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Description
Antigen (Ag): any foreign substance that elicits an
immune response (i.e., the production of specific
antibody molecules) when introduced into the tissues
of a susceptible animal and is capable of combining
with the specific antibodies formed.
AAntigens are generally of high
molecular weight and commonly
are proteins or
polysaccharides.
• Antigens that give strong immune
responses are strongly
immunogenic.
• The small site on an antigen to
which an antibody binds is called
an epitope.
• Most common antigens are
viruses and bacteria
Antibody (Ab): an immunoglobulin
capable of specific combination
with the antigen that caused its
production in a susceptible animal.
• Antibodies are produced in
response to the invasion of foreign
molecules(antigens) in the body.
• Antibodies exist as one or more copies of a Y-shaped unit.
The Difference Between
Blocking and Indirect ELISA Tests
Indirect ELISA
A broad range of Ab variants will
bind to the plate.
• The conjugate is an anti-species
IgG conjugate which will bind to
all already
bound Ab.
• Highly sensitive, but a small
possibility to have nonspecific Ab
binding to the
plate, resulting in false positive
results.
indirect ELISA Blocking ELISA
Can only be used for specific
species depending upon which
conjugate is used
in the test.
• The amount of bound conjugate is
proportional to the Ab level in the
sample.
• Quantitative results can be
calculated (S/P)
Blocking (Competitive)
ELISA
A broad range of Ab variants will
bind to the plate.
• The conjugate is a monoclonal Ab
(specific Ab) which will bind to
only a specific
epitope on the coated plate if this
space is not already occupied by an
Ab from
the sample.
• Highly specific, it doesn’t matter
if any nonspecific binding has
happened to
other parts of the plate as this will
not be detected by the conjugate
Indirectly proportional to the Ab
level in the sample (the more
antibodies present
in the sample, the less color
development in the test wells)
• Not species-dependent as the
conjugate binds directly to the Ag
on the plate
Immunoassays play a vital role in
the control and prevention of
poultry diseases. They allow for
early detection of outbreaks, which
helps to prevent the spread of
disease and minimize economic
losses. They are also used to
monitor the effectiveness of
vaccination programs and to
ensure the safety of poultry
products.
Poultry diagnostics is the process
of identifying the cause of illness or
disease in poultry. This is crucial for
ensuring the health and well-being
of birds, preventing the spread of
disease, and maintaining the safety
of poultry products for human
consumption.
Diagnostic Process:
Poultry diagnostics typically involves a
multi-step process:
1.Clinical Observation: This involves
observing the flock for signs of illness,
such as lethargy, decreased appetite,
respiratory problems, diarrhea, or
unusual behavior.
Diagnostic Process:
1.Sample Collection: Samples, such as blood, tissues, or feces, may be
collected from sick birds or the environment for further testing.
2.Laboratory Analysis: Various laboratory tests are employed to identify
the causative agent of the disease. These tests may include:
1. Immunoassays: As mentioned earlier, these tests detect the
presence of antibodies or antigens associated with specific
pathogens.
2. Bacteriology: This involves culturing bacteria from samples to
identify the specific bacterial species causing the disease.
3. Virology: This involves isolating and identifying viruses present in
the samples.
4. Parasitology: This involves identifying parasites that may be present
in the bird or its environment.
5. Necropsy: In some cases, a post-mortem examination of deceased
birds may be performed to identify signs of disease and aid in
diagnosis.
Importance of Poultry Diagnostics:
Accurate and timely poultry diagnostics is crucial for several reasons:
•Early diagnosis and treatment: Identifying the cause of illness
promptly allows for the implementation of appropriate treatment
measures, improving the chances of a successful recovery and
minimizing bird mortality.
•Disease control and prevention: Early detection of outbreaks helps
prevent the spread of disease within the flock and to other flocks. This
is particularly important for highly contagious diseases like avian
influenza.
•Food safety: Diagnosing and controlling diseases in poultry flocks
ensures the safety of poultry products for human consumption by
minimizing the risk of foodborne illnesses.
•Monitoring flock health: Regular diagnostic testing helps monitor the
overall health of the flock and identify potential problems early on.
Different Diagnostic Techniques:
Several diagnostic techniques are employed in
poultry diagnostics, each with its own advantages
and limitations:
1. Immunoassays: As discussed earlier, these are
highly specific and sensitive tests that can
rapidly detect pathogens or antibodies in
poultry.
2. Polymerase chain reaction (PCR): This is a
highly sensitive technique used to detect the
presence of specific DNA sequences of
pathogens.
3. Histopathology: This involves examining tissue
samples under a microscope to identify
microscopic changes associated with disease.
4. Serology: This involves measuring the levels of
antibodies in the blood to assess exposure to
specific pathogens
ELISA Test Kits
Rapidly detects and quantifies antigens or antibodies against bacteria, viruses
and other pathogens
• Robust and user-friendly
• One of the most sensitive and reproducible test methods available
ELISA Test Kits
Test a large number of samples at the same time
• Automate the procedure using robotics or other types of automated equipment
• Use software to calculate and report results
Test Kit Components
• Coated Plates
• Positive and Negative Control
• Sample Diluent
• Conjugate
• Substrate (TMB)
• Stop
• Wash Solution
ELISA Test Usage
• Highly sensitive screening tool
• Results need to be interpreted on a flock level with knowledge of flock health.
• ELISAs give a very low number of false-positive results.
• Unexpected positives should be confirmed by another test system (HI, Culture,
IPMA, Western Blot, Complement Fixation, PCR, VI).
• The test will not tell you if the detected Ab are maternal Ab, from vaccination or
from infection (i.e., Differentiating Infected from Vaccinated Animals [DIVA] test).
Good Laboratory Practices
• Receiving kits
– Inspect for damage
– Record date received and when used
– Store properly per kit insert instructions
– Note kit expiration date
• General reagent handling
– Follow kit insert (check often for revisions)
– Warm up reagents (2–3 hours)
– Mix reagents
– Avoid contamination (no “back pour”, use designated reagent reservoirs)
Reagent Handling
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