Immunological Techniques

BY
SUTIRTA YASA IWP

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Objective to comprehend
laboratory test is:
• Approach in the patient with immune system
disease and disorders to made diagnosis are
evidence based on :
– history of the diseases (anamnesis)
– physical examination,
– and laboratory test.
• Immunoassay can be used for the detection of
either antigens or antibodies. For antigen
detection, the corresponding specific antibody
should be prepared as one of reagents. The
reverse is true for antibody detection.
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Outcome:
• Students to comprehend the overview of
general principles , purpose of laboratories
are to assist students in (1) confirming or
rejection a diagnosis, (2) providing
guidelines in patient management, (3)
establishing a prognosis, (4) detecting
disease through case finding or screening
and (5) monitoring follow up therapy.

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Learning Task Laboratories test

1. Explain the precipitin reactions, what are antibody

excess zone, equivalence zone and antigen excess zone
2. Explain the differential of immuno-double diffusion and
immunoelectrophoresis.
3. Explain the differential of direct and indirect immuno
techniques method.
4. Mention the immunoassay using labeled reagents for
detecting antigens and antibodies.
5. Explain the competitive assay and two-site capture
assay techniques.

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Self assessment
1. Mention the principle of methods on
immunoassay techniques ?
2. What’s the meaning of equivalence zone ?
3. Mention the reaction marked on
haemagglutination methods
4. Mention the label used on the ELISA methods?

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 What is the laboratory diagnostic tests can
be used ?
What is the performance of laboratory ?
How many techniques are used commonly?
For example, the methods used to
measured :
– antigens and antibodies
– Inflammatory factor : ILs,IFNs, CRF

Most of the common used are outlined in this lecture

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 The Laboratory personal As
journalist on a war ??
As detective
•. What happened?
Damage?
Many signal?
Agent : growing, expanding Soluble mediator?
Excretion toxic substance Ab Detect by
Hematology test ?
Biochemistry test ?
Ab Immunol test ?

Sensitivity
Mikroorganisme of test
/ agent, enter/
invasion Host, defense mechanisms:
celluler & humoral
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Laboratory diagnostic tests can
be used : (Nicoll D, 2004)
– Tests can be helpful for screening
– Tests can be helpful for diagnosis
– Finally , tests can be helpful in patient
management
• Evaluate the severity of disease
• Estimate prognosis
• Monitor the course of disease ( progression,
stability, or resolution)
• Detect disease recurrence
• Select drugs and adjust therapy
(Nicoll D., McPhee, Pignone. Basic Principles of Diagnostic Test Use and Interpretation On : Pocket guide to
Diagnostic Tests .Nicoll D eds. Fourth Edition. Lange Medical Books/McGraw-Hill. New York. 2004:1-21)
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Performance of diagnostic tests (Nicoll D, 2004):
– Tests preparation : test selected
– Patient preparation (appropriate specimens)
– Specimen collection (identification , labeling,
tourniquet time > hemoconcentration, lyses of
blood cell, special handling, storage )
– Accuracy : true value; Precision : reproducibility
when repeated on the sample.
– Reference ranges are method and laboratory
specific. Often represent test results found in 95%
of small population presumed to be healthy.
– Interfering factors. External factors such as
ingestion of drugs. Internal factors such as
abnormal physiologic states.
– Sensitivity and specificity. Test sensitivity is the
likelihood that a diseased patient has positive test
(no diseased patients have negative tests). Test
specificity is likelihood that a healthy patient has a
negative test (no healthy patients have positive
tests).
(Nicoll D., McPhee, Pignone. Basic Principles of Diagnostic Test Use and Interpretation On : Pocket guide to Diagnostic Tests 9
.Nicoll D eds. Fourth Edition. Lange Medical Books/McGraw-Hill. New York. 2004:1-21)
Access is an
Immunoassay System
Chemical (Clinical Chemistry)
10-12 10-9 10-6 10-3 10-0

pg/mL ng/mL mg/mL mg/mL g/mL

Immunological (Immunoassay)
Therapeutic Drugs
Thyroid Hormones
Fertility Hormones
Cardiac Markers
Cancer Markers
Infectious Disease
Vitamins
Allergy Markers

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Laboratory on immunoassay
There are two major type measured :
– Non Specific measured :
hematology,
biochemistry,
urinary test
– Specific measured : serology test may be
developed a number of its own techniques,
particularly those based on the antigen-
antibody interaction (can be helpful for
diagnosis)
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Divide this talk into:

1. Basic concepts
2. Direct/indirect methods
3. Competitive / sandwich methods
4. Precipitation methods
5. Agglutination methods
6. Amplification methods

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 1. Basic concepts
• Based on : Ab + Ag = Ab:Ag (are not visualized)
• Ab:Ag (in proportions at or near equivalence, can be
seen by colour (enzyme, fluorecence, protein stain);
solid phase (ery, bentonite, latex); X ray (a,b,g :
detect by machinary counter), chemiluminescent as
marker.
• In vivo similar to in vitro
– Van der Waals Energy : specificity of Ag & Ab
– Columbic Energy : electricity
– Distance between antigen and antibody
– pH, hydrogen, hydrophobic, electrostatic, etc
• The immunoassay using labeled reagents are: Elisa (using
enzyme), RIA (using radioisotopes) , IF (using florescent) ,
chemiluminescent (using electron) 13
Immunoassay ( 2. Direct, indirect methods)

• Direct methods
Measured:
Ab labeled
1st stept
Ag

• Indirect methods. Ab labeled

Complement
Measured:
Antibody,
and its
responses
direct indirect
Based on methods
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Immunoassay (3. Sandwich and competitive
assay)
Serum: present Ab Px

• Sandwich Assay enzyme (color as marker)

• Competitive Assay

enzyme (color as marker)

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Immunoassay Photo
meter

• Competitive Assay

Adsorbent
Serum: Px Ab

Ab: reagent labeled Consentration

Color
of test

• Sandwich Assay

Adsorbent
Concentration
of test
Serum: Px Ab Ab: reagent labeled Color 16
Immunoassay methods
Divided three major methods:
• Precipitation methods : high concentration,
in solution
• Agglutination methods : Ag /
Immunoglobulin on solid phased
• Amplification methods : low concentration
( methods ; ELISA, RIA, IMUNOFLURESENSI) /
labeled method

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4. Precipitation methods
• We observe the antigen-antibody reactions depend on
their ability to precipitate when combined in proportions
at or near equivalence.
• By performing these reactions in agar gels
• Example:
– Simple diffusion (qualitative)
– Immunoelectrophoresis, single radial immunodiffusion
(quantitative = gram/L; mg/dl)
• Developed to quantitative methods :
immunoelectrophoresis & radial immune diffusion
• On the immunoelectrophoresis methods, antigens
separated in agar gel’s by placing an electric charge
across it before being visualized by precipitation
• These techniques operate in the range of 20 ug/ml to
2mg/ml of antigen or antibody
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 The classical illustration of
the Ag-Ab reaction invitro
is the precipitin reaction

The Ag-Ab reactions was their ability to precipitate when

combined in proportions at or near equivalence
Antibody excess zone : Ag is insufficient to react with Ab
Free Ab in the supernatant
Equivalence zone : Ag is sufficient to combine with Ab
neither free Ag and Ab can detected in the supernatan
Antigen excess zone: Ag exceeds that required to bind the A
Free Ag in the supernatant
 Precipitation reactions, the immuno-
double –diffusion technique
This technique may
be used to
determine
the relationship
between Ag(blue)
and test Ab
(yellow)

The plate are left for at least 24 hour, during which time
the antigen and antibody diffuses out to form soluble
Ag-Ab complexes, can be visualized by staining the 20
precipitin arcs with a protein stain such as Coomassie blue.
IMMUNOELECTROPHORESIS

• .

1. Antigen are separated in an agar gel by placing an electric

charge across it. The gel’s pH is chosen so that positively
charged protein move to the negative electrode and negatively
charged protein to the positive
2. A trough is then cut between the wells and filled with the
antibody, which is left to diffuse.
3. The antigen and antibody form precipitin arcs
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IMMUNOELECTROPHORESIS
• .

g glob a-2-glob
g Glob a-1-glob Pre
Albumin
Alb

ANTI SERUM

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IMMUNOELECTROPHORESIS

Albumin , a1, a2, b, g Globulin

Cirrhosis hepatic : low albumin , high g Globulin

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5. Agglutination methods
• Haemagglutination : antibody may
detected and measured by
haemagglutination (Ery)
• the ability of antibody to bind the
antigen on their surface
• As solid phase are : erythrocyte
, latex, benthonic.
Example, measure used to widal test ,
CRF, RF, HCG, blood grouping
• These techniques operate levels of less than
1 ug/ml
SDM
Ery
LATEK

LATEK
SDM

SDM

SDM
Ery 24
AGGLUTINATIONS
• Agglutination slide method

• Agglutination tube method

• Applications: Widal test, bacterial Identification 25

 Incomplete Antibody

• Incomplete antibody on surface red cells

Reagent
agglutination
Sample

Assays that detect antibodies to red cell antigens.

Application: Coomb’s test (anti –immunoglobulin) : red cells hemolytic

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6. Amplification methods
ELISA (Enzyme Linked Immunosorbent Assay)
.

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 Flow cytometry

•.

This technique used to detecting Ag in solution or detect surface molecules on cell

Ab is immobilized onto the sensing surface and the test Ag solution is passed over
the surface. Light is focused onto the gold film which is part of the sensing surface and
is reflected. The intensity of light reflected is reduced defend on Ag-Ab reaction
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Pulse Editing (Patented)
Cell flows through centre of aperture

Good Pulse

Diluent stream
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 The Coulter principle

Neutrophil A White cell passes through WBC

aperture

Oscilloscope

Sensing Zone

Oscilloscope
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Conclusion
Clinical Laboratories
– Pre analytic (yours area)
– Analytic
– Post analytic
Divided three major methods:
• Precipitation methods
• Agglutination methods
• Amplification methods
Application the theory at yours cases
• Good luck 31
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