Next, 90??l of TMB answer was added to each well, the reaction was terminated by the addition of 50?l of 1 1?M H2SO4, and absorbance at 450?nm was measured. Statistical analysis Data are expressed as the average value and standard deviation (SD). their products in GM crops is becoming increasingly important and urgent. There are numerous methods for detecting foreign genes in transgenic crops (Kamle et al. 2017; Salisu et al. 2017), and the most direct detection method is based on the gene-encoded protein. ELISA is a specific, sensitive, and convenient method for protein detection. Furthermore, the method is precise, reproducible and employs stable reagents and inexpensive gear. Therefore, ELISA is applicable for routinely detecting foreign gene-encoded proteins in GM crops and their products (Albright et al. 2016a, 2016b; Kamle et al. 2011b, 2013). Based on this information, we used overexpressed His-Vip3Aa20 as an immunogen to generate mouse monoclonal antibodies (mAbs) that recognize certain surface components of the protein. These antibodies were then employed to develop a sandwich ELISA for sensitive, direct and convenient measurement of the Vip3Aa concentration in GM crops and their products. To the best of our knowledge, this is the first quantitative monoclonal antibody-based ELISA method reported for the sensitive detection of Vip3Aa proteins. Materials and methods Reagents, strains and animals DNA markers, polymerase and (10 and BL21) chemically competent cells were obtained from TransGen Biotech. Ni-Resin, Superdex 200 and Protein A-Sepharose columns were purchased from GE Healthcare. A protein marker was purchased from Fermentas. HyClone DMEM and fetal calf serum (FCS) were purchased from Thermo. All reagents were of analytical grade. Complete and incomplete Freunds adjuvant, 50% polyethylene glycol (PEG), hypoxanthine/aminopterin/thymidine (HAT) and hypoxanthine/thymidine (HT) were obtained from Sigma-Aldrich and proteinase inhibitor cocktails from Roche. Goat anti-mouse immuno-globulin horseradish peroxidase conjugate was obtained from Univ-bio (Shanghai, China) and avidin-horseradish peroxidase conjugate from Invitrogen. BABL/c mice were obtained from SBF company (Beijing, China). His-Vip3Aa20 protein Cenicriviroc Mesylate expression The gene was amplified from GM maize MIR162 genomic DNA and subcloned into the pET28a plasmid. pET28a-and pET28a-were generated by site-directed mutagenesis(Palma et al. 2017). The and genes were chemically synthesized and subcloned into the pET28a plasmid. The reconstituted plasmids were transformed Cenicriviroc Mesylate into BL21 competent cells. Overexpressed recombinant His-Vip3Aa20, His-Vip3Aa1/19, His-Vip3Aa7/10, His-Vip3Aa14 and His-Vip3Aa8 proteins were purified with His-affinity purification (native or denature) and further fractionated by size-exclusion chromatography using a Superdex200 column with 50?mM Tris HCl, pH 7.6, 150?mM potassium chloride and 5% glycerol. Mice immunization protocols The immunization protocol followed conventional subcutaneous (s.c.) injection, with slight modifications (Li et al. 2009). Eight-week-old male BALB/c mice were immunized with immunogen [His-Vip3Aa20 dissolved in 250??L of sterile phosphate-buffered saline (PBS) adjuvant Rabbit Polyclonal to FSHR with complete Freund’s adjuvant or incomplete Freund’s adjuvant] at the nape of the neck at weeks 1, 4, 6 and 8. The immunizing dose was fixed at 50?g by subcutaneous multi-point injection per mouse each time. Three days prior to cell fusion, the mice were boosted with 50?g of adjuvant-free immunogen. Blood samples were collected from the mouse tail, and the titers were determined by ELISA. Fusion, hybridoma screening and mAb generation Seven days after the final booster immunization, a single-cell suspension was aseptically prepared in RPMI-1640 medium from mouse spleen samples. The cell suspensions were mixed with murine myeloma cells SP2/0 (Genecreate Biological Engineering Co.) at a ratio of 10:1. The mixed cell suspension was centrifuged, and the supernatant was completely removed. The combined splenocyte suspension and sp2/0 cells were fused using a modification of the method described by Galfre et al(Galfre and Milstein 1981). One milliliter of 50% PEG1450 was added dropwise to the mixed cell pellet in a 50-ml centrifuge tube over 90?s. The mixture was gently stirred while PEG1450 was added, and the mixture was then allowed to incubate for 1?min. Cenicriviroc Mesylate Fusion was stopped by dropwise.
Next, 90??l of TMB answer was added to each well, the reaction was terminated by the addition of 50?l of 1 1?M H2SO4, and absorbance at 450?nm was measured