Inhibitors may alter $V_{\max}$, the maximum rate of reaction, and $K_m$, the Michaelis-Menten constant, of HRP. Fig. 6.2 shows how reaction rate of HRP changes with substrate concentration. Complete Fig. 6.2 by drawing a curve to illustrate how a non-competitive inhibitor will affect the rate of reaction of HRP. Use the curve you have drawn to obtain an estimate of $K_m$. $K_m = \ldots\ldots\ldots\ldots\ldots\ldots\ldots\ldots\ldots\ldots$
Scientists can design synthetic DNA nucleotide sequences to make a synthetic HRP gene. These sequences will contain a start codon and a stop codon so that translation of messenger RNA (mRNA) can take place. Explain what is meant by a start codon and a stop codon.
Complete Fig. 6.4 to show the contents of the test well after step 5.
With reference to Fig. 6.3, describe one way in which the capture antibody differs from the detection antibody.
Fig. 6.5 outlines the reaction catalysed by HRP in step 6. The organic substrate, TMB, is in excess and changes colour when it is oxidised. This indicates a positive test result. $\text{TMB} + \mathrm{H_2O_2} \xrightarrow{\text{HRP}}$ oxidised TMB + water TMB(colourless) $\rightarrow$ oxidised TMB(blue-green) A student suggested that: • a low concentration of toxin may be diagnosed as a negative result instead of a positive result • using a colorimeter after step 6 in the sandwich ELISA test would provide a quantitative result and help to avoid this error. With reference to Fig. 6.5, explain why using a colorimeter would provide a quantitative measurement for detection of a low concentration of the toxin and help to confirm a positive result.