Nami - Immunology Diagnostic Test

Immunology
Diagnostic Testing
Ni Nyoman Nami Arthisari/ 1971151004
PPDS-1 Mikrobiologi Klinik FK Unud/RSUP Sanglah
Pembimbing: Dr. dr. Ni Nyoman Sri Budayanti, Sp.MK (K)
Pendahuluan
Teknik Imunodiagnostik  metode pemeriksaan imunologi
berbasis pada reaksi ikatan antigen dan antibodi yang spesifik
Teknik ini banyak dikembangkan untuk mendiagnosis penyakit

infeksi (deteksi mikroba maupun respon host terhadap mikroba)
Tujuan:
• Identifikasi agen penyebab infeksi
• Memberikan informasi prognostik
• Keperluan data epidemiologi penyakit infeksi
Prinsip Dasar Pemeriksaan Imunodiagnostik
Untuk mendeteksi antigen dari mikroba yang menginfeksi, diidentifikasi
1 dari spesimen pasien
Untuk mendeteksi antibodi yang dihasilkan sebagai respon host

2 terhadap masuknya mikroba
Subhash Chandra Parija in Textbook of Microbiology and Immunology, 2nd edition. Elsevier; 2012.
Jenis dan Metode Pemeriksaan Imunodiagnostik
Agglutination Test
• Direct dan Indirect agglutination test
• Latex agglutination, staphylococcal agglutination, hemaglutination inhibition, Liposome mediated agglutination
Precipitation Test
• Double immunodifusion, single radial imunodifusion, flocculation
Neutralization Test
• Toxin neutralization test, Virus neutralization test
Labeled Immunoassay Test

• Immunofluorescens assay, enzyme immunoassay (solid phase immune assay, enzyme linked immunosorbent
assay), membrane-bound enzyme immunoassay (immunochromatographic test), Optical immunoassay,
Complement fixation test, Western blotting, immune blot, radio immunoassay, chemiluminescent
immunoassay
Agglutination Test
 Reaksi aglutinasi  gumpalan-gumpalan yang visible yang terjadi karena adanya
reaksi antara antibodi dan antigen pada permukaan partikel mikroskopik.
 Prinsip: kombinasi pengenceran seri larutan antibody + jumlah konstan antigen
partikulat atau sebaliknya
 Tes aglutinasi dapat dilakukan pada test tube (tabung reaksi), tetapi umumnya
dilakukan pada permukaan slide kaca, plastik atau cardboard.
 Partikel yang dapat berkontribusi: eritrosit, sel bakteri, latex particles
Agglutination Test
Dibagi menjadi
 Direct Agglutination:
– Slide Agglutination Test
– Antiglobulin (Coombs test)
– Tube Agglutination Test
 Passive Agglutination Test

– Latex Agglutination Test
– Haemagglutination Test
– Coagglutination test
Direct Agglutination Test
Slide Agglutination
 Suspension of bacteria/antigen + a
drop of standardized serum or vice
versa
 Used for:
– Routine procedure to identify
bacterial strains, isolated from
clinical specimens.
– Blood grouping (hemagglutination)
and cross-matching.
Direct Agglutination ABO Typing
Tube Agglutination Test
 Also known: Standard agglutination
test or Serum Agglutination test (SAT)
 Quantitative
 Serum is diluted in a series of tubes
and constant defined amount of
antigen is added to each tube  tubes
incubated for 20h 37°C  particular
antigen clumps at the bottom of test
tube
 Example: Widal test, Brucellosis
screeninig
 Coombs’ test are used for
Antiglobulin (Coombs’ Test) detection:
 Non-Agglutinating antibody – Anti-Rh antibodies
antibodies bind to their specific – Incomplete antibodies in
antigens but do not cause Brucellosis and other disease
agglutination
Epitopes antigen too wide, Ab do
not cross link  difficult to detect
in the lab
 Anti immunoglobulin
antibodies
 Specific Ab binds at the Fc region
of non-agglutinating Ab
 anti-Ig  coombs reagent
COOMBS TEST - Direct
 Diagnosis of Hemolytic Disease

of Newborn (HDN)
 Detect anti-Rh antibodies that
already bound to fetal RBCs
Kaplan USMLE STEP 1 Lecture Notes 2018 Immunology and Microbiology; 2018.
COOMBS TEST - Indirect
 Prevention of HDN
 Detects presence of anti-Rh
antibodies in mother’s serum
Kaplan USMLE STEP 1 Lecture Notes 2018 Immunology and Microbiology; 2018.
Indirect Agglutination/Reverse Passive Agglutination
 Employs carrier particles that  Based in the carrier particles

are coated with soluble used, can be divided into:
antigens – Latex Agglutination test
 This test usually done to – Hemagglutination test
convert precipitation reactions – Coagglutination test
into agglutination reactions
Subhash Chandra Parija in Textbook of Microbiology and Immunology, 2 nd edition. Elsevier; 2012.
Passive Agglutination Test
Latex Agglutination Test
• It is a test that employs latex
particles as carrier of antigen or
antibodies
• Latex particles are inexpensive,
relatively stable.
• The large particle size of the latex
facilitates better visualization of
antigen–antibody reactions by the
naked eye observation.
• Used in:
• Rapid identification of Streptococcus
group, Staphylococcus group, etc.
Latex Agglutination
Hemagglutination Assays
 The clumping of RBCs

• Virus particles have an envelope
protein called the hemagglutinin (HA)
which binds to sialic acid receptors
+
on cells.
• The virus will also bind to
erythrocytes (RBC), causing the
formation of a lattice.
• Used to determine levels of virus RBC HA Settling
present in a sample. Suspension Virus Pattern
• Used in: e.g. virology (influenza virus)
Hemagglutination Assays
Hemagglutination Inhibition Assay
• The clumping of RBCs

• The basis of the HI assay is that
antibodies to virus will prevent
attachment of the virus to red
blood cells.
+ +
• Therefore hemagglutination is
inhibited when antibodies are
present.
• Used to determine the level of
antibodies to virus present in
serum samples. RBC Suspension HA Virus Antibody
Hemagglutination Inhibition Assays
Coagglutination Test
• Using Cowan I strain protein A (an anti-antibody) of S. aureus as
carrier particle to coat antibodies.
• In a positive test, protein A bearing S. aureus coated with antibodies
will be agglutinated if mixed with specific antigen.
• Advantage: these particles show greater stability than latex particles
• Used for: detection of cryptococcal antigen, detection of amoebic
and hydatid antigens in the serum, and grouping of streptococci and
mycobacteria.
Coagglutination Test
Bailey & Scott’s Diagnostic Microbiology Fourteenth Edition. Elsevier; 2017.

Precipitation Test
Basic Principle
• In this test, the antigen is in • Conditions necessary for
solution. precipitation reactions:
• The antibody cross-links • Antibody must have at least
antigen molecules  result two antigen binding sites
(bivalent)
in formation of immune • Antigen must be soluble
complex or lattice (insoluble) • The proportions of the antigens
 precipitate and antibodies must be equal
• Precipitation can take in liquid
media or in gels
Precipitation Prozone, Postzone, and
Test Zone of Equivalence
Precipitation Tests
• Precipitation in Solution:
• Ring test and flocculation test
Types: • Precipitation in Agar
• Precipitation in agar with an
electric field
Precipitation in Solution
Ring test
Example:
- C-reactive protein test
- Streptococcal grouping
by Lancefield methods
Marjojie Kelly Cowan, Kathleen Park Talaro in Microbiology: A System Approach Second edition. Mc Graw Hill: 2009
Precipitation in Solution
Flocculation test
 Slide Flocculation test

– VDRL, Syphilis
 Tube Flocculation test:

– Kahn test -syphilis
Precipitation in Agar
(Immunodiffusion) Single diffusion
One Dimension
Double diffusion
Immunodiffusio
n
Single diffusion
Two Dimensions
Double diffusion
Immunodiffusion
 Advantages:
– The reaction formed this method is stable and can be preserved for staining
 Applications:
– To determine relative concentrations of Antibodies / Antigens
– To compare antigen
– For disease diagnosis
– Serological surveys
Precipitation in Agar (Immunodiffusion)
-Immunodiffusion in one dimension-
Single diffusion in two dimensions (radial
immunodiffusion)
Precipitation in Agar (Immunodiffusion)
Double diffusion in two dimensions (Ouchterlony Procedure)
 Double diffusion: both antigen

and antibody allowed to diffuse
in to the gel
 Used for: comparing different
antigen preparation
 The pattern of lines can be
interpreted to determine
whether the antigen are same or
different
Ouchterlony Procedure
1. Precipitation line pattern: Arc Shaped / Single

board curve line (Identity)
– Antigen share all the epitopes and they are
identical
2. Precipitation line pattern: cross (Non-

identity)
– Antigens have no common epitopes and they are
non-identical
3. Precipitation line pattern: incomplete cross /

arc with a spur (Partial Identity)
– Antigens share one or more epitopes and they are
partially identical
Precipitation in Agar with an Electric Field
Immunoelectrophoresis
 Process of combination:
– Electrophoresis  Resolving the given
antigen mixture into separate antigens
– Double Immunodiffusion  Identifying these
antigens by precipitatin reactions
Applications:
– Detect detection of high antibody
concentrations such as albumin and
transferrin; the detection of three
immunoglobulin levels in blood: IgM, IgG,
and IgAin the serum
Electrophoresis
Immunoelectrophoresis
Ab
Complement-Dependent Serological Tests
 Basic Principle:
– Use to test for the presence of specific antibodies in patients serum
 Uses 4 components:
1. Antibody
2. Antigen
3. Complement
4. Sensitized red blood sheep
Complement Fixation Test
 The principle of the complement fixation
test is that when antigen and antibodies
of the IgM or the IgG classes are mixed,
complement is “fixed” to the antigen–
antibody complex.
 In this test: , “sensitized” red cells are then
added to the mixture. If the red cells are lysed
 no antibodies specific The complement
therefore was not consumed in the test system
and was available to be used by the anti-RBC
antibodies, resulting in hemolysis  negative
 Non-lysis of the cells indicates that patient’s

serum had antibodies specific to the antigen,
which “fixed” complement  positive
Neutralization Test
 Basic Principles:
– Neutralization is an antigen–antibody reaction in which the biological effects of viruses
and toxins are neutralized by homologous antibodies known as neutralizing antibodies.
– These tests are broadly of two types:
(a) Virus neutralization tests (i.e antibodies to influenza A virus in swine) and
(b) Toxin neutralization tests (i.e: Antibody to Streptolysin O) .
Neutralization Tests
Immunofluorecence
Antigens in the patient specimens are immobilized and fixed onto glass slides with
Tests formalin, methanol, ethanol, or acetone
Basic Principles
• Immunofluorescent
assays are frequently
used for detecting
Antibodies conjugated (attached) to fluorescent dyes are applied to the specimen
bacterial and viral
antigens and for
detection of specific
antibodies in the serum
and other body fluids.
• This technique is After appropriate incubation, washing, and counterstaining (staining of the
frequently used to background with a nonspecific fluorescent stain such as rhodamine or Evan’s blue),
the slide is viewed using a microscope equipped with a high-intensity light source
diagnose syphilis (FTA- (usually halogen) and filters to excite the fluorescent tag.
ABS) and various viral
infections.
Types
Indirect immunofluorescence
Direct immunofluorescence test
test
Radioimmunoassay (RIA)
Basic Principles
- Radioisotope is used as a tag or
label (radioisotope is link to
antigen or antibody) for the
detection of antigen-antibody
complex.
- Most popular: 125I
- This method is used for the
quantitation of antigens that can
be radioactively labelled.
- Measured by the amount of
radioactivity using gamma counter.
- The test is used for quantitation of
hormones, drugs, HBsAg, and
other viral antigens.
Enzyme Immunoassays
Basic Principles Types
• Enzyme immunoassays (EIAs) can be 1. Indirect ELISA.

used for detection of either antigens 2. Sandwich ELISA.
or antibodies in serum and other body 3. Competitive ELISA.
fluids of the patient. 4. ELISPOT assay.
• In EIA techniques, antigen or antibody
labelled with enzymes are used.
• The commonly used EIAs are enzyme-
linked immunosorbent assays (ELISAs).
Used for the quantitative estimation of antibodies in the serum
Indirect ELISA and other body fluids
Sandwich ELISA Used for the detection of antigen
Western Blot
Basic Principles
• The Western blot is an analytical technique used to detect specific proteins in a given sample of
tissue or extract.
• Specific proteins separated by electrophoresis and detected by use of labelled antibodies.
• Can detect more types of antibodies and are less subject to misinterpretation than other tests.
• Southern Blot  to detect specific DNA

• Northern Blot  to detect specific RNA
Western Blot
 Steps:
1. Protein gel electrophoresis
2. Protein transfer
3. Blocking
4. Antibody probing
5. Detection
Abbas, AK. In Cellular and Molecular Immunology 9th edition. Elsevier; 2018.
Flow Cytometry
Basic Principles
• Commonly used to measure the number of various types of immunologically active

blood cells.
• In this test, the patient’s cells are labeled with monoclonal antibody to the protein
specific to the cell of interest.
• Used for: e.g. determine the number of CD4+ T Cells
Warren Levinson in Review of Medical Microbiology and Immunology 14 th edition. McGrawHill; 2016.
Flow Cytometry
Abbas, AK. In Cellular and Molecular Immunology 9 th edition. Elsevier; 2018.

Flow Cytometry –
FACS
FACS: Fluorescence-Activated Cell Sorting
Abbas, AK. In Cellular and Molecular Immunology 9 th edition. Elsevier; 2018.

Immunochromatographic Test
Referensi
 Abbas, AK. In Cellular and Molecular Immunology 9th edition. Elsevier;
2018.
 Warren Levinson in Review of Medical Microbiology and Immunology
14th edition. McGrawHill; 2016.
 Subhash Chandra Parija in Textbook of Microbiology and Immunology,
2nd edition. Elsevier; 2012.
 Buku Ajar Pemeriksaan Mikrobiologi pada Penyakit Infeksi
 Marjojie Kelly Cowan, Kathleen Park Talaro in Microbiology: A System
Approach Second edition. Mc Graw Hill: 2009

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Immunology

Teknik ini banyak dikembangkan untuk mendiagnosis penyakit

Untuk mendeteksi antibodi yang dihasilkan sebagai respon host

Labeled Immunoassay Test

 Passive Agglutination Test

 Diagnosis of Hemolytic Disease

 Employs carrier particles that  Based in the carrier particles

 The clumping of RBCs

• The clumping of RBCs

Bailey & Scott’s Diagnostic Microbiology Fourteenth Edition. Elsevier; 2017.

 Slide Flocculation test

 Tube Flocculation test:

 Double diffusion: both antigen

1. Precipitation line pattern: Arc Shaped / Single

2. Precipitation line pattern: cross (Non-

3. Precipitation line pattern: incomplete cross /

 Non-lysis of the cells indicates that patient’s

• Enzyme immunoassays (EIAs) can be 1. Indirect ELISA.

• Southern Blot  to detect specific DNA

• Commonly used to measure the number of various types of immunologically active

Abbas, AK. In Cellular and Molecular Immunology 9 th edition. Elsevier; 2018.

FACS: Fluorescence-Activated Cell Sorting

Abbas, AK. In Cellular and Molecular Immunology 9 th edition. Elsevier; 2018.

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