6_Immunoassays.pdfImmunoassays unlabeled techniques

1. Immunoassays
Dr. Amany M. Elshamy
Lecturer of Biochemistry and
Molecular Diagnostics

2. Immunoassays
In immunoassays, an Ag binds to
an Ab. The Ag–Ab interactions may
involve unlabeled reactants in less
analytically sensitive techniques or
a labeled reactant in more
sensitive techniques.
The design, label, and detection
system combine to create many
different assays, which enable the
measurement of a wide range of
molecules.

3. • In an immunoassay, an antibody (Ab) molecule recognizes and binds
to an antigen (Ag).
• The molecule of interest may be either an Ag or an Ab.
• Immunoassays types:
A.Unlabeled Immunoassays
B.labeled Immunoassays
Immunoassays

4. Unlabeled Immunoassays
1.Immune Precipitation in Gel
unlabeled Ab was layered on top of unlabeled Ag (both in
the fluid phase); during the incubation period, the Ab and
Ag diffused and the presence of precipitation was recorded.

5. Immune Precipitation in Gel
• Precipitation reactions in gel are not as commonly performed in
the clinical laboratory today.
• Gel is dilute agarose (typically less than 1%) dissolved in an
aqueous buffer. This provides a semisolid medium through
which soluble Ag and Ab can easily pass.

7. Unlabeled Immunoassays
• The precipitation occurred because each Ab recognized an
epitope and the multivalent Ags were cross-linked by multiple
Abs.
• When the Ag–Ab complex is of sufficient size, the interaction
with water is limited so that the complex becomes insoluble and
precipitates

9. • there is an optimal ratio of the concentration of Ab to the
concentration of Ag that results in the maximal
precipitation; this is the zone of equivalence.
• When Ab concentration is in excess and cross-linking is
decreased, the assay is in prozone.
• when Ag concentration is in excess and crosslinking is
decreased, the assay is in postzone.

11. Radial immunodiffusion (RID)
• Radial immunodiffusion (RID) or Mancini method, is an
immune precipitation method used to quantitate protein
(the Ag).
• Immuno-diffusion is a technique for the detection or
measurement of antibodies and antigens by their
precipitation which involves diffusion through a substance
such as agar or gel agarose.

12. Radial immunodiffusion (RID)
• Radial immunodiffusion (RID) is a quantitative
test, and is often used in serology laboratories
to quantify the concentration of a certain
antigen or antibody class in a patient’s serum.

13. Procedure of Radial Immunodiffusion
1.An agar containing an appropriate antiserum (antibody) is poured in plates.
2.Carefully circular wells are cut and removed from the plates.
3.A single or series of standards containing known concentration of antigen are
placed in separate wells, while control and “unknown” samples are placed in
other remaining wells.
4.As the antigen diffuses radially, a ring of precipitate will form in the area of
optimal antigen – antibody concentration.
5.The ring diameters are measured and noted.

15. Antigen–antibody complexes precipitate, forming a ring
( precipitin ring ) around the sample well

16. Radial Immunodiffusion
• The ring size is the equivalence point where the antigens
or antibodies concentration in the sample and antibodies
in the agarose gel are equally proportioned. The
concentration of antigens or antibodies is proportional
to the diameter of the precipitin ring.
• The most common RID in serology is one that measures
IgG, IgA, and IgM concentrations.

17. Results Interpretation
1.1.After incubation, measure the diameter of the precipitin ring of the
calibrators, control and samples.
2.2.Plot the diameter size of the precipitin ring of the calibrators onto a
graph.
3.3.To determine the immunoglobulin concentration in patient serum using
the calibration curve, draw a horizontal line out of the y-axis from the
diameter value of the precipitin ring to reach the calibration curve.
4.4.From the point of intersection, drop a second vertical line to the x-axis.
5.5.Record the antibody concentrations.

19. Applications of Radial Immunodiffusion
•Immuno-diffusion techniques are mostly used
in immunology to determine the quantity or
concentration of an antigen in a sample.
•Estimation of the immunoglobulin classes in sera.
•Estimation of IgG, IgM antibodies in sera to
influenza viruses.

20. Applications of Radial Immunodiffusion
•To determine relative concentrations of antibodies
in serum.
•Estimate serum transferrin and alpha-feroprotein.
•To compare properties of two different antigens.
•Serological surveys

21. Advantages
of
Radial
Immunodiffusion
1.Simple and Cost-effective
2.Precipitation in gels is believed to provide more specific
and sensitive results than other methods available.
3.The reaction is in the form of bands of precipitation and
can be stained for better viewing as well as preservation.
4.If a large number of antigens are present, each antigen-
antibody reaction will give rise to a separate line of
precipitation.

22. Limitations
of
Radial
Immunodiffusion
1.Long reaction time (18-48 hours)
2.It has also been proposed that the results of Mancini’s test is
influenced by the presence bound metal cations in the test
samples (protein).
3.The test has been recently replaced by more sensitive and
automated methods, such as nephelometry and enzyme-linked
immunosorbent assays.

23. Counter immunoelectrophoresis (CIEP)
Counterimmunoelectrophoresis is an immune
precipitation method that uses an electrical
field to cause the Ag and Ab to migrate
toward each other.

24. • Ab is placed in one line and Ag is placed in the other. Ab
will migrate to the cathode and the Ag to the anode; a
precipitin line forms where they meet.
• This qualitative test is useful to detect bacterial antigens
in cerebrospinal fluid and other fluids when a rapid
laboratory response is needed.
Counter immunoelectrophoresis (CIEP)

27. Other methods
• Immunoelectrophoresis (IEP) and
immunofixation electrophoresis (IFE)
are two methods used in the clinical
laboratory to characterize
monoclonal proteins in serum and
urine.

28. Immunoelectrophoresis (IEP)
• Immunoelectrophoresis (IEP) was the first practical method that
combined electrophoresis and -immunoprecipitation for identifying
and characterizing proteins within complex mixtures.
• Evaluate the protein, the heavy chain class and light chain type.
• technique that involves electrophoresis and antigen-antibody
precipitin reaction for proteins.
• For example : multiple myeloma.

32. Others
Turbidimetry measures the light transmitted and
nephelometry measures the light scattered.
Turbidimeters (spectrophotometers or colorimeters)
are designed to measure the light passing through a
solution so the photodetector is placed at an angle
of 180 degrees from the incident light.

33. Qs

34. The RID method depends on:
1.(a)Precipitation of antigens– antibodies complexes.
2.(b)Agglutination of latex particles.
3.(c)Agglutination of RBCs.
4.(d)None of the above.

35. RID requires:
1.(a)Gel electrophoresis.
2.(b)Agarose gel.
3.(c)Polyacrylamide gel.
4.(d)Cellulose acetate membrane.

36. The antigen concentration in RID is:
1.(a)Proportional to the diameter of the precipitin ring .
2.(b)Double the diameter of the precipitin ring.
3.(c)Half the diameter of the precipitin ring.
4.(d)None of the above.

37. The incubation time required for IgM measured by RID is:
1.(a)12 h.
2.(b)24 h.
3.(c)72 h.
4.(d)96 h.

38. The incubation time required for IgG measured by RID is:
1.(a)12 h.
2.(b)24 h.
3.(c)72 h.
4.(d)96 h.

39. 1.RID plates should be:
1. (a)Dried out.
2.(b)Kept in a moist environment.
3.(c)Heated before results measurement.
4.(d)Incubated at 4 °C.
2.6.RID results measurements require:
1. (a)A calibration curve.
2.(b)A plate reader.
3.(c)A microscope.
4.(d)A rotator.