3. Using Labeled Antibodies to
Detect Interactions: Flow cytometry
Technology that simultaneously measures and
then analyzes multiple physical characteristics of
single particles, usually cells, as they flow in a
fluid stream through a beam of light (usually
laser).
The properties measured include a particle’s
relative size, relative granularity or internal
complexity, and relative fluorescence intensity.
4. Flow Cytometry
In the flow cytometer, particles are carried to the
laser intercept in a fluid stream. Any suspended
particle or cell from 0.2–150 um in size is suitable
for analysis.
Cells from solid tissue must be disaggregated
before analysis.
When particles pass through the laser intercept, they scatter laser light. Any
fluorescent molecules present on the particle fluoresce. The scattered and
fluorescent light is collected by appropriately positioned lenses. A combination
of beam splitters and filters steers the scattered and fluorescent light to the
appropriate detectors. The detectors produce electronic signals proportional to
the optical signal striking them.
6. Flow cytometry
Fluorescence Activated Cell Sorter (FACS)
Special version of flow cytometry counts cells
labeled with fluorescent antibodies
Used to count subsets of T cells, e.g.
CD4 and CD8 cell especially
Antibodies are attached to the CD4 and CD8 markers
Cells with fluorescently labeled markers are counted
Can also be used to separate cells based on their
phenotype
8. Flow cytometry:
Immunophenotyping
normal cells express a variety of cell surface
markers, (each marker assigned a CD number)
dependent on the specific cell type and degree of
maturation.
However, abnormal growth may interfere with the
natural expression of markers resulting in
overexpression of some and under-representation of
others.
Flow cytometry can be used to immunophenotype
cells and thereby distinguish between healthy and
diseased cells.
10. Complement fixation test
Test uses four components
Antigen, antibody, complement, and sensitized
sheep RBCs
Used to test for the presence of specific
antibodies in patient serum
Has been replaced by other tests such as
ELISA
12. Immunoelectronmicroscopy
uses antibodies to detect the intracellular
location of structures of particular proteins by
electron microscopy. Ultra thin sections are
labeled with antibodies against the required
antigen and then labeled with gold particles
Visualization done using TEM
Can be used to identify viral infections
13. immunofiltration and
immunochromatography assays
Are qualitative rapid diagnostic tests
Immunofiltration assays are rapid ELISAs
based on the use of antibodies bound to a
membrane filter rather than to plates.
Because of the large surface area of a
membrane filter, reactions proceed faster and
assay times are significantly reduced as
compared to a traditional ELISA.
14. Immunochromatographic assays are still faster and easier to
read
antigen solution flowing through a porous material encounters
antibody labeled with either pink colloidal gold or blue colloidal
selenium.
Where antigen and antibody bind, colored immune complexes
form in the fluid, which then flows through a region where the
complexes encounter antibody against them, resulting in a
clearly visible pink or blue line, depending on the label used.
These assays are used for pregnancy testing, which tests for
human chorionic growth hormone and for rapid identification of
infectious agents such as HIV, Escherichia coli group A
Streptococcus, respiratory syncytial virus (RSV), and
influenzaviruses.
15.
16. Vaccination and Immunity
Immunity is the state of protection against
infectious disease conferred either through an
immune response generated by immunization
or previous infection or by other non-
immunological factors
Immunisation is the process of introducing
weakened or killed pathogen or part of
pathogen (vaccines) into the body to elicit
protective immune responses against the
pathogen
17. Principles of Immunization
Naturally acquired immunity is
acquisition of adaptive immunity
through natural events
Immunization mimics these events
by inducing artificially acquired
immunity
Natural or artificial immunity can
be divided into
Active immunity
Passive immunity
18. Principles of Immunization
Active immunity
Result from immune response
upon exposure to an antigen
Active immunity can develop
naturally
Following illness
Or artificially
After immunization
19. Principles of Immunization
Passive Immunity
Occurs naturally during pregnancy
IgG from mother crosses placenta
Inferres protection to the baby
Occurs naturally as result of breast
feeding
IgA antibodies in breast milk given
to child
Artificial passive immunity involves
transfer of antibodies produced by
another person or animal
Can be used to prevent disease
before or after likely exposure
21. Vaccines
The word “vaccine” originates from the Latin
Variolae vaccinae (cowpox), which Edward
Jenner demonstrated in 1798 could prevent
smallpox in humans.
Today the term ‘vaccine’ applies to all biological
preparations, produced from living organisms,
that enhance immunity against disease and
either prevent (prophylactic vaccines) or, in
some cases, treat disease (therapeutic
vaccines)
22. Vaccines 22
Edward Jenner and the
origin of vaccination
Small pox caused by ‘variola virus’
Induced immunity dates to ancient Chinese
-- practiced ‘Variolation’
-- brought to England in 1700s
Jenner discovered protective effect of cow pox
-- ‘vaccinia virus’
-- ‘vacca’ Latin for cow - vaccination
WHO erradicated small pox in 1970s
23.
24.
25.
26.
27.
28.
29.
30.
31. Vaccination
Vaccination prevents and control such diseases as
cholera, rabies, poliomyelitis, diphtheria, tetanus,
measles, and typhoid fever
Vaccines can be:
a- prophylactic (e.g. to prevent or ameliorate the
effects of a future infection by any natural or
"wild" pathogen
b- Therapeutic (e.g. vaccines against cancer are
also being investigated
33. Vaccines and Immunization-Types
Attenuated vaccines
Weakened form of pathogen
Generally unable to cause disease
Strain replicates in vaccine recipient
Causes infection with undetectable or mild
symptoms
Results in long lasting immunity
modified live viral vaccines trigger a cellmediated
immune response dominated by type 1 helper T cells
(Th1) and cytotoxic T cells (Tc).
34. Vaccines and Immunization
Attenuated vaccines
Advantages
Single dose usually
sufficient to induce long-
lasting immunity
Due to multiplication of
microbe in body
Continued stimulation
of immune system
Vaccine as added potential
for being spread
“Disease” after
immunization could be
spread to un-immunized
individuals inadvertently
Disadvantages
Have potential to
cause disease in
immunocompromised
individuals
Pregnant women
should also avoid
immunization with
attenuated vaccine
Attenuated vaccines in
use include
Sabin polio vaccine
MMR
Yellow fever
35. Attenuated vaccines:
- Pprepared by:
a-repeated subculture in unsuitable
condition (chemical or media)
e.g BCG vaccine against T.B and
17 D vaccine against yellow fever.
b-growing at high temp. (above optimum
temp) e.g Pasteur anthrax vaccine
c-selection of mutant strains of low
virulence
e.g Sabin vaccine against poliomylitis.
36. Inactivated vaccines
Unable to replicate in vaccinated individual
Retains immunogenicity of infectious agent
Immunogenic not pathogenic
Inactivated vaccines fall into two categories
Whole agents
Contain killed organisms of inactivated virus
Does not change epitopes
Cholera, plague, influenza and Salk polio are whole agents
Fragments
Portions of organisms or agents including toxins proteins and
cell wall components
Includes toxoids, protein subunit vaccines and polysaccharide
vaccines
Vaccines and Immunization
37. Toxoids, which are inactivated toxins,
are vaccines directed at the toxins produced by
a pathogen.
The tetanus and diphtheria toxoids have
long been part of the standard childhood
immunization series.
They require a series of injections for full
immunity, followed by boosters every 10 years.
Many older adults have not received boosters;
they are likely to have low levels of protection.
38. 38
Modification of
Toxin to Toxoid
(example)
Modification of
Toxin to Toxoid
(example)
toxin moiety antigenic determinants
chemical and temperature
modification
Toxin Tetanus Toxoid
Extracellular Toxin
of Clostridiun tetani
39. Subunit vaccines use only those
antigenic fragments of a microorganism that best
stimulate an immune response. Subunit vaccines that
are produced by genetic modification techniques,
meaning that other microbes are programmed to
produce the desired antigenic fraction, are called
recombinant vaccines.
For example, the vaccine against the hepatitis B virus
consists of a portion of the viral protein coat that is
produced by a genetically modified yeast.
Types of Vaccines and Their Characteristics
40. Recombinant vaccines:
prepared by recombinant DNA technology for improvement
vaccines e.g:
a- subunit vaccines
in which microbial polypeptides are isolated from the infective
material hepatitis B and influenza viruses
B- Recombinant DNA-derived antigen vaccines:
in which Ag are synthesizing by inserting the coding genes into
E. coli or yeast cell as HBV vaccines
C- Recombinant DNA avirulent vector vaccines:
in which the genes coding for the Ag is inserted into genome of
an avirulent vector such as BCG vaccine
D-Synthetic peptide vaccines:
synthesis of short peptides that corrospond to antigenic
determinants on a viral or bacterial proteins e.g cholera toxins
and poliovirus to produce Ab response.
41.
42. Polysaccharide vaccines
Unique type of inactivated subunit vaccine
composed of long chains of sugar molecules
that make up the surface capsule of
certain bacteria.
Available for Pneumococcal disease,
meningococcal disease and
Haemophilus influenzae type b
43. Vaccines and Immunization
Combination Vaccines
combine antigens from several toxoids and
inactivated pathogens that are administered
simultaneously.
Examples include MMR—vaccine against
measles,mumps, and rubella—and Pentacel,
which is a vaccine against diphtheria,
tetanus, pertussis (whooping cough), polio,
and diseases of Haemophilus influenzae
44. Adjuvants
Adjuvants enhance the immune effect of the
vaccine antigen, but do not themselves act as
antigens.
Aluminum salts are the most commonly used
adjuvant for vaccines.
Adjuvanted vaccines may have a slightly
higher rate of adverse reactions, including
pain at the injection site, malaise and feve
47. BCG (bacillus Calmette-Guerin)
Content:– living or
attenuated, liophilic dried
up culture unpathogenic
strain of M. tuberculosis,
was found by the French
scientists Calmette and
Guerin. Used for the
active specific prophylaxis
of tuberculosis. Plugged in
the calendar of
inoculations. Contra-
indicated people with
violation of cellular link of
immunity.
What type of immunity
(originally) is created in an
organism after
introduction?
Postvaccine
Active
Cellular
Antibacterial
General
Specific
