Lab 12: ELISA Questions

Lab 12: ELISA Questions Why do we need to determine which cell lines produced the highest recombinant FIX? - To reduce the cost of hemophilia treatment. The cell lines produce recombinant FIX and other proteins. Which biomolecule can specifically detect the presence of a certain protein, such as recombinant FX, in a sample full of other proteins? - Immunoglobins In a Direct ELISA, which components are directly attached to the plate? - Antigens (by positive absorption) Which component of Sandwich ELISA is required in pairs? - antibodies in a Competitive ELISA, how would the signal measured by the detector react to an increase of antigen in the sample? - Decrease What type of ELISA is shown in the diagram? - Indirect In this coding step, the capture antibodies are immobilized on the surface of polystyrene microplate wells. What type of force causes the passive absorption of the captured antibody due to the interaction between amino acid side chains on the antigen used for coding, and the plastic surface? - Hydrophobic What are antibodies with high specificity that only detect one epitope called? - Monoclonal The basic blocking buffer contains 5% bovine serum albumin (BSA) dissolved and PBS. What is the purpose of adding a blocking buffer? - To reduce ELISA background signal Twin 20 is used in our wash buffer. What type of compound is Tween 20? - Surfactant Why do we have to load the sample into specific wells? - To achieve valid results. What is the purpose of a standard? - To create a standard curve. The first well contains 200 μL of standard with a concentration of 90ng/mL. If we want to use a dilution of 1:2 dilution factor, how much sample should be transferred from the first well to the second well that contains 100 μL dilutant? - 100 μL Serial dilution with a 1:2 dilution factor. Concentration of the standard in the first well is 90ng/mL. The aliquot volume is 100 μL. The volume of dilutant in well 2-8 is 100 μL each. What is the concentration of the standard in well 8? Take into account that we are diluting the concentration to half in each step. - .07 ng/μL A positive control is a sample known to give positive results for the given test period what is the purpose of the positive control? - Verify that the negative results are valid. What is a group of samples where no response is expected called? This group contains all buffers and reagents except the substance of interest. - Negative control What is the purpose of agitating the ELISA plate? - Increasing the rate of binding Which step do we have to do next? - Adding TMB substrate TMB (tetramethyl benzene) is a substrate for horseradish peroxidase (HRP). What is the proper container for storing TMB? - Light protected container. A stop solution is added to provide a fixed