You are running your first ELISA and make some mistakes. What would happen if each of the following happened in isolation? Would the mistake inherently make the data unusable? (Tip: You might find the trouble shooting table from an ELISA protocol helpful, one is posted to ICON) a) You only add 50ul instead of 100 ul of both standards and experimental samples b) You use the same pipette tips to add the HRP-detection antibody and TMB substrate. c) You forget about your plate after adding your sample to a pre-coated plate and it incubates for an extra 4 hours. d) You forget about your plate after adding the TMB substrate and it incubates for an extra 4 hours. e) After adding the HRP-Detection antibody, you are so excited to see the result, you decide to wash just once and then add the TMB reagent.
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- What are the ordered steps of an ELISA protocol? A. Add primary antibody->wash-> Bind sample to a surface ->Add substrate ->Add secondary antibody-enzyme conjugate ->wash B. Bind sample to support -> Add substrate -> Add primary antibody -> wash -> Add secondary antibody-enzyme conjugate -> wash C. Bind sample to a surface -> Add primary antibody -> wash -> Add secondary antibody-enzyme conjugate -> wash -> Add substrate D. Add secondary antibody-enzyme conjugate -> wash -> Add primary antibody -> wash -> Add substrate -> Bind sample to surfaceExplain why a semi-log plot should be used for determining antigen concentration by ELISA.a) did the positive and negative controls work correctly in this ELISA? Explain. B) in the sample wells( wells3-7) some are darker blue than others. Explain what happened here and how you'd interpret the results?
- Many ELISAs that are used to identify the presence of antibody to viruses have a 3rd control (along with negative and positive). In between the rows of wells coated with antigen, are rows of cells coated with tissue culture used to grow the virus. In other words, patient serum is added to wells that contain antigen along with wells that contain tissue culture. Why do you think this might be necessary?ELISA tests usually use a primary and secondary antibody. Why? What are the necessary controls one would need to perform to ensure that the antibody specificities are valid (i.e., no false-positive or falsenegative reactions)?Recombinant Protein G from Streptococcus are covalently attached in an oriented fashion to magnetic beads. Bovine Serum is added and the antibodies are captured by the beads. Using the magnetic device, beads are attached and unbound material is washed away. Antibodies are eluted using a lower pH buffer and the solution is neutralized. The equilibration step is adding the serum sample to the buffer-equilibrated (pH 6) magnetic Protein G beads. What was expected to occur if this step was missed? When we mix the samples on the mixer, it was crucial that there was no foam present in the sample - why is this and what part of the mixture would be "foamy" at this step? Lastly, we use an neutralizing buffer after eluting the samples from the column. Why is this step important?
- What are ELISA assays used for in labs? Give at least three examples.A medical technology intern was assigned to the serology section of a clinical laboratory. His supervisor asked him to prepare a 5% red cell suspension, which will be used to run a hemagglutination inhibition assay. The intern, being aware of the importance of preparing the desired concentration of red cells recommended in the assay, considers the factors enumerated on the table below during the sample processing. If you are the intern assigned to the task, explain why these factors are vital to the procedure and determine their effect on the sensitivity of the test. Factors to Consider Importance Effect 1. Tonicity of the suspending saline solution 2. Overfilling of the tube 3. Proper washing of red cells 4. Too heavy or too diluted red cell suspensionELISA is a vital diagnostic tool. Reagents required for this technique include Thermostable DNA polymerase, RNA primers, and antibodies latex beads, antibodies, glass slide, serum Antigen, Antibodies, microtiter plate, and detection substrate. Thermostable DNA polymerase, DNA primers, and nucleotides
- ELISAs require a number of steps to be completed in order for the test to work. Why do you need to wash your samples at each step? When you added primary antibody to the wells, what happened if your sample contained the antigen? What if it did not contain the antigen? When you added secondary antibody to the wells, what happened if your sample contained the antigen? What if it did not contain the antigen?What are the four steps of an Elisa protocolConjugated enzymes are a key component of ELISA. To what are these enzymes conjugated, and why is that important? To the antigen so the enzymes are only present when the molecule of interest is also present To the plastic of the reaction well so the enzymes can concentrate the molecule of interest To the capture antibody so the enzymes are only present when the molecule of interest is also present To the detection antibody so the enzymes are only present when the molecule of interest is also present