ELISA
FOR THE ESTIMATION OF
AFLATOXINS




PROCEDURE SUMMARY


ICRISAT
  
AFLATOXINS

Agricultural products are often contaminated with fungi that can produce toxic metabolites refereed to as “mycotoxins”. Among these, aflatoxins have assumed economic importance because of their influence on the health of human beings and livestock and on the marketability of agricultural products. Aflatoxin is a Group 1 carcinogen proven to cause liver cancer and also suppresses the immune system. In most developing countries limited or no facilities exist for monitoring these toxins in foods and feeds. They are based on phisicochemical methods as TLC and to a limited extent high performance liquid chromatography (HPLC) are used for the estimation of aflatoxins. Immunological methods are preferred over analytical methods because of their simplicity and cost-effectiveness. However, commercial kits based on immunological methods are expensive, and may be difficult to import them. To develop cost effective and simple technologies for the estimation of aflatoxins, United Kingdom Department for International Development (DFID) granted a project in 1998 (project no R7083) to ICRISAT and SCRI. This funding helped in the development of immunochemical methods. High quality antibodies were produced for aflatoxins and the methodologies developed to use antibodies for aflatoxin estimation in different agricultural commodities. The results were comparable with those of HPLC. Costs for performing this test procedure were compared with those of TLC and HPLC and found to be the least expensive of all the procedures and permitted analysis of up to 200 samples per day. The method developed is therefore simple, robust and cost-effective. Constant monitoring of food and feed will contribute to improvement of health of humans and livestock and will also enhance export potential leading to increased income for poor farmers in developing countries.



  
DEFINITION OF TERMS

Antigen: A substance which can elicit production of antibodies when introduced into warm blooded animals.
Antibodies:Glycoproteins that are produced as a result of an immune response following introduction of antigens leading to the production of a specific antigen-antibody complex.
Conjugate:A compound molecule prepared by linking two molecules.
AFB1-BSA:A conjugate consisting of aflatoxin molecules linked to bovine serum albumin. This is required to induce antibodies.
AFB1-BSA-Enzyme: AFB1-BSA attached to an enzyme molecule (alkaline phosphatase or penicillinase or horseradish peroxidase).
Enzyme-Linked Immunosorbant Assay (ELISA):Detecting and quantifying the presence of an antigen (aflatoxin) in a sample using an enzime labelled toxin and antibodies specific to aflatoxin
Direct competitive ELISA:The antibody is coated on to the wells of the ELISA plate (Maxisorp or equivalent). The test sample and the enzyme-labelled aflatoxin B1-BSA are added to the wells. If no toxin is present in the sample, the enzyme labelled toxin will bind to the capture antibody coated to the wells. If toxin is present in the sample, it will compete with the labelled toxin for binding to the antibody. During washing procedures any unbound labelled enzyme will be washed away. On the addition of substrate, a colour will develop, the intensity of which is proportional to the amount of AFB1-BSA-enzyme bound to the well; i.e., the colour intensity decreases with increasing concentrations of the toxin in the sample.

 


 

SAMPLE EXTRACTION

Triturate the seed powder in 70% methanol (v/v - 70 ml absolute methonal in 30 ml distilled water) containing 0.5% KCL (proportion used is 5 ml for 1g seed) in a blender for 2 min. Transfer the extract to a conical flask and shake it for 30 min at 300 rpm. The extract was filtered through Whatman No. 41 filter paper and diluted 1:10 in PBS - Tween. 
 
ELISA PLATE FORMAT
(Fig1)
 
DIRECT COMPETITIVE ELISA
Coat each well of an ELISA plate by using 150 µl of AFB1 antiserum diluted at 1:80,000 in coating buffer
  
Incubate 1 h at 37 oC or overnight at 4 oC
Wash the plate with PBS-T
Add 100 µl of AFB1 standards at concentrations ranging from 100 ng to 0.09 ng. This cover upper two rows of the late. Add samples (100 µl) diluted to 1:10 in the lower part of the plate Use two replicates per sample (see Fig.1)
Coating buffer
Na2CO31.59g
NaHCO32.93g
Distilled H2O1 L
 
Phosphate buffered
saline Tween (PBS-T)
Na2HPO42.38g
KH2PO40.4 g
KCl0.4 g
NaCl16g
Tween201 ml
Distilled Water2 L
 
PBS-T-BSA
Dissolve 200 mg BSA in
100 ml PBS-T
 
Distilled water Tween
Distilled Water2 L
Tween202 ml
    SUBSTRATES
  • ALP system
     Prepare 10% diethanolamine (v/v) in distilled water and adjust pH to 9.8 with conc. HCL. Store this in a dark colored bottle. Add substrate para-nitro-phenylphosphate at the rate of 1 mg/ml buffer before use.
  • PNC system
    Dissolve 15mg bromothymol blue (BTB) in 100 ml of 0.01 M NaOH. Neutralise the alkali by adding 0.1 N HCL drop wise until the pH of the solution is 7.2. Add sodium penicillin-G at 0.5 mg/ml (w/v) concentration.

 

 
 

Calculations:

Using the OD values obtained for AFB1 standards draw a curve, taking AFB1 concs. on the X-axis and OD values on the Y-axis.

AFB1(µg/kg) : (AxDxE)/G

A= AFB1 concentration in sample extract (ng/ml)

D= Times dilution with buffer

E = Extraction solvent volume used (ml)

G = Sample weight (g)







For more information

Dr Farid Waliyar
Principal Scientist
F.Waliyar@cgiar.org
http://aflatoxin.info

Add 50 µl of AFB1-BSA-labelled with ALP at a dilution of 1: 2000
  
Add 50 µl of AFB1-BSA-labelled with PNC at a dilution of 1:10,000
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Incubate at 37 0C for 1 h
  
Incubate at 37 0C for 1 h
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Wash with PBS-T
  
Wash with distilled water-Tween
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Add 150 µl of ALP substrate and keep the plate in dark for 1 h at room temperature
  
Add 150 µl PNC substrate and keep the plate at room temperature for 1/2 h
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Read the plate at 405 nm
  
Read the plate at 620 nm
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Positive reaction:Toxin standards show gradual increase from no colour to pale yellow to deep yellow. No colour indicates high toxin and the deep yellow, no toxin
  
Positive reaction:Toxin standards show gradual change of colour from deep blue to apple green to orange yellow. Deep blue indicates high toxin and orange colour indicates no toxin