Q4. You are running your first ELISA and make some mistakes. What would happen i
ID: 3513697 • Letter: Q
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Q4. 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? a) You only add 50ul instead of 100 ul of both standards and experimental MC 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 reagentExplanation / Answer
a) ELISA technique is very sensitive and reliable for detection of quilitative and quantitative data for antigene detection.For this we have to first make the standard curve via detection of known conc of antigen and than we can detect the unknown conc( experimental) of the sample. if we can take sample conc for both 50 ul instead of 100 ul will not create any problem.
B) In ELISA process ,we first put the antigen conc and then put the antibody tagged with HRP- a enzyme that convert TMB courless subtract into coloured form.Than we perform washing to remove unbound antibody.so only antigen-antibody complex will remain. When we put TMB and measure the intensity of the coloured substrate,we can find antigene conc.But if we use same tip for HRP antibody and TMB than aleady remaining antibody can convert TMB into coloured product and it can give false result.
c) generally it is recommended to don't keep plates long with antigen but it will not harm you data beacuse you have only antigen and that you can't ca detect.
d) it is recommended to standardized the incubation time for TMB substart .if you incubate long time .it might produced more background and that will not give a stabdars result .
E)in Elisa .it is recommended that put enough antibody. when antigen bind with the antibody .nexr step is to remove unbound antibody.if you will not do proper washing.it might possible that some unbound antibody will remain there and it will might give false result
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