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Elisa: Enzyme-Linked Immunosorbent Assay: Advances in Drug Delivery

The document discusses enzyme-linked immunosorbent assay (ELISA), a common analytical biochemistry technique used mainly to detect the presence of antibodies or antigens in a liquid sample like blood or serum. It describes the basic principles and different types of ELISA, including direct ELISA, indirect ELISA, immobilized antigen ELISA, sandwich ELISA, and competitive ELISA. The document also covers applications of ELISA in medical diagnostics, food allergen testing, drug testing, and its advantages and limitations.

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alvindo devi
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134 views32 pages

Elisa: Enzyme-Linked Immunosorbent Assay: Advances in Drug Delivery

The document discusses enzyme-linked immunosorbent assay (ELISA), a common analytical biochemistry technique used mainly to detect the presence of antibodies or antigens in a liquid sample like blood or serum. It describes the basic principles and different types of ELISA, including direct ELISA, indirect ELISA, immobilized antigen ELISA, sandwich ELISA, and competitive ELISA. The document also covers applications of ELISA in medical diagnostics, food allergen testing, drug testing, and its advantages and limitations.

Uploaded by

alvindo devi
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Advances in Drug Delivery

23rd of May 2011

ELISA:
Enzyme-Linked Immunosorbent Assay

Slides by Mathias Bader and Simon Loew


ELISA: Enzyme-Linked Immunosorbent Assay

ELISA detects substances with antigenic


properties (mainly proteins)

Based on enzymatic color-reaction

Slides by Mathias Bader and Simon Loew 2


Basic principle of ELISA

Enzyme is used to detect the binding of Antibody - Antigen

Enzyme converts colorless substrate into colored product, indicating


the presence of Antibody - Antigen complex

ELISA can be used to detect either presence of Antigens or Antibodies

Slides by Mathias Bader and Simon Loew 3


Direct and Indirect ELISA

Direct ELISA Indirect ELISA

Slides by Mathias Bader and Simon Loew 4


Direct ELISA

Slides by Mathias Bader and Simon Loew 5


Antibody is adsorbed
onto microtiter plate

Add serum to test for


antigen

Specific antigen binds if


contained

Slides by Mathias Bader and Simon Loew 6


Remove test fluid and
unbound antigen
Binding of antigen is
strong enough to
withstand rinsing

Slides by Mathias Bader and Simon Loew 7


Remove test fluid and
unbound antigen
Binding of antigen is
strong enough to
withstand rinsing

Slides by Mathias Bader and Simon Loew 8


Slides by Mathias Bader and Simon Loew 9
Add enzyme labeled
E E antibody

E E Labeled antibody binds


to antigens
E E

E E

Slides by Mathias Bader and Simon Loew 10


Wash sample to remove
unbound antibodies

E
E

E
E E

E E E

Slides by Mathias Bader and Simon Loew 11


Wash sample to remove
E unbound antibodies

E E

E E E

Slides by Mathias Bader and Simon Loew 12


Wash sample to remove
unbound antibodies

E E E

Slides by Mathias Bader and Simon Loew 13


Add substrate

Measure color change

Positive Negativ

E E E

Slides by Mathias Bader and Simon Loew 14


Direct and Indirect ELISA

Direct ELISA Indirect ELISA

Slides by Mathias Bader and Simon Loew 15


Indirect ELISA
Antigen is adsorbed onto
microtiter plate

Add serum to test for


antibody

Slides by Mathias Bader and Simon Loew 16


Antigen is adsorbed onto
microtiter plate

Add serum to test for


antibody

Slides by Mathias Bader and Simon Loew 17


Antigen is adsorbed onto
microtiter plate

Add serum to test for


antibody

Wash sample

Slides by Mathias Bader and Simon Loew 18


Antigen is adsorbed onto
microtiter plate

Add serum to test for


antibody

Wash sample

Slides by Mathias Bader and Simon Loew 19


Slides by Mathias Bader and Simon Loew 20
Add secondary antibody
E E

E E
Primary antibody binds
to secondary anitbody
E E

E E

Slides by Mathias Bader and Simon Loew 21


Add secondary antibody

Wash unbound antibodies

E
E
E

E
E
E E E

Slides by Mathias Bader and Simon Loew 22


Add secondary antibody
E
E
Wash unbound antibodies
E

E E E

Slides by Mathias Bader and Simon Loew 23


Add secondary antibody

Wash unbound antibodies

E E E

Slides by Mathias Bader and Simon Loew 24


Add substrate

Measure color change

Positive Negativ

E E E

Slides by Mathias Bader and Simon Loew 25


Direct vs. Indirect ELISA
Advantages of Indirect ELISA:

Immunoreactivity of primary
antibody is not affected by labeling E E E
Wide variety of secondary
antibodies available on the market
Signal amplification (several epitopes)

Advantages of Direct ELISA:

Quick methodology – only one AB


E E E
Cross-reactivity of second AB
eliminated

Slides by Mathias Bader and Simon Loew 26


Immobilized Antigen vs. Sandwich

Immobilized Mainly used for antibody


Antigen: detection E E E

Sandwich Mainly used for antigen


ELISA: detection
E E E

Slides by Mathias Bader and Simon Loew 27


Competitive ELISA

Step 1: Step 2: Step 3: Step 4:


Add sample Add antibodys Washing Add substrate

E E E

Quick methodology – just one washing step

Slides by Mathias Bader and Simon Loew 28


Applications

Medical diagnostic to detect presence of antibodies in patient

HIV Test

West Nile Virus

Detection of potential allergens in food

Test kits for eggs and peanuts

Drug tests

But: high material costs

Slides by Mathias Bader and Simon Loew 29


HBsAg (ELISA)
Langkah 1 : persiapan reagen
Pengenceran wash solution 1 : 20 ( 8 wells(1 blangko,1 kontrol negative, 1
kontrol positif,5 sampel (1-5) 5 kali pencucian sebanyak 50 µL : 2000 µL)
Langkah 2 : memberi identitas
Beri identitas pada well (No 1: blangko, No 2 : kontrol negative, No 3 :
kontrol positif, No 4 – 8 sampel 1-5
Langkah 3 : menambahkan sampel dan HRP conjugated
Masukkan pada masing masing well kecuali blangko dengan (No 2 : 50 µL
kontrol negative, No 3 : 50 µL kontrol positif, No 4 – 8 : @50 µL sampel 1-5)
Tambahkan pada masing- masing well kecuali blangko dengan 50 µL HRP
Conjugated.
Langkah 4 : inkubasi
Inkubasi pada suhu 37 ºC selama 1 jam pada termostat

Slides by Mathias Bader and Simon Loew 30


HBsAg (ELISA)
Langkah 5 : mencuci
Cuci masing - masing well dengan menggunakan wash solution sebanyak 5
kali. Masing – masing pencucian membutuhkan waktu perendaman selam
30-60 detik.
Langkah 6 : mewarnai
Tambahkan pada masing- masing well 50 µL larutan chromogen A dan 50
µL larutan chromogen B. Homogenkan dengan cara mengetuk – ngetuk
pada bagian tepi dari well. Inkubasi pada suhu 37 ºC selama 15 menit
terhindar dari cahaya terang. Hasil positif ditunjukkan dengan terbentuknya
warna biru.
Langkah 7 : stopping reaction
Tambahkan pada masing- masing well 50 µL stop solution Hasil positif
ditunjukkan dengan terbentuknya warna kuning.
Langkah 8 : membaca absorbansi
Kalibrasi plate reader dengan menggunakan balngko di well dan baca pada
absorbansi 450 nm. Jika menggunakan dua jenis filter , maka atur panjang
gelombang antara (600-650 nm). Kalkulasi nilai cut off dan baca hasilnya
Slides by(pembacaan dilakukan
Mathias Bader and Simon Loew setelah 10 menit dari penambahan larutan stop 31
solution)
Thank you

Slides by Mathias Bader and Simon Loew 32

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