ANTIGEN ANTIBODY REACTIONS-III

By Dr. Dinesh Jain

 ENZYME IMMUNOASSAY(EIA)
• It is an immunoassay where estimation of enzyme
labelled antibody, antigen or hapten is done
• Enzyme labelled conjugates were first introduced in
1966
• Most widely used procedure in clinical serology due to
their versatility, sensitivity, simplicity, economy and
absence of radiation hazard.
• EIAs are of 2 basic types:
i) Homogenous EIA
ii) Heterogenous EIA
 Homogenous EIA
• Test can be completed in one step with all reagents added
simultaneously
• No need to separate bound and free fractions
• Can be used only for assay of haptens such as drugs and not
for microbial antigens and antibodies.
• Example: Enzyme multiplied immunoassay technique(EMIT)
used for small molecule drugs like opiates, cocaine,
barbiturates or amphetamine in serum.
• Advantages: Simple and one step assay, so can be performed rapidly
• Disadvantage: Microbial antigens and antibodies cannot be
assayed
 Heterogenous EIA
• Multistep procedure with reagents added
sequentially.
• Separation of bound and free fraction required
either by
a)centrifugation or
b)by absorption on solid surfaces and washing

• Enzyme linked immunosorbent assay (ELISA) is major

type of Heterogenous EIA
 Enzyme Linked Immunosorbent Assay (ELISA)

• It is a hetrogenous EIA which relies on an immunosorbent

i.e. an absorbing material that is specific for one of the
components of the reaction, to affect separation of free
and bound antigens.

• Principle:
It is a solid phase immunoassay where one of the reactants (Ag or
Ab) is bound onto a solid phase. The sample in which the Ab or Ag
has to be detected is added. If it is present, immune complex is
formed and free Ab or Ag can be removed by washing. The bound
Ag-Ab complex can be detected using Enzyme labelled Ab or Ag.
 Requirements
– Solid phase: supporting system to fix Ag or Ab. It can be polystyrene,
polyvinyl or polycarbonate microwells, beads or tubes or nitrocellulose
membrane. ELISA is done usually using 96 well microtitre plate

– Antibodies: Enzyme labelled monoclonal antibodies are used because of

high specificity
– Enzymes: some enzymes used are horseradish peroxidase(HRP), alkaline
phosphatase(AP), ß-D- galactosidase, glucose oxidase, carbonic anhydrase,
lysozyme etc.
– Substrate: should be chromogenic, safe and easy to use

Enzyme Substrate
1. Alkaline phosphatasePara-nitrophenol phosphate(PNP)
2. Horse radish peroxidase Diaminobenzidine/Tetramethyl benzidine(TMB)
3. ß-galactosidase Lactose or Lactosyl ceramides

TMB>OPD>ABTS: sensitive response to limiting amounts of peroxidase

– Washing solutions : phosphate buffered saline containing 0.05% tween
20
– Diluent buffers and blocking reagents: PBS/T used as diluent buffer.
Sodium casein , non fat milk(skimmed milk),gelatin, newborn calf,
bovine serum albumin etc. are used as blocking reagent {concentration
1-2%}
– ELISA Reader: a spectrophotometer
 Types of ELISA
1.Direct ELISA
2.Indirect ELISA
3.Sandwich ELISA
1.Direct Sandwich ELISA
2.Indirect Sandwich ELISA
4.Competitive ELISA
5.Modifications of ELISA
1.IgM Capture ELISA
2.Membrane bound or Cassette ELISA
3.ELISPOT assay
4.Biotin avidin linked immunosorbent assay( BALISA)
 Direct ELISA
• This test is used to detect the presence of specific
antibodies in the patient’s serum

• Main problem encountered with this test is that antibodies

for detection of each antigen needs to be linked to an
enzyme which makes the test very cumbersome
 Indirect ELISA
• In this test, to the antigen coated wells ,
patient’s serum containing specific
antibodies which is unlabeled is added
to each well (known as primary
antibody or Ab1)
• Then enzyme linked second antibody or
Ab2 (Antihuman immunoglobulin
antibodies made in rabbit, sheep or
goat) is added.
– This reagent can be used as common
reagent for all human antibodies.
• On addition of substrate color develops
which can be detected by
spectrophotometer.
 Contd…

• The intensity of colour is directly

proportional to the concentration of the
primary antibody in the patient’s serum.

• The problem of labelling antibodies from

each serum sample is circumvented in
indirect ELISA
 Sandwich ELISA

• This test is developed specifically for

the detection of antigen in the
patient’s sample , especially when the
quantity of antigen is expected to be
too low (e.g. hormones and growth
factors)
• Here Ag is sandwiched between 2
antisera raised in 2 different species
against different epitopes of the same
antigen
• The OD measurement of the intensity
of colour quantify the Ag in the test
sample
 Indirect Sandwich ELISA
• Although sandwich ELISA can detect nanogram quantities
of Ag, the second antibodies(Ab2) directed to each Ag need
to be labelled with the enzyme . So it is cumbersome
• To circumvent this problem indirect sandwich ELISA is used
which introduce a 3rd Ab called Ab3, directed against the Ig
of the species of Ab2.
• That is, if Ab1 is a rabbit Ab and Ab2 is a mouse antirabbit
Ig MAb, the enzyme labelled Ab3 against mouse Ig can be
made in goat.
• Ab3 serves as a common reagent for MAbs of any
specificity
 Competitive ELISA
• Steps:
1. Specific Ag to the Ab being detected
is bound to a solid phase
2. Enzyme labelled Ab and the Ab (test
sample) to be assayed are added to
the solid phase Ag and incubate
3. A control well containing just
enzyme labelled Ab is also set up
4. Specific Ab if present in the sample
will compete with the labelled Ab
for the binding site on the Ab/Ag
5. The unbound Ab is washed away
and the substrate for enzyme is
added
6. Addition of substrate does not
produce a colored product in case if
positive sample
 Contd…

• Thus, the amount of hydrolysis of substrate is

inversely proportional to the amount of Ab
present in the sample.

• Advantage : crude or impure samples can be

analyzed for the presence of Ag by this
technique.
 IgM Capture ELISA
• EIA methods for antibodies detect primarily IgG. However,
specific IgM detection methods can be used to differentiate
recent from past infections because only IgM is present early in
the course of most infections. EIA formats for IgM detection
used an enzyme labelled anti-IgM antibody as conjugate.
• Drawbacks of IgM detection by conventional techniques:
1. Presence of excessive IgG may give false negative result for IgM
antibodies.
2. Presence of autoantibodies( rheumatoid factor ) gives false positive
results
• Uses of IgM capture ELISA:
1.Used to differentiate recent from past infections
2.In congenital infections, detection of specific IgM in fetal/neonatal
blood indicates active infection rather than transplacental antibodies.
E.g. cong rubella syndrome
 Cassette ELISA
• Simple modification of ELISA which maybe used for rapid serological
diagnosis of infectious diseases( takes only 10min)
• Each specimen is tested in a separate disposable cassette.
• For validation of test procedure there is inbuilt +ve and –ve controls.
• Principle: It is used to detect antibodies. The specific antigens are
spotted onto a nitrocellulose paper in a cassette. Beneath the
nitrocellulose paper, there is absorbing material. The patient’s serum is
added which is sucked through the nitrocellulose paper by absorbing
material.
If the serum contains antibodies ,they bind to the Ag on the
nitrocellulose paper . Enzyme labelled antiglobulin is added to detect the
bound antibody followed by addition of substrate. Coloured product
develops only on the spot where Ag-Ab complex is formed.
 Contd..

Uses:
1.Used for detection of HIV type 1 and 2 antibodies.
2.Used for direct detection of Group A streptococci in throat
specimen, RSV and influenza in lower respiratory tract
secretions, rotavirus in stool specimen, chlamydia trachomatis
in endocervical samples.
Advantages:
3.Can be used for testing one sera at a time
4.Rapid (takes 10min) as compared with 2-4 hours for micro-ELISA
5.Result can be read visually
6.Has inbuilt controls
7.Does not require washing
 Applications of ELISA
ELISA can be performed to detect the presence of Ag or Ab in a
sample.
• Detection of antibodies:
1.Viral infections: HIV, Influenza, Rubella, Type 2 herpes virus, viral
hepatitis
2.Parasitic infections: Toxoplasmosis, Kala azar(Leishmaniasis)
3.Bacterial infections: Tuberculosis, Chlamydial infections
• Detection of antigens:
1.Viral infections: Detection of rotavirus in stool samples, RSV in lower
respiratory tract specimens, detection of p24 Ag in serum during acute
HIV infection
2.Parasitic infections: Detection of Entamoeba histolytica/ dispar, Giardia
lamblia and Cryptosporidium parvum Ags in stool specimens.
 .

3. Fungal infections: Histoplasma Ag test,

Cryptococcal Ag test
4. Bacterial infections: N. meningitides in CSF,
H.influenza type b, legionella and S. pneumoniae
Ag in urine, Capsular Ag of S. pneumoniae, Shiga like
toxins produced by enterohemorrhagic E.coli,
Clostridium difficile toxins in stool specimens and
Chlamydial Ag in endocervical specimens
Modifications of ELISA maybe used to detect
specific IgM antibodies e.g.. Detection of mumps
specific IgM antibody.
 .

• Other uses:
1.It can be performed in detecting potential food allergens
in the food industry
2.It can also be used in toxicology
3.It can also be used in detection of hormones and enzymes
• Merits of ELISA:
1.ELISA is safe compared to RIA as it has no radioactive
hazard.
2.Can be automated using 96 well plate
3.Highly sensitive
4.Cost effective and accurate method for assessing
hormone levels
 Limitations of ELISA
1.It is a multistep technique-

2.Antiglobulinemic factors and especially

rheumatoid factors which occur in certain
sera may interfere in EIA
3.Specific Ab should have high affinities to
achieve sensitive and specific immunoassays
 CHEMILUMINESCENCE IMMUNOASSAY (CLIA)
• Chemiluminescence refers to a chemical reaction
emitting energy in the form of light.
• It is an extension of ELISA where the chromogenic
substrate is replaced by luxogenic (light emitting)
substrate such as luminol or acridine esters.
HRP conjugated Ab + Ag HRP.Ab.Ag + H 2O2 Development of colour

HRP.Ab.Ag + luminol + H2O2 Generation of light

• The light generated can be quantitatively measured
on a luminometer.
• This test is 10 times more sensitive than a
chromogenic assay
 .

Thank You