helmapazhouhankia (2)

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?? ??? ???ELISA(Enzyme-Linked Immunosorbent
Assay)

• Dr Darvishi
• Presented by H.Pazhouhankia

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Simple ELISA protocol
1. Coat antigen onto microplate
2. Allow protein adsorption and block unoccupied
sites with neutral protein
3. Add antibody solution into each well
4. Add enzyme conjugated secondary antibody into
each well and develop colorimetric reaction
with appropriate substrate
5. Read absorbance in spectrophotometer
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Enzyme label

• Horseradish peroxidase (HRP)
• Alkaline Phosphatase (Alk-phos)

• ß-Galactosidase (ß-gal)

• Urease

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Substrate

• Ortho Nitro Phenylene Diamine hydrochloride
  (ONPD)
• Yellow, 450 nm
• (p-Nitrophenyl Phosphate, Disodium Salt (
  PNPP)

Orange, 492 nm

• Tetra Methyl Benzidine
  (TMB)

• 2,2'-Azinobis 3-ethylbenzothiazoline-6-sulfonic
  acid-diammonium salt

(ABTS)
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ELISA

• Enzyme-linked immunosorbent assay
• ELISA kit components
• Antibody
• Allows for specific detection of analytic of
  interest
• Solid phase (sorbent)
• Allows one to wash away all the material that is
  not specifically captured
• Enzymatic amplification
• Allows you to turn a little capture into a
  visible color change that can be quantified using
  an absorbance plate reader

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Non-competitive
Y

• - Ag detection
• Immobilize Ab
• Incubate with sample
• Add labeled antibody
• Amount of labeled Ab bound is proportional to the
  amount of Ag in the sample
• Quantitative

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Non-competitive
Y

• Ab detection
• Immobilize Ag
• Incubate with sample
• Add labeled anti-Ig
• Amount of labeled Ab bound is proportional to
  amount of Ab in the sample
• Quantitative

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• 1-Antibody Capture
• Ag Sandwich??Direct Ab Capture
• 2- Ag Capture
• Ab sandwich

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Sandwich Elisa
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Competitive ELISA

• Ag Detection

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Competitive ELISA

• Ab Detection

Labeled-Ab
Ab in Patients sample
Y
Y
Y
Y
Y
Y
Ag
Ag
Ag
Ag
Ag
Immobilized
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Reference
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Journal 1Journal of Food Composition and
Analysis Available online 25 November 2021,
104303 A novel ELISA test to detect soy in highly
processed foods Author links open overlay
panelSegura-GilIsabelaGalan-MaloPatriciabMataLuisb
Ana P.TobajasaAlbaCiveraaLourdesSánchezaMaría
D.Péreza https//doi.org/10.1016/j.jfca.2021.10430
3Get rights and content Under a Creative Commons
license open access 2Food Chemistry , 1 March
2022, 131338 Development of sandwich ELISA and
lateral flow immunoassay to detect almond in
processed food Author links open overlay
panelAlbaCiveraaPatriciaGalan-MalobIsabelSegura-Gi
labLuisMatabAna P.TobajasaLourdesSánchezaMaría
D.Péreza https//doi.org/10.1016/j.foodchem.2021.1
31338Get rights and content Under a Creative
Commons license open access 3Talanta Available
online 3 December 2021, 123125 In Press, Journal
Pre-proof What are Journal Pre-proof
articles?   Development of a biotinylated
nanobody for sensitive detection of aflatoxin B1
in cereal via ELISA Author links open overlay
panelTingtingYanabJiangZhuabYingLiabTingHea Yunhua
ngYangab MailiLiuab https//doi.org/10.1016/j.tala
nta.2021.123125Get rights and content
49
4Journal of Microbiological MethodsVolume 154,
November 2018, Pages 127-133 Development of
duplex PCR-ELISA for simultaneous detection of
Salmonella spp. and Escherichia coli O157 H7 in
foodAuthor links open overlay panelJinqiangHuabcd
RunnaHuangaYiWangaXiangkeWeiaZhangcunWangaYaoGeng
abJianzhouJingabHuiGaoabXinchengSunabCaiwenDongaCh
unpengJiangahttps//doi.org/10.1016/j.mimet.2018.
10.017Get rights and content5Analytical
BiochemistryVolume 635, 15 December 2021,
114448 Development of a sensitive
sandwich-ELISA assay for reliable detection of
fish residues in foodsAuthor links open overlay
panelBinaka PrabashiniDasanayakaaJinlongZhaoaJiuka
iZhangbYuhaoHuangaMati UllahKhanaHongLinaZhenxingL
iahttps//doi.org/10.1016/j.ab.2021.114448Get
rights and content6Nanobody multimerization
strategy to enhance the sensitivity of
competitive ELISA for detection of ochratoxin A
in coffee samplesAuthor links open overlay
panelKunluBaoa1XingLiua1QiXuaBencaoSuaZilongLiuaHo
ngmeiCaoaQiChenabhttps//doi.org/10.1016/j.foodco
nt.2021.108167Get rights and content
50
7Re-modeling ELISA kits embedded in an
automated system suitable for on-line detection
of algal toxins in seawaterAuthor links open
overlay panelKonstantinosPetropoulosaSergio
F.BodinibLauraFabianiaLauraMicheliaAlessandroPorc
hettaaSilviaPiermariniaGiuliaVolpeaFabio
MassimoPasquazzibLucaSanfilippobPompeoMoscettabSal
vatoreChiavarinicGiuseppePalleschiahttps//doi.or
g/10.1016/j.snb.2018.12.083Get rights and
content8Chapter 9 - Food traceabilityAuthor
links open overlay panelBurcuGuldiken1SimgeKarliga
2EsraCapanoglu2PerihanYolci-Omeroglu3SenemKamilogl
u4https//doi.org/10.1016/B978-0-12-819493-5.0000
9-1Get rights and content9Chapter 8 -
Innovations in analytical methods for food
authenticityAuthor links open overlay
panelM.Esteki1M.J.Cardador2N.Jurado-Campos2A.Martí
n-Gómez2L.Arce2J.Simal-Gandara3https//doi.org/10
.1016/B978-0-12-819493-5.00008-XGet rights and
content
51
BOOK 101 Enzyme-ImmunoassayShow Path
ChapterEnzyme-Linked Immunosorbent Assay (Elisa)
Theoretical and Practical Aspects ByBrian R.
Clark, Eva EngvallBookEnzyme-ImmunoassayEdition
1st EditionFirst Published 1980Imprint CRC
PressPages 14eBook ISBN 978135107184011Food
Microbiology Third EditionMartin R. Adams and
Maurice O. MossUniversity of Surrey,
Guildford,uk, Chapter10Methods for the
Microbiological Examination of Foods12Molecula
r Techniques in Food BiologySafety,
Biotechnology, Authenticity and
TraceabilityEdited byAly Farag El SheikhaMcMaster
University CanadaRobert LevinUniversity of
Massachusetts USAJianping XuMcMaster University
CanadaSection V Foodborne Pathogens and Food
Safety 34115 Rapid Detection of Food Pathogens
Using Molecular Methods ,page34313Modern Food
Microbiology Seventh Edition James M. Jay
University of Nevada Las Vegas Las Vegas, Nevada
Martin J. Loessner Institute of Food Science and
Nutrition Zürich, Switzerland David A. Golden
University of Tennessee Knoxville,
Tennessee11Chemical, Biological, and Physical
Methods.page 24114Handbook ofFood
AnalysisThird Edition - Volume IEDITED BYLeo M.L.
Nollet Fidel Toldrá17.7 ELISA Methods .page 364
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