The Aflatoxins(AF) are a group of toxic and carcinogenic secondary fungal metabolites produced Aspergillus spp. It is the main monohydroxylate derivative of aflatoxin B1 (AFB1), formed in the liver by means of cytochrome P450-associated enzymes AFM1 in dairy product serious risk for public health in all age group

2. Soniya Ranveer
Dairy Microbiogy Division
National Dairy Research Institute, Karnal- 132001 (Haryana), India
Protein Based Approach for Detection of Aflatoxin
(immunobased)

3. Introduction
Importance of Aflatoxin detection
Methods Of detection Aflatoxin in food
Drawback of Conventional method
Rapid method for detection of Aflatoxin
Conclusion
Content

4. Introduction
• The Aflatoxins(AF) are a group of toxic and carcinogenic secondary fungal
metabolites produced Aspergillus spp. (Ito et al., 2001)
• It is the main monohydroxylate derivative of aflatoxin B1 (AFB1), formed in
the liver by means of cytochrome P450-associated enzymes
• Aflatoxin M1 (AFM1), is a hepatocarcinogen classified in group 1
(carcinogenic to humans) by the IARC (IARC, 2002)

5. AFM1 is mainly soluble in the aqueous phase of milk or adsorbed to casein
particles (Iqbal et al., 2015)
It is metabolized in the liver and kidneys and secreted in urine, faces, and
milk of lactating animals
There is a linear relationship between the concentration of AFM1 in milk
and of AFB1 in feed for dairy cattle (Bakirci, et al., 2014)
AFM1 in dairy product serious risk for public health in all age group
(Nile et al.,2016)

6. The European Community(EC) and Codex Alimentarius Commission(CAC) given
maximum limit of AFM1 in milk and milk products should not exceed 50 ng /L or
50 ng/kg
(CAC, 2001; Creppy, 2002)
It could inhibit RNA transcription and protein synthesis (Prandini et al.,2009)
Aflatoxin M1 is less poisonous than aflatoxin B1 (Govaris et al., 2002)

7. Pulmonary
cancer
Hepatitis
with vomiting
Abdominal
pain
Jaundice
Liver cancer
Acute
Aflatoxicosis
Chronic
aflatoxicosis
Impaired
food
conversion
lower rates
of growth
Dilute bile
Histologic
signs of fatty
changes in the
hepatocytes,
Bile duct
proliferation
Paulín et al,.2016

8. The effects of aflatoxin on human health
Matabaro et al, 2017

9. Stable
Pasteurization
Ionizing
radiation
UHT
cooking
Sterilization

10. Methods of detection Aflatoxin in food
High-performance liquid chromatography
Thin-layer chromatography
Gas chromatography
ELISA - competitive enzyme immunoassay
Ultra Violet Absorption

11. Complicate
Time-consuming
Quantitative difference
Need skilled person
Samples need to be thorough and effective purification
treatment
Not suitable for the detection of large numbers of sample
Drawback of Conventional Method

12. Rapid Method for detection of Aflatoxin
Strip Assay
Method
Immunosensors
Method

13. Principle
It is an analytical method based on an antibody antigen reaction.
Reactant is normally marked and other one is immobilized on a platform
Use of specific antibodies to detect immunogens, which contain the targeted
chemical structures.
AFs are smaller molecules which are not immunogenic ,So there is need
chemical modification into haptens.
AF hapten includes a spacer arm terminating with a reactive group (–COOH
and –NH2) for combining with the carrier protein

14. Antibodies

15. Carrier protein used in detection of Aflatoxin
Bovine serum albumin
Egg albumin
Transthyretin
Human serum albumin
Hemocyanin
KLH

16. Fluorescent microsphere immunchromatographic
test strip assay
Magnetic nanogold microspheres-based lateral-flow
immunodipstick
Techniques for detection of Aflatoxin

17. Fluorescent Microsphere
Immunochromatographic Test Strip
assay(FM-ICTS)

18. It is developed for the detection of aflatoxin M1 (AFM1) residues in milk.
Ultrasensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared
and identified
The MAb was covalently conjugated with carboxylate-modified FMs, and
the FMs were used as the label in a competitive immunochromatography
assay
Moderate hapten-to-protein coupling ratio in the coating antigen used in
improved immunoassay sensitivity
FM-ICTS assay is easy to conduct, rapid, highly sensitive
Principle

20. Magnetic Nanogold Microspheres-based lateral-flow
immunodipstick for rapid
detection

21. Move unbound labeled antibodies along the membrane and bind to
immobilized mycotoxin–protein conjugate on membrane surface at the test
zone
The intensity of the appeared color is indirectly proportional to the
concentration of the mycotoxin in the sample.
Quantitative detection by digital image processing or by measuring properties
such as fluorescence, electroconductivity or magnetism
Principle

22. a. Fabrication process of the synthesized MnGM
c. Principle of the detection method
b. schematic illustration of the immunodipstick (left) and readout (right)

23. Immunosensors

24. Optical
Immunosensors
Electrochemical
Immunosensors
Immunosensors
Matabaro et al,.2017

25. Electrochemical immunosensor

26. It is based on direct ELISA, the enzyme conjugate is free, while
a specific Ab or Ag is immobilized on the electrode
Competitive reaction between enzyme and substrate generates
the current, which is proportional to the enzyme density
Detection range was between 0.015 and 1 ng/mL of AFM1 in
different samples
Principle

27. Matabaro et al,.2017

28. Optical Immunosensors

29. Principle
It use sensitive surface such as a layer of gold or a glass surface is used to
immobilize Ab or Ag.
Amount of AFM1 binding to the Au layer or glass surface increases, the
angle of spectral power distribution (SPD) also increases
Plasmon-enhanced fluorescence spectroscopy used to detect the intensity
of the fluorescence signal produced by resonant excitation of surface
plasmons.
Immunosensor with lower LOD of 0.006 ng/mL
Matabaro et al,.2017

31. Simplicity
Wide application
range
Reproducibility
Low cost
Rapidness
Advantage

32. FLUORESCENCE-BASED DETECTION OF AFLATOXIN M1 IN MILK
USING IMMOBILIZED SPORES
Immoblized aptamer on gold electrode senses trace amount of
aflatoxin traces
Detection of aflatoxin M1 in milk using aptasensor
Singh 2015
Pandey, 2016
Gosewade , 2017
Workdone at
NDRI

33. Conclusion
Presence of AFM1 in dairy product serious risk for public health since all age
groups including infants and children’s consume milk worldwide
The EU and CAC given maximum limit of AFM1 in milk and milk products
should not exceed 50 ng /L or 50 ng/kg
Basic principle of protein based approach is antibody antigen (Ab-Ag) reaction
These method available in both type qualitative and quantitative by small
modification
It is User-friendly tests, Ultrasensitive, Cheaper and less time consuming

34. FLUORESCENCE-BASED DETECTION OF
AFLATOXIN M1 IN MILK USING IMMOBILIZED
SPORES
Immoblized aptamer on gold electrode senses trace
amount of aflatoxin traces
Detection of aflatoxin M1 in milk using aptasensor
Singh 2015
Pandey, 2016
Gosewade , 2017
Workdone at NDRI