Study On ELISA For Detection Of Toxins

Transgenic protein Cry1Ab and aflatoxin B1(AFB1) is the primary ingredient of genetically modified organisms (GMOs) and the most carcinogenic mycotoxin, respectively. It is of great significance to develop fast detection technology against these two toxins according to the increasing demands for agricultural products and food safety as well as environmental monitoring. The project focuses on the development of new immunoassay methods. Besides the importance of Cry1Ab protein and AFB1, they are chosen as the model analytes since CrylAb protein and AFB1are typical big molecule and the small molecule, respectively. Thus, the resulting methods may be readily extended to the detection of other similar analytes such as pathogenic bacteria and pesticides, and the serving of agriculture, food and environmental safety and so on may be attained.Involving knowledge from optics, immunology, agriculture and food science, material science, nanotechnology, biochemistry, organic chemistry and analytical chemistry etc, this project, taking the big molecule endotoxin (transgenic protein Cry1Ab) and small molecule mycotoxin (aflatoxin B1, AFB1) as target analytes, developed new enzyme-linked immunosorbent assay (ELISA) for toxins. Home-made magnetic beads (MBs) and the commercial nucleic acid purification system was used to improve the automation of immunoassay and consequently a MBs-transfer based semi-automated ELISA (Mts-ELISA) was established. The Mts-ELISA was subsequently successfully applied for the detection of CrylAb protein, providing methodological foundation for development of high-throughput automated immunoassay device with independent intellectual property. Besides, two new ELISA formats were exploringly proposed:the sandwich (based) competitive ELISA and the indirect competitive ELISA with polyHRP for signal enhancement. The proposed formats may provide theoretical foundation for the development of new ELISA kits and diagnostic strips etc.The main contents and results are summarized as follows.(1) A magnetic beads (MBs) transfer based semi-automated ELISA (Mts-ELISA) was proposed with the α-fetoprotein (AFP) as model protein. The Mts-ELISA was compared with both the microplate-based classical ELISA and the MBs based manual magnetic ELISA. The results indicated that:The MBs with diameter of about440nm, synthesized by hydrothermal method, retained good magnetic responsivity after being coated with silicon layer and further animated. The optimal ratio between antibody and aminated MBs (20mg/mL) for bioconjugation by the two-step glutaraldehyde method was20μg antibodies per75μL MBs. It was found feasible to use the MBs-transfer function of the commercial nucleic acid purification system for automation of the immunoassay, and consequently the sensitive MBs-transfer based semi-automated ELISA (Mts-ELISA) was developed. The sensitivity of Mts-ELISA (0.104OD·mL/ng) was better than that of the classical ELISA, with labor saved and shorter dection time (50min). The Mts-ELISA has comparable sensitivity to the manual magnetic ELISA but it is superior to the latter format, since the Mts-ELISA is both time-saving and labor-saving with capacity for high-throughput detection.(2) The Mts-ELISA for transgenic protein CrylAb detection was established. The results indicated that:The aminated MBs are superparamagnetic with the saturation magnetization of56.8emu g-1. It was feasible to extend the direct sandwich Mts-ELISA for AFP protein to the indirect sandwich Mts-ELISA for CrylAb protein. The detection time of Mts-ELISA for Cry1Ab protein was no more than2h, with the capacity to quantitate as low as1ng/mL Cry1Ab. Its performance was comparable to the commercial Cry1Ab ELISA kits.(3) A new ELISA format (sandwich based competive ELISA) for small molecule detection was created, and systematically compared with the conventional direct competitive ELISA. The results indicated:1) It was feasible to detect the AFB1protein conjungate (BSA-AFB1) in the sandwich way with the same AFB1monoclonal antibody; the BSA-AFBl was imagined to be a new intact big molecule antigen and the linked AFB1to be the new epitope of the BSA-AFB1antigen. When the biotinlytated antibody (Biotin-Ab) was used as the secondary antibody, the resulting sandwich ELISA has a LOD of0.05ppb for BSA-AFB1.2) On the basis of the sandwich detection for BSA-AFB1, further introduction of AFB1led to the signal inhibition to BSA-AFB1. The consequent sandwich competitive ELISA for AFB1was achieved.3) The secondary antibody using the anti-AFB1either biotinlytated or HRP-labeled can enable the sandwich competitive ELISA. The HRP-label format is superior to the biotinlytated one since it led the shorter detection time and better calibration curves with smaller background noise and coefficient of variability (CV).4) The optimized sandwich competitive ELISA (2nd batch HRP-anti-AFB1,1:1000dilution; capture antibody,2ppm; BSA-AFB1sandwiched,100ppb) and direct competitive ELISA (2nd batch HRP-anti-AFB1,1:1000dilution; BSA-AFB1coated,100ppb) were compared; both formats were comparable in terms of sensitivity, IC50and linear range etc. The sandwich competitive ELISA has the IC50of0.06-0.07ppb, LOD of19.4ppt and linear range of34.1-166.6ppt; the direct competitive ELISA has the IC50of0.06-0.07ppb, LOD of29.0ppt and linear range of40.2-135.8ppt.5) The intra-assay and inter-assay CVs of sandwich competitive ELISA were4.9-19.3%and3.6-16.8%, respectively; the intra-assay and inter-assay CV of direct competitive ELISA were6.1-11.5%and4.2-9.9%, respectively.(4) PolyHRP was introduced to enhance the signal as signal reporter. The bioAb-SA indirect competitive ELISA, using the biotinlytated anti-AFB1(bioAb) and streptavidin-PolyHRP40(SA), was developed and compared systematically with the traditional indirect competitive ELISA. The results indicated that:1) The optimal concentration for bioAb-SA format was0.005-0.025ppm.2) In the traditional indirect competitive ELISA where anti-AFB1worked as detection antibody (DeAb) and the HRP-anti-mouse IgG as tracer antibody (TrAb), the optimal concentration for DeAb-TrAb was0.005-0.2ppm.3) The comparison between bioAb-SA and DeAb-TrAb formats under optimal concentrations for AFB1detection in different matrix indicated:For PBS, MeOH/PBS and barley matrix in bioAb-SA, the IC50were4.1ppt,8.0ppt, and12.5ppt, respectively; LOD0.7ppt,1.3ppt and0.8ppt, respectively;(O.D.)max1.12,1.27and1.27, respectively. For PBS, MeOH/PBS and barley matrix in DeAb-TrAb, the IC50were4.2ppt,4.4ppt and6.8ppt, respectively; LOD2.1ppt,0.7ppt and2.8ppt, respectively;(O.D.)max0.52,0.55and0.40, respectively. The two formats have comparable performance in terms of IC50and LOD. However, the bioAb-SA ELISA achieved sensitivity2-3times as high as the DeAb-TrAb format. It can be inferred that the signal-reporting capacity for streptavidin-PolyHRP40is16-20times as strong as the HRP-anti-mouse IgG at the same concentration.