1.Development Of Colliodal Gold Immunochromatographic Assay For Rapid Detection Of Aflatoxin B₁ 2.Panning Mimotopes Of Aflatoxin B₁ By Phage Display Peptide Library

Aflatoxins (AF) are toxic secondary metabolites mainly prouduced by the fungi Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius on foods and feedstuffs. AFB₁ has been evaluated by the International Agency for Ressarch on Cancer of WHO as a class I human carcinogen in 1993 because of their toxic and carcinogenic potentials to humans and animals. Among 20 different types of aflatoxins identified, the major ones are aflatoxin B₁ (AFB₁), AFB₂, AFG₁, AFG₂2, AFMi, AFM₂ and so on, and AFB₁ is the most potent hepatocarcinogen known in mammals. Many courtries have established regulatory limits for AFBi in foods. In order to avoid foods and foodstuffs which are high contaminated by AFB₁ enter human food chain. So it is very important to develop convenient operation method for rapid detecting AFB₁.The main results are as follows: 1 Development of colloidal gold immunochromatographic assay for rapid detection of AFB₁(1) One ascitic IgG monoclonal antibody (McAb) was purificated by caprylic acid-ammonium sulfate precipitation (CA-AS).Activities and purity of monoclonal antibody were monitored by indirect ELISA and SDS-PAGE, respectively. The antibody titers and ascetic are l:1.0×l0⁶and I:2.0×l0⁶, respectively. The antibody only occur light and weight chains but ascetic occur many band in SDS-PAGE.(2) Tri-sodium citrate with aqueous gold chloride were warmed up and mixed to make gold sol. Colloidal gold were labeled with monoclonal antibody against AFB₁ and were jetted onto the glass fiber by XYZ3000 system after the mixure wered purified. AFB₁-BSA and donkey anti-mouse immunoglobulins were jet-positioned onto a nitrocellulose membrane which the distance was 4 mm. Nitrocellulose membrane, gold conjugate pad, sample application pad, filter paper and absorbent pad were assembled onto a sheet and cut into individual strips(4 mm X 55 mm) using a strip cutter CM4000 supplied by Biodot. Then strips were cased into card and sealed conservation.(3) Parameters of strips were optimized by single factor analysis: diameter of colloidal gold particles was 40 nm, colloidal gold labeled antibodies was 0.5 μg/mL, optical density (OD) of labeled antibodies was 6.0, material of NCM was CN140 and concentration of AFB₁-BSA and donkey anti-mouse IgG were 0.3 mg/mL and 0.5 mg/mL, respectively.(4) The visual detection of strip limit is 5 ng/mL and have no cross-reacted with Ochratoxin A, zearalenone, Deoxynivalenol, Citrinin and patulin mycotoxins. Repetition of strips was 98%. According to expert experience that the quality of the strips conserved in 4°C for fifteen months were no declined.(5) 11 samples were detected by strip and ELISA, respectively. MeOH/H₂O(80:20, V/V, 0.4% NaCl) was used for the extraction of AFB₁. Mixure centrifuged several minutes and pipeted the...