ELISA (Enzyme Linked Immunosorbent Assay)

Type of binding assay that depends on Ag-Ab reaction base and

reaction as marker
as
enzyme
Simple, versatile and highly sensitive test and needs only microlitre
quantities of test reagents
Used for detection of Ags and Abs
Done on a solid phase (absorbing material)
Macro- ELISA
Micro-ELISA
Ifperformed in polystyrene tubes If performed in polyvinyl microtitre
plates

Enzyme
Horseradish peroxidase Specific substrate
Alkaline phosphatase O-phenyl-diamine dihydrochloride
When the substrate acts upon the P-nitrophenyl phosphate
enzyme, it produces a specific color, detected
by spectrophotometer (ELISA reader)

if:te

r ietecid by spect:opnotoneter
(iISAreader)

Types of ELISA
1. Direct ELISA (for Ag detection)
2. Indirect ELISA (for Ab &
Ag detection)
3. Sandwich ELISA (for
Ag detection)
4. Competitive ELISA
(for Ab deteution)
 Direct ELISA Indirect ELISA
For Ab detection For Ag detection
ntigen (test serum) Ag coated well Antigen (test serum)
+ +
Primary Ab (Ab to AAg) Serum (Ab to be detected) Primary Ab
labelled with enzyme +

Secondary Ab labelled with Secondary Ab lablled with

Substrate enzyme enzyme
+ +
Color detection Substrate Substrate

Color detection Color detection

Direct ELISA I n d i r e t FLISA
(FOr antigen deteclion) (orlg deler.r
Indireci ELISA
drtrcien)
ntldy
(
Step 1

Patient Serurn
(antigeri) L Patienl e
(anten

Step 2

.
Step 3

Step 4

CoMGUr (Posilive) Colour (Positive)

Colour (Positive)

Note : At every step of ELISA test, incubation and washing is to be done to wash off
unbound reagents
 Sandwich ELISA
Ab coated well Competitive ELISA
Competition b/w 2 antibodies (one
Serum (Ag)
enzyme & other
present in serum -
conjugated with
if +ve for
+ same Ag
Abs) for
Primary Ab (Conjugated) Ag coated well
+ +

Test serum (if having Abs)

Substrate
wash
Add enzyme labelled HIV
Color detection specific Abs
wash
Substrate added
No color seen Positive
Color seen Negative (i.e. No Abs in test
serum, so
enzyme labelled HIV specific Abs got bound to
Ag,
enzyme acts on substrate to produce color)
Sandwich ELISA
For antion toection) Compctitive ELISA
antithdy detrctiot)

Step

Step 2

Conjugate is washed
Step 3
oui as antigen 5
not free to bna the
Conjugate

Step 4 L Substratenzyme(tagged
to conjujate) is
not there to act On
Colour (Positive) SLubstrate
No Colour (Positive)
 test/POCT

es

ateral flow through assay eg. Immunochrom atographic test

Fow through assay eg. TRI-DOT assay
(ICT)

aral flow through assay / Immunochrmaography

Combination of chromatography and immunoassay
.Depending on the assay, either antigen (for detection of antibody) or antibodies (for
detection of antigens) are immobilized on a nitrocellulose membrane in discreet spots in
a cartridge device.
Strip contains a chromatographic pad with 3 zones Sample pad, Coniugate pad,

Capture line
Conjugate pad has : Colloidal gold/ dye /latex beadsas conjugate which produces
signal
When the liquid sample is dropped on the sample pad, it flows laterally by capillary action,

antigen in the sample forms an immune complex with the Ab labelled with the colloidal gold.

This Ag-Colloidal gold labeled Ab complex moves along and forms complex with the Ab(on the
est line) immobilized on the nitrocellulose membran2, resulting in generation of red / purple
co'ored ine (indicates the presence-of Ag of interest in the sample)

Positive control line: to check ifthe test was properly performed. The free conjugate (colloidal
Gold labeled Ab) moves further and binds to Anti-Human Immunoglobulin to form a color
control line

Saniple

FOSITIVE
SAMPLE

Centrol
Test line positive Capture
Sampe pao Conjugate pad line zore ine

Sample

EGATIVE
SAIAPLEE

Sampie pad Conjugate pad Test line Capture line Control

negative Zone line

Artigen Conjugate jantibody to antigen (&) Antibody to antigen () Antibody to antibody(

Fig 23.10 hmmunochronatography stip for antigen detection in sample
 te
id test/POCT
nciples
ateral flow through assay eg.
2. Flow through assay eg.
Immunochrom stographic test (ICT)
TRI-DOT assay
ateral flow through assay /
Immunochrmaography
.Combination of chromatography and immunoassay
Depending on the assay, either antigen (for detection of
detection of antigens)
antibody) or antibodies (for
are immobilized on a nitrocellulose membrane in discreet spots in
a cartridge device.
Strip contains a chromatographic pad with 3 zones :Sample pad, Coniugate pad,
Capture line
Conjugate pad has: Colloidal gold/ dye/ latex beads as conjugate which produces
signal
When the liquid sample is dropped the sample pad, it flows
on
laterally by capillary action,
antigen in the sample forms an immune complex with the Ab labelled with the colloidal gold.

This Ag-Colloidal gold labeled Ab complex moves along and forms complex with the Ab(on the
test line) immobilized on the nitrocellulose membran2, resulting in generation of red/ purple
co'ored line (indicates the presence-of Ag of interest in the sample)

Positive control line: to check if the test was properly performed. The free conjugate (colloidal
Gold labeled Ab) moves further and binds to Anti-Human Immunoglobulin to form a color
control line
Sample

POSITIVE

Test line positive Capture Control

Sampit pao Conjugate pad line zore line

Sample

NEGATIVE
SAIMPLE

Sampie ped Conjugate pad Test line Capture line Control

negative Zone ine

Antgen Conjugate jantibody to antigen ()) Antibody to antigen (4) Antibody to antibody
Fig 23.10 Immunochromatography strip for antigen detection in sample
 te

Ough Assay
iffers from ICT:
oProteinA used to label Ab instead of colloidal gold

a Specimen flows vertically through the nitrocellulose membrane instead

of Lateral
flow in ICT

Can detect Ag or Ab
Test present in a cassette format, consists of Nitrocellulose membrane and absorbent

pad
o Eg. HIv-TRI DOT (detects Abs against HIV-1& 2 separately in pt's serum)
.Nitrocellulose membrane has 3 regions :2testregions for HIV-1&2 Ags, a control

region(coated withAnti-human lg
Specimen and buffer agents added sequentially.
Serum passes through nitrocellulose membrane, if HIV Abs +nt, bind to HIV Ags
Test dots Control dots
Protein A conjugate (present Control region is coated with Antihuman Ig. Protein A can bind
in buffer) binds to Fc portion to any IgG present in serum and this lgG-Protein A complex can
of HIV Abs to give pinkish bind to Anti-human lg present in control region to give a
purple DOT(s) for HIV1 & pinkish purple DOT
HIV2 separately NOTE for validity of a test, control dot must always be
present irrespective of whether HIV Abs in serum are present
or not
- -

Protein A
Contro Conjugate

Psitive HIV antigen

HIV
antibody
Nitrocellulose
membrane
(a) (b)

Fig 23.11 fa) HIV-TRI DOTtest (b) Diagramatic representation