Application of ELISA in food analysis

1. APPLICATION OF NANO-ELISA IN
FOOD ANALYSIS: RECENT
ADVANCES AND CHALLENGES
Reza Joia
Teacher: Albana vaseli
FACULTY OF
CHEMISTRY
ANDCHEMICAL
TECHNOLOGY

2. Introduction 2
A sensitive biochemical or immunochemical assay method that uses analyte (antigen or
antibody) and color change to detect and identify a substance and its quantification
Also called solid-phase enzyme immunoassay as it employs an enzyme linked antigen
or antibody as a marker for the detection of specific protein
An enzyme conjugated with an antibody reacts with a colorless substrate to generate a
colored reaction product
A number of enzymes labels have been employed for ELISA, including alkaline
phosphatase (AP), the most commonly used horseradish peroxidase (HRP), and β-
galactosidase

3. Brief history of ELISA
• Health and environmental concerns
associated with radioactive elements led to
the search for non-radioactive alternatives.
• In 1966, Avrameas and Pierce introduced enzyme-labeled
antibodies, addressing some issues but creating new
challenges.
• The development of immunosorbents and the
emergence of nano-ELISA in 2012, utilizing gold
nanoparticles, marked a transformative shift in
immunoassay technology.
• Radioimmunoassay (RIA) with iodine-131
labeled antibodies was introduced in 1960
by Yalow and Berson.

4. Structure of ELISA
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Sorbent Substrate: The foundation of ELISA involves the use of antigen or antibody as the
sorbent substrate. This component binds to a supporting material, forming the basis for
bioconjugation.
Immuno-Recognition: The framework of ELISA is built upon immuno-recognition, where the
sorbent substrate interacts with complimentary biomolecules labeled with enzymes. This
interaction is a crucial step in the assay.
Enzyme Label: Serving as the signal transverter, enzymes are used as labels in ELISA. They
facilitate the formation of bioconjugates between the sorbent substrate and labeled biomolecules,
contributing to the detection process.
Chromogenic Reagent: The generation of the detection signal in ELISA is achieved by
introducing a chromogenic reagent. This step produces a measurable output signal, completing
the four fundamental components of ELISA – sorbent substrate, immuno-recognition, enzyme
label, and chromogenic reagent.

5. Principle 5
• Immuno-Recognition Principle:
• The core of ELISA revolves around specific immune reactions between antibodies and antigens.
• Antibodies or antigens are initially coated on a solid substrate through physical adsorption effects.
• Specific recognition captures and immobilizes the antibody or antigen on the supporting substrate.
• Process Steps:
• Substrate surface is blocked to prevent non-specific adsorption in subsequent stages.
• Enzyme-labeled antibodies are introduced and incubated with the antigen, forming a bioconjugation.
• Washing steps are crucial in each coating or incubation to remove excess proteins and prevent false positives.
• Chromogenic Reaction:
• Chromogenic reagents catalyze the enzymatic reaction, leading to color variations in the microplate.
• Color intensity serves as a qualitative judgment via the naked eye or a quantitative measurement using a
microplate reader.
• Detection Methods:
• The direct method involves measuring the antibody or antigen directly.
• An alternative method includes detecting the antibody or antigen through an enzyme-labeled secondary
antibody.

6. General Procedure
Antibody (Ab) or
immunoglobulin (Ig)
a large, Y-shaped protein
produced mainly by
plasma cells that is used
by the immune system to
neutralize pathogens
such as pathogenic
bacteria and viruses
Antigen
a toxin or other foreign
substance which induces
an immune response in
the body, especially the
production of antibodies.

7. Type of ELISA
• Direct ELISA:
• Coat solid surface with target antigen.
• Add enzyme-labeled primary antibody.
• Allow direct binding.
• Measure signal after enzymatic reaction.
• Indirect ELISA:
• Coat wells with antigen.
• Add sample containing primary antibody.
• Introduce enzyme-conjugated secondary
antibody.
• Measure signal after enzymatic reaction.
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• Sandwich ELISA:
• Coat wells with capturing antibody.
• Add sample containing antigen.
• Introduce enzyme-linked detection antibody.
• Measure signal after enzymatic reaction.
• Competitive ELISA:
• Incubate antibody with antigen-containing
sample.
• Add mixture to antigen-coated well.
• Introduce enzyme-conjugated secondary
antibody.
• Measure signal, inversely related to antigen
concentration.

8. Type of ELISA 8

9. Sandwich ELISA
 the most powerful ELISA assay format is the sandwich assay.
This type of capture assay is called a “sandwich” assay because
the analyte to be measured is bound between two primary
antibodies – the capture antibody and the detection antibody.
 sandwich format is used because it is so sensitive and robust

10. Direct ELISA
Indirect ELISA

11. Indirect ELISA Detection
Advantages • A wide variety of labeled secondary
antibodies
are available commercially.
• Versatile - many primary antibodies can be
made in one species and the same labeled
secondary antibody can be used for detection.
• Maximum immunoreactivity of the primary
antibody is retained because it is not labeled.
• Sensitivity is increased because each
primary
antibody contains several epitopes that can
be
bound by the labeled secondary antibody,
allowing for signal amplification.
• Different visualization markers can be used
with the same primary antibody.
Disadvanta
ges
• Cross-reactivity might occur with the
secondary
antibody, resulting in nonspecific signal.
• An extra incubation step is required in the
procedure.
Direct ELISA Detection
Advantages • Quick because only one antibody
and fewer steps are used.
• Cross-reactivity of secondary
antibody is eliminated
Disadvantag
es
• Immunoreactivity of the primary
antibody might be adversely
affected by labeling with enzymes or
tags.
• Labeling primary antibodies for
each specific ELISA system is time
consuming and expensive.
• No flexibility in choice of primary
antibody label from one experiment
to another.
• Minimal signal amplification

12. Reza Joia 12
•Introduction of nanomaterials addresses
traditional ELISA limitations, making it more
widely applicable in research and various
applications.
•Improved strategies for the "four
fundamentals" of ELISA involve nanomaterials
to enhance stability, sensitivity, and detection
range.
Improvement of traditional ELISA using nanomaterials

13. ADSORBENT SUBSTRATE IMPROVEMENT
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• Nanomaterials for Substrate Enhancement:
• Nanomaterials with large specific surface area or strong
adsorption capacity are introduced to enhance substrate
quality.
• Nanofibers, such as poly(MMA-co-MAA) coated PHB
fibers, address substrate issues, providing stability and
high sensitivity for virus detection.
•Molecularly Imprinted Polymers (MIPs):
• MIPs, created through Molecular Imprinting Technology
(MIT), offer specific target identification and translucency
of plate wells in ELISA.
• Glass bead surface imprinting technique simplifies
coating steps and improves adsorption performance in
ELISA.
•Magnetic Nano-Beads for Substrate and Separation:
• Magnetic nano-beads serve dual roles in separating and
enriching samples.
• They act as substrates to capture absorbents, providing a
solution to nonspecific adsorption and offering more
binding sites for coating antibodies or antigens.
• Magnetic bead-based ELISA demonstrates specificity
and sensitivity in detecting Salmonella in beef and poultry

14. Recognition element improvement
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•Aptamer Advancements:
• Aptamers overcome antibody challenges, providing a versatile alternative.
• Aptamer-based ELISA introduces a new recognition model for surface antigens, allergens,
and contaminants, enhancing specificity.
•Nanobody Excellence:
• Nanobodies, resistant to organic solvents and high temperatures, exhibit superior affinity.
• Nanobody-based ELISA demonstrates heightened sensitivity, notably in cancer biomarker and
soluble epoxide hydrolase detection.
•Antigenic Hapten Success:
• Antigenic hapten conjugates effectively generate selective antibodies, especially for small
molecules like pesticides.
• Monoclonal antibodies from hapten immunogens enable simultaneous detection of multiple
residues, showcasing high affinity and selectivity.
•Collective Enhancements:
• Overall improvements in ELISA's recognition elements provide enhanced specificity,
sensitivity, and versatility for detecting a diverse range of targets, from small molecules to
complex biomarkers.

15. 15

16. ENZYME LABEL IMPROVEMENT
•Nanomaterial Carriers: Nanoparticles like poly-
styrene, PAMAM polymers, graphene, and SiO2
enhance ELISA sensitivity by providing stable carriers
for loading abundant enzyme-labeled signals, amplifying
traditional ELISA's detection capabilities.
•Peroxidase-Mimicking Nanomaterials:
Nanomaterials with peroxidase activity, such as Au-
Pt/SiO2 nanocomposites, CeO2 nanoparticles, and
rGO/PEI/Pd nanohybrids, replace natural enzymes in
ELISA, overcoming limitations like separation
challenges and low resistance to environmental
conditions.
•Dual-Function Nanomaterials: Some nanomaterials,
serving as both carriers and signal labels in nano-ELISA,
exhibit synergistic effects, as demonstrated by three-in-
one antibody-HRP-Cu3(PO4)2 nanocomposites. This
approach enhances enzymatic activity, leading to
improved signal amplification and sensitivity.
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17. APPLICATION OF ELISA IN FOOD ANALYSIS
FOOD CONTAMINANT DETECTION
•Microbial Contaminant Detection:
• Nano-ELISA offers a sensitive and rapid method for detecting harmful bacteria like Salmonella, Listeria
monocytogenes, and Escherichia coli in food.
•Pesticide Residue Analysis:
• Nano-ELISA is applied for the detection of pesticide residues in food, providing enhanced sensitivity.
• Examples include quantum-dot-based LFIA and Au NPs-loaded bio-bar-code ELISA for rapid
identification of neonicotinoid insecticides and triazophos pesticide residues.
•Veterinary Drug Residue Detection:
• Nano-ELISA techniques, such as Au NPs-based indicators and nanomagnetic beads-based assays, enable
sensitive detection of veterinary drug residues in poultry and chicken tissues.
• Contributes to food safety monitoring.
•Heavy Metal Detection:
• Nano-ELISA plays a role in detecting heavy metals in food.
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18. Nano-ELISA in food quality control
•Rapid Determination of Physicochemical Properties:
• Addressing the need for swift and robust methods in food processing and storage, ELISA
provides a valuable tool for assessing physicochemical properties.
•Meat Product Identification and GMO Detection:
• PCR assays prove reliable and cost-effective for identifying beef and pork in processed meat
products, while ELISA offers less time-consuming alternatives.
• ELISA shows promise in detecting genetically modified organisms (GMOs) in food, with
methods such as silicon-coated magnetic nanoparticles and electrochemical genosensors.
•Irradiated Food Detection and Allergen Analysis:
• ELISA demonstrates potential in detecting irradiated food, with an immuno-rotary biosensor
for 2-dodecylcyclobutanone.
• Nano-ELISA emerges as an effective tool for allergen analysis, with examples like Au NPs-
labeled antibody LFIA for glycinin and an enzyme-free method for gliadin detection.
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19. NANO-ELISA IN FOOD NUTRIENT ANALYSIS
•Visual Detection of Glucose and Cholesterol:
•Nano-ELISA has been applied for colorimetric glucose inspection
using Fe-core/carbon shell nanoparticles (Fe,C NPs) with peroxidase
mimics, allowing for the detection of glucose in juice samples.
•Vitamin B1 Detection:
• Nano-ELISA method for colorimetric detection of vitamin
B1.
• Utilizes single-crystalline akaganeite (b-FeOOH) nanorods.
•Protein Immunoassays:
• Application of nano-ELISA for protein detection.
• Magnetic beads (MBs) and Au NPs used for b-casein
detection in bovine milk samples.
•Trace Minerals Detection:
• Nano-ELISA applied for the detection of trace minerals,
including selenium (Se), iron (Fe), and zinc (Zn).
• Methods utilize N-acetyl-L-cysteine (NALC) capped Ag NPs
for Fe3+ detection and label-free Ag NPs for Zn2+ ion
detection.
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20. Conclusion
ELISA has undergone remarkable evolution, progressing from
radioimmunoassay to modern applications, cementing its significance in
various fields. Continuous advancements, particularly the integration of
nanomaterials, have amplified ELISA's sensitivity and broadened its utility in
food analysis and safety. Challenges persist, including cost and procedural
complexities, yet the emergence of nano-ELISA addresses these issues.
However, hurdles in modifying nanomaterials for comprehensive ELISA
enhancement remain. The collaborative application of nano-ELISA with
techniques like PCR and smartphones shows promise for rapid analyte
detection. Looking forward, the ongoing pursuit of novel nanomaterials and
methodologies with superior performance is essential to further advance
stable and reliable ELISA methods in food analysis.
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21. THANK YOU FOR YOUR
ATTENTION
Reza Joia – 2023 Almaty
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