ELISA (Enzyme-Linked Immunosorbent Assay) is a common laboratory technique used to detect and measure antibodies or antigens in a sample. In the poultry industry, ELISA can be used for a variety of applications, such as:

• Diagnosing infectious diseases: ELISA can be used to detect antibodies to common poultry diseases like Newcastle disease, Avian Influenza, and Infectious Bronchitis Virus.

• Monitoring vaccination programs: ELISA can be used to measure the levels of antibodies in poultry after vaccination to ensure that the vaccination program is effective.

• Detecting foodborne pathogens: ELISA can be used to detect bacteria like Salmonella and E. coli in poultry products.

This course will provide students with a comprehensive understanding of the ELISA technique and its applications in the poultry industry. Students will learn about the principles of ELISA, different types of ELISA assays, and how to interpret ELISA results. The course will cover the use of ELISA for diagnosing infectious diseases in poultry, monitoring vaccination programs, and detecting foodborne pathogens.

Prerequisites:

• Basic knowledge of poultry diseases and immunology

Learning Outcomes:

• Upon successful completion of this course, students will be able to:

  • Explain the principles of ELISA

  • Describe different types of ELISA assays

  • Interpret ELISA results

  • Apply ELISA techniques for diagnosing poultry diseases

  • Evaluate the use of ELISA in monitoring vaccination programs

  • Discuss the application of ELISA for detecting foodborne pathogens in poultry products

immunology

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

Who this course is for:

• veterinerian , all interested in poultry industry ,lab technician, lab scientists