Mouse mab isotyping kit

The extracellular domain of ErbB2 (ErbB2-ECD) was prepared as described previously,^{15 (link)} except that we used the pcDNA3.1(+)-expressing vector (Invitrogen, Waltham, MA, USA) and the FreeStyle 293 expression system (Invitrogen). Female BALB/c mice were repeatedly immunized with recombinant human ErbB2-ECD protein. Three days after the final immunization, spleens were collected and the splenocytes were fused to NS-1 mouse myeloma cells. The fused cells were cultured in hypoxanthine/aminopterin/thymidine medium. Culture supernatants from the resulting hybridomas were tested by ELISA for specific antibody reactivity to ErbB2-ECD. Antibody isotypes were determined by using a mouse mAb isotyping kit (Sigma, St Louis, MO, USA). Finally, the mouse anti-ErbB2 mAbs were purified by protein G affinity chromatography from hybridoma culture supernatants.

Six-to-eight-weeks-old Balb/c mice were purchased from the Animal Laboratory Center of Sun Yat-sen University (Guangzhou, China). All experimental procedures were in compliance with the Guide for Care and Use of Laboratory Animals (NIH version, revised 1996). All animals were allowed free access to food and water.
MAbs were produced as described by Kohler and Milstein [19 (link)] and screened by indirect ELISA described below. The IgG fractions were prepared by ammonium sulfate precipitation and purified on Protein A column. The titers were determined by indirect ELISA and mAb isotyping was carried out using mouse mAb isotyping kit purchased from Sigma-Aldrich (St. Louis, MO, USA) according to the manufacturer’s instructions. The specificity of monoclonal antibodies was determined by their binding affinity to both rHRP 2 and P. falciparum infected blood samples. SDS-PAGE was carried out on 12% separating and 5% stacking gels, followed by protein transfer to polyvinylidene fluoride (PVDF) membranes. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., USA) was applied for visualization. Determination of antibody affinity was carried out by a noncompetitive ELISA as described by David Beatty [20 (link)] with little modifications.

The positive hybridoma clones expressing antibodies against His-IpaJ were screened using the previously established indirect ELISA method, with MBP-IpaJ recombinant protein as the coating antigen (9 (link)). Positive clones were sub-cloned three times by the limiting dilution method. The Ig sub-class of MAbs were identified by using a mouse mAb isotyping kit (Sigma, USA) according to the manufacturer's instruction. The positive hybridoma cell lines secreting anti-IpaJ MAbs were injected intraperitoneally into BALB/c mice to grow and proliferate. Ascites fluids containing abundant anti-IpaJ MAbs were collected from the immunized mice and purified by protein A chromatography (GE Healthcare). The purified MAbs were send to GenScript Biotechnique Company (Nanjing, China) for biotinylation with HRP.