Enzyme linked immunosorbant assay (ELISA) is a popular biomedical technique, developed around 40 years back, mainly used in clinical diagnosis to detect disease biomarkers. It takes the advantage of specific interaction between antigen and antibody. The enzyme used in this technique convert substrate molecules into product molecules that are detected in a variety of ways. Because the reaction is enzyme catalyzed, product molecules are generated continuously, it amlifies the assay signal so that low concentrations of analytes can be measured . Basics of ELISA can be found in wiki.
There are two different ways to monitor the assay signal in ELISA. They are end point ELISA and kinetic ELISA. In end point of ELISA, the enzyme reaction is stopped after certain time (usually 30 min) by adding some quenching agents (generally acids or bases or some other enzyme inhibitors) and the signal generated is recorded. This gives just one data point for one sample. Most of the commercial microtiter plate formate ELISA kits follow end point ELISA. But this system has several disadvantages as listed below.
- Even after the addition of quenching solution the reaction can continue, if the contents are not mixed well.
- Optical properties of product molecule may change (less colored) by adding acid or base for quenching.
- Main disadvantage for quantitative analysis is that it has to rely on single data point and this can lead to wrong result.
- If the background is high, it would give high signal for sample. Also, variations in background signal among the assay chambers lead to inaccurate results.
Most of the disadvantages listed above can be overcome by using kinetic ELISA (k-ELISA). In my PhD research, I am working with kinetic ELISA on microfluidcs chips. In this case, the enzyme reaction is monitored continuously (in our lab weI take readings every 5 min). A graph is plotted as assay time on x-axis and signal on y-axis. The slope of the linear line correspond to the concentration of the analyte. Such linear lines are obtained for each different dilutions or concentrations of sample. This type of ELISA depends on several data points for each assay which is more quantitative. And also even the background is different, it doesn't really matter. What matters is the slope of the line. It also eliminates quenching step.