ELISA technique application in poultry industry

1. **Coated Plates**: These are the plates that have been treated with antibodies or antigens specific to the pathogen you're testing for. Each well in the plate is designed to capture and retain only those pathogens if they are present in the sample being tested. 2. **Positive and Negative Controls**: These are samples that are known to contain or not contain the target pathogen, respectively. They serve as a reference to ensure the test is working correctly. A positive control should show a positive result, and a negative control should show a negative result. 3. **Sample Diluent**: This reagent is used to prepare the samples from the specimen in a concentration that is compatible with the ELISA assay. It often contains buffers and substances to block non-specific binding sites. 4. **Conjugate**: This is a complex molecule composed of an antibody specific for the pathogen's antigen, linked to an enzyme or other label that can produce a detectable signal. The conjugate binds to the antigen if it is captured by the coated plate. 5. **Substrate (TMB)**: TMB (tetramethylbenzidine) is a chromogenic substrate used with peroxidase-labeled conjugates in ELISA assays. It reacts with the enzyme to produce a colored product, which can be quantified as an indication of antigen or antibody presence. 6. **Stop Solution**: This solution stops the reaction between the substrate and the enzyme, halting the color development at the optimal time for reading the results. 7. **Wash Solution**: This reagent is used to remove unbound materials from the wells of the plate after each step of the ELISA process. It ensures that only bound substances remain in the wells, which is crucial for obtaining accurate results. **Usage in Laboratory**: - **Screening Tool**: ELISA test kits are used as a first line of screening to quickly identify the presence of pathogens within a population. - **Confirmation**: Positive results from ELISA tests should be confirmed using additional diagnostic techniques such as Hemagglutination (HI), Culture, Immunoprecipitation Assay (IPMA), Western Blot, Complement Fixation (CFC), Polymerase Chain Reaction (PCR), and Viant (VIV) tests. - **DIFFERENTIATING INFECTED FROM VACCINATED ANIMALS (DIVA) Test**: ELISA test kits are not designed to differentiate between animals that have been infected by a pathogen and those that have been vaccinated against that pathogen. To achieve this, additional specific diagnostic tests would need to be performed. **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 the kit insert (check often for revisions), warm up reagents (2–3 hours), mix reagents, avoid contamination (no "back pour", use designated reagent reservoirs), and proper storage. When performing ELISA test kits, it's crucial to follow the manufacturer's instructions meticulously, as these guidelines ensure the accuracy and reliability of your test results. Always refer to the insert for specific instructions tailored to the kit you are using. Proper handling, storage, and execution of each step in the ELISA process are key to obtaining accurate and reliable test results.