| Fumonisin(FB)is a secondary metabolite produced by several fungal species of the genera Fusarium moniliforme and Fusarium proliferatum,which especially contaminates maize and maize products.Currently,more than 20 Fumonisins and their derivatives are known,with fumonisin B1(FB1)being the most abundant and toxic.To ensure efficient control of Fumonisin B1 in cereal and cereal products,accurately and easily performed analytical methods will be required,such instrumental techniques and immunoassays.Immunoassays are sensitive,specific and cost effective encourage themselves to become point of use tools for detecting a large number of samples.Determination of small molecules such FB1 has been based on the competitive immumoassay.In the synthesized procedures,large amounts of FB1 standard and organic solvents which are expensive and may pose a threat to human health are involved,as well as in the development of an immunoassay,which causes potential hazardous to analysts and the environment.In this study,the anti-fumonisin B1 monoclonal antibody(anti-FB1 m Ab(3F11))was used as the target for biopanning from a na?ve alpaca nanobody(Nb)phage display library.One anti-idiotypic nanobody(B26)was isolated.A competitive immunoassay for FB1 was performed based the anti-idiotypic nanobody as coating antigen to substitute the synthetic antigen.Besides,the anti-idiotypic nanobody based immunoassay was more sensitive than the conventional ELISA based on using the FB1-BSA antigen as the coating antigen.Moreover,the anti-idiotypic nanobody–alkaline phosphatase(Ab2 Nb-AP)was expressed.One–step ELISA and One–step CLIA were performed using the Ab2 Nb-AP fusion protein as a probe.Finally,affinity maturation of the B26 by mutational hotspot randomization was constructed.Determination of FB1 immunoassays based on surrogate standard which has higher affinity were developed.The main contents are as follow:1 Biopanning,identification and application of the anti-idiotypic nanobody1.1 Biopanning of FB1 antigen mimeticThe anti-FB1 m Ab(3F11)was used as the target for biopanning from a na?ve alpaca nanobody phage display library.One anti-idiotypic nanobody named B26 was isolated,which showed specific binding to anti-FB1 m Ab(3F11).B26 can be inhibited by FB1 on the competitive phage ELISA with the IC50 of 1.07 ± 0.2 ng/m L.1.2 Expression and application of FB1 antigen mimeticThe expression vector pET-B26 was constructed,which encodes the Nb fragment into the vector p ET25 b.A competitive immunoassay for detecting FB1 was performed using the Ab2 Nb as the coating antigen.The Ab2 Nb was produced in the auto-induction media and was purified using the His Pur Ni-NTA column.The sensitivity of Nb-ELISA was improved 20 times than that of ELISA based FB1-BSA under the optimal conditions.The specificity of the assay was tested,but negligible cross reactivity was observed with AFB1,ZEN,DON,and OTA,except for FB2 which exhibited 4.93%.Ab2 Nb only specific bind to the variable region of anti-FB1 m Ab(3F11)showing it is the ? type anti-idiotypic antibody.Spike and recovery analysis with Nb-ELISA was conducted with corn,rice,and feedstuff samples to evaluate effectiveness of the assay for FB1 analysis.Recovery rates from71.90% to 112.15% were shown.The Nb-ELISA and the commercial ELISA kit were used to detect 30 cereal samples.The correlation(R2 = 0.992)was acquired between the Nb-ELISA and commercial ELISA kit.1.3 Determination of binding kinetics of antigen and antibodyAffinity measurement of the purified Nb fragment was developed on a label-free biosensor.The equilibrium dissociation constants(KD)measured for Ab2 Nb :anti-FB1 m Ab(3F11)and FB1-BSA : anti-FB1 m Ab(3F11)were 164.6 n M and0.62 n M,respectively.Owing to the lower binding affinity between Ab2 Nb and anti-FB1 m Ab(3F11),the sensitivity of Nb-ELISA was better than that of ELISA based FB1-BSA.2 Expression,activity analysis and application of the anti-idiotypic nanobody–alkaline phosphatase fusion protein2.1 The One-step competitive ELISAThe Ab2 Nb-AP protein was produced with good binding capacity and alkaline phosphatase activity.The One-step competitive ELISA was performed using Ab2Nb-AP as the enzyme-labelled antigen.The IC50 of the assay was 2.69 ± 0.25 ng/m L.The LOD of the assay was calculated to be 0.35 ng/ m L,and the linear range,calculated by 20%–80% inhibition of Nb-ELISA,was 0.93–7.73 ng/m L.These samples spiked with a series of known concentrations of FB1(10–1000 ?g/kg)exhibited recovery rates from 71.20% to 114.03%,with relative standard deviations(RSD)rates from 2.43% to 12.50% by One-step competitive ELISA.2.2 The One-step competitive CLIAThe One-step competitive CLIA was performed using Ab2?-Nb-AP as the enzyme-labelled antigen.The LOD of the assay was 0.12 ng/m L,and the IC50 was0.89 ± 0.09 ng/m L with a linear range of 0.29–2.68 ng/m L.The sensitivity of the One-step competitive CLIA was improved by nine times compared to the two-step competitive ELISA(IC50 = 7.22 ± 0.52 ng/m L).The cross reactivity(CR)against FB2,AFB1,ZEN,DON,and OTA was tested,and the results demonstrated CR toward FB2(5.11 %)and negligible CR toward other mycotoxins.These samples spiked exhibited recovery of 83.50% to 112.68% with RSD of 2.47% to 14.86% by One-step competitive CLIA.The One-step competitive immunoassay correlated well with the LC-MS/MS method(R2=0.97).3 Affinity maturation of the anti-idiotypic nanobody3.1 Affinity maturation of the anti-idiotypic nanobody by mutational hotspot randomizationA mutant phage display library based on an AGY/RGYW mutational hotspot mutagenesis in CDR3 of B26 was constructed.After three rounds of selection,four clones were isolated,Named B1 D,B2E,B3 I and B4 L.The equilibrium dissociation constant(KD)measured for B1 D,B2E,B3 I and B4 L : anti-FB1 m Ab(3F11)was 10.4n M,25 n M,49.5 n M and 55 n M,respectively.Compared with B26,the affinity of B1 D was improved 15.8-fold.The thermal stability of B1 D,B2E,B3 I and B4 L was improved as compare to B26 and the FB1-BSA antigen.3.2 The development of green ELISA based on anti-idiotypic nanobodiesTwo competitive ELISAs were developed based on FB1 standard and surrogate standard of nanobody B1 D,respectively.The concentration of nanobody B1 D showed excellent linear relationship with FB1 concentration at the same inhibition ratio.The linear equation is y = 1.7x + 3.2(R2 = 0.998),in which y is FB1 concentration and x is the concentration of nanobody B1 D.The concentration of FB1 was calculated by a two-step calculation: the concentration of nanobody B1 D was first achieved by a logistic regression,and then converted to FB1 concentration by a linear equation.The average recoveries of FB1 spiked from corn samples were in the range of72.80 %-115.44 %.The inter-and intra-laboratory reproducibility was in the range of 2.70 %-9.66 % and 3.17 %-11.04 %,respectively.The assay was compared to a LC-MS/MS method for derermination of 20 corn samples.The correlation was R2=0.986. |
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