Critical reagents immunoassay development

The expense of an analytical procedure depends upon much more than the cost of the final analysis. Much of the expense of an assay is related to sample preparation, and for many applications immunoassays have tremendously reduced the time needed for sample preparation. Another consideration is the amount of time needed for the development of an assay. The additional expertise which must be developed in an analytical laboratory before immunoassays can be used with confidence may seem formidable, and waiting for an animal to develop antibodies may lead to unacceptable delays in assay development. On the other hand, once a usable antibody titer is obtained, the development of a workable assay is usually straightforward. It is also likely, if immunoassays become accepted for some aspects of pesticide analysis, immunoassay kits or at least critical reagents will become commercially available. Such kits already exist for many pharmaceutical products and hormones, and numerous companies will supply antibodies to a user supplied hapten on a contract basis (83). 

As for all bioanalytical methods applied to support of drug development, validation of immunoassays is important. However, several validation issues need special attention for immunoassays. These include stability of the critical reagents, the curvilinear nature of the calibration curve, the greater variability of immunoassays, and, particularly important, the specificity of the assay. 

The antibody or pair of antibodies selected for the assay format is the primary critical reagent for an immunoassay because it will ultimately define the specificity and sensitivity of the assay. Therefore, whenever possible, as many antibodies or pairs should be tested hand-in-hand with the selection of the assay format before moving into further development. This may include whole or fragmented mono-clonals, polyclonals, and combinations as the assay format demands. The time it takes to develop and purify unique reagent antibodies demands that this start as early in the drug development process as possible. 

Immunoassays are inherently sensitive and specific. However, with continued need to develop increasingly sensitive assays to support preclinical and clinical studies, there have been ongoing efforts to enhance the capabilities of these techniques. Advances in critical binding reagents, detection systems, new assay formats and automation have resulted in improved immunoassay technology. 

Although most immunoassays have used polyclonal antibodies as the critical binding reagents, development of monoclonal antibodies by Kohler and Milstein in 1975,has resulted in their widespread use, particularly in assays for macromolecules. Their unique epitope specificity conveys advantages in double antibody immunoassays for proteins, where one monoclonal antibody may be used to capture the protein by a specific subunit or epitope, and another, directed against a... 

In the development of lab-on-a-chip technology, a key is to develop the ability to pump the liquids and transport sample/reagent molecules as well as biological cells in a microchannel network. This can be achieved by using the electroosmotic flow and electrophoresis. Mixing of different solutions and dispensing a specified amount of one solution from one microchannel into another microchannel are important to many microfluidic chips. There are extensive research works done in these areas [1]. Furthermore, precise control of temperature is often critical to on-chip biochemical reactions. In the following the PCR lab-on-a-chip, flow cytometer lab-on-a-chip and immunoassay lab-on-a-chip will be reviewed. 

---