted with PBA (0.01 M PBS [pH 7.4], 0.1% BSA) at the concentration of 0.2 mg/mL in a 200 μL volume were mixed with the cell for 2 h at 4°C. Then the cell was washed with PBS twice and incubated with FITC-conjugated goat anti-mouse (diluted with PBA at 1:100, ZSGB) for 30 min at 4°C. After washing twice with PBS, the cells treated with MAb and SP2/0 were analyzed by flow cytometry.⁽¹⁶⁾

Purification of goose serum immunoglobulin as immunogen

SDS-PAGE was used to analyze the purified protein. The bottom of the band was the light chain, which is noted by the arrow at the location of 26 kDa in Figure 1A. The protein concentration of purified Ig was about 1.5 mg/mL.

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FIG. 1.

SDS-PAGE analysis of protein for MAb preparation and Western blot analysis of the MAb 1B11. (A) SDS-PAGE analysis of protein purified from serum. Lane M, protein molecular weight marker; lane 1, purified goose serum Ig. (B) SDS-PAGE analysis of recombinant protein rGoCL. Lane M, protein molecular weight marker; lane 1, negative control of empty vector pET-30a (+); lane 2, thalline of rGoCL; lane 3, sedimentation of rGoCL; lane 4, supernatants of rGoCL; lane 5, purified rGoCL. (C) Western blot analysis of MAb 1B11 against GoIgCL. Lane M, EasySee Western marker; lane 1, goose serum; lane 2, thalline of rGoCL; lane 3, goose bile.

Expression and purification of rGoCL

rGoCL was mainly expressed as a soluble form in the bacterial cytosol after induction with 0.8 mM IPTG at 16°C for 24 h; the expressed rGoCL was 20 kDa, as expected. Then rGoCL was purified using a nickel-charged column and the result revealed a single band (Fig. 1B), whose concentration was tested as 0.3 mg/mL.

Production and basic characterization of MAb

After the fusion, approximately 20% (75 hybridoma cultures) of the wells yielded hybridomas. One hybridoma, designated 1B11, was selected following subcloning by limiting dilution until the secreting antibody was stable. Isotyping results classified the MAb as the subclass IgG1 with κ light chain. The number of chromosomes of hybridomas 1B11 ranged from 85 to 95, which is consistent with the chromosome analysis of hybridoma. The ascites produced by hybridoma 1B11 has a high titer (Table 1), which can be used for further study.

Analysis of MAb by Western blot, ELISA, and flow cytometry

Western blot analysis showed that MAb 1B11 has a clear reaction with goose serum and bile at a molecular weight of 26 kDa (Fig. 1C), which is the location of the light chain of goose Ig. It also shows a specific reaction with the thalline of rGoCL at a molecular weight of 20 kDa, which corresponds with the expression analysis of rGoCL. The result of I-ELISA showed that the MAb 1B11 only has reactivity with goose serum, and not with the serum of other animals tested (Fig. 2). Flow cytometry analysis demonstrated that MAb 1B11 can recognize the majority of peripheral blood B lymphocytes; the B lymphocytes that reacted with MAb 1B11 took up about 20.5% in the total lymphocytes isolated from peripheral blood, while SP2/0 had no reactivity with the peripheral blood B lymphocytes (Fig. 3). They both demonstrated that the produced MAb has a high specificity with the light chain of goose Ig.

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FIG. 2.

Reactivity of MAb 1B11 with serum from different species. The mean of the OD450 nm for each serum sample was used to analyze the histogram. Error bars were generated by the function of STDEV.

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FIG. 3.

Flow cytometry analysis of the reactivity of MAb 1B11 with B lymphocytes from goose peripheral blood. (A) Flow cytometry analysis of SP2/0 control. (B) Flow cytometry analysis of MAb 1B11 against GoIgCL.