SEROLOGI

ELISA

Engla Merizka
Serologic diagnosis : why ?

– Some microorganisms cannot be cultivated on artificial media

– Isolation of obligate intracellular organisms requires animal inoculation or the
use of cell culture
– Toxin detection
– Cultivable organisms
– Labile in transport condition
– Long incubation period
 Important requirements
– Adequate collection, transport, and processing of clinical specimens

– Cooperation among medical team members :

– Clinical practitioners
– Nurses
– Laboratory practitioners

– Ongoing communication and education among all members of the team

 SEROLOGI INFEKSI

Serologi  Area diagnostik dari

imunologi klinis yang meliputi deteksi,
identifikasi, kuantifikasi dan kualifikasi
antibodi atau antigen di dalam serum.
 SEROLOGI INFEKSI

Digunakan untuk 2 tujuan:

1. Deteksi antibodi dg menggunakan
antigen yg diketahui
2. Deteksi antigen dg menggunakan
antibodi yg diketahui

Sifat antibodi  bereaksi spesifik dengan

antigen yang menimbulkannya
 SEROLOGI INFEKSI

1.Immunostaining
2.Immunoblotting
3.Immunoaglutination.
4.Reaksi fiksasi komplemen
5.ELISA
 1. Immunostaining

• Tujuan: untuk mendeteksi sel terinfeksi

• Menggunakan penanda:
- FITC (Fluorescin Isothiocyanate)
immunofluorescence
- Peroxsidase immunoperoxsidase
 1. Immunostaining

Direct Indirect
 IMMUNOFLUORESCENCE ASSAY

un-infected cells Infected cells

Gambar milik Beti Ernawati Dewi
Gambar milik Beti Ernawati Dewi
 IMMUNOPEROXIDASE ASSAY

un-infected cells Infected cells

Gambar milik Beti Ernawati Dewi

Gambar milik Beti Ernawati Dewi
 ELISA
– ELISA, or enzyme-linked immunosorbent assay, is an immunoassay
technique involving the reaction of antigen and antibody in vitro.
– ELISA is a sensitive and specific assay for the detection and quantitation of
antigens or antibodies.
– ELISA tests are usually performed in microwell plates.
– The ELISA test, or the enzyme immunoassay (EIA), was the first screening
test commonly employed for HIV. It has a high sensitivity.
 COMPONEN of ELISA

– Antigen: Elisa plate coated with antigen

– Primary antibody: Human Serum IgG , IgA , & IgM
– Secondary antibody: Peroxidase-labelled anti-human IgA , IgG , or IgM
antibodies that bind to the antibobdy complexes Enzyme : Horse Radish
Peroxidase (HRP) MW 44,000, glycoprotein with 4 lysine residues
COMPONEN of ELISA

– Substrate : TMB (3,3',5,5', tetramethylbenzidine ) The enzyme acts as a catalyst

to oxidize substrate in the presence of Hydrogen peroxide to produce a blue
colour .
– Reaction stopped with dilute acid to cause complex to turn yellow colour . Stop
Solution : Sulphuric Acid 0.5N (H 2 So 4)
Substrate

– PNPP ( p -Nitrophenyl Phosphate, Disodium Salt); When alkaline phosphatase

and PNPP are reacted, a yellow water-soluble reaction product is formed. This
reaction product absorbs light at 405nm.
– OPD (o-Phenylenediamine ) OPD is a water-soluble substrate for horseradish
peroxidase (HRP) . produces a yellow-orange product. detectable at 492nm by
ELISA plate readers
Enzymes Used in Elisa : 

– Enzymes Used in Elisa Horseradish peroxidase (most commonly used), Alkaline

Phosphatase, B-galactosidase Lactoperoxidase, Tetra Methyl benzidine
PRINCIPLE of ELISA

Antibody is immobilized on micro-plate wells

Competition between in sample and labeled
enzyme for antibody binding sites

The unbound material is washed out

Chromogenic substrate added to develop

colour

Resulting colour is determined in a

spectrophotometer
 5. E L I S A
Enzyme-Linked Immunosorbent Assay

1. ELISA pendeteksi antibodi

- Indirect ELISA
- Double antibody-sandwich ELISA
- Indirect cellular ELISA
2. ELISA pendeteksi antigen
- Direct competitive ELISA
- Antibody sandwich ELISA
- Direct cellular ELISA

Current protocols in molecular biology

Antibody detection
 Add substrate
Incubate with antibody-enzyme conjugate
Incubate with Antigen
Anti-Human IgM Precoated Observe colo

Block dengan blocking solution

96 well plate
Kontrol Negative Serum Pasien
(Serum tanpa IgM) (Mengandung IgM)

Double antibody sandwich

IgM capture ELISA (Mac ELISA)
 Peroksidase
OPD OPD
H2O2 H 2O + O 2
 Perbedaan warna yang dramatik antara hasil ELISA
yang positif and negatif

Blank
Neg
control

Pos
control
Mesin Pencuci ELISA
Unit Pembaca Hasil ELISA
ANTIBODY SANDWICH ELISA
Coat with antibody

Add substrate and observe

color change

Incubate with patient’s sera

Incubate with
antibody-enzyme conjugate

Current protocols in molecular biology

3. ANTIBODY-SANDWICH

Coat with antibody

Add substrate and observe

color change

Incubate with antigen

Incubate with
antibody-enzyme conjugate

Current Protocols in Molecular Biology

False-Negative Serologic test results

Negative result for a patient who really is infected

– May not have an intact immune system, and therefore may

not be able to respond to an antigenic stimulus
– Congenital or acquired immunodeficiency diseases
– Receiving either immunosuppressive therapy after organ
transplantation or cancer chemotherapy

– Neonates may not always respond to an infectious agent

because their immune system are not fully mature

– For some infections (e.g. legionaires’ disease) antibody titers

may not rise until months after acute infection
DIRECT ELISA

– The direct ELISA uses the method of directly labeling the antibody it self.

– Microwell plates are coated with a sample containing the target antigen, and
the binding of labeled antibody is quantitated by a colorimetric,
chemiluminescent , or fluorescent end-point.
ADVANTAGE OF DIRECT ELISA

– Quick methodology since only one antibody is used. Cross-reactivity of

secondary antibody is eliminated.
– Disadvantages of Direct Detection Immunoreactivity of the primary antibody
may be reduced as a result of labeling.
– Labeling of every primary antibody is time-consuming and expensive. No
flexibility in choice of primary antibody label from one experiment to another.
INDIRECT ELISA

– The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a

labeled secondary antibody.
– Since the labeled secondary antibody is directed against all antibodies of a
given species (e.g. anti-mouse), it can be used with a wide variety of primary
antibodies (e.g. all mouse monoclonal antibodies).
ADVANTAGE INDIRECT ELISA

– Wide variety of labeled secondary antibodies are available commercially.

Versatile, since many primary antibodies can be made in one species and the
same labeled secondary antibody can be used for detection.
– Immunoreactivity of the primary antibody is not affected by labeling.
– Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing for
signal amplification.
SANDWICH ELISA

– Plate is coated with a capture antibody Sample is added, and any antigen
present binds to capture antibody
– Detecting antibody is added, and binds to antigen
– Enzyme-linked secondary antibody is added, and binds to detecting antibody
Substrate is added, and is converted by enzyme to detectable form
COMPETITIVE ELISA

– Primary antibody (unlabeled) is incubated with sample antigen.

– Antibody-antigen complexes are then added to 96-well plates which are
pre-coated with the same antigen.
– Unbound antibody is removed by washing the plate. (The more antigen
in the sample, the less antibody will be able to bind to the antigen in the
well, hence "competition.")
– The secondary antibody that is specific to the primary antibody and
conjugated with an enzyme is added.
– A substrate is added
ELISA Reverse method & device
(ELISA-R m&d )
– A newer technique uses an solid phase made up of an immunosorbent
polystyrene rod with 4-12 protruding ogives .
– The entire device is immersed in a test tube containing the collected sample
and the following steps (washing, incubation in conjugate and incubation in
chromogenous ) are carried out by dipping the ogives in microwells of standard
microplates pre-filled with reagents
ADVANTAGES

– The ogives can each be sensitized to a different reagent, allowing the

simultaneous detection of different antibodies and different antigens
for multi-target assays.
– The sample volume can be increased to improve the test sensitivity in
clinical (saliva, urine), food (bulk milk, pooled eggs) and
environmental (water) samples.
APPLICATIONS

– Screening donated blood for evidence of viral contamination by HIV-1 and HIV-
2 (presence of anti-HIV antibodies). hepatitis C (presence of antibodies).
hepatitis B (testing for both antibodies and a viral antigen)
– Measuring hormone levels HCG (as a test for pregnancy). LH (determining the
time of ovulation). TSH, T3 and T4 (for thyroid function).
APPLICATIONS

– Detecting infections sexually-transmitted agents like HIV, syphilis and

chlamydia, Hepatitis B and C
– Detecting allergens in food and house dust Measuring "rheumatoid factors"
and other autoantibody in autoimmune diseases like lupus erythematosus
Measuring toxins in contaminated food Detecting illicit drugs
ELISPOT

– ELISPOT The Enzyme-linked immunosorbent spot ( ELISPOT ) assay is a common

method for monitoring immune responses in humans and animals.
– It was developed by Cecil Czerkinsky in 1983 . It is used to detect cytokines
secreted from different cells.
ELISPOT
ELISPOT
 False-Positive Serologic test results
Positive result for a patient who is not infected by the specific
agent for which the test is design

– Production of cross-reacting antibody

Some antigen associated with different microorganisms are closely
related and a host may respond by producing antibody not only
to the invading organism but also to antigenically closely related
organism

– Reactivation of a latent organism due to infection by a

different organism

– Receiving intravenous immunoglobulin