IMUNOASSAY

dr. Danis Pertiwi, M.Si. Med., SpPK

Bagian Patologi Klinik FK Unissula/ RSI Sultan Agung Semarang
 INTRODUCTION TO IMMUNOASSAYS

Objectives:

• To Understand the basic definitions in ImmunoAssays.

• To Understand the most important concepts used in ImmunoAssays
 Potential sources of diseases

Pathogenic agents Physiological imbalance

=
microbes • too many or not enough
circulating substances
• virus
• bactéries • malfunctioning of an organ
...
• parasites
• fungi

Infectious Metabolic
diseases diseases
 Clinical diagnostics
 virology  clinical chemistry
 bacteriology

 parasitology  hemostasis

 mycology
 "immunochemistry"
. hormonology
. allergy
. tumor markers

Infectious Metabolic
diseases diseases
 Some examples of disease
 virology  biochemistry
hepatitis diabetes
AIDS cholesterol
influenza gout
 bacteriology  hemostasis
sore throat hemorrhages
gastro-enteritis thrombosis

 parasitology
toxoplasmosis "immunoassays"
malaria infertility
allergy
 mycology cancer
candida

Infectious Metabolic
diseases diseases
The immune system differentiates between
« self » and « non-self »

 « self » « non self »


recognizes and recognizes and
tolerates rejects
its own substances. foreign substances =
= Antigens
 To fight against foreign agents,
we produce antibodies

Virus

Parasite

Bacteria

Antibodies
produced by
white blood cells :
lymphocytes
 Antibodies are the body's defenders

Antibodies

Virus

the neutralized attacker will be

destroyed by white blood cells White blood cell
How detect infectious diseases?
 To diagnose an infectious disease

we study …

either directly or the body's

the microbe response

Antibody
 Antibodi

• Nama lain : Immunoglobulin

• Merupakan protein (globulin) yang dihasilkan oleh tubuh
akibat masuknya antigen.
• Bersifat spesifik.
• Macam Antibodi:
– Immunoglobulin M (IgM)
– Immunoglobulin G (IgG)
– Immunoglobulin A (IgA)
– Immunoglobulin E (IgE)
– Immunoglobulin D (IgD)
 Antibodi

• Fungsi utama Immunoglobulin adalah untuk

melawan antigen  pertahanan tubuh
• Tidak semua Immunoglobulin yang terbentuk
dapat melawan antigen, tetapi ada yang hanya
berfungsi sebagai “Marker” walaupun tujuan
diproduksinya merupakan pertahanan.
To diagnose an infectious disease

the microbe is studied directly ...

 How detect bacteria ?

by direct by studying its

observation programming

morphological genetic material

properties

using the composition of its

by studying
"packaging"
what it eats or evacuates
antigenic
biochemical properties
properties
 How detect bacteria ?
by direct by studying its
observation programming

Molecular
biology
Bacteriology

using the composition of its

by studying "packaging"
what it eats or evacuates
Immuno-assays
IMMUNOLOGY?

IMMUNOASSAYS?
 IMMUNOLOGY?

• Study of the body's reactions to foreign substances

• Study of immune system disorders

• Many different applications in several medical disciplines

 IMMUNOASSAYS?

• in vitro diagnosis techniques

• based on the specific reaction between antigens and

antibodies.

• Identification and/or quantification of micro-organisms,

hormones and markers specific to certain diseases
 THE IMMUNE SYSTEM

• Collection of mechanisms that protects against diseases by

identifying and neutralizing invading agents.

• Immune system differentiates «SELF» from «NON-SELF».

• SELF: What belongs to the body: healthy cells and tissues

To be recognized and tolerated.

• NON-SELF: Foreign substances and defective tissues

To be recognized and rejected.
 THE IMMUNE SYSTEM

• The immune system is composed of cells

(cell mediated response)
and soluble molecules in the biological fluids
(humoral response).

• White blood cells or Leukocytes (B-cells, T-cells, phagocytes...)

are responsible of Cell-mediated response

• Humoral response involve antibodies and other soluble

molecules (cytokines, complement system...)
 ANTIGENS? ANTIBODIES?

• Foreign substances are called ANTIGENS (Ag),

when introduced in the body they induce the production of
ANTIBODIES (Ab).

• ANTIBODIES help to recognize and neutralize ANTIGENS

• ONE ANTIBODY recognizes ONLY ONE ANTIGEN:

SPECIFICITY OF ANTIGEN-ANTIBODY REACTION

 ANTIBODIES?

• Antibody = Immunoglobulin (Ig)

• Proteins produced by cells of the immune system to identify

and neutralize foreign objects

• Antibodies are produced by B cells (B-Lymphocytes)

• Each type of antibody is unique and defends the body

against one specific type of antigen.
BASIC STRUCTURE OF ANTIBODIES
 5 CLASSES OF ANTIBODIES

FIVE DIFFERENT TYPES OF HEAVY CHAINS

FIVES ISOTYPES ANTIBODIES

Five different isotypes:

 Play different roles

 help to direct the appropriate immune response for each different

type of antigen.
 5 ISOTYPES OF ANTIBODIES

Name Description

IgA Usually dimeric (2 Igs bound by the secretory piece)

Found in mucosal areas (gut, respiratory tract, eyes, uro-genital tract…)
Prevents colonization by pathogens. Also found in saliva, tears, and breast milk.

Monomeric (1 single Ig)

IgD
Works mainly as an antigen receptor on B lymphocytes that have not been exposed to antigens.
Its function is not well defined yet.

Monomeric
IgE
Involved in allergy, binds to allergens and triggers histamine release from mast cells and basophils.
Also protects against parasitic infections.

Monomeric,
IgG 2nd Ab class to be produced after Ag introduction in the organism.
Provides the majority of antibody-based immunity against invading pathogens.
The only antibody capable of crossing the placenta to give passive immunity to fetus.

IgM Pentameric (5 Igs)

1st Ab class to be produced after Ag introduction in the organism. Indicate a recent infection.
Eliminates pathogens in the early stages of B lymphocytes mediated immunity before there is sufficient IgG.
 ANTIGENS

• An antigen is a foreign substance in the organism,

capable of triggering an immune response.

• Virtually any foreign macro-molecule can act as an antigen.

• Particular sites on the antigen's surface fit and bind to specific

antibodies: These sites are called epitopes
 THE IMMUNE RESPONSE

• Immune Response is an extremely complex process

• Involving several types of cells: lymphocytes, macrophages...

• Involving many different substances: antibodies, cytokines,

complement system....

• Immune Response can be USEFUL: Protection against infections

• BUT sometimes it can be NOXIOUS:

– When the response is exaggerated: ALLERGIES
– When the immune system attacks normal cells: AUTO IMMUNE DISEASES
 IMMUNOASSAYS

• The antigen-antibody reaction applied in vitro = Immunoassays

• The antigen-antibody reaction is interesting due to its SPECIFICITY

ONE ANTIBODY FOR ONE ANTIGEN!

• Lock and Key concept:

 IMMUNOASSAYS

2 types of applications for the Ag-Ab reaction:

• Detection of antigens using antibody-based methods.

Example: - detection of Chlamydia in genital collections,

- detection of hormones, tumor markers...

• Detection of antibodies produced in response to an antigen

in patient serum.

Example: - detection of antibodies against syphilis agent,

- detection of antibodies against HIV...
 KONSEP DASAR IMUNOASAI

 Reaksi antigen dan antibodi membentuk ikatan kompleks

(Ab:Ag)
Ab + Ag  Ab:Ag
Reaksi bolak balik, kompleks akan berdisosiasi:
Ab:Ag  Ab + Ag
Pd saat tertentu terjadi keseimbangan bag berdisosiasi dg yg berasosiasi
(equilibrium):
Ab + Ag ↔ Ab:Ag
 Bukan hanya antibodi yg dpt ditentukan menggunakan
antigen yg diketahui tetapi sebaliknya ≈ reverse serology, mis:
Reverse Passive Hemagglutination test
 KONSEP DASAR IMUNOASAI…con’t…

 Afinitas antibodi: kekuatan interaksi antara antibodi dg epitop

tunggal (monovalen)
 Aviditas antibodi: kekuatan interaksi antibodi heterogen dg
antigen multivalen.
Aviditas mrpk refleksi dari afinitas dalam jumlah binding sites
 Ikatan Ag-Ab dipengaruhi bbrp faktor:
 Ikatan hidrogen
 Daya elektrostatik
 Ikatan van der Waals
 Ikatan hidrofobik
 KONSEP DASAR IMUNOASAI…con’t…

Daya Coulombic/elektrostatik:
Daya tarik menarik antara gugusan molekul antigen dengan
antibodi yang mempunyai muatan listrik berlawanan.
Makin dekat jarak antigen antibodi makin cepat keduanya
berikatan
Daya Van der Waals:
Ikatan karena adanya kesesuaian antara antibodi dengan
permukaan “determinant antigen” (epitop).
Ikatan antigen dengan antibodi merupakan ikatan
reversibel & mudah berdisosiasi
 KONSEP DASAR IMUNOASAI…con’t…
Ukuran kuantitas Antibodi:
 Kualitatif; Menyatakan ada/tidaknya antibodi dalam bahan yg
diperiksa dg melihat adanya perubahan dari bahan yg diperiksa.
Contoh: uji VDRL mikro, perubahan warna pd penentuan HBs antigen
secara ELISA
 Semikuantitatif: Kadar antibodi atau bahan lain dalam bahan yang
diperiksa ditentukan dengan cara pengenceran bahan pemeriksaan
secara progresif. Kuantitas dinyatakan dlm bentuk titer. Titer adalah
harga kebalikan dr pengenceran bahan pemeriksaan yg masih
memberi reaksi (+).
 Kuantitatif: Ditentukan dg menggunakan bbrp sera baku yg telah
diketahui kadar bahan yg akan ditentukan, misal antibodi & dibuat
kurva baku
Pemeriksaan Imunologi/ immunoassay

• Prinsip
– Mendeteksi Antigen /Antibodi
• Metode
– Aglutinasi
– Immunochromatography
– Enzyme Immuno Assay (EIA)
– Radio Immuno Assay (RIA)
– Chemiluminescence
 MACAM-MACAM IMUNOASAI
 IMUNOASAI TAK BERLABEL
 Faktor dasar yang mempengaruhi:
 Sifat antigen
 Elektrolit & PH
 Waktu & suhu
 Perbandingan Ag & Ab (hasil reaksi bisa dilihat apabila terjadi zona
ekuivalen. Jk Ag>Ab  prozone. Jk Ag<Ab postzone)
 Mekanisme daya tahan tak spesifik (misal: produk sampingan flora
normal masuk dlm cairan tubuh/serum, bahan mikrobisidal yg
normal terdpt pada cairan tubuh, interferon yg dikeluarkan oleh
limfosit T pd infeksi virus, dll)
 JENIS IMUNOASAI TAK BERLABEL

 Uji Presipitasi
Ag yg larut bereaksi dg Ab-nya akan terjadi presipitasi
 Uji presipitasi lempeng (slide), contoh: Uji VDRL mikro
 Uji presipitasi tabung (tube test), contoh: Uji VDRL makro
 Uji presipitasi tabung kapiler, contoh: penentuan CRP
 Uji presipitasi cincin, contoh: Penentuan Ab thd albumin telur
 Uji Difusi agar (Gel diffusion test), contoh: Penentuan bbrp Ag atau Ab
(Difusi tunggal, jarang dipakai), Penentuan Ab thd jamur (Difusi ganda
sederhana), Difusi ganda majemuk
 Imunoelektroforesis
 Imunodifusi radial (RID), contoh: Penentuan kadar Ig kelas tertentu
(IgM, IgG, IgA), penentuan HBs antiugen, Ab thd parasit.
 Counterimmunoelectrophoresis, contoh: Deteksi HBs Ag atau Ab
 Elektroimunodifusi (EID), contoh: Penentuan Ig
 JENIS IMUNOASAI TAK BERLABEL…con’t…

 Uji Aglutinasi
Reaksi antara Ab dg Ag seluler atau Ag pd permuk sel.
(Aglutinatnya mrpk agregasi dari banyak sel)
 Uji aglutinasi lempeng (slide), contoh: Uji Widal Lempeng
 Uji aglutinasi tabung, contoh: Uji Widal, uji Weil Felix, identifikasi
bakteri, uji aglutinasi fungi, dll
 Uji hambatan aglutinasi, contoh: Uji konfirmasi RPHA untuk penentuan
HBsAg
 Aglutinasi tak langsung
• Aglutinasi pasif, contoh: Uji kehamilan, uji aglutinasi lateks
• Ab inkomplet, contoh: Uji Rose-Waaler utk deteksi faktor
Rematoid
 JENIS IMUNOASAI TAK BERLABEL…con’t…

 Uji Hemaglutinasi
: Uji aglutinasi sel darah merah (domba)
 Uji Hemaglutinasi Slide & tabung
 Uji Hemaglutinasi langsung
 Uji Hemaglutinasi pasif
 Uji Hambatan Hemaglutinasi
 Uji Hambatan Hemadsorbsi
 Uji Hemagregasi
 Uji Hemaglutinasi campuran
 JENIS IMUNOASAI TAK BERLABEL…con’t…

 Lisis Imun & Fiksasi Komplemen

Uji fiksasi komplemen, contoh: Uji Wasserman, deteksi
antibodi terhadap virus, fungi, parasit, dll
Uji Hambatan Fiksasi Komplemen
 JENIS IMUNOASAI TAK BERLABEL…con’t…

 Uji Netralisasi
:Berbagai pengenceran serum dicampur dengan sejumlah tertentu
toksin atau suspensi kuman. Campuran tsb kemudian dibiarkan
bereaksi kemudian diuji reaktivitas toksinnya atau viabilitas
kumannya.
ANTITOKSIN + TOKSIN  NETRALISASI TOKSIN
 Netralisasi toksin:
• Uji proteksi hewan (Penentuan toksin antidifteri, antitetanus)
• Uji Antistreptolisin O
 Netralisasi Virus:
• Uji in vivo
• Uji in Ovo
• Pock Reduction Test
• Uji perbenihan jaringan
• Plaque Reduction Test
• Uji hambatan metabolik
  IMUNOASAI BERLABEL

 ASAI BERLABEL FLUOROSENS

 ASAI BERLABEL RADIOISOTOP
 Radioimunoasai (RIA)  radiolabelisasi dilakukan pada antigen
 Imunoradiometrik asai (IRMA)  radiolabelisasi dilakukan pada
antibodi
 LUMINESCENT IMUNOASAI (LIA)
Label: bahan luminescent
 ASAI BERLABEL ENSIM (EIA)
 EIA homogen
 EIA Heterogen = ENZYME-LINKED IMMUNOSORBENT ASSAY
(ELISA)
Prinsip Reaksi EIA
 JENIS TEKNIK ELISA

A. Pemberian Label ENZIM pd ANTIGEN

1. ELISA kompetitif ( Antigen)
2. ELISA titrasi ( Antigen)
3. Solid phase Anti Ig M Elisa ( Antibodi)

B. Pemberian Label ENZIM pd ANTIBODI

1. Double antibodi sandwich ELISA ( Antigen)
2. Inhibition ELISA ( Antigen)
C. Pemberian Label ENZIM pd Anti-imunoglobulin
1. Indirect ELISA ( Antibodi)
2. Indirect sandwich ELISA ( Antigen)
3. Indirect inhibition ELISA ( Antigen)
A.1. ELISA Kompetitif
 Prinsip:
Ag (e) dicampur bahan pemeriksaan yg juga
mengandung Ag (s) yg sama yg akan ditentukan shg
terjadi kompetisi dlm mengikat sejumlah yg terbatas Ab
spesifik yg terikat fase padat

Aktivitas enzim yg terikat berbanding terbalik dg kadar

Ag dlm bahan yg diperiksa
 COMPETITIVE ELISA

For detection of small molecules (haptens)

Solid Substrate: TMB (Tetra Methyl

support Immobilize Benzidine)
d Ab

Alkaline phosphatase- labeled

hapten (Conjugate)

Hapten to be
detected
Competitive EIA

substrat
Antibodi + enzyme
E
Antibodi

Ag Ag Ag
A.2. ELISA Titrasi
 Prinsip:
Ag (s) ditambahkan dahulu pd Ab spesifik yg terikat fase
padat. Setelah inkubasi bagian yg tak terikat dibuang &
dicuci.
Selanjutnya ditambahkan sejumlah tertentu Ag (e).
Kemudian ditambahkan substrat (Cr)
Aktivitas enzim yg terikat berbanding terbalik dg kadar
Ag dlm bahan yg diperiksa
A.3. Solid phase Anti Ig M ELISA

Prinsip:
AHG Ig M diikatkan fase padat lalu ditambahkan serum yg
akan diperiksa shg semua Ig M dlm serum diikat oleh
Antihuman Ig M yg terdapat pd fase padat.
Bagian yg tak terikat dibuang & dicuci , ditambahkan Ag (e)
shg Ig M spesifik thd Ag tsb saja yg dapat mengikat Ag (e)
yg ditambahkan.
Ditambahkan substrat (Cr)
Eg; Ig M anti HBsAg
B.1. Double Antibodi Sandwich ELISA
Prinsip:
Ag (s) direaksikan dg Ab spesifik I (fase padat).
Ditambahkan Ab spesifik II (e)
Ditambahkan substrat
Aktivitas enzim yg terikat berbanding lurus dg Ag (s)
Eg; Ig E total, HBsAg, hormon (BM besar)
 DIRECT ELISA (Sandwich)

For antigens detection

Substrate: TMB (Tetra Methyl
Solid
Benzidine)
Support

Immobilized Alkaline phosphatase-

Ab labeled Ab (Conjugate)

Ag to be detected
B.2. Inhibition ELISA
Prinsip:
Ag (s) ditambahkan Ab spesifik (e)= konjugat dlm
jumlah tertentu yg berlebihan, shg sebagian akan
mengikat Ag (s) sisanya bebas
Campuran tsb di atas ditambahkan pada Ag sama yg
terikat fase padat. Cuci.
Maka sisa konjugat yg masih bebas akan terikat pd Ag
Cuci, tambahkan substrat
C.1. Indirect ELISA
Prinsip:
Ab (s) direaksikan dg Ag yg terikat fase padat.
Ditambahkan Antihuman globulin(e)
Ditambahkan substrat

Aktivitas enzim yang terikat berbanding lurus dg kadar Ab

(s)
 Indirect EIA

Anti-human Ig+enzyme E
substrat

Antibodi

Ag
 INDIRECT ELISA

For antibodies detection

Substrate: TMB (Tetra Methyl
Solid Benzidine)
support
Ab to be
detected

Alkaline phosphatase-
labeled Ab (Congugate)
Immobilized
Ag
 C.2. Indirect Sandwich ELISA

= Double Ab sandwich Antiglobulin ELISA

Prinsip:
Ag (s) direaksikan dg Ab spesifik I (fase padat).
Ditambahkan Ab spesifik II
Ditambahkan Antihuman globulin (e)
Ditambahkan substrat
Aktivitas enzim yang terikat berbanding lurus dg kadar Ag (s)
 C.3. Indirect Inhibition ELISA
Prinsip:
Gabungan inhibition ELISA & Indirect ELISA
Ag (s) ditambah Ab spesifik (berlebihan) thd Ag (s).
Sebagian Ab spesifik akan terikat, sebagian lagi bebas.
Campuran tsb ditambahkan pd Ag sama yg terikat fase
padat.
Antibodi bebas akan terikat Ag fase padat
Bagian yg tak terikat fase padat dibuang & dicuci,
ditambahkan AHG(e) mk konjugat terikat Ab spesifik di
fase padat
Tambahkan substrat (Cr)
* Antigen sandwich EIA

Antigen + enzyme E
substrat

Antibodi

Ag
 * Capture EIA

Antigen + enzyme substrat

E

Antibodi

Anti-human Ig
G/M
 *IMMUNOCAPTURE

For IgM detection

Solid Substrate: TMB (Tetra Methyl

Support Benzidine)

IgM to be
detected Alkaline phosphatase-
labeled Ab (Congugate)
Anti-IgM
Antigen
 Tahapan Penting Pemeriksaan EIA

 Persiapan Reagent
 Penambahan Sample
 Inkubasi
 Pencucian
 Penghentian Reaksi
 Pendeteksian
Thank You……..
• TUGAS;
RIA (2)
ELISA (3)
IMUNOELEKTROFORESIS (3)