480177
Description
Flashcards by sophietevans, updated more than 1 year ago
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Created by sophietevans
over 10 years ago
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| Question | Answer |
| Which antibody was being investigated in this lab? | HIV antibody. |
| Which antigen was used to coat the plate? | HIV P120 surface protein. |
| Which enzyme-linked antibody was used to bind to any anti-HIV IgG? Which enzyme was attached? | Goat anti-human IgG linked to alkaline phosphatase was used. |
| Which chromogenic substrate was used? | Para-nitrophenylphosphate (PNP phosphate). |
| What is a chromogen? | Any chemical that can change colour in the presence of an enzyme. |
| What type of technique is an ELISA? | It is both qualitative and quantitative, and it is highly specific. |
| What type of dilutions were performed on the serum samples? | Doubling dilutions. |
| What is the importance of washing and blotting in an ELISA? | The plates must be washed in between steps to maintain the specificity of the technique. Ideally, one antigen should bind one antibody attached to one enzyme so that the result is proportional to the antigen present. Ultimately, all non-specific antibody and all unbound antibody/substrate will be removed. The blotting is significant in that it helps remove the above, but also helps remove PBS-Tween to prevent the detergent from affecting binding, and it removes any bubbles that might be present from detergent/pipetting which would interfere with the binding and the reading. |
| What was the point of the positive control wells? | These confirmed that a colour change was taking place (in case there was something wrong with the substrate/enzyme, which would not be clear from just a plate without results) and also confirmed if the pipetting had been consistent - there should have been a constant amount of antibody and therefore enzyme present, so the absorbance should be constant, or pipetting variability could have made the other results inaccurate. |
| What was the point of the negative control wells? | These detect 'background' absorbance, and also that which results from inefficient plate washing, and the value produced should be subtracted from each sample value to demonstrate how much of this absorbance is a result of other factors, and to give a true reading. |
| What is the limit of detection? | The point at which a chromogenic compound begins to be detected by the spectrophotometer, the smallest concentration of antibody/antigen required for a visible result to be recorded. |
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