The Study On Preparation And Development Of Monoclonal Antibody For Fast Detection Aflatoxin M₁

Aflatoxins(AFs) are highly toxic and carcinogenic substances produced by Aspergillus flavus and Aspergillus parasiticus on a variety of agricultural commodities.Aflatoxins M₁(AFM₁) is the hydroxylated metabolites of aflatoxin B₁(AFB₁) and may be found in the milk of animals that are fed with AFB₁ contaminated feeds.AFM₁ is known to be hepatotoxic and carcinogenic,therefore its toxicity,initially classified by WHO-International Agency for Research on Cancer(IARC) as a Group 1 human carcinogen(IARC,2002).Foods and foodstuffs which are highly contaminated with AFM₁, especially dairy products can enter human food chain.So it is very important to develop the fast,exact and sensitive detection method to prevent food chains from AFM₁ contamination.The main results from our research are as follows:1.Eight weeks Balb/C mice were immunized by Aflatoxin M₁-bovine serum albumin(BSA) conjugate(AFM₁-BSA).After four immunizations,the mice had a higher immune response,and the spleen cells were fused with mouse Sp2/0 myeloma cells to produce hybridoma.Positive hybridoma was detected by indirect ELISA.Finally,one hybridoma cell line that stably secretes monoclonal antibody against AFM₁was selected.Chromosome average number is 90±10 pairs;the titer of culture medium supernate is 1:6,400;the titer of purified ascites is 5,000×2⁸;heavy chain antibody isotype is IgG1 and light chain antibody isotype isλ,respectively;and the affinity of monoclonai antibody is 0.4×10^-9 mol/L.After 30 generations its antibody titer is stable.2.Using this antibody,an indirect competitive ELISA method for the detection of AFM1 was developed.The best concentration of antigen and monoclonal antibody dilution that were selected by phalanx experiment were 1.0μg/mL and 1:8,000;the best antigen coating condition was 4℃overnight; the best blocking liquid was 5%skim milk;the best concentration dilution of second antibody marked with HRP was 1:5,000;the best reaction time of substrate was 15min,the competitive mode was inter-plate.The linear range for developed indirect competitive ELISA was 0.04-5ng/mL AFM₁,The limit of detection was O.04ng/mL,the recovery of Afatoxin M₁ was between 80.0%-128.3%.The anti-AFM₁ monoclonal antibody concentration required for 50%inhibition of binding for AFM₁,AFB₁, aflatoxin G₁(AFG₁)and deoxynivalenol(DON) was 0.62 ng/mL,4.46 ng/mL,9.25 ng/mL and 0 ng/mL, and the cross-reactivity of the monoclonal antibody clone was 100%,13.9%,6.7%and 0%against AFM₁,AFB₁,AFG₁ and DON.The detection method developed by the optimal conditions,was highly specific and sensitive for detecting AFM₁,showing high practical application value.3.Totally 135 milk samples collected from different big supermarkets and milk factories in three provinces of Northeasten China and were tested by indirect competitive ELISA and all samples were negative and have lower level than 0.5ng/mL for AFM₁.In anyway,only 38(28%) samples were not contaminated or have AFM1 at lower level as detection limit for developed ELISA(0.04ng/mL),but 55 (41%),24(18%) and 18(13%) milk samples have AFM₁ in ranges:0.32～50;0.16～0.32 and 0.04～0.16ng/mL,respectively.