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To generate anti-CHIKV-E2 mAb-secreting hybridomas, the recombinant CHIKV-E2 protein was used as an immunogen. As shown in Fig. 1A, a coomassie brilliant blue dye-stained SDS-PAGE gel showed that the size of the recombinant CHIKV-E2 protein was approximately 40 kDa. Immunoblotting also confirmed that the recombinant CHIKV-E2 protein was detected by the commercial anti-CHIKV-E2 mAbs. Thus, mice were immunized 3 times with recombinant CHIKV-E2 protein mixed with adjuvants in their footpads at 2-week intervals. Two weeks after each immunization, serum samples were obtained from the immunized mice and assessed by ELISA to monitor the production of anti-CHIKV-E2 Abs. The level of the anti-CHIKV-E2 Abs in the serum was four-fold higher after the third immunization compared with that after the first immunization (Fig. 1B), indicating that repeated immunization with recombinant CHIKV-E2 protein drastically induces production of anti-CHIKV-E2 Abs. Next, lymphocytes were obtained from the popliteal lymph nodes of the mice 2 weeks after the last immunization, and the lymphocytes were then fused with FO myeloma cells to generate hybridomas that secrete anti-CHIKV-E2 mAbs. ELISAs were performed using the culture supernatants from the hybridomas to confirm the generation of Ab-secreting hybridomas. Among 58 primary clones, 10 clones had higher OD values to the CHIKV-E2 protein compared with the values obtained using BSA as a negative control (Fig. 1C). In particular, the 4 primary clones called 19, 21, 22, and 26 showed OD values above 2.5, suggesting that these hybridomas effectively produce antiCHIKV-E2 Abs. Thus, we performed subcloning and selected 4 monoclones, designated 19-1, 21-1, 22-2, and 26-4, for further experiments.
Figure 1. Production of anti-CHIKV-E2 mAb-secreting hybridomas. A CHIKV-E2 protein was separated by 12% SDS-PAGE and visualized by Coomassie blue staining. For immunoblotting, the protein was transferred from the gel onto a PVDF membrane and probed with commercial mAbs (Chk265 or 16A12), followed by incubation with HRP-conjugated anti-mouse IgG. Lane M indicates a protein marker (kDa). B, C BALB/c mice were immunized 3 times with CHIKV-E2 protein via footpad injection at 2-week intervals. Serum samples were collected 2 weeks after each immunization (B), and the culture supernatant was harvested from the hybridomas (C). Binding affinities against CHIKV-E2 protein were determined by ELISA. Corrected OD values are shown.
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We firstly investigated whether the newly generated mAbs recognize a linear or conformational epitopes of the CHIKV-E2 protein. As shown in Fig. 2A, immunoblotting showed that all of the anti-CHIKV-E2 mAbs detected CHIKV-E2 protein, suggesting that the mAbs can recognize linear epitopes in the CHIKV-E2 protein (Fig. 2A). The isotypes of the mAbs were determined using antibody isotyping kits (Table 1). The 19-1 and 22-2 mAbs were IgG2b kappa-chain isotypes, while 21-1 and 26-4 were IgG1 kappa-chain isotypes. To compare the binding affinities of the anti-CHIKV-E2 mAbs, we performed ELISAs using various amounts of either the mAbs or CHIKV-E2. Two commercially available anti-CHIKV-E2 mAbs, 16A12 and Chk265, were used for comparison. When various amounts of the generated mAbs were applied on CHIKV-E2 protein-coated plates, the 21-1 mAb showed a higher OD value than those of both the commercial mAbs, and the 19-1 mAb had similar OD values with those of Chk265 (Fig. 2B). Two other mAbs, 22-2 and 26-4, showed lower OD values compared with those of both commercial mAbs. Next, we performed ELISAs using the generated mAbs on the plates coated with serially diluted CHIKV-E2 protein, and all of the mAbs exhibited higher OD values compared with those obtained using the commercial mAbs (Fig. 2C). Notably, the 19-1 mAb showed the highest OD value on the plates coated with CHIKV-E2 protein, ranging from 1.2 to 312.5 ng/mL. As a spiking experiment, we performed ELISA using CHIKVE2 protein in PBS containing 1% human serum. The mAbs, 19-1 and 21-1, showed higher reactivity to CHIKV-E2 protein in the human serum than other commercial mAbs (Fig. 2D). These results indicate that the 19-1 and 21-1 mAbs have higher binding affinities to CHIKV-E2 protein compared with those of the commercial mAbs and recognize linear epitopes in the CHIKV-E2 protein.
Figure 2. Comparison of the binding affinities of the anti-CHIKV-E2 mAbs to the CHIKV-E2 protein. A CHIKV-E2 protein was separated by 12% SDS-PAGE and transferred onto a PVDF membrane. The membranes were probed with the anti-CHIKV-E2 mAbs, followed by incubation with HRP-conjugated anti-mouse IgG. Lane M indicates a protein marker (kDa). B ELISA plates were coated with CHIKV-E2 protein (5 μg/mL), The plates were blocked with 5% skim milk in PBS and treated with serially diluted anti-CHIKV-E2 mAbs, followed by incubation with HRP-conjugated anti-mouse IgG. C CHIKV-E2 protein was coated onto ELISA plates in a dose-dependent manner, blocked, and treated with the anti-CHIKV-E2 mAbs (10 μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG. D As a spiking experiment, ELISA plates were coated with CHIKV-E2 protein (5 μg/mL) in PBS containing 1% human serum, blocked, and treated with the anti-CHIKV-E2 mAbs (10 μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG. Commercial antiCHIKV-E2 mAbs (Chk265 and 16A12) were used for comparison. Corrected OD values are shown.
Clone Isotype Light chain EC50 against CHIKV (ng/mL) 19-1 IgG2b κ 13.86 21-1 IgG1 κ 20.45 22-2 IgG2b κ – 26-4 IgG1 κ – 16A12 IgG1 – 24.58 Chk265 IgG1 λ 22.75 Table 1. Isotypes and EC50 values of the anti-CHIKV-E2 mAbs.
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To investigate whether the newly generated mAbs recognize CHIKV or other mosquito-transmitted viruses, we performed ELISAs using the 19-1 and 21-1 mAbs that bound tightly to the CHIKV-E2 protein. First, inactivated CHIKV was serially diluted and coated onto ELISA plates. When consistent amounts of the anti-CHIKV-E2 mAbs were applied to the plates, the ELISA results showed that the 19-1 mAb had the highest OD value compared with those of the 21-1 mAb and the commercial mAbs (Fig. 3A). The 21-1 mAb had OD values similar to those of the commercial Chk265 mAb. In contrast, the commercial 16A12 mAb showed the lowest OD value. Next, we performed ELISAs using various amounts of the mAbs on the inactivated CHIKV-coated plates. Consistently, the 19-1 mAb had the highest OD value compared with those of the other mAbs, while the 21-1 mAb showed similar OD values with the commercial Chk265 mAb (Fig. 3B). Next, we quantitatively analyzed the binding affinities of the mAbs by calculating the half-maximal effective concentration (EC50). As shown in Table 1, the EC50 of the 19-1 mAb (13.86 ng/mL) was the lowest compared with those of the other mAbs (20.45 ng/mL for the 21-1 mAb, 22.75 ng/mL for the Chk265 mAb, and 24.58 ng/mL for the 16A12 mAb). Additionally, immunoblotting showed that the 19-1 and 21-1 mAbs detected inactivated CHIKV at a bind size of approximately 50 kDa, which matched the predicted molecular mass of the CHIKV-E2 (Warter et al. 2011). For the commercial mAbs, Chk265 recognized the viral CHIKV-E2 protein, while 16A12 barely detected the protein (Fig. 3C). These results indicate that the 19-1 mAb efficiently binds to CHIKV.
Figure 3. Binding affinities of the anti-CHIKV-E2 mAbs against inactivated CHIKV. A Serially diluted inactivated CHIKV was coated onto ELISA plates. The plates were blocked with 5% skim milk in PBS, incubated with the anti-CHIKV-E2 mAbs (10 μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG. B ELISA plates were coated with the inactivated CHIKV (90 μg/mL), blocked, and incubated with serially diluted anti-CHIKV-E2 mAbs, followed by incubation with HRP-conjugated anti-mouse IgG. Corrected OD values are shown. C Inactivated CHIKV proteins were separated by 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were probed with the anti-CHIKV-E2 mAbs, followed by incubation with HRP-conjugated anti-mouse IgG. Commercially available anti-CHIKV-E2 mAbs (Chk265 and 16A12) were used for comparison. Lane M indicates a protein marker (kDa).
Because CHIKV is one of the known arboviruses that cause mosquito-borne diseases, we lastly investigated whether our newly generated mAbs can discriminate CHIKV from other mosquito-transmitted arboviruses, including ZIKV, JEV, and DENV types 1–4. ELISA plates were coated with inactivated arboviruses, and the binding affinities were compared using the anti-CHIKV-E2 mAbs followed by incubation with HRP-conjugated anti-mouse IgG. Chk265 was used for comparison, and PBS was used as a negative control. The ELISAs showed that the 19-1 mAb showed significantly higher OD value only against CHIKV (Fig. 4A). In contrast, the 21-1 mAb showed significant increases in the OD value against DENV types 1–4, as well as CHIKV, compared with the value obtained with the PBS control (Fig. 4B). The commercial Chk265 mAb showed significantly high OD value against CHIKV and DENV types 2–4. To further confirm the lack of binding to other arboviruses of the 19-1 and 21-1 mAbs, we performed ELISAs using the plates coated with serially diluted inactivated arboviruses. As expected, the 19-1 and 21-1 mAbs showed higher OD values against the inactivated CHIKV and barely bound to various amounts of the inactivated ZIKV, JEV, and DENV types 1–4 (Fig. 4D–J). These results indicate that the 19-1 mAb binds significantly to CHIKV but not to the other arboviruses.
Figure 4. Specificity of the anti-CHIKV E2 mAbs. Inactivated arboviruses (CHIKV, ZIKV, JEV, and DENV types 1–4) were coated onto ELISA plates. After blocking with 5% skim milk in PBS, the plates were incubated with A the 19-1 mAb (1 μg/mL), B the 21-1 mAb (1 μg/mL), and C the Chk265 mAb (1 μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG. D–J ELISA plates were coated with serially diluted viral lysates such as inactivated CHIKV (D), ZIKV (E), JEV (F), and DENV type 1 (G), DENV type 2 (H), DENV type 3 (I) and DENV type 4 (J). After blocking with 5% skim milk in PBS, the plates were incubated with the 19-1 and 21-1 mAb (1 μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG. Corrected OD values are shown. Significant differences were analyzed by one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.