Twenty-eight hybridoma cell lines were generated using a hybridoma technique with immunization of HSET peptides (Table 2). To get high concentration of mAb, we injected hybridoma cells into abdominal cavity of mice and collected the Ab-rich ascitic fluids. On the other hands, Abs were purified from the supernatant of hybridoma cells by affinity chromatography. The ascitic fluids and the Abs purified from hybridoma culture supernatant were analyzed by western blotting and SDS-PAGE (Fig. 6 and 10).
Four different forms of HSET antigens were prepared to analyze the binding activity of twenty-eight anti-HSET mAbs. Three antigens including 12 amino acid-length peptides used for mice immunization, 50 amino acid-length synthetic peptide (Met1 - Lys50), and 100 amino acid-length HSET fragment (Met1 - Ala100) displayed on the yeast surface were used for ELISA to analyze the binding activities of mAbs in ascitic fluids. The purified whole HSET antigen was used to analyze the binding of the purified IgG mAbs.
First, we analyzed the binding of mAbs in ascitic fluids to 12 amino acid-length peptides.
Most of clones bound to each peptide which had been used for immunization with high extent, except for clone 1C272, 2C279, 2C280, and 2C281 (Fig. 7). To confirm whether mAbs in ascitic fluids bind to longer synthetic peptide (Met1 - Lys50), ELISA was performed. Anti-HSET mAbs in ascitic fluids were incubated with the peptide-coated wells and then detected with AP-conjugated anti-mouse IgG Ab. Clone 6C407, 8C346, 9C352, and 9C353 showed high binding, compare to positive control that commercial anti-HSET mAb (Fig. 8). MAbs in ascitic fluids, which target epitopes between Met1 - Ala100 of HSET, were analyzed by ELISA using the yeast displayed HSET fragment antigen (Met1 - Ala100). Clone 8C346 showed relatively
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high binding and clone 6C407 and 7C315 bound 1.7-fold greater than negative control to HSET N-terminus (Met1 - Ala100) displayed on yeast cells (Fig. 9).
In ELISA using ascitic fluids, 2 µl of ascitic fluids was treated per well. Considering that the amount of mAbs in 3 µl of ascitic fluids was similar to 1 µg of positive control mouse IgG (Fig. 6), about 1 µg of mAbs were treated to each well. Although concentration of mAbs was different between clones, it seems that binding of mAbs to antigen was mainly determined by the affinity of mAbs. It was because that 8C346 Ab amount in ascitic fluids was smaller than 8C344, but 8C346 showed higher binding to 50 amino acid-length peptide and yeast displayed HSET fragment in ELISA (Fig. 8 and 9).
When the purified mAbs were stored in 50% of glycerol/ 0.05% of sodium azide, aggregation of the purified mAbs was observed, making us unable to determine accurate concentration of the purified mAbs. The concentration of the purified mAbs used for ELISA was compensated based on SDS-PAGE result (Fig. 10). The binding activity of the purified mAbs to whole HSET (Met1 - Lys673) protein was confirmed by ELISA. Clone 1C274, 2C280, 2C281, 6C407, 9C352, and 9C353 bound to whole HSET protein, to similar extent with a positive control (Fig. 11).
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Figure 6. Western blotting of the ascitic fluids containing anti-HSET mAbs. The aliquots (3 µl) of ascitic fluids that had been obtained by i.p. injection of the hybrioma cells were run on 12% acrylamide gels under denature condition. Then, the mAbs were detected by Western blotting using AP-conjugated goat anti-mouse IgG (Fc-specific). Pure mouse IgG of 1µg was loaded as a positive control to compare the concentration of mAbs in ascitic fluids.
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1C272 1C274 1C275 2C279 2C280 2C281 3C288 3C289 3C290 3C292 4C295 4C298 5C303 5C304 6C404 6C406 6C407 7C315 7C316 8C342 8C344 8C346 9C350 9C352 9C353 10C358 10C362 11C308 N.C PBS
0.0 0.5 1.0 1.5 2.0 2.5
Immunogen peptide coating PBS coating
Hybridoma clone
Abs at 405 nm
Figure 7. ELISA for the binding of ascitic fluids containing anti-HSET mAbs to peptide used for immunization. Eleven kinds of immunogen peptides (0.05 μg/well) were coated on the 96-well plate, followed by treatment of 50-fold diluted ascitic fluids. MAbs that bound to immunogen were detected by AP conjugated goat anti-mouse IgG. As negative controls, irrelevant ascitic fluid (N.C) or PBS was used. The first digit of the clone name stands for peptide antigen number. Data represent the mean ± S.D. of triplicate wells.
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4C295 4C298 6C404 6C406 6C407 7C315 7C316 8C342 8C344 8C346 9C350 9C352 9C353 P.C N.C PBS
0.0 0.5 1.0 1.5 2.0 2.5
Hybridoma clone
Abs at 405 nm
Figure 8. ELISA for the binding of ascitic fluids containing anti-HSET mAbs to synthetic HSET peptide (Met1 - Lys50). Synthetic HSET peptide (1 µg/ml) was coated on 96-well plate and then 50-fold diluted ascitic fluids were treated. MAbs were detected by AP conjugated goat anti-mouse IgG. As negative controls, irrelevant ascitic fluid (N.C) or PBS was used.
Commercial anti-HSET mAb (from Abcam) was used as a positive control. The first digit of the clone name stands for peptide antigen number. Data represent the mean ± S.D. of triplicate wells.
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2C279 2C280 2C281 4C295 4C298 6C404 6C406 6C407 7C315 7C316 8C342 8C344 8C346 9C350 9C352 9C353 11C308 P.C N.C PBS
0.0 0.5 1.0 1.5 2.0 2.5
Hybridoma clone
Abs at 405 nm
Figure 9. ELISA for the binding of ascitic fluids containing anti-HSET mAbs to yeast displayed HSET fragment (Met1 - Ala100). HSET fragment displaying yeast cells (5 × 105 cells/well) were coated on 96-well plate and then 50-fold diluted ascitic fluids were treated. The bound mAbs were detected by AP conjugated goat anti-mouse IgG. As negative controls, irrelevant ascitic fluid (N.C) or PBS was used. Commercial anti-HSET mAb (from Abcam) was used as a positive control. The first digit of the clone name stands for peptide antigen number.
Data represent the mean ± S.D. of triplicate wells.
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Figure 10. SDS-PAGE of the purified anti-HSET mAbs. Anti-HSET mAbs collected from hybridoma cell culture supernatant were purified using protein G column. About 20 µg of each protein was loaded on 12% acrylamiade gels and visualized with coomassie blue. In the reducing condition, heavy chain and light chain protein are localized near 50 kDa and 25 kDa size respectively.
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1C272 1C274 1C275 2C279 2C280 2C281 3C288 3C289 3C290 3C292 4C295 4C298 5C303 5C304 6C404 6C406 6C407 7C315 7C316 8C342 8C344 8C346 9C350 9C352 9C353 10C358 10C362 11C380 anti His Ab anti HSET 1-100 Ab anti HSET 625-673 Ab mouse IgG
0.0 0.5 1.0 1.5 2.0 2.5
Hybridoma clone
Abs at 405 nm
Figure 11. ELISA for the binding of the purified anti-HSET mAbs to whole HSET protein antigen. The anti-HSET mAbs (5 µg) purified from culture supernatants were added to the wells coated with whole HSET (Met1 - Lys673) protein (2.5 µg). The bound IgG mAbs were detected using AP-conjugated anti-mouse Ab. Irrelevant polyclonal mouse IgG was used as a negative control. As positive controles, commercial mAbs against N-terminus (1-50 aa) and C-terminus (625-673 aa) of HSET, and His tag were used as positive controls. The first digit of the clone name stands for peptide antigen number. Data represent the mean ± S.D. of triplicate wells.
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