4.40: Competitive Binding

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A laboratory has been performing a competitive immunoassay for the quantitative measurement of hCG in urine. The physicians of the hospital have complained that the lower limit of sensitivity is not adequate for their needs. The assay employs 0.5 mL of urine, 0.5 mL of antibody reagent (diluted 1:10 from the stock), and 0.1 mL of labeled $\beta$-HCG. Under these conditions, approximately 70% of the label is bound to antibody in the absence of competing HCG (% B0 = 70%, see Table 13-9, p. 255).

QUESTION

How can the laboratory increase the sensitivity of the assay?

Questions to Consider

What factors affect the sensitivity of a competitive binding immunoassay?
How do proportional changes in the concentration of antibody and labeled ligand affect the sensitivity of the assay? The precision of the assay?
If only one reagent is diluted, what will be the effect on the binding and assay sensitivity?
If additional sample volume is added, how might this alter the reaction in an unacceptable manner?

Answer

The laboratory can increase the sensitivity of the assay by diluting antibody and labeled, antigen proportionately, or by adding more specimen.

Answers to Questions to Consider

The factors that affect sensitivity are the concentrations of labeled antigen, antibody, and antigen from sample, and the affinity of the antibody for binding the antigen (pp. 248, 251-252).
Decreasing the concentrations of both labeled antigen (or ligand) and antibody proportionately usually changes the binding equation equilibrium (Eq. 13-3 and p. 248) $$Ab + Ag^{*} \leftrightarrow AbAg^{*}$$by shifting the reaction to the left, i.e. dissociation with consequently less total binding.

In this circumstance, the percent labeled antigen bound at zero exogenous ligand (B₀) will become significantly smaller with lower concentrations of Ab and labeled Ag. In this case, less exogenous, unlabeled Ag is required to displace the bound labeled Ag and give 50% binding. This shifts the binding curve to the left, increasing the sensitivity of the assay. The upper limit of linearity will also become less and the lower limit will be extended to a new lower range. Usually, the assay becomes less precise, and there will be an increase in the standard deviation.
Usually it is not possible to increase or decrease the concentration of only one of the two reactants, antibody or labeled ligand, without making the reaction less sensitive. The antigen and antibody are usually titered to be at equivalence. Increasing the concentration of labeled antigen in relationship to the amount of antibody present in the reaction results in the need for a greater amount of unlabeled ligand to yield the same amount of displacement, and the percent bound labeled ligand will decrease in proportion to the excess added. Decreasing the amount of labeled antigen or increasing the amount of antibody results in available binding sites remaining on the antibody. Added unlabeled antigen will bind to these available sites rather than displace bound, labeled Ag; that is, no displacement. This will shift the binding curve to the right. In either circumstance there is a decrease in sensitivity.
Addition of a larger volume of sample to the reaction mixture would theoretically increase the sensitivity of the assay (see #2 above). However, substances such as urine can adversely affect the matrix of the reaction by changing pH, salt concentrations, etc. Therefore, it is not always possible to increase sensitivity by increasing the amount of test sample.