VIROLOGICAL TECHNIQUES Introduction of Serology Types of Serology Radioimmunoassay (RIA) Techniques Enzyme Linked ImmunosorbentAssay (ELISA) Techn Agglutination Techniques Immunofluorescence techniques Complement Fixation Techniques Problems With Serology Summary Reference

1. University Of Hargeisa
College: Applied Science
Faculty: Medical Laboratory
Class: 3B
Course: Medical Virology
Presented: Abdikadir Daahir (Gees)
TeL: +252634992499 Date: 12-12-2018

2. VIROLOGICAL
TECHNIQUES
Serological Techniques
For Virology
Presentation

3. Continents
Introduction of Serology
 Types of Serology
1) Radioimmunoassay (RIA) Techniques
2) Enzyme Linked ImmunosorbentAssay (ELISA) Techn
3) Agglutination Techniques
4) Immunofluorescence techniques
5) Complement Fixation Techniques
Problems With Serology
Summary
Reference

4. Introduction of
Serological Techniques
 Serological Techniques: is a diagnostic methods
that used to identify antibodies and antigen in
patients same which is serum and plasma. There are
some classical serological methods like agglutination
and complement Fixation Techniques that are used
to identify infectious disease and human blood
grouping. There are several serology techniques.
 Other definition serology; detection of rising titer of
antibody between acute and convalescent stages of
infection, or the detection of IgM in Primary infection

5. Note that during reinfection, IgM may be absent or present at a low level transiently
Typical Serological Profile After Acute
Infection

6. 4
Immuno-
Fluorescent
3
Agglutination
5

7. 1. Radioimmunoassay
(RIA)
Radioimmunoassay (RIA) : is a very sensitive technique used to
measure concentrations of antigens (for example hormone levels in
the blood). As the technique couples radioactivity and immune
function, it is called “Radioimmunoassay”.
There Are Two types RIA:
1)Competitive Binding: The labeled and Unlabeled
antigen (Ag) compete with limited number of binding
antibody sites
Ag* Unbound Ag washed out
Ag+Ag* + Ab AgAb+ Ag*Ab+Ag

8. Continue…
2. Non-Competitive : the concentration of antigen is directly
proportional to bound labeled antibody concentration
Unbound Ag washed out
No competition exist because concentration of antibody is
more
Ag+Ab* +Ab Ag*Ab+Ag

9. History
RIA Was developed in 1960, by endocrinologist
Rosalyn Yalow and Solomon Berson. In 1977,
Rosalyn Yalow was awarded Nobel Prize for Medicine.

10. Advantages And Disadvantages
Advantage
•Great sensitivity
•Possible to detect a few picograms of antigen.
•Greater specificity of the assay.
Disadvantage
•Expensive
•Hazards in preparing & handling the radioactive Agents
•Requires special counting equipment
The body concentrates iodine atoms- radioactive or not- in
the thyroid gland where they are incorporated in thyroxin

11. Application
Epidemiology
•Hepatitis B
Clinical immunology
•Antibodies for Inhalant Allergens
•Allergy Diagnosis
Endocrinology
• Insulin, HCG, Vasopressin
• Detects Endocrine Disorders
• Physiology of Endocrine function

12. 2. Enzyme-Linked Immunosorbent Assay
(ELISA) Techniques
Definition
 ELISA: is techniques used for antibody
or infectious agents in a samples
 ELISA: is plate-based assay techniques
designed for detecting and quantifying
substances such as peptides, protein,
Antibodies and Hormones.

15. Types of ELISA
1) Direct ELISA: When the presence of an antigen is
analyzed, the name "direct ELISA" refers to an ELISA in
which only a labelled primary antibody is use.
2) Indirect ELISA: refers to an ELISA in which the antigen
is bound by the primary antibody which then is detected
by a labeled secondary antibody.
3) The Sandwich ELISA: is a sensitive and robust method
which measures the antigen concentration in an unknown
sample.
4) Competitive ELISA: is a competitive binding process
executed by original antigen (sample antigen) and add-in
antigen. The procedures of competitive ELISA are
different in some respects compared with Indirect
ELISA, Sandwich ELISA and Direct ELISA

16. Advantage
 Relatively cheap and have long shelf
life
 Highly sensitive and specific
 ELISA Are widely available
 Can be used a variety infection such as
HIV

17. Disadvantage
 Enzyme activity may be effected by
plasma constituents
 Very specific to particular antigen

18. Application
 The ELISA Can be performed to evaluate either the presence of
antigen or presence of antibody in a sample.
 It is useful tool for determining Serum antibody concentration
 It was first screening test widely used for HIV because of its
high sensitivity
 Other application
 Detection of mycobacterium antibodies in tuberculosis
 Detection of Rotavirus in feces
 Detection of Hepatitis B Markers in serum
 Detection of enterotoxin of E.coli in feces

19. 3. Agglutination Techniqu
Definition
Agglutination is used to describe antibodies that agglutinate
particulate antigens.
When antigen is an erythrocyte the term Haemagglutination is used
All antibody can theoretically agglutinate particulate antigens but IgM
do to its high valance, is particularly good agglutinin and one
sometimes conclude that antibody may be of the IgM class if it is a
good agglutinating antibody

20. Types Agglutination Techn
 Qualitative Agglutination Techniques
this Technique is used to identify the presence of antigen or
antibody in the serum of the patients.
 a patient’s RBCs can be mixed with antibody to blood group
antigens to determine a person’s blood type
 A patient’s serum is mixed with RBCs of unknown blood type to
assay for the presence of the antibodies to that blood type in the
patients serum.
 Quantitative Agglutination techniques
it is used to measure to level antibodies to particulate antigens.
 Serial dilutions are made of a sample to test for antibody.
 Then fixed number of RBCs or bacteria is added

21. Advantages and Disadvantage
 Advantage of agglutination test
 Large variety of test antigens can be used
 Reading is easy (lysis, no lysis)
 More sensitive than other serological test
 Disadvantage of agglutination test
 Demand on equipment and reagents are large
 Some of components need to be fresh (RBCs,
complement)

22. Applications
 Determination of blood types or antibodies to blood group
antigen
 To asses bacterial infections e.g. arise in titer of an antibody to
particular bacterium indicates an infection with that bacterial
type.
 It can be used to type blood cells for transfusion, to identify
bacterial culture, and to detect the presence and relative
amount of specific antibody in a patient’s serum

23.  Definition
 Is a laboratory technique used to detect the presence of specific
target antigen in the sample by using antibody tagged with
fluorescence.
 This simplifies to measure the amount present of an antigen or
antibody or immunocomplexes in the system.
 Fluorochrome or fluorophore is a fluorescent chemical
compound that absorbs ultraviolet and emits visible light.
 Commonly used immunofluorescent are:
 Fluorescein (green)
 Rhodamine (red)
4. Immunofluorescence

24. Immunofluorescense
Positive immunofluorescence test for
rabies virus antigen. (Source: CDC)(Virology Laboratory, Yale-New
Haven Hospital)

25. Types immunofluoresce
 Direct immunofluorescent:
 Staining in which the primary antibody is labeled with fluorescence dyes
 Can be used to detect viral, parasitic, tumor antigens from patient
specimens
 Steps involved
 Fixation of smear on the slide
 Treating with labeled antibody
 Incubation
 Washing to remove unbound excess labeled antibody and visualization
under fluorescent microscope
 When viewed under fluorescent microscope, the field is dark and areas
with bound antibody fluoresce green.

26. Continue…
 Indirect immunofluorescent
 It is double layered technique
 It is considered as the standard technique for the detection of
the auto antibodies
 Staining in which secondary antibody is labeled with
Fluorochrome is used to recognize primary antibody
 Steps involved
 The antigen on smear are made to react with specific unlabeled
antibody (raised in mouse) and incubated.
 The unbound antibody gets washed off.
 The presence of specific mouse antibody bound to the antigen
on smear is detected by adding another antibody.

27. Advantage
 Very sensitive Techniques
 required only one labeled antibody to
identify many proteins.
Disadvantage
 Short shelf life
 Expensive equipment necessary

28. 5. Complement Fixation
 Definition
complement fixation: Are immunological
Techniques that can be used to detect the presence
of either specific antibody or specific antigen in a
patient’s serum, based or whether complement
fixation occurs.
it was widely used to diagnose infection, particularly
with microbe that are not easily detect by culture
methods, and Rheumatic diseases

29. Complement Fixation
Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement
fixation obtained with acute and convalescent phase serum specimens,
respectively. (2-fold serum dilutions were used) The observed 4-fold increase is
significant and indicates recent infection.

30. Types Complement Fixation
 Step 1:
 A known antigen and inactivated patient’s serum are
incubated with a standardized, limited amount of
complement.
 If the serum contains specific, complement activating
antibody the complement will be activated or fixed by
the antigen-antibody complex.
 However., if there is no antibody in the patient’s
serum, there will be no formation of antigen-antibody
complex, and therefore complement will not be fixed,
but will remain free.

31. Continue…
 Step 2:
 The Second step detects whether complement has been utilized in the
first step or not, this is done by adding the indicator system.
 If the complement is fixed in the first step owing to the presence of
antibody there will be no complement left to fix to the indicator system
 However., there is antibody in the patient’s serum , there will be no
antigen-antibody complex, and therefore, complement will be present
free or unfixed in the mixture.
 This unfixed complement will now react with the antibody-coated red
blood cells to bring about their lysis.
 Thus, no lysis of red blood cell (Positive CFT) indicates the presence of
antibody in the test serum.

32. CFT antibodies
 Used mainly for the diagnosis of herpes simplex and VZV
encephalitis
 CFT normally contain little or no antibodies
 presence of antibodies suggest meningitis or
meningoencephalitis
CFT antibody titre > _1_ is indicative of meningitis
Serumantibodytitre 100
 Diagnosis depends on the presence of an intact blood-brain
barrier

33. Advantages and Disadvantages
Advantages
 Result available quickly, usually within a few hours.
Potential Problems
 Often very much reduced sensitivity compared to cell culture,
can be as low as 20%. Specificity often poor as well.
 Requires good specimens.
 The procedures involved are often tedious and time-
consuming and thus expensive in terms of laboratory time.

34. Problems with Serology
 Long period of time required for diagnosis for paired acute and convalescent
sera.
 Mild local infections such as HSV genitalis may not produce a detectable
humoral immune response.
 Extensive antigenic cross-reactivity between related viruses e.g. HSV and
VZV, Japanese B encephalitis and Dengue, may lead to false positive results.
 immunocompromised patients often give a reduced or absent humoral
immune response.
 Patients with infectious mononucleosis and those with connective tissue
diseases such as SLE may react non-specifically giving a false positive
result.
 Patients given blood or blood products may give a false positive result due to
the transfer of antibody.

35. Summary
 Serology techniques is a detection of rising titer of
antibody between acute and convalescent stages of
infection, or the detection of IgM in primary.
 Types of serology techniques such as RIA, ELISA,
Agglutination, Immunofluorescent And Complement
Fixation Techniques.
 Problems with serology : long period of time required
for diagnosis for paired acute and convalescent sera.

36. Reference
 Waran Livason Edition Seven for microbiology
 Pharmaceutical analysis – Ashutoshkar
 Biochemistry – Vasudevan
 Pharmaceutical analysis Vol.2 – G.Devalarao
 www.wikepedia.com/Elisa
 Baron, S. (1996) Medical Microbiology, Medical
Microbiology. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/21413252.
 Desikan, S. and Narayanan, S. (2015) ‘Genetic
markers, genotyping

37. Thank for your Attention