Serological tests in Science that you can think of.

Every serological test you can think of.

1. Serological tests
(Antigen antibody interactions)
SAM ASAMOAH SAKYI

2. ANTIGEN-ANTIBODY REACTIONS
Refers to the interaction between antigens and
antibodies.These reactions occurs either in vivo or
in in-vitro reactions.
•In vivo Ag-Abreactions:
These Ag-Abreactions occurs inside the host where
they form the basis of AMI in infectious diseases or
of tissue injury in some types of hypersensitivity and
autoimmune diseases.Functions of antibodies are
also included under;
a. Agglutination
b. Neutralization
c. Opsonization(IgG1 and 3)
d. Complement fixation (IgMand IgG)
e. Induction of inflammation (IgE)

3. ANTIGEN-ANTIBODY REACTIONS
•In vitro Ag-Ab reactions:
These Ag-Ab reactions occurs outside host. they help
in the diagnosis of infections, in epidemiological
surveys, in the identification of infectious agents and
of noninfectious antigens such as enzymes.
In-vitro Antigen-antibody reactions are known as
serological reactions. All immunological techniques
about to be studied are of in vitro type.

4. ANTIGEN-ANTIBODY REACTIONS
•The reactions between antigens and antibodies occur
in three stages.
•The primary stage is the initial interaction between
the two, without any visible effects.This reaction is
rapid, occurs even at low temperatures and obeys
the general laws ofthermodynamics
•The reaction is reversible and it is effected by the
weaker intermolecular forces such as Van derWaal’s
forces, ionic bonds and hydrogen bonding, rather
than by the firmer covalent bonding.
•The primary reaction can be detected by estimating
free and bound antigen or antibody separately in the
reaction mixture by a number of physical and
chemical methods, including the use of markerssuch
as radioactive isotopes, fluorescent dyes or ferritin.
•

5. ANTIGEN-ANTIBODY REACTIONS
•In most instances, but not all, the primary stage is
followed by the SECONDARY STAGE
•Secondary stage leads to demonstrable events such
as precipitation, agglutination, lysisof cells, killing of
live antigens, neutralization of toxins and other
biologically active antigens, fixation of complement,
immobilization of motile organisms and
enhancement of phagocytosis.
•Some antigen-antibody reactions occurring in-vivo
initiate chain reactions that lead to neutralization of
destruction of injury antigens or to tissue
damage.These are the Tertiary Reactions and
include humoralimmunity against infectious disease,
allergy and other immunological disease.

6. SEROLOGICAL REACTIONS
•PRECIPITATION
•AGGLUTINATION
•COMPLEMENT FIXATION TEST
•OPSONIZATION
•IMMUNOFLUORESCENCE
•RADIO-IMMUNO ASSAY
•ELISA
•IMMUNOELECTROBLOT
•IMMUNOCHROMATOGRAPHY
•PCR
•NEPHELOMETRY/TURBIDITY

7. Classification of antigen-antibody
interactions:
1.Primary serological tests: (Marker techniques) e.g.
–Enzyme linked immunosorbent assay (ELISA)
–Immuno flurescent antibody technique (IFAT)
–Radio immuno assay (RIA)
2.Secondary serological tests: e.g.
–Agglutination tests
–Complement fixation tests (CFT)
–Precipitation tests
–Serum neutralization tests (SNT)
–Toxin-antitoxin test
3.Tertiary serological test: e.g.
–Determination of the protective value of an anti serum in an animal.

8. ELISA
•Enzyme-linkedimmunosorbentassayusesanenzyme
asalabel.Anenzymeconjugatedwithanantibody
reactswithacolourlesssubstratetogeneratea
colouredreactionproduct.
•Suchasubstrateiscalledachromogenicsubstrate.A
examplesofenzymesemployedforELISA,include
alkalinephosphatase,horseradishperoxidase,andb-
galactosidase.
•Theseassaysaresensitiveandhavetheadvantageof
beingsaferandlesscostly.ThreetypesofELISAs
commonlyusedareIndirect,Sandwichand
CompetitiveELISA.

9. INDIRECT ELISA
•Antibody can be determined quantitatively with an
indirect ELISA. Serumcontaining primary antibody
(Ab1) is added to an antigen-coated microtiter well and
allowed to react with the antigen attached to the well.
•After any free Ab1 is washed away, the presence of
antibody bound to the antigen is detected by adding an
enzyme-conjugated secondary anti-isotypeantibody
(Ab2), which binds to the primary antibody.
•After washing, a substrate for the enzyme is added. The
amount of colored reaction product that forms is
measured by specialized spectrophotometricplate
readers, which can measure the absorbance of all of
the wells of a 96-well plate in seconds.

10. INDIRECT ELISA
For Ab detection,
Ag coated wells used
If Ab present, it binds to Ag
Add goat anti-human
Ig antibody conjugated with
enzyme
Add a substrate. Enzyme
acts & colour produced
Indirect ELISA is used to
measure Abs in HIV

11. ELISA PLATE

12. ELISA PLATE READER/WASHER

13. SANDWICH ELISA
•In this technique, theantibody (rather than the
antigen) is immobilized on a microtiter well. A
sample containingantigen is added and allowed
to react with the immobilized antibody.
•After the well is washed; a second enzyme-linked
antibody specific for a different epitopeon the
antigen is added and allowed to react with the
bound antigen, substrate is added, and the
colouredreaction product is measured.
•

14. SANDWICH ELISA
For Ag detection, wells coated
with Sp. Ab.
Ag in specimen binds to
coated Ab.
Add enzyme conjugated
antiserum.
Add substrate colour produced

15. COMPETITIVE ELISA
•Competitive ELISA, antibodyis first incubatedin
solution with a sample containing antigen.
•
•The antigen-antibody mixture is then added to an
antigen coated microtiter well. The more antigens
present in the sample, the less free antibody will be
available to bind to the antigen-coated well.
•Addition of an enzyme-conjugated secondary
antibody (Ab2) specific for the isotypeof the primary
antibody can be used to determine the amount of
primary antibody bound to the well as in an indirect
ELISA.
•In the competitive assay, however, the higher the
concentration of antigen in the original sample, the
lower the absorbance.

16. SUMMARY OF TECHNIQUES

17. COMPETITIVE ELISA
•Ag coated wells
•Two specific Abs,
(enzyme conj.& test Ab) added
simultaneously
•More specific test Abs attach
to Ag
•Substrate added and colour
produced

18. PCR
•PCRprovides an extremely sensitive means of
amplifyingrelatively large quantities of DNA
•First described in 1985, Nobel Prize for Kary Mullis in
1993
•The technique was made possible by the discovery
of Taqpolymerase, the DNA polymerase that is used
by the bacterium Thermus aquaticus

19. PCR
•The primary materials used in PCR are:
-DNA nucleotides, the building blocks for the
new DNA
-Template DNA, the DNA sequence that you
want to amplify
-Primers, single-stranded DNAs between 20 and
50 nucleotides long that are complementary
to a short region on either side of the
template DNA
-DNA polymerase, a heat stable enzyme that
catalyzes, the synthesis of new DNA

20. The cycling reactions
•There are three major steps in a PCR, which are repeated for 20
to 40cycles. This is done on an automated Thermo Cycler,
which can heat and cool the reaction tubes in a very short
time.
•Denaturationat around 94°C: the double strand melts open to
single stranded DNA, all enzymatic reactions stop
•Annealing at around 54°C:
Hydrogen bonds are constantly formed and broken between the
single stranded primer and the single stranded template. If the
primers exactly fit the template, the hydrogen bonds are so
strong that the primer stays attached
•Extension at around 72°C:
The bases (complementary to the template) are coupled to the
primer on the 3' side (the polymerase adds dNTP'sfrom5' to
3', reading the template from 3' to 5' side, bases are added
complementary to the template)

22. DNA SEQUENCING
•Every cycle results in a doubling of the number of strands
DNA present
•After the first few cycles, most of the product DNA strands
made are the same length as the distance between the
primers
•The result is a dramatic amplification of a the DNA that
exists between the primers. The amount of amplification is
2 raised to the n power; n represents the number of cycles
that are performed. After 20 cycles, this would give
approximately 1 million fold amplification. After 40 cycles
the amplification would be 1 x 10
12

23. DNA SEQUENCING
•The process of determining the order of the
nucleotide bases along a DNA strand is called
DNA sequencing
•This method is based on the principle that
single-stranded DNA molecules that differ in
length by just a single nucleotide can be
separated from one another using
polyacrylamide gel electrophoresis.

24. VERIFICATION OF PCR PRODUCT

25. Turbidity and Nephelometry
•PRINCIPLES:
•Light scattering is the physical phenomenon resulting
from the interaction of light with a particle(s) in
solution.
Dependent on:
•Particle size
•Wavelength
•Distance of observation,
•Concentration of particles
•MW of particles

26. Turbidity and Nephelometry
•Chemical analysis based on the phenomenon whereby
light, passing through a medium with dispersed
particles is attenuated in intensity by scattering
•In turbidimetry, the intensity of light transmitted
through the medium, the unscattered light, is
measured.
•In nephelometry, the intensity of the scattered light
is measured, usually, but not necessarily, at right
angles to the incident light beam.

27. Turbidity and Nephelometry
•Turbidity can be measured on most routine
analysers by a spectrophotometer(absorbed
light)
•Reducedsensitivity and precision.
•Extent of light scattering increases as
wavelength increases
•The intensity of scattered light is normally
measured by nephelometer

28. METHODS
•Immunoprecipitation. Aggregation of immune
complexes (Aband Ag)-only if the antigen was
originally soluble. Used forquantativedetermination
•Agglutination. Aggregation of immune complexes (Ab
and Ag)-if antigen was originally particulate or
cellular. Used forqualitative determination.
•Antibody + Antigen forms immune complex.
•Immune complex causes light scattering (greater the
aggregation, greater the light scattering)
•Measure the rate of formation of immune complexes
against std curve

29. APPLICATIONS
•Immunoglobulins,
•Specific proteins: Haptoglobin, Transferrin,
Alpha 1 AT, Lipoproteins, Albumin
•Coagulation factors: Antithrombin III
•Theraputic drugs

30. IMMUNOFLUORESCENCE
•Described by Coons & Colleagues (1942)
•Flourescent dyes conjugated with Abs
identifies:
•Tissue antigens
•Antigens of infecting organisms
•Antibodies, Autoantibodies
•Antigen-Antibody complexes

31. DYES
Fluorescent dyes commonly used:
•Fluorescein isothiocyanate -blue green
•Lissamine rhodamine-orange red
•IF may be direct or indirect
•Direct IF-for unknown Ags like bacteria,
viruses in specimens Rabies virus)
•Indirect IF-for Abs in serum/ body fluids.

32. Direct IF
•for unknown Ags like bacteria
•Separate fluorescent labeled Ab required

34. Indirect IF
•Need only single antihuman globulin
fluorescent conjugate

37. RADIOIMMUNOASSAY
•Described by Bensonand Yallow(1959)
•Measurespicogram quantities of :
•Hormones
•Drugs
•Tumour markers
•IgE
•Viral antigens
•RIA is a competitive binding assay

38. RIA
•Competitionfor fixed amounts of specific Ab
between:
•a known radio labeled antigen &
unknown unlabeled (test) antigen
•After Ag-Ab reaction, Ag separated into free &
bound fractions & their radioactivity
measured

39. RIA