Development Of Aequorin-based Mast Cell Sensor For Rapid Identification Of Botulinum Neurotoxin

The diagnosis of infectious diseases, the monitoring of environment and detection ofpotential bioterrorism agents greatly require a pathogen identification method with bettercombined speed, accuracy and sensitivity. Traditional methods that are based on biochemistry,immunology and genotype are either hyposensitive or time-consuming. Nucleic acid-baseddetection platforms are in general more sensitive than antibody-based detection platforms.However, PCR requires a clean sample and is unable to detect protein samples such as toxins.Cell-based sensors are emerging for the identification of microorganisms, protein toxins andeven nucleic acid. This type of biosensor has many potential advantages in terms of combinedsensitivity, accuracy and speed.In this study, the aim is to develop an aequorin-based mast cell sensor, and evaluate thepotential factors affecting the sensitivity and photon yeild of the sensor. Finally, the detectiontechnique was established and applied in rapid identification and detection of protein toxins.The sequence of the apoaequorin gene (AeqD) was obtained from NCBI nuclear database.DNA of the sequence was synthesized by JINSITE biotechnology (Nanjing, China) aftercodon optimization and then cloned into the pET-his expression vector. The pET-AEQplasmid were transformed into BL21(DE3) competent cells. The apoaequorin was expressedin E. coli, and purified by affinity chromatography. The protein sample containing10mMEDTA was incubated with5μM coelenterazine for2h, and then activated by100mM CaCl2and read in the GloMax96microplate luminometer. The light-emitting charicteristic ofaequorin was studied and evaluated.Aequorin was highly expressed in E. coli in a soluble form with a robust activity, andachieved a acceptable purity after purified by affinity chromatography. It is found that2-mercaptoethanol plays an important part in liberating an efficient signal yield, which canreduce the background and enhance the signal value of aequorin simultaneously. In addition,the linear relation between photon yeild and concentration of aequorin were good (r2=0.993)at the concentration rang from0.16ng to1.6μg. It is indicated that aequorin can bequantified by photon counts from a luminometer. Aequorin can hardly be quantified bymethods based on immunology because aequorin is itself a fairly poor immunogen and thusvery good antibodies are not available. It is suggested that the problem of quantification of aequorin can be resolved by luminescence assay.The apoaequorin gene was cloned into the pEGFP-C1expression vector. The pEGFP-Aeqplasmid was transfected into RBL-2H3cells using lipofectamine2000reagent. Forty-eighthours after transfection, cells were split into fresh medium containing800mM G418until thecells were no more than10%confluent. The cells were fed with selective medium containing400mM G418until G418-resistant cells were stable. Stable clones were selected by limitingdilution. An antigen (DNP-BSA) and coordinate antibody (anti-DNP IgE) were used toevaluate the detection capacity of the biosensors. Many factors, including antibody-bindingduration, coelenterazine concentration and incubation period, were evaluated to findout theeffect of these factors on the eventual signal and sensitivity of cell sensors.Stable transformants were achieved after15-day selection, from which eight clones weregenerated by limiting dilution assay. Aequorin in mast cells behaved as a calcium probe thatcould monitor the signal generated by antigenic stimulation. Constructs with or without GFPfusion partners were developed, and the fusion protein indeed showed significant increases inquantum yield. The incubation period of coelenterazine plays an important part in liberatingan efficient signal yield and sensitivity. Concentrations of coelenterazine ranging from2.5μM to10μM were incubated with cells at37oC, and5%CO2for2h. The cells incubatedwith10μM coelenterazine yielded higher signals but with a higher background, and the LODattained10ng/mL. However,0.5μM coelenterazine could not provide effective substrates toyield robust signals, and the cells could thus only respond to100ng/mL DNP-BSA. Thegroup treated with5μM coelenterazine provided the highest signal-to-noise ratio (S/N) andattained an LOD of1ng/mL. It is shown that1h to4h incubation provided the highest andsimilar signals, and the LOD attained a level of1ng/mL. To select an appropriateantibody-binding duration, cells were incubated with anti-DNP IgE for a period of time thatranged1h to8h. We found that the antibody-binding duration was significantly important toachieve optimal signal intensity and sensitivity. The quantum yield of cells with1h bindingtime was about four times that of cells with an8h binding time. The highest ratio of signalover background of the1h group reached upwards of74folds, while the8h group onlyachieved an increase of8folds. In addition, the sensitivity was improved and the LOD of the1h group achieved0.1ng/mL.A chimeric protein (p21-Fcε) was developed. The mRNA sequence of the CH3and CH4domain of mouse IgE was obtained from the NCBI nuclear database. The sequence of the21-amino acid peptide (P21) was derived from residues40-60of Syt II as describedpreviously. The sequence of the CH3and CH4domain were fused downstream of the P21fragment linked by a (G4S)3linker. The chimeric sequence was synthesized and cloned into pPICZα. The vector pPICZα-p21-Fcε was linearized before transformation of the P. pastoriswild-type strain X-33. The chimeric IgE antibody was expressed, and purified using a Ni2+affinity chromatography column and a gel filtration column.The purified protein wasconfirmed by Western blotting, and ELISA was used to assess the specificity and the bindingactivity of chimeric antibody.The chimeric protein p21-Fcε was highly expressed in P. pastoris in a soluble form, andachieved a considerably high purity after purified by affinity chromatography and gelfiltration. SDS-PAGE analysis and immunoblotting confirmed that the p21-Fcε was indimeric form under native condition and existed in the monomeric form when the disulfidebond was disrupted following β-mercaptoethanol (2-ME) treatment. ELISA showed that thechimeric protein bound strongly to BoNT/B, but it also minimally bound to BoNT/A.Diaminodipropylamine-functionalized magnetic beads were coated with anti-BoNT/Bantibodies to capture BoNT/B molecules in liquid to make targets multimer and concentrated.Adherent cells were loaded with growth medium containing p21-Fcε, followed by growthmedium that was supplemented with coelenterazine-h. Twenty microliters of bead suspensionwas mixed with quantified BoNTs in1.0mL of PBS in microcentrifuge tubes. The beadswere added into the prepared cells in a96-well plate and then read in the GloMax96microplate luminometer.To evaluate the capacity of mast cell sensors for detection of protein toxins, we choseBoNT/B as the model target. To determine whether the recognition of BoNT/B was specific,we used two non-target toxins, BoNT/A and Clostridium perfringens epsilon toxin (ETX) totest RBL cells that pre-sensitized with p21-Fcε. The result showed BoNT/B can active thecells and yeild great signals, and ETX toxins hardly produced any obvious responses.BoNT/A, in part, could stimulate cells to partially emit light. To determine the LOD of thesensor in BoNT/B detection, ten-fold serial dilutions of BoNT/B from0.1nM to10nM wereanalyzed and quantification of photon signal changes was made. As antigen concentrationincreased, photon signals peaked or showed an abrupt increase within60seconds, and the lagtime to the onset of a response decreased. The detection limit for BoNT/B was100pM.In conclusion, the sequence of aequorin gene was confirmed to be correct by the successfulexpression in E. coli, which underlies the expression of aequorin in mammalian cells. And weapplied aequorin in mast cell sensor and investigated several factors affecting the signaloccurring and the sensitivity of the cell sensors. The sensitivity of detection of DNP-BSA wasimproved to0.1ng/mL, which was probably due to the aequorin application and protocoloptimization. In addition, The chimeric protein bound strongly to BoNT/B, which indicateschimeric antibodies is a significant attempt and the expression system of P. pastoris is another way to express antibodies in addition to mammalian cell expression system. Westudied the applicability of RBL-2H3cell sensors in the detection and identification ofBoNT/B. This assay detected as little as0.1nM BoNT/B in less than1h, which included theinitial sample preparation time. It is a meaningful exploration, and different from othercurrent methods for BoNTs detection, even though it was no dominant in terms of sensitivityas compared with other techniques. The success of detecting BoNT/B lays a foundation fordetecting other protein toxins by a mast cell sensor.