Construction Of Label-free Impedimetric Immunosensor For AFB₁Detection And Application Of Anti-idiotypic Nanobody On AFB₁Molecular Mimicry Study

Aflatoxin B1(AFB1), an identified aflatoxin, is extremely toxic among mycotoxinsin contaminated foods and thus poses as a serious hazard to public health. Although adiversity of analysis methods, including thin layer chromatography, enzyme-linkedimmunosorbent assay (ELISA) and high-performance liquid chromatography havebeen developed for AFB1determination, they are often associated with limitationsfrom low sensitivity, high cost, unreliable results, complicated procedures and/orsophisticated instruments. Moreover, the AFB1as reference substance inevitably doesharm to the operater and causes environmental pollution. The present study has twocontents: First, a label-free and reporter-free impedimetric immunosensor based onPPy/PPa/rGO film was developed for ultra-sensitive detection of AFB1. Thedeveloped impedimetric immunosensor provides a potentially powerful tool forreal-time monitoring of antibody-antigen interactions and for the development ofportable devices for food safety screening. Second, the anti-idiotypic nanobodytechnology was applied for AFB1molecular mimicry research with expect to avoidthe injury to operater and damage to environment. The main research content isdivided into the following several aspects:1Construction of label-free impedimetric immunosensor for AFB1detection1.1The deposition of PPy/PPa/rGO nanocomposite film was carried out bycopolymerization of Py and Pa in the presence of rGO with a constant currenttechnique for300s. The molar ratio of Py and Pa in the polymerization solution wasfixed as4:1and the rGO concentration was optimized as16μg/mL. Thenanocomposite was characterized by cyclic voltammetry, electrochemical impedancespectroscopy, scanning electron microscope, e.g., and the results indicated that thePPy/PPa/rGO film had good electrochemically activity and stability, as well as uniquemorphology.1.2The immunosensor was constructed by covalently immobilizing anti-AFB1mAbvia EDC/NHS activation of the carboxyl groups on the PPy/PPa/rGO film and the detection was carried out by monitoring the impedance change of the immunosensorin PBS solution. Its detection limit reaches10fg/mL with a wide dynamic range of10fg/mL to10pg/mL. The AFB2, AFG1and AFG2have5.0%,30.6%and20.1%cross-reactivity with the immunosensor compared with AFB1at the same levelconcentration, respectively. While either DON or OTA has less than1%cross-reactivity with the immunosensor. AFB1spiked real corn samples wereanalyzed and the average recoveries are in the range of80.0±5.0%to112.0±2.5%.In the sensing platform, pyrrolepropylic acid (PPa) provides covalent linkers forantibody attachment while enhancing the hydrophilicity of the film, polypyrrole (PPy)endows the film electroactivity due to its inherent reversible electrochemicaldoping-depoding property, for which no exogenous electrochemical reporter isrequired during impedance measurements. Due to its label-free and reporter-freecharacteristic, the developed impedimetric immunosensor provides a potentiallypowerful tool for real-time monitoring of antibody-antigen interactions and for thedevelopment of portable devices for food safety screening.2Application of anti-idiotypic nanobody on AFB1molecular mimicry study2.1An alpaca was immunized with the F(ab′)2fragment of anti-AFB1mAb and animportant anti-idiotypic (anti-Id) response was developed. Total RNA was preparedfrom the mononuclear cells which were isolated from the whole blood samplescollected7days after the fourth immunization, and then a phagemid librarycontaining2×108clones with100%diversity and90.9%inserted was constructed.2.2The phage display library was panned against anti-AFB1mAb and three rounds ofpanning using Gly-HCl elution method was adopted. The competitive phage-ELISAshowed that11out of the48clones from the third round can be inhibited by AFB1standard when the anti-AFB1mAb coated. The AFB1specific elution method wasalso carried out during panning against anti-AFB1mAb, and13out of48identifiedclones from the second round showed competitive with AFB1standard to anti-AFB1mAb. The results indicate that the use of an antigen-specific elution method duringthe selection of anti-ld Ab from an alpaca VHH library has high efficiency. Moreover,the library was panned against anti-AFB1F(ab′)2using AFB1specific elution method, and8out of48identified clones from the second round showed competitive withAFB1standard to anti-AFB1F(ab′)2.2.3The selected VHHs were expressed, refolded, purified, and then identified by acompetitive ELISA. The results showed that the expressed protein possess wellbioactivity. Immunization the Balb/c using m7showed that it could elicit an Ab3response that recognized the nominal antigen AFB1.