Immunoassays are easy to run, but the path to develop these tests can be tricky. To make your own reliable, sensitive, and specific ELISA tests requires knowledge of immunology and protein chemistry, not to mention an understanding of all the different components of the test (buffers, stabilizers, stop solutions, diluents, and more). To optimize your immunoassay development, keep in mind these five critical steps:
Minimize background and promote higher specific signal
There are a variety of methods to promote a strong signal while minimizing nonspecific binding and background noise. First, use an efficient plate coating buffer to provide a stabilized coating of the antibody or antigen on the microtiter plate. These plate coating buffers stabilize coated proteins by maintaining their tertiary three-dimensional structure, allowing for greater binding reactivity with the detection molecule and enhancing the specific signal. Next, use a blocking buffer to block assay wells. This blocking of unoccupied space in the plate wells prevents nonspecific binding of sample and assay components and reduces the overall background signal. Similarly, using the proper sample diluents will dilute the samples to read within the functional range, minimize sample matrix effects, block nonspecific conjugate binding, and inhibit complement and thrombin activity, all of which reduce background noise and optimize signal. Conjugate stabilizer diluents will also inhibit nonspecific binding, minimizing background signal. Finally, effective wash buffers are needed to rinse microtiter plates between each reagent addition step in an immunoassay. The use of these methods provides an overall improvement in assay sensitivity by reducing background noise.
It is necessary to reduce plate to plate variability when preparing immunoassays. Protein stabilizing buffers allow plates to be prepared in batches to be used over time, which increases consistency and provides increased plate to plate precision over extensive storage periods.
Increase assay reproducibility
To increase the reproducibility of any custom ELISA or immunoassay, it is important to source reagents from a consistent and reliable supplier. Look for established companies that offer strong customer support, an easy-to-use purchasing system, and fast shipping options.
Stabilize protein conjugates
Not only do conjugate stabilizer diluents inhibit nonspecific binding to reduce background noise, but they also preserve enzymatic activity and protein conformation of enzyme conjugates. In addition, stabilizing conjugates enables you to store conjugated proteins and antibodies for future use, prepare batches of diluted, ready-to-use conjugate aliquots, and reconstitute lyophilized conjugates while preserving native protein configuration and activity.
Using dependable stabilizing and blocking buffers to stabilize proteins allow you to store your prepared microtiter plates. Depending on the type of protein, properly prepared plates can be stored under proper conditions for several months or even years if prepared using reliable reagents.
ICT’s Antibody-Sandwich ELISA Development Kit provides all the necessary reagents to assist you in developing and optimizing your immunoassays. From coating buffer to substrate stop solution, this kit contains enough reagent to construct ten 96-well ELISA plates. In addition, a comprehensive ELISA development manual is supplied with protocols for assessing initial assay feasibility and optimizing ELISA performance parameters, which will be beneficial to both novice or experienced users. This kit is convenient, economical, and backed by the dedicated ICT technical support team.