Prepared by Mr Dejene G 08/28/2023

1 COMPETITIVE ELISA TO
DETECT AG
Provides another extremely sensitive variation for measuring amounts
of antigen

Antibody is first incubated in solution with a sample containing

antigen

The antigen-antibody mixture is then added to an antigen-coated

microtiter well

The more antigen present in the initial solution-phase sample, the less
free antibody will be available to bind to the antigen-coated well
 Prepared by Mr Dejene G 08/28/2023

2 COMPETITIVE ELISA…

After washing off the unbound antibody, an enzyme-

conjugated Ab2 specific for the isotype of the Ab1 can
be added to determine the amount of Ab1 bound to the
well

In the competitive assay, the higher the concentration of

antigen in the original sample, the lower the final signal
 Prepared by Mr Dejene G 08/28/2023

3 COMPETITIVE ELISA TO
DETECT AB
 Prepared by Mr Dejene G 08/28/2023

4 ENZYMES USED
 Peroxidase (horse raddish)=HRP
 Alkaline phosphatase = ALP
 Beta glactosidase
 Glucose oxidase
 Catalase
 Glucoamylase
SUBSTRATES USED
Prepared by Mr Dejene G 08/28/2023

5
TMB (3,3',5,5'-tetramethylbenzidine)=HRP
OPD (o-Phenylenediamine)= HRP
ABTS (2,2´-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-
diammonium salt) = HRP

PNPP (p-Nitrophenyl Phosphate) =ALP

BCIP (Bromo-Chloro-Indolyl Phosphate) =ALP

IPTG (isopropyl beta-D-thiogalactoside) = Beta- galactosidase

ONPG (o-Nitrophenyl-beta-galactopyranoside) = Beta- galactosidase
X-GAL (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)
Beta- galactosidase
READING THE RESULT
Prepared by Mr Dejene G 08/28/2023

6
If it is qualitative ELISA
o Read by the naked eye

• The presence or absence of antigen is seen as a

simple color change
If it is quantitative antigen/antibody techniques
o Read either

• by measuring the intensity of color in a

spectrophotometer or ELISA reader
• By testing dilutions of the test serum and
determining the highest dilution that shows a
color change
 Prepared by Mr Dejene G 08/28/2023
ELISA Positive and negative samples after substrate
7 and incubations
addition
 Prepared by Mr Dejene G 08/28/2023
ELISA Positive and negative samples after adding
8 reagent
stopping
 ADVANTAGES AND DISADVANTAGES OF
Prepared by Mr Dejene G 08/28/2023

DIRECT
9 ELISA
ADVANTAGES DISADVANTAGES

 Quick methodology since only one  Immunoreactivity of the primary

antibody is used antibody may be reduced as a result of
labeling
 Labeling of every primary antibody is
 Cross-reactivity of secondary antibody time-consuming and expensive
is eliminated
 Limited sensitivity –Little signal
amplification.
 Prepared by Mr Dejene G 08/28/2023

10 ADVANTAGES AND DISADVANTAGES

OF SANDWICH ELISA

 Complex workflow – requires more

ADVANTAGES DISADVANTAGES
incubation steps
 High sensitivity; it is 2-5 times
more sensitive than direct  Requires more optimization – cross-
 High specificity as two reactivity between the different
antibodies are used to detect the antibodies must be checked
antigen
 Require more reagents(expensive)
 It offers flexibility since both
direct and indirect methods can
be used
 Prepared by Mr Dejene G 08/28/2023

11 ADVANTAGES AND
ADVANTAGES
DISADVANTAGES OF INDIRECT
DISADVANTAGES
ELISA DETECTION
A wide variety of labeled secondary
METHODS
 Cross-reactivity may occur with the
antibodies are available commercially. secondary antibody, resulting in a
nonspecific signal

Versatile, since many primary antibodies

can be made in one species and the same  An extra incubation step is required in
labeled secondary antibody can be used the procedure
for detection

Immunoreactivity of the primary

antibody is not affected by labeling

 High sensitivity, since the use of secondary antibodies makes

it possible to amplify the signal.
 Prepared by Mr Dejene G 08/28/2023

12 ADVANTAGES AND DISADVANTAGES

OF COMPETITIVE ELISA

 High sensitivity  Requires the use of labeling antigen

ADVANTAGES DISADVANTAGES
 Allows the detection of  Requires two antibody reagents, and one
small-size antigens in low antigen reagents(expensive)
concentrations
SUMMARY OF ELISA
Prepared by Mr Dejene G 08/28/2023

13
 RADIO IMMUNO
Prepared by Mr Dejene G 08/28/2023

14
ASSAY(RIA)
One of the most sensitive techniques for detecting antigen or
antibody

1st developed by two endocrinologists called

o S.A. Berson and

o Rosalyn Yalow, in 1960 to determine levels of insulin-anti-

insulin complexes in diabetics
RADIO
Prepared by Mr Dejene G 08/28/2023

15
o The conc. of Ag or Ab is measured with the use of radioisotopes

IMMUNOASSAY…
o Radioactive isotopes are molecules with unstable nuclei and therefore emit
radiation spontaneously
o The activity of radioisotopes is detected/quantitated by gamma counters or scintillators based on the type
of rays emitted in counts per minute (CPM) by the isotopes used

o Radio isotopes commonly used:-

Isotope Type of ray Half life

125
I γ 30 days

60
CO β,γ 5-6 years

51
Cr β,γ 27.8 days

131
I β,γ 8.1 days

14
C β 5-6 years

32
P β 14.3 days
 RADIO IMMUNO ASSAY(RIA)…
Prepared by Mr Dejene G 08/28/2023

16
Two types
Conventional

Immunoradiometric Assay (IRMA)

CONVENTIONAL RIA
Prepared by Mr Dejene G 08/28/2023

17
is a competitive immunologic procedure

It measures very low concentrations of Ags or

Abs

 It is a highly sensitive method to detect low

concentrations of unknown (unlabeled) antigen
 CONVENTIONAL…
Prepared by Mr Dejene G 08/28/2023

18
RIA is used to assay:
Hormones
Drugs
Enzymes
Microbial antigens
e.g.
• hepatitis B antigen,
• carcinoembryonic and α-feto protein antigen
It also used for the detection of antibody
 PRINCIPLE OF RADIOIMMUNOASSAY
Prepared by Mr Dejene G 08/28/2023

19 Uses an immune reaction [Antigen – Antibody reaction] to estimate a

 Principle:
ligand

Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ag*

• Unbound Ag* and Ag washed out

• Radioactivity of bound residue measured
• Ligand conc is inversely related to radioactivity

[Ag: ligand to be measured; Ag* radiolabelled ligand]

CONVENTIONAL…
Prepared by Mr Dejene G 08/28/2023

20
• RIA technique utilizes three components

• Patient antigen

the specific compound we wish to determine

• Labeled antigen

the same compound as above to which is

attached a radioactive label

• Antibody

specific for the sample and labeled antigen

COMPETITIVE RIA/ELISA FOR AG
Prepared by Mr Dejene G 08/28/2023

21
• Method
• Determine amount of Ab needed to bind to a known
amount of labeled Ag
Prior to Test

– Use predetermined
+ ↔
amounts of labeled Ag Labeled
Ag
and Ab and add a
sample containing Test
unlabeled Ag as a
competitor + + +
↔
Labeled Patient’s
Ag sample
 Prepared by Mr Dejene G 08/28/2023

22
IRMA- IMMUNO RADIO METRIC ASSAY

In this technique, after the initial solid

phase Ag and Ab reaction and separation
of free from bound, a second radio labelled
Ab is reacted to the bound and the activity
counted

A kind of sandwich technique

 SOLIDPrepared
PHASE by Mr Dejene NON-COMPETITIVE
G 08/28/2023

RIA/ELISA(IRMA)
23
• Ab detection
• Immobilize Ag
• Incubate with sample
• Add labeled anti-Ig Labeled
Anti-Ig
• Amount of labeled Ab
Ab in
bound is proportional Patient’s
to amount of Ab in sample
the sample Immobilized Ag

Solid
• Quantitative Phase
 Prepared by Mr Dejene G 08/28/2023

General
24
procedure for RIA:
 A known quantity of an antigen is made radioactive,
frequently by labeling it with gamma-radioactive isotopes of
iodine attached to tyrosine

 This radiolabeled antigen is then mixed with a known amount

of antibody for that antigen, and as a result, the two chemically
bind to one another

 Then, a sample of serum from a patient containing an

unknown quantity of that same antigen is added. This causes the
unlabeled (or "cold") antigen from the serum to compete with
the radiolabeled antigen for antibody binding sites
 Prepared by Mr Dejene G 08/28/2023

25the concentration of "cold" antigen is increased, more of it

As
binds to the antibody, displacing the radiolabeled variant, and
reducing the ratio of antibody-bound radio labeled antigen to free
radio labeled antigen

 The radioactivity falls because unlabelled antigen dilute it

 The count obtained from the radioactivity are used to determine

the hapten concentration in the sample, the interpretation being
done on the standard curve
 ASSAY PROCEDURE
Prepared by Mr Dejene G 08/28/2023

26
 Add known amounts of the test sample + labelled antigen
into the microtitre wells
 Incubate  allow the reaction to reach completion
 Decant & wash contents of the well  removes all
unbound antigens
 Radioactivity remaining in the Microtitre wells measured
by a Counter [GM counter , Scintillation counter etc]
 Intensity of radioactivity is inversely correlated with the
conc of antigens in the test sample
 Sensitive to very low conc of antigens
REQUIREMENTS FOR RIA
Prepared by Mr Dejene G 08/28/2023

27

1. Preparation & characterisation of the

Antigen [Ligand to be analysed]
2. Radiolabelling of the Antigen
3. Preparation of the Specific Antibody
4. Development of Assay System
 Prepared by Mr Dejene G 08/28/2023

28 PREPARATION & RADIOLABELLING OF THE

ANTIGEN

• Antigens prepared by..

• Synthesis of the molecule
• Isolation from natural sources

• Radiolabelling [Tagging procedure]

• 3
H 14
C 125
I are used as radioactive tags
• Antigens are tagged to 3 H 14
C 125
I
• Tagging should NOT affect Antigenic specificity & Antigenic activity !
 Prepared by Mr Dejene G 08/28/2023

29PREPARATION OF THE SPECIFIC

ANTIBODY

• Antigen injected intradermally into rabbits or guinea pigs  antibody

production

• Antibodies recovered from the serum

• Some ligands are not Antigenic

• Hormones, Steroids, Drugs  HAPTENS
• Eg: Gastrin, Morphine,
• Haptens conjugated to albumin  antigenic
 Prepared by Mr Dejene G 08/28/2023

INSTRUMENTATION:
30

1)Centrifuge :
swing bucket rotor : 1200-2500 rpm.
Fixed angle head rotor : 3500-4000 rpm.

2) Radioactive counter
A) Gamma counter : which is used for a gmma energy emiting isotopes. E.g. I 125
B) Scintilation counter : It is used for beta energy emitting isotopes . Eg. Tritium 3H
and 14 C isotopes
ADVANTAGES & DISADVANTAGES OF RIA
Prepared by Mr Dejene G 08/28/2023

31
• Advantages
• Highly specific: Immune reactions are specific
• High sensitivity : Immune reactions are sensitive

• Disadvantages
• Radiation hazards: Uses radiolabelled reagents
• Requires specially trained persons
• Labs require special license to handle radioactive material
• Requires special arrangements for
• Requisition, storage of radioactive material
• radioactive waste disposal.
IMMUNOFLORUCENCE TEST(IFT)
Prepared by Mr Dejene G 08/28/2023

32
Two type of fluorescent antibody tests (FAT)
A.Direct and

B.Indirect
 Prepared by Mr Dejene G 08/28/2023

DIRECT
33 FLUORESCENT ANTIBODY
TESTS
 is used to(DIRECT
detect and FAT)
identify unknown antigen in
specimens
• E.g. Viral, bacterial, and parasitic antigens

• It is called direct test because the fluorescent

dye is attached, or labeled, directly to the
antibody
 Prepared by Mr Dejene G 08/28/2023
DIRECT FLUORESCENT ANTIBODY TESTS (DIRECT FAT)
34

 Ab to tissue Ag is labeled with fluorochrome

Fluorochrome
Labeled Ab

Ag
Tissue Section
 Prepared by Mr Dejene G 08/28/2023

35 DIRECT FLUORESCENT ANTIBODY TESTS

(DIRECT FAT)…

 The Known Ab is soluble

o Fluorocein (Fluor) is attached to Ab via cyanate radical
• Provides emitted UV light

 The Unknown Ag is particulate – virus, parasite, or cell

from the patient
 Prepared by Mr Dejene G 08/28/2023

DIRECT FLUORESCENT ANTIBODY TESTS (DIRECT FAT) …

36

 The fluorochrome used is usually fluorescein

isothyocynate (FITC), which gives a yellow-green
fluorescence

 A fluorescent substance is one that, when absorbing light

of one wavelength, emits light of another (longer)
wavelength
 Prepared by Mr Dejene G 08/28/2023

DIRECT FLUORESCENT ANTIBODY TESTS (DIRECT FAT)…

37

Procedure
 A tissue or smear containing the organism (antigen) is fixed to a glass slide and
incubated with the fluorescent antibody (antibody chemically linked to FITC)
 It is then washed to remove unbound antibody.
 Examined by dark-field illumination under a microscope with UV light source
o The antigenic particles that have bound the labeled antibodies are seen to fluoresce brightly
 Prepared by Mr Dejene G 08/28/2023

DIRECT FLUORESCENT ANTIBODY TESTS (DIRECT FAT)…

38

 Direct FAT can be used;

o To identify bacteria when the numbers are very low

o It may also be used to detect

 viruses growing in tissue culture
 or tissues from infected animals such as rabies virus in the brains of infected animals
 or antigens of HIV on the surface of infected cells
 Prepared by Mr Dejene G 08/28/2023

FLUORO LABELED (AB) ANTI-RABIES

39 VIRUS

Fluor attached
to Anti-rabies
antiserum

Rabies virus

Brain section of rabid skunk

 Prepared by Mr Dejene G 08/28/2023

40

Phosphate buffered saline

 Prepared by Mr Dejene G 08/28/2023

41
Rabies visible
in
brain section
because of
fluoro
labeled anti-
rabies
attached to
virus

Unattached fluor labeled

anti-rabies are washed off
 Prepared by Mr Dejene G 08/28/2023

42

                                                   

              
                                        

• positive test for rabies • negative test for rabies

 Prepared by Mr Dejene G 08/28/2023

INDIRECT FLUORESCENT ANTIBODY TEST

43

In the IFAT, unlabelled antibody combines with antigen

and the antigen antibody complex is detected by attaching a

fluorescent-labeled anti species globulin to the antibody

The antibody, therefore, is labeled indirectly

Fluorescent-labeled antihuman globulin is used if the antibody is

of human origin
 Prepared by Mr Dejene G 08/28/2023

INDIRECT FLUORESCENT ANTIBODY TEST…

44
 Ab to tissue Ag is unlabeled
 Fluorochrome-labeled anti-Ig is used to detect
binding of the first Ab
Fluorochrome
Labeled Anti-Ig
Unlabeled
Ab

Ag
Tissue Section

 Qualitative to Semi-
Quantitative
 Prepared by Mr Dejene G 08/28/2023

45 INDIRECT FLUORESCENT
ANTIBODY TEST…
 Detect patient’s Ab to specific structures or organisms
o Known organism or cells Ag attached to the slide
o Patient’s serum is added
o Unattached Ab removed by washing
o Examples: test for Rocky Mountain Spotted Fever or antinuclear
antibodies (ANA)
 Prepared by Mr Dejene G 08/28/2023

INDIRECT FLUORESCENT ANTIBODY

46 TEST…
Conjugate [fluoro labeled anti-Human IgG] is added
Free Conjugate is washed off

Slide is viewed with flourecent microscope

 Positive reactions - fluorescing structures

Sandwich technique
 Prepared by Mr Dejene G 08/28/2023

47
 Prepared by Mr Dejene G 08/28/2023

48 WASHING WITH BUFFERED

SALINE
 Prepared by Mr Dejene G 08/28/2023

49
Conjugate
 Prepared by Mr Dejene G 08/28/2023

50 INDIRECT FLUORESCENT
ANTIBODY TEST…
 The indirect FAT is used in two main ways:

oTo detect and identify unknown antigen in

specimens

oTo detect antibodies in a patient's serum using a

known antigen (microorganism)
 Prepared by Mr Dejene G 08/28/2023

51 INDIRECT FAT TO
DETECT ANTIGEN
A slide preparation of the specimen is made and unlabelled specific antibody is
added

After allowing time for the antigen and antibody to combine, the preparation is
washed leaving only antibody that has combined with the antigen on the slide

A fluorescent labeled anti-species globulin is added and allowed to combine

with the antibody. The excess is washed from the slide

• The preparation is examined by fluorescence microscopy using the correct

filters. The antigen antibody complex will be seen fluorescing brightly
 Prepared by Mr Dejene G 08/28/2023

52 INDIRECT FAT TO DETECT

ANTIBODY
 In this test, a known antigen is placed on the slide and the patient's serum is
added

 The preparation is then washed and fluorescent-labeled antihuman globulin

is added to demonstrate the antigen-antibody reaction. The preparation is
examined by fluorescence microscopy using the correct filters
 Prepared by Mr Dejene G 08/28/2023

INDIRECT VERSUS DIRECT IMMUNOFLUORESCENCE

53

 Direct: particulate Ag in Unknown

o Conjugate fluor + specific antiserum (Ab)

 Indirect: particulate Ag in Known tissue or cell; soluble Ab (free floating) in

Unknown
o Conjugate fluor + Anti-Human IgG
 Prepared by Mr Dejene G 08/28/2023

IMMUNOBLOT (WESTERN BLOT)

54
A most useful technique in the analysis of proteins

The immunoblot is often used to detect viral proteins with specific

antibodies that bind to these proteins on the nitrocellulose blot

Then a second antibody, which is either enzyme conjugated or

radiolabeled, is used to detect the antigen–antibody band

The enzyme causes a localized color reaction that reveals the

location of the antigen band, or the radiolabel is detected by
exposing the nitrocellulose to photographic film

08/28/2023 54
 Prepared by Mr Dejene G 08/28/2023

55 Immunoblot (Western Blot)

 Prepared by Mr Dejene G 08/28/2023

56
PROCEDURE/STEPS:

Extraction of protein
Gel electrophoresis: SDS PAGE
Blotting: electrical or capillary blotting
Blocking: BSA
Treatment with primary antibody
Treatment with secondary antibody( enzyme labelled anti Ab)
Treatment with specific substrate; if enzyme is alkaline phosphatase,
substrate is p-nitro phenyl phosphate which give color
 Prepared by Mr Dejene G 08/28/2023

57

Western blot test (WB)…

Used as a confirmatory test
 Very specific for HIV
 Samples that give a negative result are reported as negative
 Antibodies to only a selection of viral proteins may yield an indeterminate Western blot
 Bands corresponding to p24 and p55 are detected early in sero conversion followed by glycoprotein
bands

08/28/2023 57
 CREATING WESTERN BLOT STRIPS
Prepared by Mr Dejene G 08/28/2023

58

HIV lysate Proteins are

proteins are transferred
separated by (blotted) onto
size using gel the surface of a
electrophoresis membrane

Strips are
The membrane is incubated with
cut into strips patient serum
and antihuman
IgG conjugated
with an enzyme
(and
chromagen)

Diagram reproduced from Commercial Methods in Clinical Microbiology, ASM Press

HIV WESTERN BLOT BANDING PATTERN
Prepared by Mr Dejene G 08/28/2023

59
env gp160
gp120
gp 41

gag p55
p18
p24

pol p65
p51
p31

Image reproduced from Commercial Methods in Clinical Microbiology. 2000. ASM Press.
WESTERN BLOT BANDING
Prepared by Mr Dejene G 08/28/2023

60

Image reproduced from Commercial Methods in Clinical Microbiology, 2000. ASM Press.
 Western Blot
Prepared by Mr Dejene G 08/28/2023

61
ADVANTAGES

Specific interaction of antibody and antigen can be directly visualized.

Disadvantages
Technically demanding
Expensive
Subject to interpretation
Presence or absence of bands
Intensity of those bands
 Prepared by Mr Dejene G 08/28/2023

62

Southern blotting: principle

 Named in E.M. Southern, who 1st described it in 1975
 Used to detect specific DNA sequences

 Used in clinical lab to detect gene mutations known to cause disease

 Used to detect carriers of the fragile X syndrome and sickle cell anemia for genetic counseling

08/28/2023 62
 Prepared by Mr Dejene G 08/28/2023

63
PROCEDURE/ STEPS

 Restriction digest: by RE enzyme and

amplification by PCR
 Gel electrophoresis: SDS gel
electrophoresis
 Denaturation: Treating with HCl and NaOH
 Blotting
 Baking and Blocking with casein in BSA
 Hybridization using labelled probes
 Visualization by autoradiogram
 Prepared by Mr Dejene G 08/28/2023

64

Northern blotting

Very similar with southern blotting

Used to detect RNA

Not used in clinical laboratory mostly

08/28/2023 64
 Prepared by Mr Dejene G 08/28/2023

65 PROCEDURE

 RNA Isolation
 Separation of RNA using gel
Electrophoresis
 Transfer of RNA to a membrane
 Hybridization and Washing
 Visualization