Since antigen and antibody reactions are specific, they can be used to identify each other. These diagnostic tests are particularly useful in diagnosing for examples: infectious diseases, autoimmune diseases, and in typing of blood and tissues prior to transplantation.

1. Medical Microbiology
(Serology)

2. Antigen-Antibody Reactions in the Laboratory
• Outline:
• Introduction.
• Agglutination techniques.
• Precipitation techniques.
• Immunostaining of tissues.
• Antibody assays based on molecule binding to solid-phase supports.

3. • Since antigen and antibody
reactions are specific, they
can be used to identify each
other.
• These diagnostic tests are
particularly useful in
diagnosing for examples:
infectious diseases,
autoimmune diseases, and
in typing of blood and
tissues prior to
transplantation.

5. • In serological tests, it is useful to measure the antibody titer, that is, the
highest dilution (lowest concentration) of serum that yields positive reaction.
• In most cases, the mere presence of Ab to a specific pathogen doesn’t indicate
active, ongoing infection.
• To diagnose an ongoing infection serologically, we can either: 1- demonstrate
seroconversion, 2- demonstrate the presence of IgM, or, 3- demonstrate a 4-fold
or more rise in total Ab titer.
• In addition to being retrospective, serology also suffers from cross-reactivity.

6. Agglutination Reactions
• Di or multivalent Abs can cross-link bacterial or other cellular (particulate)
Ags, forming clumping “agglutination”.
• These reactions, mediated by agglutinins, can be seen by the naked eye.
• Direct agglutination occurs when the Ag is naturally found on surface of a cell
or other insoluble particle, eg: ABO grouping (hemagglutination), and Widal
test.

10. • In passive agglutination, Ags or Abs have been adsorbed to cells, or insoluble particles
such as latex beads, charcoal particles, and RBCs, e.g. VDRL, Anti-streptolysin O (ASO).

12. Precipitation Reactions
• The precipitation technique requires the formation of a visible lattice that
results from the combination of soluble antigen to its soluble antibody.
• It results from interaction of a soluble Ab (typically di or multivalent) with a
soluble Ag forming insoluble immune complex.
• It occurs maximally only when there are optimal proportions of the two
reacting substances (see the precipitation curve).
• These reactions can be accomplished in liquid-phase, eg: Lancefield grouping of
β-hemolytic streptococci.
• Or, it can be performed in agarose gels, called immunodiffusion tests, resulting
in formation of precipitin line.
• When coupled with electrophoresis, more rapid techniques, such as
immunoelectrophoresis and counter immune electrophoresis (CIE), yield results
in 30-60 mins.

18. Immunostaining of Cells and Tissues
• Cells and tissue Ags can be detected in tissue sections using enzyme-labeled Abs
( as in Immunohistochemistry or IHC).
• Fluorescent-labeled Abs (as in immunofluorescence or IF) can be used to detect
Abs in serum or Ags on cells and are visualized using a fluorescent microscope.

22. Fluorescence microscopy can be used to quantify the percentage
of infected cells. Green: Zika virus infected cells; Blue: cell nuclei.

24. Antibody Assays on Solid-phase
• Ags or Abs in these techniques are bound to solid-phase supports such as
microtiter plates, microscope slides, or beads of different kinds.
• Radioimmunoassays (RIAs) are used to measure the concentration of
biologically relevant proteins and hormones in body fluids, it was invented by
Yalo in 1960.
• Ags or Abs are bound to solid-phase, and one of them is labeled radioactively
(125I).
• Enzyme-linked immunosorbent assay (ELISA or EIA) uses Abs or Ags covalently
linked to enzymes, and these enzymes react with chromogenic, fluorogenic, or
chemiluminescent substrate.

25. Enzyme Immunoassay (EIAs) and Enzyme-
Linked Immunosorbent Assay (ELISA)
• There are four major principles for ELISA methods: direct ELISA, indirect
ELISA, sandwich ELISA and competitive ELISA.
• In general, all ELISAs are quantitative tests to determine the concentration of
unknown specific antibodies or antigens.

26. • Direct ELISA requires the immobilization of
patient antigens on a microplate well, and
complementary antibodies to the patient
antigens.
• Complementary antibodies are conjugated to an
enzyme that reacts with a substrate.
• Its simple, with less time than others.
• Low sensitivity.

27. • Indirect ELISAs require the immobilization of
patient antigens on the surface of microplate
well.
• 1. Primary antibodies, which are complementary
and specific to the patient antigen, and form
complexes with the patient antigen on the
surface of the microplate well.
• 2. Secondary antibodies are specific to the Fc
region of the primary antibodies and conjugated
to an enzyme that reacts with a substrate to
produce detectable products.
• More sensitive.

28. • Specific antibodies (called capture antibodies or
complementary antibodies) to the antigen of
interest are adsorbed to the microplate well.
• Therefore, the sandwich ELISA overcomes the
issue of reduced sensitivity that is associated
with both direct and indirect ELISA owing to
various proteins that could be adsorbed onto the
microplate well.

29. • This method depends on the competition
between two antigens for binding on limited
available antibodies site.
• The first antigen is the patient antigen, while
the second one is usually similar to that
antigen in patient serum but labeled with
biotin or directly labeled with an enzyme.
• Here, the antigens compete for the same
binding sites on the antibodies, and the
number of antigens in the sample is inversely
proportional to the level of signals
generated.

30. Immunochromatography tests (ICTs), Rapid diagnostic
tests (RDTs)
In vitro Ag.Ab RXN, which done on nitro cellulose paper in which conjugated molecular
with chromogen (colloidal gold, HRP) that interact correspondingly to molecular in the
patient sample resulting in appearance of red-pink color or band in the test area in
addition to other red band in the control area indicating positive results.
• ICT may detect Ags (malaria-HRP2) or Ab (lieshmania-anti-k39)
• ICT device:
• Cassette.
• Nitro-cellulose paper.
• Sample well.
• Buffer well.
• Test area.
• Control area.
• Absorbed pad.

31. • The labeling and detection zones
are set between the two ends.
• As the sample is loaded, it
reconstitutes the labeled Ag or Ab
and the two form a complex that
migrates toward the detection
zones.
• An Ag or Ab immobilized in the
detection zone captures the
immune complex and forms a
colored line for a positive test.

32. Interpretation

33. Complement Fixation Test
• Patient serum usually contains complements; therefore, the patient samples
should be pre-heated (56C) to destroy all the complements without affecting
the antibodies and the antigens in the serum.
• Pre-heated serum is mixed with standard antigens and complements.
• Patient antibodies opsonize the standard antigens, and complements become
fixed on the surface of the opsonizing antibodies.
• Complement remains unfixed when antibodies are absent from patient serum.
• Sheep red blood cells (sRBCs) sensitized with specific antibodies to sRBCs are
used as an indicator system to detect any residual of the unfixed complement
remaining in the reaction.
• Positive: no hemolysis.
• Negative: hemolysis.

34. • Sheep red blood cells (sRBCs) sensitized
with specific antibodies to sRBCs are used
as an indicator system to detect any
residual of the unfixed complement
remaining in the reaction.

35. Immunoblotting
• A technique used to detect the presence of a specific protein in a
complex protein mixture.
• Used in cell biology, molecular biology, virology.
• Three types: Western blot (WB), Southern blot (SB) and
Northern blot (NB).
• The WB is the most common analytical tools for the detection
of viral proteins, confirmatory test of HIV.

37. Alyazeed Hussein, BSc, SUST
This has been a presentation of Alyazeed Hussein
Thanks for your attention and kind patience
@elyazeed7
@Alyazeed7ussein