21107957
| Question | Answer |
| Measuring affinity: Radio-binding assays | 1. drug is labelled with a radioactive isotope, allowing us to detect and measure the amount of drug bound in tissue samples, as well as the number of specific binding sites, and the affinity of the drug for these sites. 2. Brain is homogenised (blended up) & divided equally in 2 test tubes. 3. Brain is then incubated with varying concentrations of radiolabelled drug or varying concs of radiolabelled drug plus an excess of non-labelled, (cold) drug. 4. The samples are then filtered and washed, leaving only the drug that is bound to the brain 5. The amount of radioactivity can then be measured for each sample. 6. For the 1st set of tubes, plot the amount of binding against the concentration of labelled drug, giving a plot of total binding. - Non-specific binding can be plotted using the set of tubes with the unlabelled drug. A measure of specific binding can be found by subtracting the number of non-specific binding from total binding, using a saturation plot, can then be used to calculate the max number of receptors per tissue (Bmax) and the affinity of the radio-labelled drug (Kd) |
| Measuring antagonist affinity: Dose ratio | - The extent to which agonist concentration is shifted by antagonist relates to the affinity of the antagonist for the receptor - The shift in curves can be measured using the 'dose ratio' - Dose ratio: ratio of the concentration of agonist producing the same response in the absence and presence of antagonist - For a reversible competitive antagonist, the dose-ratio should increase linearly with the conc of the antagonist |
| Measuring antagonist affinity: PA2 of antagonist | PA2= how the affinity of a reversible competitive antagonist is quantified; the negative log of the molar conc of antagonist that requires you double the agonist conc to produce the same response. - Obtaining PA2 by experiment: 1. Construct log conc-response curves for agonist in the presence of several concs of antagonist 2. Form the above, calculate the PA2 using schlid analysis Schlid equation: log(dose-ratio-1)=log(antagonist conc)-logKB KB= dissociation equilibrium constant measured using schlid analysis (similar to Kd) - A reversible competitive antagonist in a plot of log (dose ratio-1)vs log antagonist conc (schlid plot) should have a gradient of 1 and intercept the x axis at (logKB) PA2=PKB=-logKB |
| Steps in producing a Schlid plot | 1. construct log conc response to agonist in the presence of varying levels of antagonist 2. Use curves to read EC50 values for agonist in the absence and presence of different concs of antagonist 3. Calculate EC50 values for the agonist in the presence of varying concs of antagonist 4. Calculate dose ratio for each conc using: EC50 in presence of antagonist/EC50 in absence of antagonist 5. Plot log(dose ratio-1) versus log (antagonist) |
| Important points | - If the slope of a schlid plot does not equal 1, suggests antagonism is either non-competitive or non-reversible -PA2 for an antagonist is independent of agonist used, as it is only a measure of antagonist affinity for receptor. |
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