The use of absorbance values for dose-response curves

Visual inspection is still widely used for titration. This method is very simple, but relies heavily on the skill of the operator due to subjectivity. Results of this nature are at best semi-quantitative and generally acceptable only for qualitative investigations. 

In quantitative determinations usually the optical density of the product is measured. Absorbance values, obtained for different dilutions of the antibody or antigen of known concentration, mostly yield sigmoidal calibration dose-response curves. Although this procedure seems rather simple and straightforward, it is not. Enzyme kinetics may be influenced by any of the factors discussed in Chapter 9. Correlation of absorbance with sample dilution beyond the linear 

After prolonged reaction periods, dose-response curves for solid-phase EIA become steeper, although the detection limit remains virtually unchanged. After the optimum sensitivity has been reached, a steeper slope rapidly increases the coefficient of variation of the response ( = ACx d/ /dC), and so decreases the span within which reliable results may be obtained. 

Difference between dose-response curves for antigens and for antibodies in activity amplification assays 

Concentration of the antigen can be readily determined from a standard dose-response curve. The range of antigen concentration rarely exceeds two logic dilutions and is, therefore, much smaller than for different antisera. Microcomputers or desk-top programmable calculators, interfaced with microplate readers, may help to linearize rapidly the dose-response curves and to transform the values obtained for the test-samples into concentrations. In contrast, dose-response curves for antibodies are complicated by a variety of factors  

---