Good information about immunoassay technique

1. Radioimmunoassay

2. • Radioimmunoassay (RIA) involves the
separation of a protein (from a mixture) using
the specificity of antibody - antigen binding
using radioactivity.

3. • The technique of radioimmunoassay has
revolutionized research and clinical
practice in many areas, e.g.,
–blood banking
–diagnosis of allergies
–endocrinology

4. • The technique was introduced in 1960 by
Berson and Yalow as an assay for the
concentration of insulin in plasma.
• It represented the first time that hormone
levels in the blood could be detected by an in
vitro assay.

5. Principle
• Based on competition between unlabelled
antigen and finite amount of corresponding
labeled antigen for a limited number of
antibody binding sites in a fixed amount of
antiserum.
• At equilibrium in the presence of an antigen
excess there will be both free antigen bound
to antibody.

6. • Under standard conditions the amount of
labelled antigen bound to the antibody will
decrease as the amount of unlabelled antigen
in the sample increases.

7. • At increasing concentrations of unlabeled
antigen, an increasing amount of radioactive
antigen is displaced from the antibody
molecules.
• The antibody-bound antigen is separated from
the free antigen in the supernatant fluid, and
the radioactivity of each is measured.

9. Gamma Counter

10. Applications of RIA
• In medicine, it is especially useful in diagnosing
autoimmune diseases such as Hashimotos thyroiditis
and systemic lupus erythematosus.
• RIA has many uses, including narcotics detection,
early cancer detection, measurment of growth
hormone levels and diagnosis and treatment of
peptic ulcers.

11. • The main drawbacks to radioimmunoassay are the
expense and hazards if preparing and handling the
radioactive antigen.
• Both 125I or 131I emit gamma radiation that requires
special counting equipment;
• The body concentrates iodine atoms — radioactive
or not — in the thyroid gland where they are
incorporated in thyroxine (T4).

12. • Despite these drawbacks, RIA has become a major
tool in the clinical laboratory where it is used to
assay
• plasma levels of:
– most of our hormones;
– digitoxin or digoxin in patients receiving these drugs;
– certain abused drugs
• for the presence of hepatitis B surface antigen
(HBsAg) in donated blood;
• anti-DNA antibodies in systemic lupus erythematosus
(SLE).

13. Immunoprecipitation
• The multivalency of antibodies has allowed
the development of techniques in which
antibody-bound molecules can be precipitated
from solution

14. Principle
• Immunoprecipitation (IP) is the technique
of precipitating a protein antigen out of
solution using an antibody that specifically
binds to that particular protein. This process
can be used to isolate and concentrate a
particular protein from a sample containing
many thousands of different proteins.

15. Immunoprecipitation Can Be
Performed in Solution
• When antibodies and soluble antigen are
mixed in solution, the bi- or multivalent
nature of immunoglobulins allows for a single
antibody molecule to bind to more than one
antigen.
• If the antigen is polyvalent (has more than one
antibody binding site per antigen molecule), it
may in its turn bind multiple different
antibodies.

17. Immunoprecipitation of Soluble Antigens Can
Be Performed in Gel Matrices
• Immune precipitates can form not only in solution
but also in an agar matrix.
• When antigen and antibody diff use toward one
another in a gel matrix, a visible line of precipitation
will form.
• As in a precipitation reaction in solution, visible
precipitation occurs when the concentrations of
antibody and antigen are equivalent to one another.

19. • In the Ouchterlony method, the most frequently employed
variation of gel immuno precipitation, both antigen and
antibody diff use radially from wells toward each other,
thereby establishing a concentration gradient.
• At the relative antibody-antigen concentrations at which
lattice formation is maximized, termed “equivalence,” a visible
line of precipitation, or “precipitin line,” forms in the gel.
• Ouchterlony assays are still occasionally used in the clinic.
presents a comparison of the sensitivity, or minimum amount
of antibody detectable, of a number of immunoassays.

20. ELISA
[Enzyme Linked Immuno-Sorbent Assay]

21. Principle
• ELISA assays are similar in principle to RIAs but, instead
of using antibodies or antigens conjugated to radioisotopes,
they use antibodies or antigens covalently bound to enzymes.
• The conjugated enzymes are selected on the basis of their
ability to catalyze the conversion of a substrate into a colored,
fluorescent, or chemiluminescent product.

22. Types of ELISA
• A number of variations of the basic ELISA assay
have been developed.
• Each type of ELISA can be used qualitatively to
detect the presence of antibody or antigen.
• Alternatively, a standard curve based on known
concentrations of antibody or antigen can be prepared
and used to determine the concentration of a sample.

23. Indirect ELISA
• Antibody can be detected, or its concentration
determined with an indirect ELISA assay.
• Serum or some other sample containing primary
antibody (Ab1) is added to an antigen-coated
microtiter well and allowed to react with the antigen
attached to the well.

24. Applications
• This version of ELISA is the method of choice to
detect the presence of serum antibodies against
human immunodefi ciency virus (HIV), the causative
agent of AIDS.
• In this assay, recombinant envelope and core proteins
of HIV are adsorbed as solid-phase antigens to
microtiter wells.
• Individuals infected with HIV will produce serum
antibodies to epitopes on these viral proteins.
• Generally, serum antibodies to HIV can be detected
by ELISA within 6 weeks of infection.

25. Sandwich ELISA
• Antigen can be detected or measured by a sandwich
ELISA.
• In this technique, the antibody (rather than the
antigen) is immobilized on a microtiter well.
• A sample containing unknown amounts of antigen is
allowed to react with the immobilized antibody. Aft er
the well is washed, a second enzyme-linked antibody
specific c for a diff erent epitope on the antigen is
added and allowed to react with the bound antigen.
• Aft er any free second antibody is removed by washing,
substrate is added, and the colored reaction product is
measured.

26. • Sandwich ELISAs have proven particularly useful for the
measurement of soluble cytokine concentrations in
tissue culture supernatants, as well as in serum and
body fluids.
• Note that, for this assay to work, the two antibodies
used for the antigen immobilization (capture) and
detection phases respectively must bind to different
determinants (epitopes) on the antigen.
• Sandwich ELISAs therefore routinely use a pair of
monoclonal antibodies specific for different regions on
the antigen.

27. Competitive ELISA
• The competitive ELISA provides another extremely sensitive
variation for measuring amounts of antigen.
• In this technique, 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.
• 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.

29. Agglutination Reactions
• The cross-linking that occurs between di- or
multivalent antibodies and multivalent, bacterial, or
other cellular antigens can result in visible clumping of
the complexes formed between cells bearing the
antigens and the antibody molecules.
• This clumping reaction is called agglutination, and
antibodies that produce such reactions are called
agglutinins.
• Agglutination reactions are identical in principle to
precipitation reactions; the only diff erence is that the
cross linked product is visible to the naked eye because
of the larger size of the antigens.

31. Complement Fixation Test
• The complement fixation test is one of the major
traditional tests for the demonstration of
presence of specific antigens or antibodies.
• First step (Complement fixation stage): a known
antigen and inactivated patient’s serum are
incubated with a standardized, limited amount of
complement.
• Second step (Indicator Stage): The second step
detects whether complement has been utilized in
the first step or not. This is done by adding the
indicator system.

34. Applications
• Ability to screen against a large number of viral
and bacterial infections at the same time.
• Economical
• Disadvantages of Complement Fixation Test
• Not sensitive – cannot be used for immunity
screening
• Time consuming and labor intensive
• Often non-specific e.g. cross-reactivity between
HSV and VZV

35. Immunodiffusion
• When the antigen and antibody are allowed to meet each
other in a gel like agar or agarose, they will precipitate in
the form of a line. This line is called precipitation line or
precipitin line.
• Normally diffusion is performed in 1% agar or agarose.
• There are four types of immuno diffusion in gel. They are:
• 1. single diffusion in one dimension (old in technique)
• 2. single diffusion in two dimensions (Mancini technique)
• 3. double diffusion in one dimension (Oakly-Fulthorpe
technique)
• 4. double diffusion in two dimensions (Ouchterlony
technique)

36. Single diffusion in one dimension
(Oudin technique)
• In 1952, Oudin introduced the technique in which
the antibody is mixed in molten agar and allowed
to set in a test tube.
• After the agar solidifies, the anitgen solution is
overlaid on the agar gel.
• The antigen solution will diffuse into the agar gel
and when the concentration reaches the region
of equivalent zone, a precipitation ring is formed
in the tube. It is called single diffusion test
because only antigen is allowed to diffuse in the
gel.

37. Applications
• It is used to find unknown antigen.
• It is used to find the approximate concentration of antigen as
the thickness of the precipitation line.
• It is also used to find the number of antigenic components
present in the solution against the polyclonal antiserum.

45. Single Diffusion in Two Dimension
(Mancini Technique)
• This technique is also called as radial immuno
diffusion (RID) or Single radial immuno
diffusion ( SRID).
• This technique was first introduced in 1965 by
Mancini and his co-workers to estimate the
concentration of serum proteins.
• In this technique, only the antigen is allowed
to diffuse in the gel that has been
incorporated by the antibody.

46. Applications
• This technique is routinely used in hospitals
and laboratories to measure the concentration
of IgG, IgM and IgA in a patient serum.

47. Double diffusion in one dimension
(Oakley-Fulthorpe Technique)
• This technique is rarely used nowadays.
• It is similar to Oudin tube method.
• However, plain agar is overlaid on the
antibody incorporated agar.
• Thus the both antigen and the antibody are
allowed to diffuse into the plain agar ‘(double
diffusion).

48. Double diffusion in two dimensions
(Ouchterlony Technique)
• It is one of the widely used immunological
techniques.
• In this technique, plain agar is overlaid on a
glass slide.
• Small wells are punched in the gel.
• The antigen and antibody are allowed to
diffuse towards each other. When they meet
each other at the equivalent zone, they will
form a precipitation line on the gel.

49. Applications
• To find the identity of two given antigens
against the polyclonal antiserum.
• To find the titre of the antiserum