ELISA
Aflatoxin
Fungal contamination of agricultural products often leads to the production of toxic secondary metabolites known as mycotoxins. Among these, aflatoxins are particularly significant due to their serious impact on both human and animal health, as well as on the economic value and marketability of food and feed products. Classified as Group 1 carcinogens, aflatoxins are known to cause liver cancer and suppress immune function.
In many developing countries, there is a lack of infrastructure to effectively monitor aflatoxin contamination. While physicochemical methods such as Thin Layer Chromatography (TLC) and, to a lesser extent, High-Performance Liquid Chromatography (HPLC) are commonly used, they are often resource-intensive. Immunological techniques offer a more practical alternative due to their simplicity and cost-efficiency. However, the high cost and limited accessibility of commercial immunoassay kits pose additional challenges.
To address this issue, the UK’s Department for International Development (DFID) funded a project in 1998 (Project No. R7083), led by ICRISAT and SCRI, aimed at developing affordable and user-friendly aflatoxin detection methods. This initiative resulted in the production of high-quality antibodies and the development of immunochemical protocols for the detection of aflatoxins in various agricultural commodities. The accuracy of these methods proved comparable to that of HPLC, while offering a much lower cost per test. In fact, the developed technique allows for the analysis of up to 200 samples per day and is more economical than both TLC and HPLC.
Regular monitoring using these improved methods can significantly enhance food and feed safety, contribute to better public and animal health, and increase the export capacity of agricultural products ultimately improving the livelihoods of small-scale farmers in developing regions.
DEFINITION OF TERMS
Antigen :
A foreign substance (like a toxin or part of a virus) that, when it enters the body of a warm-blooded animal, triggers the immune system to produce antibodies.
Antibodies:
Special proteins made by the immune system in response to an antigen. Each antibody can recognize and attach to a specific antigen, forming an antigen-antibody complex.
Conjugate:
A single compound created by chemically linking two different molecules together, often used in laboratory tests.
AFB1-BSA:
A molecule made by attaching aflatoxin B1 (a toxin) to bovine serum albumin (a protein from cow blood). This combination helps the immune system recognize the aflatoxin and produce antibodies against it.
AFB1-BSA-Enzyme:
The AFB1-BSA molecule connected to an enzyme (such as alkaline phosphatase, penicillinase, or horseradish peroxidase). This allows the detection of aflatoxin during ELISA tests through a color change reaction.
Enzyme-Linked Immunosorbent Assay (ELISA):
A laboratory method used to detect and measure the amount of a specific substance (like aflatoxin) in a sample, by using antibodies and enzymes that produce a visible color when a reaction occurs.
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.
DIRECT COMPETITIVE ELISA
Steps of 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 °C
- 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
5.Add 50 µl of AFB1-BSA-labelled with ALP 5. Add 50 µl of AFB1-BSA-labelled with PNC at a dilution of 1:10,000
at a dilution of 1: 2000
6. Incubate at 37 ° C for 1 h
7.Wash with distilled water-Tween
8.fff Add 150 µl PNC substrate and keep the plate at room temperature for 1/2
9. Read the plate at 620 nm
10. Positive reaction:Toxin standards show gradual change of colour from blue to apple green to orange yellow.
Deep blue indicates high toxin and orange colour indicates no toxin
7.Wash with PBS-T
8.Add 150 µl of ALP substrate and keep the
plate in dark for 1 h at room temperature
9. Read the plate at 405 nm
10.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
More details
| Coating buffer | |
| Na2CO3 | 1.59g |
| NaHCO3 | 2.93g |
| Distilled H2O | 1 L |
| Phosphate buffered saline Tween (PBS-T) | |
| Na2HPO4 | 2.38g |
| KH2PO4 | 0.4 g |
| KCl | 0.4 g |
| NaCl | 16g |
| Tween20 | 1 ml |
| Distilled Water | 2 L |
| PBS-T-BSA | |
| Dissolve 200 mg BSA in 100 ml PBS-T | |
| Distilled water Tween | |
| Distilled Water | 2 L |
| Tween20 | 2 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)