Elisa Technique Application In Poultry Industry

What you'll learn
immunoassay and poultry diagnostics
introduction indirect Elisa blocking
key player in immune response
ELISA test kits
Requirements
immunology basics ,poultry diseases basics, ELISA basics
Description
Antigen (Ag): any foreign substance that elicits animmune response (i.e., the production of specificantibody molecules) when introduced into the tissuesof a susceptible animal and is capable of combiningwith the specific antibodies formed.AAntigens are generally of highmolecular weight and commonlyare proteins orpolysaccharides.• Antigens that give strong immuneresponses are stronglyimmunogenic.• The small site on an antigen towhich an antibody binds is calledan epitope.• Most common antigens areviruses and bacteriaAntibody (Ab): an immunoglobulincapable of specific combinationwith the antigen that caused itsproduction in a susceptible animal.• Antibodies are produced inresponse to the invasion of foreignmolecules(antigens) in the body.• Antibodies exist as one or more copies of a Y-shaped unit.The Difference BetweenBlocking and Indirect ELISA TestsIndirect ELISAA broad range of Ab variants willbind to the plate.• The conjugate is an anti-speciesIgG conjugate which will bind toall alreadybound Ab.• Highly sensitive, but a smallpossibility to have nonspecific Abbinding to theplate, resulting in false positiveresults.indirect ELISA Blocking ELISA Can only be used for specificspecies depending upon whichconjugate is usedin the test.• The amount of bound conjugate isproportional to the Ab level in thesample.• Quantitative results can becalculated (S/P)Blocking (Competitive)ELISA A broad range of Ab variants willbind to the plate.• The conjugate is a monoclonal Ab(specific Ab) which will bind toonly a specificepitope on the coated plate if thisspace is not already occupied by anAb fromthe sample.• Highly specific, it doesn't matterif any nonspecific binding hashappened toother parts of the plate as this willnot be detected by the conjugateIndirectly proportional to the Ablevel in the sample (the moreantibodies presentin the sample, the less colordevelopment in the test wells)• Not species-dependent as theconjugate binds directly to the Agon the plate Immunoassays play a vital role inthe control and prevention ofpoultry diseases. They allow forearly detection of outbreaks, whichhelps to prevent the spread ofdisease and minimize economiclosses. They are also used tomonitor the effectiveness ofvaccination programs and toensure the safety of poultryproducts.Poultry diagnostics is the processof identifying the cause of illness ordisease in poultry. This is crucial forensuring the health and well-beingof birds, preventing the spread ofdisease, and maintaining the safetyof poultry products for humanconsumption.Diagnostic Processoultry diagnostics typically involves amulti-step process:1.Clinical Observation: This involvesobserving the flock for signs of illness,such as lethargy, decreased appetite,respiratory problems, diarrhea, orunusual behavior.Diagnostic Process:1.Sample Collection: Samples, such as blood, tissues, or feces, may becollected from sick birds or the environment for further testing.2.Laboratory Analysis: Various laboratory tests are employed to identifythe causative agent of the disease. These tests may include:1. Immunoassays: As mentioned earlier, these tests detect thepresence of antibodies or antigens associated with specificpathogens.2. Bacteriology: This involves culturing bacteria from samples toidentify the specific bacterial species causing the disease.3. Virology: This involves isolating and identifying viruses present inthe samples.4. Parasitology: This involves identifying parasites that may be presentin the bird or its environment.5. Necropsy: In some cases, a post-mortem examination of deceasedbirds may be performed to identify signs of disease and aid indiagnosis.Importance of Poultry Diagnostics:Accurate and timely poultry diagnostics is crucial for several reasons:•Early diagnosis and treatment: Identifying the cause of illnesspromptly allows for the implementation of appropriate treatmentmeasures, improving the chances of a successful recovery andminimizing bird mortality.•Disease control and prevention: Early detection of outbreaks helpsprevent the spread of disease within the flock and to other flocks. Thisis particularly important for highly contagious diseases like avianinfluenza.•Food safety: Diagnosing and controlling diseases in poultry flocksensures the safety of poultry products for human consumption byminimizing the risk of foodborne illnesses.•Monitoring flock health: Regular diagnostic testing helps monitor theoverall health of the flock and identify potential problems early on.Different Diagnostic Techniques:Several diagnostic techniques are employed inpoultry diagnostics, each with its own advantagesand limitations:1. Immunoassays: As discussed earlier, these arehighly specific and sensitive tests that canrapidly detect pathogens or antibodies inpoultry.2. Polymerase chain reaction (PCR): This is ahighly sensitive technique used to detect thepresence of specific DNA sequences ofpathogens.3. Histopathology: This involves examining tissuesamples under a microscope to identifymicroscopic changes associated with disease.4. Serology: This involves measuring the levels ofantibodies in the blood to assess exposure tospecific pathogensELISA Test KitsRapidly detects and quantifies antigens or antibodies against bacteria, virusesand other pathogens• Robust and user-friendly• One of the most sensitive and reproducible test methods availableELISA Test KitsTest 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 SolutionELISA 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 orfrom 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
Overview
Section 1: Introduction
Lecture 1 introduction instructor
Lecture 2 Elisa how it works
Lecture 3 introduction indirect ELISA Blocking ELISA
Lecture 4 introduction indirect ELISA Blocking ELISA
Lecture 5 Introduction
Lecture 6 serology
Lecture 7 samples
Lecture 8 key player in humoral immune response
Lecture 9 key player in humoral immune response
Lecture 10 time line of viral infection
Lecture 11 primary secondary immune response and vaccination
Lecture 12 immune assays poultry diagnostics
Lecture 13 immunoassays and poultry diagnostics
Lecture 14 detection methods
Lecture 15 detection methods
Lecture 16 protocol
Lecture 17 protocol
Lecture 18 step by step how to perform Elisa to detect Antibody titer for IBD
Lecture 19 ID test kits step by step
Lecture 20 ID test kits step by step ELISA
Lecture 21 New castle disease virus test kits
Lecture 22 Avian influenza test kits
Lecture 23 calculation
Lecture 24 ELISA calculations
Section 2: equipment
Lecture 25 ELISA test kits
Lecture 26 plates
Lecture 27 pipette
Lecture 28 ELISA washer
Lecture 29 washer manual
Lecture 30 ELISA reader
Lecture 31 Reader manual
Lecture 32 software manual
veterinerian , all interested in poultry industry ,lab technician, lab scientists
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