ELISA Techniques PDF

## ELISA Techniques ### ELISA DIRECTE * **NO COMPETITIVE** * **Rapid (1 Ac)** * **Eliminates reactivity created between Ac** * **Inflexible and expensive** * **Amplified signal for Ag detection** **Steps:** 1. **Sample Problem:** The sample with Ag is fixed to the bottom and remains immobilize...

## ELISA Techniques ### ELISA DIRECTE * **NO COMPETITIVE** * **Rapid (1 Ac)** * **Eliminates reactivity created between Ac** * **Inflexible and expensive** * **Amplified signal for Ag detection** **Steps:** 1. **Sample Problem:** The sample with Ag is fixed to the bottom and remains immobilized. 2. **Conjugate (Marked Ac):** The marked Ac is added with the euziene and Ag. 3. **Substrate:** The substrate is added. 4. **Detection:** After washing to remove unbound Ac, a specific substrate is added to produce a color. The intensity of the color is proportional to the quantity of Ag. ### ELISA INDIRECTE * **NO COMPETITIVE** * **For Ac detection** **Steps:** 1. **Sample Problem (fixed Ag):** The sample containing the fixed Ag is immobilized at the bottom. 2. **Addition of Primary Ac:** The primary Ac is added. This binds to the Ag. 3. **Conjugate (Anti-Ac*):** The Anti-Ac is added. This binds to the primary Ac. 4. **Substrate:** The substrate is added. 5. **Detection:** After washing to remove unbound Ac, a specific substrate is added to produce a color. The intensity of the color is proportional to the quantity of Ac. ### ELISA SADWITX * **NO COMPETITIVE** * **For Ag detection** **Types**: * **DAS (Direct Antigen Sandwich)** * **HADAS (Heterogeneous Antibody Double Antigen Sandwich)** **Steps:** * **DAS** 1. **Prepare the Well (Specific Ac):** A specific Ac is added to the well bottom. 2. **Add Sample Ag:** The sample containing Ag is introduced to the well. It binds to the specific Ac at the bottom 3. **Add Specific Conjugate (Marked Ac):** The marked specific Ac is added. It binds to the Ag. 4. **Add Substrate:** The substrate is added. 5. **Detection:** After washing to remove unbound Ac, a specific substrate is added to produce a color. The intensity of the color is proportional to the quantity of Ag. * **HADAS** 1. **Prepare the Well (Marked Specific Ac):** A marked specific Ac is added to the well bottom. 2. **Add Sample Ag:** The sample containing Ag is introduced to the well. It binds to the specific Ac at the bottom 3. **Add Anti-Anti-Ac:** The Anti-Anti-Ac is added. It binds to the specifically marked Ac at the bottom. 4. **Add Substrate:** The substrate is added. 5. **Detection:** After washing to remove unbound Ac, a specific substrate is added to produce a color. The intensity of the color is proportional to the quantity of Ag. ### ELISA Competitiv/d'Inhibició: * **COMPETITIVE** * **For Ag or Ac detection** **Steps:** 1. **Pre-incubation of the sample with Ac*:** * The sample containing Ag is pre-incubated with the marked Ac. * The mixture is then added to a well with fixed Ag. * The fixed Ag and sample Ag compete for binding to the marked Ac. 2. **Add Substrate:** The substrate is added. 3. **Detection:** * After washing to remove unbound Ac, a specific substrate is added to produce a color. * The intensity of the color is INVERSELY proportional to the quantity of Ag in the sample. ### EMIT: * **Homogeneous** * **COMPETITIVE** * **Enzyme Multiplied Immunoassay Technique** **Steps:** 1. **Sample Ag is mixed with marked Ac (Ac*) and substance.** * If the sample Ag is *present* in the mixture (*forms part of the immunocomplex*) then the Ac* is inactive because its binding site is occupied. * If the sample Ag is *absent* then the Ac* is active and will be added to the substrate. 2. **Addition of Substrate:** * The substrate is added. * If Ac* is active, the enzymatic reaction occurs and a color change is detected. 3. **Detection:** * The intensity of the color is INVERSELY proportional to the quantity of sample Ag. ### RADIOINMUNOASSAJOS * **Use radioactive isotopes as markers.** * **Isotopes should have high specific activity, high energy production, intermediate half-life, easy access and no interference with the Ag-AC reaction.** **Types:** * **Radioimmunoanalysis (RIA)** * **Heterogeneous** * **Precipitates the immunocomplex with insoluble reagents**. * **Classic Method.** **Steps** 1. **Sensitization:** The specific Ac is added and binds to the solid support. 2. **Incubation and Competition**: The sample containing Ag is added along with the radioactively marked Ac. 3. **Washing:** Unbound Ag and marked Ac are washed away. 4. **Counting:** The amount of radioactivity bound to the solid support is measured. * This can be done by counting the radioactivity of the solid support directly, or by using a scintillation counter to measure the radioactivity of the liquid after removal from the solid support. * The amount of radioactivity is inversely proportional to the amount of Ag in the sample. * **Radioimmunometric Analysis (IRMA)** * **Heterogeneous** * **Solid-phase based.** * **Two types: Competitive and sandwich** **Steps** * **Competitive** 1. **Pre-incubate:** The sample containing Ag is incubated with the radioactively marked Ac. 2. **Add to the Solid Phase:** The mixture from step 1 is added to a solid support containing a specific Ac bound to the support. This Ac is for binding with the sample Ag. 3. **Washing:** Unbound Ac* is washed away. 4. **Counting:** The amount of radioactivity bound to the solid support is measured. * The amount of radioactivity counted is inversely proportional to the amount of sample Ag. * **Sandwich** 1. **Sample Ag:** The pre-incubated sample containing Ag is added to the well with fixed Ac. 2. **Add Specific Ac*:** The radioactively marked specific Ac is added to the well. It will only bind if the Ag and the fixed Ac are already bound together. 3. **Washing:** Unbound Ac* is washed away. 4. **Counting:** The amount of radioactivity bound to the well is measured. * The amount of radioactivity counted is directly proportional to the amount of sample Ag. ### FLUOROINMUNOASSAJOS (FIA) * **Use fluorescent dyes for marking.** * **Fluorescent compounds produce a high intensity signal but are still weak in the presence of a strong light source.** **Types:** * **Fluoroimmunoanalysis of Polarization of Fluorescence (FPIA)*** * **Homogeneous** * **Competitive** * **Quantifies low levels of compounds such as drugs and hormones.** **Steps:** 1. **Sample Ag is mixed with a fluorescent marked Ac (fluorophore).** 2. **Ag competes with the fluorophore for binding to the specific Ac.** 3. **The fluorescence emitted will be higher if the Ag in the sample is high and lower if the Ag in the sample is low.** ### INMUNOASSAIG MICROPARTICULAT (MEIA) **Steps:** 1. **Add sample Ag to latex microparticles coated with specific Ac.** 2. **Incubate to allow the formation of Ag-Ac complex.** 3. **Wash away unbound components of the complex.** 4. **Add an enzyme-linked specific Ac (Ac*).** 5. **Add substrate, such as MUP, which is catalyzed by Ac* to produce a colored product.** * The intensity of the color is proportional to the amount of Ag in the sample. ### INMUNOASSAJOS QUIMIOLUMINISCENTS (CIA) * **Use chemical compounds that produce chemiluminescence.** **Types:** * **Magnetic Chemiluminiscent Immunoassay (CMIA)** 1. **Magnetic particles coated with specific Ac.** 2. **Capture Ag from the sample.** 3. **Add a second chemiluminescent Ac* (Ac* conjugated with a chemiluminescent substance).** * The chemiluminescent substance can be a luminol derivative or a luciferin derivative. 4. **Measure the chemiluminescence signal.** * **Immunochromatographic Assay (Lateral Flow Assay)** **Steps:** 1. **Add a sample containing Ag to a sample pad.** 2. **The Ag will be transported, along with specific Ac** and **the chemiluminescent dye (Ac*)** through a porous membrane by capillary action. 3. **The Ac* will bind to the specific Ac to form an immunocomplex** * This will be visualized as a colored line on the test strip. ### INMUNO TRANSFERENCIA * **Used with DNA, RNA or Proteins.** * **Results are more sensitive and specific.** **Steps:** 1. **Prepare the sample (lysed cells/ tissue samples).** 2. **Separate proteins by electrophoresis according to charge and size.** 3. **Transfer the separated proteins to a membrane.** * This is done by using an electric current to move the proteins from the gel to the membrane. 4. **Block membrane to reduce non-specific binding.** 5. **Add specific antibody (primary antibody) to the membrane** * This will bind to the target protein. 6. **Add a secondary antibody (conjugate)** * This antibody is specific to the primary antibody and will bind to the primary antibody even more strongly. * The conjugate is often tagged with a fluorescent dye or enzyme. 7. **Detect the presence of the signal** * A fluorescent dye will emit a signal when exposed to a specific wavelength of light. * An enzyme will catalyze a reaction that produces a color change.