Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody

Yinli Zhao, Guoxi Li
2015 Journal of immunoassay & immunochemistry  
Crystal proteins (Cry) from Bacillus thuringiensis (Bt) are well-known pesticidal proteins in genetically modified (GM) crops and often used as the target proteins in detection of GM food. Instead of the classic mouse hybridoma, new rabbit monoclonal antibodies (McAb) were first produced in detection of GM food. Rabbit McAb and polyclonal antibodies (PcAb) were prepared using Bt-Cry1Ac protein as the immune antigen (Ag). Sandwich ELISA with either PcAb as the coating antibody and peroxidase
more » ... eradish (HRP) labeled McAb as the detecting antibody (PcAb-Ag-McAb), or McAb as the coating antibody and HRP labeled PcAb as the detecting antibody (McAb-Ag-PcAb), was developed for detecting Bt-Cry1Ac protein. The analytical sensitivity of McAb-Ag-PcAb was higher with the linear detection range of 0.97-62.50 ng mL -1 and the limit of detection (LOD) of 0.24 ng mL -1 . There was no cross-reaction with Cry1Ab and Cry1c proteins and the mean recovery was 103.35% in corn leaves. The LOD and the linear detection range of the proposed ELISA are more satisfactory than other Published by Francis Academic Press, UK -26 -ELISA systems using an anti-Cry1Ac PcAb or two Cry1Ac-specific McAb for detection of the target protein and the research provides an effective instrument for detecting Cry1Ac protein in GM crops and their derivatives. In the optimal ELISA, assay specificity, recovery and precision were evaluated. Extraction of whole corn leaves protein: All the corn leaves were collected at the seedling stage and the protein extract buffer was PBST with 2.0 mg mL -1 vitamin C. Clean and dry leaf samples of 23.0 ± 2.0 g were mashed and soaked in 500 µL extract buffer for 60 min on ice. The extracts were centrifuged at 6000 g for 5 min and the supernatant was stored at 4°C until use [17] . Specificity: Another two Bt proteins (Cry1Ab and Cry1c) were used to validate the specificity of the optimal ELISA reaction with the positive control Cry1Ac in this assay. The concentrations of these proteins (Cry1Ac, Cry1Ab, Cry1c) were 2000, 1000, 500, 250, 125, 62.50, 31.25, 15.62, 7.81, 3.90 ng mL -1 . Recovery: To determine the accuracy of the optimal sandwich ELISA method,
doi:10.1080/15321819.2015.1050108 pmid:26023821 fatcat:v36hovaooba77jcpz6epzxwzum