This document provides information about the course CODE 320 on Virology & Parasitology. It discusses several diagnostic techniques used to detect viruses including hemagglutination, hemagglutination inhibition, complement fixation, immunodiffusion, and immuno-electrophoresis tests. The techniques detect viruses or antibodies in serum by mechanisms such as agglutination, precipitation, or complement binding. The document also provides examples of viruses detected by these methods and includes sample multiple choice questions.

1. COURSE CODE: 320
Pre-requisite: 301
Ms. ZAINAB MASOOD
SEMESTER: 6
VIROLOGY & PARASITOLOGY
REFERENCE TEXT BOOK:
Howley, P.M., Roizman , B., Straus, S.E., Martin , M.A., Griffin, D.E., 2001.
Fundamental Virology, Lippincott Williams & Wilkins.

2. Principles of viral diagnostic
procedures

3. Quantitative Micro
Hemagglutination Test (HA)
12/16/2023
M Asif Shaheen
Haemagglutination Test (HA)

4. Agglutination

5. Contents:
 Hemagglutination test
 Hemagglutination Inhibition
 Complement fixation test
 Immunodiffusion
 Immunoelectrophoresis

6. Haemagglutination
 Some viruses and microbes contain proteins which bind to erythrocytes (red blood
cells) causing them to clump together

7. Examples
NDV ( Newcastle Disease Virus)
Adenovirus III
AIV (Avian influenza Virus)
Mycoplasma

9. 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
 Titer dilution contains (1 HA unit) (one haemagglutinating
unit)

10. In the absence of anti-virus antibodies
Erythrocytes
Virus
Virus agglutination of
erythrocytes

11. Haemagglutination Inhibition Test
(HAI)

12. Viral Haemagglutination Inhibition
Tests
 The presence of virus can be detected by hemagglutination
 The identity of the virus or of antibodies in a patient’s serum
can be determined by specific inhibition of that
hemagglutination

13. Haemagglutination Inhibition
Tests

14. In the presence of anti-virus antibodies
Erythrocytes
Virus Anti-virus
antibodies
Viruses unable to bind to
the erythrocytes

15. Significance
 Useful for viruses that are not cytopathic
 Based on viral hemagglutination
 Some viral surface proteins can clump red blood cells
 A serum sample that contains antibodies against a specific virus will
inhibit viral Haemagglutination
 Commonly used to detect antibodies against influenza, measles, and
mumps

16. Complement fixation test

17. Complement system
 A part of the immune system that enhances (complements)
• the ability of antibodies and phagocytic cells to clear microbes and damaged cells
from an organism
• promotes inflammation
• attacks the pathogen's plasma membrane.
or
 A system of plasma proteins
 can be activated directly by pathogens or indirectly by pathogen-bound antibody,
leading to a cascade of reactions that occurs on the surface of pathogens and
generates active components with various effector functions.

19. Complement fixation test
 Complex assay used to detect the presence of specific antibodies in
an individual's serum.
 The test can detect the presence of small amounts of antibody---
amounts too small to detect by agglutination
 Principle
It is the nature of the complement to be activated when there is formation
of antigen-antibody complex.

21. Results
 In the positive test : The available complement is fixed by Ag-Ab
complex and no haemolysis of sheep RBCs occurs. So the test is
positive for presence of antibodies.
 In the negative test : No Ag-Ab reaction occurs and the complement is
free. This free complement binds to the complex of sheep RBC and it’s
antibody to cause haemolysis, causing the development of pink colour.

22.  Examples
 Newcastle disease virus. (NDV)
 Rubella virus
 Avian leukosis virus

23. Immunodiffusion
 Definition
 It is a simple and direct means of demonstrating the Ag-Ab by the
precipitation reaction (Ag and Ab are soluble)
o Radial Immunodiffusion (Mancini method).
o Double Immunodiffusion

24. Radial immunodiffusion assay
 also known as the Mancini method
 A protein assay that detects the amount of specific viral antigen
 The medium contains antiserum specific to the antigen of interest and the antigen
is placed in the well cut into agarose gel
 The Ags (in the serum) diffuses radially and produces a ring of precipitation.
 The area enclosed by the ring is proportional to the conc. of the Ag in the serum

26. Double Immunodiffusion
 In double I.D both reactants are free to move towards each
other and Precipitate
 Movement in either form may be linear or radial

27. The density of the line reflects the amount of immune complexes formed

28. Immuno-electrophoresis
 Double-diffusion technique that utilizes electric current to enhance results.
 Immunoelectrophoresis is a qualitative method that combines
electrophoresis and immunoprecipitation.
Electrophoresis separates molecules according to differences in their electrical charge
and their sizes.

29. Principle
Antigen from
serum
electrophoresed
Trough is cut
in the gel
parallel to the
line of
separation
Antiserum is
placed in the
trough
Incubate: 18-
24 hours
•double diffusion
occurs at right
angles to the
electrophoresis
separation
•Precipitin lines
develop where
Ag-Ab
combination takes
place.
Lines or arcs shape, intensity and
location: compared with normal
serum control
2 step process

32. Uses
 to identify different disease entities
 to aid in monitoring the course of the disease
 therapeutic response of the patient with such conditions as
immune deficiencies, autoimmune disease, chronic infections,
chronic viral infections etc.

33. MCQs
 One principal function of complement is to
A. inactivate perforins
B. mediate the release of histamine
C.Bind antibodies attached to cell surfaces and to lyse these cells
D.phagocytize antigens E. cross link allergens

34. MCQs
 One principal function of complement is to
A. inactivate perforins
B. mediate the release of histamine
C. Bind antibodies attached to cell surfaces and to lyse these cells
D. phagocytize antigens E. cross link allergens

35. MCQs
 Antigen-antibody reactions can result in the following:
A. Agglutination
B. complement fixation
C. Precipitation
D. All of the above

36. MCQs
 Antigen-antibody reactions can result in the following:
A. Agglutination
B. complement fixation
C. Precipitation
D. All of the above

37. MCQs
 The double diffusion method allows for disease identification and diagnosis
and is an application of what immunological technique?
A. Agglutination
B. Precipitation
C. Immunoassay
D. Fluorescent antibody test
E. Complement fixation

38. MCQs
 The double diffusion method allows for disease identification and diagnosis
and is an application of what immunological technique?
A. Agglutination
B. Precipitation
C. Immunoassay
D. Fluorescent antibody test
E. Complement fixation

39. MCQs
 Hemagglutination test utilizes:
A. Red blood cells
B. White blood cells
C.Platelets
D.Lymphocytes

40. MCQs
 Hemagglutination test utilizes:
A.Red blood cells
B. White blood cells
C.Platelets
D.Lymphocytes