| ObjectiveTo explore the effects of monoclonal antibody against α-2(840-877) of Na+/Ca2+exchanger on Na+/Ca2+exchange current (INa/ca).Effects of the antibody on L-type calcium current (ICa-L), voltage-gated sodium current (INa), inwardly rectifier potassium current (Ik1) and transient outward potassium current (Ito) were also investigated in rat ventricular myocytes.Methods1. Acquisition and purification of the monoclonal antibody against a-2<840-877):BALB/c mice were actively immunized with Chemical synthesized a-2polypeptide of Na+/Ca2+exchanger (amino acid sequence840algtsvpdtfaskvaatqdqyadasignvtgsnavnvf877) and monoclonal anti-body was prepared using traditional hybridoma technique. The antibody was purified by antigen affinity column, and titers of the antibody were determined by SA-ELISA.2. Isolation of single ventricular myocyte:Single calcium-tolerant ventricular myocyte was obtained by enzymatic dissociation with Langendorff-perfused system from the ventricular free wall of SD rats weighed250-300g.3. Whole-cell patch clamp technique:Whole-cell patch clamp technique was used to investigate the effect of the monoclonal antibody against α-2(840-877) of Na+/Ca2+exchanger on INa/Ca in enzymatically isolated rat ventricular myocytes. To evaluate the functional specificity of this antibody, its effects on ICa-L, INa, Ik1and Ito were observed.Results1. Establishment of hybridoma cell lines:The positive hybridoma cell lines were screened by ELISA and subcloned three times by limiting dilution culture.Three stable hybridoma cell lines were obtained and named (1E7,2D2,3F10), and the monoclonal antibodies secreted by them were called1E7antibody,2D2antibody and3F10antibody.2. Titers and concentrations of the antibodies against α-2(840-877):ELISA showed that the titers and concentrations of the three monoclonal antibodies(lE7,2D2,3F10) were1:243000,1:243000,1:81000and0.81mg/ml,0.86mg/ml,0.70mg/ml.3. Effects of the antibodies against α-2(840-877) of Na+/Ca2+exchanger on INa/Ca in rat ventricular myocytes:The whole-cell patch clamp results showed that both1E7antibody and3F10antibody had no significant effect on INa/Ca, which suggested neither of them had biological activity to Na+/Ca2+exchange.2D2antibody decreased INa/Ca concentration-dependently:the results showed that both outward and inward Na+/Ca2+exchange currents decreased dose-dependently by the2D2antibody in the concentration range from10to40μg/mL in rat ventricular myocytes. The outward INa/Ca decreased from (3.54±0.09) to (3.05±0.05)(P<0.05),(2.24±0.10)(P<0.01),(1.11±0.08)(P<0.01) pA/pF; the inward INa/Ca decreased from (-3.06±0.07) to (-2.45±0.08)(P<0.05),(-1.54±0.05)(P<0.01),(-0.62±0.04)(P<0.01) pA/pF.4. Investigations on the functional specificity of2D2antibody indicated that at the concentration range of10-40μg/mL, it had no significant effects on Ica-L, INa, Iki and Ito in rat ventricular myocytes (P>0.05).Conclusions1. Chemical synthesized α-2polypeptide of Na+/Ca2+exchanger was used as the antigen, and monoclonal antibody against α-2(840-877) was successfully obtained by traditional hybridoma technique. The monoclonal antibody had significant biological activity.2. At concentrations of10-40μg/mL, the antibody against α-2(840-877) of Na+/Ca2+exchanger specificly inhibited both outward and inward components of INa/Ca in a concentration-relative manner without significant effects on Ica-L, INa/Ca, Iki and Ito in rat ventricular myocytes.Therefore, the monoclonal antibody has higer specificity than the currently existing Na+/Ca2+exchanger inhibitors and it may become a potent tool drug for investigation on Na+/Ca2+exchanger in the future. 3. The monoclonal antibody against α-2(840-877) of Na+/Ca2+exchanger specificly inhibited INa/Ca, which support the idea that a-2repeat region is thought to play an important role in Na+/Ca2+exchanger molecular structure. |
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