| Aflatoxins, the most probable human carcinogen, are toxic, mutagenic, andcarcinogenic secondary metabolites produced primarily by Aspergillus flavus and A.parasiticus. Aflatoxin B1(AFB1) commonly contaminates agricultural commodities,foods and animal feedstuffs, which is an important problem all through. Howeveractual methods (such as TLC, HPLC, ELISA and so on) are time consuming and needexpensive instruments. Thus development of a rapid and convenient detection methodfor local AFB1 analysis is extremely desirable. An immunochromatographic stripsystem involving the use of colloidal gold-labeled monoclonal antibody(McAb)specific to AFB1 as the marker was developed for the analysis of AFB1 in foods. Andthe correlative mechanism was also discussed.The conjugation of AFB1 and bovine serum albumin BSA (AFB1-O-BSA, theimmuo-antigen of AFB1) was produced according to the method of EDC catalyze.Mole ratio of the conjugation of 9.34 mole AFB1-0 (AFB1-Oxime) to BSA wasobtained with the reaction mole ratio of 20:1 and mixture solution system of DMF andPBS. And the utilizing ratio of AFB1-O was 46.7%. The characterizations of BSAwere also studied by fluorescence specectroscopy. Results showed that AFB1-0 hadquenched the fluorescence of BSA and had effect on its conformation. Ba1b/c micewere immunized by intrasplenic injection of AFBi-O-BSA. Two hybridoma cell lines3A7 and 5D3 capable of secreting specific monoclonal antibodies (McAb) againstAFB1 were obtained through cell fusion, clone and cultivation. Subtype of the twoMcAb was IgG2a-Titer was 20,0000 and 7,0000 respectively. The affinity constantwas 3.81 xlO7 and 3.43xl07L/mol respectively. Cross reactivity with AFB1 analogswas showed to be very low.The optimum preparation parameters of monodispersional colloidal gold wereattained by correctitude experiment through magnetic force mixer. Average diameterof the colloidal gold was 10.7nm and coefficient variation (CV) wasl4.2%. Reactionkinetics process of the solution behavior of colloidal gold formation in differentdiameters was performed at different time by UV-visible spectrophotometer andtransmission electric microscope (TEM). Results showed that the absorptionmaximum wavelength at 200nm markedly decreased with the reduction beginning andan obvious visible light absorption occurred at 560nm as the color changing. With thetime continuing, the visible light absorption increased and maximum wavelengthdecreased continuously. The formation process of big particles is much shorter thansmall gold solutions but showed inferior dispersion stability. Five colloidal goldparticles (average diameter of 10.7nm, 14.4nm, 25.1nm, 36.9nm, and 67.4nmrespectively) were synthesized. TEM imagines indicates gold colloid are inmono-dispersion with a narrow diameter distribution (<15%).McAb specific to AFB1 was conjugated to the gold nanoparticles under friendly andoptimal condition. Optimum pH for conjugation was determined to be 7.4, reactiontime was 5 min, and the least stable content of McAb to colloidal gold in fivediameters(l0.7-67.4nm) was 0.07, 0.051, 0.033, 0.019, 0.012 mg per milliliter goldsolution, respectively. Combination of McAb with nanogold particles was alsocharacterized by TEM, IR spectra and immunoreactivity. Results showed that titer ofMcAb increased from 20,0000 to 22,0000 respectively. The affinity constantincreased from 3.81×107to 4.14×107L/mol. And conjugation probe exhibited a smallincrease of 11 % in stability to temperature.The interaction of colloidal gold and McAb was firstly studied by fluorescencespectroscopy. Results showed that colloidal gold has a powerful ability to quench theMcAb fluorescence via a nonradiative energy transfer mechanism. The fluorescencequenching data were analyzed according Stern-Volmer equation. The bindingconstant of reaction was obtained. The data of enthalpy and entropy proved that themain interactions were London-van der Waals force and hydrogen bond betweencolloidal and McAb. From the synchronous spectrum colloidal gold has not obviouseffect on the conformation of McAb. Results of circular dichrism(CD) spectra showedthat a-helix content increased from 52.5% to 54.2%, p-pucker content decreased from21.0% to 19.8%, corner content increased from 4.6% to 7.3%, rueless curl contentincreased from 20.2% to 20.5%. It was confirmed that the small change ofconformation of McAb was the reason of increase of titer, affinity and stability.A rapid, signal step and sensitive colloidal gold labeledimmunochromatographic(GICA) test for detection of AFB1 in foods was developed.Conditions for the analysis of AFB1 to be completed in less than 10 min wereoptimized. With visual observation, the lower limit was found to be around 1.0 ppb.For quantitative analysis by adopting a photometric strip reader, the lower detectionlimit was 0.01-2.5ug/kg. The detection limit (ID10) for AFB1 was around0.019ug/kg.The analytical recoveries for AFB1 added to rice, corn, and wheat rangein 2 to 50(ig/kg were found to be 80.54 %~108.81 %. The CVs of all the assays werebetween 6.07%~14.63%. The shelf life of immunostrips constructed with preserverwas also examined to retain for 120 days at room temperature. Sixty seven naturallycontaminated food samples were subjected to GICA and commercial ELISA kit test forAFB. Results showed a good correlation between these two methods with a square ofcorrelation coefficient of 0.92 and slop of 0.98. Effects of salt density , metal ions (Fe,Cu, Pb, As) and oil content on the testing results of GICA were also certified in theresults. These effects could be diminished when the extraction was leached with certainsolvents before testing. The results would provide theory information to the foundationof extraction methods of AFB1 in different food samples.
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