serology presentation Serology is the scientific study of blood serum and other bodily fluids such as semen and saliva. In practical immunological terms, serology is the diagnostic identification of antibodies in the serum. Antibodies are typically formed in response to; An infection, (against a given microorganism), Other foreign proteins (blood transfusion) Or to one’s own proteins (autoimmune disease).

1. DIAGNOSTIC
IMMUNOLOGY
Serology
AGGLUTINATION and PRECIPITATION TESTS
Abdirahman Jibril Warsame
Mcs, Microbiology and clin immunology

2. WHAT IS SEROLOGY?
• Serology is the scientific study of blood serum and other
bodily fluids such as semen and saliva.
• In practical immunological terms, serology is the diagnostic
identification of antibodies in the serum.
Antibodies are typically formed in response to;
• An infection, (against a given microorganism),
• Other foreign proteins (blood transfusion)
• Or to one’s own proteins (autoimmune disease).

3. Affinity, Avidity and Cross Reactivity

4. Serum, Plasma and Antiserum

5. How do you get Antiserum

6. Precipitation reactions
• Precipitation reactions are serological assays for the
detection of immunoglobulin levels from the serum of a
patient with infection.
• Antibodies when mixed soluble antigens in equal
proportions
• Resulting in insoluble lattice or immune complex formation.
• Precipitin: Any antibody which reacts with an antigen to
form a precipitate.

7. Conditions necessary for precipitation
reactions
• Antibody must have at least two antigen binding sites
(bivalent).
• Antigen must be soluble, either bivalent or multivalent.
• The proportions of the antigens and the antibodies must be
equal.
• The reaction occurs in the presence of a electrolyte at
suitable temperature and PH.
• Precipitation can take place in liquid media or In gels.

8. How the relative proportions of antigen and
antibodies influence the formation of precipitate?

9. How the relative proportions of antigen and
antibodies influence the formation of precipitate?

10. Three test sample results
• Test sample 1
• Antibodies are in excess
• No cross-linkages
• No lattice formation
• Test Sample 2
• Antibodies and antigen are in equal proportion
• Cross-linking occur
• Lattice formation takes place
• Insoluble visible precipitate
• Test sample 3
• Antigen are in excess
• No cross-linkages
• No lattices formation

11. Precipitation curve
• Graphic representation of precipitation reactions.
• Concentration of one is kept constant
• Concentration of second reactant is increased serially.

12. Immunotechniques Based on
Precipitation Reactions
Precipitation methods include;
Radial immunodiffusion (semi-quantitation of proteins
by gel diffusion using antibody incorporated in agar)
• Double immunodiffusion (qualitative gel technique
that determines the relationship between antigen and
antibody)
• Electroimmunodiffusion (variation of the double
immunodiffusion method reaction that uses an electric
current to enhance the mobility of the reactants
toward each other).

13. Radial immunodiffusion
•Radial immunodiffusion (RID) is also known as
Mancini immunodiffusion or single radial immunodiffusion
assay. It is a single diffusion technique whereby
a solution containing the antigen is placed into wells in a gel
or agar surface evenly impregnated with antibody.
•The diameter of the ring that precipitates around the well as
a result of antigen antibody reaction corresponds to the
amount of antigen in the solution

14. Materials Required
• An antigen solution of unknown concentration (X)
• A solution containing antibody molecules against the antigen X
• three antigen standard solutions (known concentration).The
concentration increasing in order, means first containing
minimum and third containing maximum antigen concentration
• Agar slides

15. Procedure of Radial Immunodiffusion
• Firstly solution containing antibody molecules is added to the
molten agar
• The mixture is then poured onto a glass to set. wells are cut out
in the agar.
• First three wells standard antigen solution is added and then
fourth well is added antigen solution of unknown concentration.
• Slide is left for 24/72 hrs, during that time antigens diffuse out
of their wells in all directions.
• The precipitation reaction is visible as precipitation ring
surrounding the wells.
• More the antigen concentration, wider the ring

16. Result Interpretation of Radial
Immunodiffusion

17. 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.

18. Double immunodiffusion (Ouchterlony
Double Diffusion
• Ouchterlony double immunodiffusion is an
immunological technique used in the detection,
identification and quantification of antibodies and
antigens.
• In this method, both the antigen and antibody diffuse
independently through agar gel in two dimensions,
horizontally and vertically.

19. Materials Required
• Glass plate or Petri plate
• Agarose
• Gel borer
• Buffer
• Antiserum
• Antigen solutions

20. Procedure of double
immunodiffusion
• In the immune diffusion both antibody and antigen diffuse
radially from the wells towards each other and at the zone
of equivalence visible line of precipitation is formed. This
technique is mainly used for the comparison of the
antigens.
• The pattern of precipitation line in the double immune
diffusion technique tells whether the given antigens are
• Identical
• Partially identical
• Non identical

21. given two identical antigens
• Two identical antigens and one antibody are added
respectively in well. two precipitation lines will be formed
and they will fuse Because the antigens are identical and
antibody can not distinguish between them. In result are an
arch shaped precipitation band. This precipitation line is
known as pattern of identity.

22. non identical antigens
• Add two non identical antigens in antigen well A
and B. in the antibody well add two types of
antibodies each specific to the antigen A and B.
• two precipitation line well be formed but they will
not fuse they cross each other without any
interaction because each antigen has its own
antibody.
• This pattern is known
as pattern of non identity.

23. two antigens are partially identical
• In Case three two antigens are partially identical they share one
or more common epitopes.
• For example antigens AX and BX.
• antibody specific antigen AX will react it and precipitation line
will be formed at the zone of equivalence but antigen BX also
react with antibody X and cross reaction will occur.
• This pattern is known as partial of identity.

24. Uses of double immunodiffusion test
• To detect antigen-antibody complexes.
• Describe the circumstances under which
antigen-antibody complexes precipitate out.
• Detect the presence of an antigen-specific
antibody.
• To test the similarity between antigens.

25. DIRECT AGGLUTINATION
• These reactions
can be performed
on slides (rapid
tests) or on
microliter plates
or tubes for
Antibody titration
if required.

26. Positive Negative
Ag-Ab complex
Direct agglutination
Principle
• combination of an insoluble
particulate antigen with its soluble
antibody
– forms antigen-antibody
complex
– particles clump/agglutinate
• used for antigen detection
Examples
– bacterial agglutination tests
for sero-typing and sero-
grouping e.g., Vibrio cholerae,
Salmonella spp

27. Glass Agglutination Test

28. Tube Agglutination Test
• Also known as the standard agglutination test or
serum agglutination test (SAT)
• Test serum is diluted in a series of tubes (doubling
dilutions)
• Constant defined amount of antigen is then added to
each tube and tubes incubated for ~20h @37°C
• Particular antigen clumps at the bottom of the test
tube
• Test is read at 50% agglutination
• Quantitative
• Widal Testing

29. Tube Agglutination Test

30. No agglutinationAgglutination
1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl
In this case, the titre is 1/40
Tube Agglutination Test

31. Passive Agglutination
• An agglutination reaction that employs
particles that are coated with antigens or
antibody not normally found in the cell
surfaces
• Particle carriers include:
– Red blood cells
– Polystyrene latex

32. Haemagglutination
RBC

33. Viral Haemagglutination
• Some viruses contain proteins which bind to
erythrocytes (red blood cells) causing them to
clump together

34. Viral Hemagglutination
• the attachment of viral particles by their receptor sites to
more than 1 cell.
• As more and more cells become attached in this manner
agglutination becomes visible

35. Readings The results
• Titer: The maximum dilution that gives visible
agglutination.
• The end point: is the well with the lowest
concentration of the virus where there is
haemagglutination
2 4 8 16 32 64 128 256 512 1024 2048 4096
The HA titer of this virus in this row is 256 or 28
(1:256 dilution contains (1 HA unit) (one
haemagglutinating unit)

36. Titer = 32 HA units/ml
Hemagglutination test: method
1:8
1:2 1:21:21:21:2
8 16 32 64 128 256
virus
serial dilution
mix with red
blood cells
side view
top view
One HA unit :minimum amount of virus that causes
complete agglutination of RBCs

37. What is Antibody Titer
• Is the lowest
concentration
of antibodies
against a
particular
antigen.
Figure 18.6

38. Latex Agglutination
• Antibody molecules can be bound to each
latex beads
• It will increase the potential number of
exposed antigen-binding sites.
• When an antigen is present in test specimen,
it may bind to the latex bead thus forming
visible cross-linked aggregates.
• Latex particles can be coated with antigen
(pregnancy testing, rubella antibody testing)

39. Thank you