Al Rafidain University College

IMMUNOLOGICAL TEST
INTRO

Prepared by
Dr. Ammar H. Abbas
PHD. Immunology
 SEROLOGICAL TESTS
Serological Tests: types of tests where serum is
used to measure the amount of antibodies or
Antigens present in it.
 ANTIGEN -ANTIBODY
REACTIONS
 Antigen – antibody reactions are performed to
determine the presence of either the antigen or
antibody. (serological tests ).
 Either the antigen or the antibody have to be known.

e.g. with a known antigen, such as

influenza virus , a test can determine
whether antibody to the virus is
present or not .
 1. AGGLUTINATION

 In this test the antigen is particulate (visible, big and

insoluble) (e.g. bacteria and red blood cells) or an
inert particle (latex beads) coated with antigen.
 Antibody is divalent and cross links the multivalent
antigen to form a lattice network or clumps
(agglutination).
 This reaction can be performed in a tube or on a
glass slide e.g. ABO blood grouping.
AGGLUTINATION TEST
POSITIVE.
NEGATIVE.

antigen
Antibody
2. HAEMAGGULTINATION TESTS:
 It is a type of agglutination test performed on
RBCs.
 It has two types:
 Active: the antigen is the RBC itself.
 Viruses can clump red blood cells from one species or
another (active hemagglutination)
 This can be inhibited by specific anti-viral antibodies.
 Another example is the test used in ABO grouping.
 Passive: the antigen here is not the RBC. The RBC
absorbs it and expresses it on the surface.
 It will form clumps when mixed with antibodies.
 i.e. red cells are passive carriers .
HAEMAGGULTINATION TESTS

active passive
Immunochromatography
Introduction
 Immunochromatographic
assays, also called lateral
flow dipstick immunoassay
or simply strip tests

• Are simple devices intended to detect the presence (or

absence) of a target analyte in sample (matrix) without
the need for specialized and costly equipment.
• A widely spread and well known application is the
home pregnancy test.
 Principle of Immunochromatography Kit:
 The liquid sample is dropped on the sample pad, the
antigen in the sample forms an immunocomplex with the
antibody labeled with colloidal gold.
 Its complex moves along with the liquid sample, and
makes a contact with the antibody immobilized on the
membrane, followed by forming an immunocomplxes
with the immobilized antibody, resulting in generating a
colored red purple line
Principle of Immunochromatography Kit:

 Appearance of red purple line on the membrane

indicates the presence of antigen in interest in the
sample. Since the liquid of the sample migrates
through the membrane very fast, it makes it possible
to detect the presence or absence of antigen within
15 minutes.

 An Immunochromatographic unit is completed by

attaching the sample pad at the end of the
membrane.
How they work:
 Step 1: Sample placement
◦ To perform the test, a sample is placed on
the sample pad at the end of the strip.
◦ The sample may be used alone as
commonly done with urine or serum or
whole blood or plasma compatible tests,
or it may be mixed with a buffer specific
to the test
 Step 2: Molecules solubilize

◦ with the addition of the sample, the

detector molecules are solubilized.
◦ When solubilized the detector molecules
mix with and bind to the analyte in the
sample (if analytes is present).
 Step 3: Capillary action
◦ Then capillary action draws the fluid mixture up the
sample pad and into the membrane.
◦ The sample/detector molecule mix continues to move up
the membrane until it reaches the analyte capture
molecule.
◦ In these lines a second (and third) antibody or antigen,
immobilized as a thin stripe in the nitrocellulose will then
capture the complexes if it is positive for the target
analytes.
◦ The control line should always show as a visible line,
otherwise the test is invalid and must be repeated. If the
test is positive, a colored (typically pink or purple ) line
develops along with the control line
 Step 4: Excess absorbed
◦ Excess buffer along with any reagent not captured
at the test of control line will then moves into the
absorbent wicking pad.
Advantages:
1. Can detect antigen or antibody
2. Commercially available.
3. Easy to perform
4. Limited / no instrumentation.
5. Single use, rapid test.
6. User friendly format
7. Very short time to get test result.
8. Long-term stability over a wide range of
climates
9. Relatively inexpensive to make.
Disadvantages:
1. Results are qualitative.
2. Rapid tests can be less sensitive and less
accurate.
Applications of
Immunochromatographic assays:
 It can be applied for multiple test platforms
for liver, sexually transmitted diseases,
cardiac markers, as well as women’s and
men’s health (hCG, VDRL, CMV, PSA, H. Pylori,
Troponin I, TORCH, HIV, HBs Ag, HBV, HCV,
RA, CRP, ASO, SLE, IM, salmonella IgM & IgG,
…)
ELISA
 INTRODUCTION TO 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.
A 96-WELL MICROTITER PLATE USED FOR ELISA
 COMPONENTS OF AN ELISA

 Antibody: IgG fraction of serum purified by

affinity chromatography
 Enzyme: Horse Radish Peroxidase (HRP) MW 44,
000, glycoprotein with 4 lysine residues
 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 color. Reaction stopped with dilute acid to
cause complex to turn yellow.
 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 color
 Resulting color is read in a spectrophotometer
 TYPES OF ELISA

 DIRECT ELISA
 INDIRECT ELISA
 SANDWICH ELISA
 COMPETETIVE ELISA
 DIRECT ELISA

The direct ELISA uses the method of directly labeling

the antibody itself. 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.
DIRECT ELISA

Advantages of Direct Detection

 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.
 Little signal amplification.
 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).
 INDIRECT ELISA

Advantages of indirect detection

 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.
Disadvantages of indirect detection
 Cross-reactivity may occur with the secondary antibody,
resulting in nonspecific signal.
 An extra incubation step is required in the procedure.
 SANDWICH ELISA

1. Plate is coated with a capture antibody

2. Sample is added, and any antigen present binds to
capture antibody
3. Detecting antibody is added, and binds to antigen
4. Enzyme-linked secondary antibody is added, and binds
to detecting antibody
5. Substrate is added, and is converted by enzyme to
detectable form.
 COMPETITIVE ELISA
In this Unlabeled antibody is incubated in the presence
of its antigen.
 These bound antibody/antigen complexes are then
added to an antigen coated well.
 The plate is washed unbound antibody is removed.
 The secondary antibody, specific to the primary
antibody is added. This second antibody is coupled to
the enzyme.
 A substrate is added, and remaining enzymes elicit a
chromogenic or fluorescent signal.
 For competitive ELISA, the higher the original antigen
concentration, the weaker the eventual signal.
 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.
 One ogive is left unsensitized to measure the non-
specific reactions of the sample.
 The use of laboratory supplies for dispensing sample
aliquots, washing solution and reagents in microwells
is not required, facilitating ready-to-use lab-kits and
on-site kits.
 PRECAUTIONS

 Negative control with strong signal

The excessive background signal can be caused by
inadequate rinsing of plates, reagents not sufficiently
diluted, inadequate blocking of plates or non-specific
binding of enzyme conjugate. The appearance of color in
negative control wells may also indicate cross-reactivity of
secondary antibody with components in the antigen
sample.
Positive control with no signal

 Microwell plates not coated properly.

 Reagents applied in wrong order or step omitted.
 Secondary antibody not matched to the species of primary antibody.
 Enzyme conjugate defective or inhibited by contaminant.
 Detector antibody not compatible with capture antibody (for sandwich
assays).
ELISA with weak signal
 Wash buffer not adequately drained after every wash step.
 Inadequate incubation times.
 Detection reagents too dilute. Perform checkerboard titrations.
 Enzyme conjugate defective or inhibited by contaminant.
 Substrate defective or contaminated.
 Microwell plates poorly coated.
 Loss of capture antibody during blocking/washing. Decrease or
eliminate use of Tween-20.
 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)
 Detecting infections
 sexually-transmitted agents like HIV, syphilis and chlamydia
 hepatitis B and C
 Toxoplasma gondii

 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, e.g.,
 cocaine
 opiates