The Best Pair package is designed to select the best possible antibody pair for the development of a sandwich immunoassay. This option is particularly suitable for metabolite or biomarker immunoassays, providing the analyte to be detected is large enough to allow the binding of two antibodies. For instance, the conversion of a sandwich ELISA to an HTRF® format may require the antibodies used in the original assay to be reassessed and combined with additional ones in order to further improve the performance of the detection.
The diagram below provides a general description of the process' main phases:
The initial phase of the Best Pair package process is the selection of up to 10 antibodies to be tested. This phase is carried out in close collaboration with the customer to ensure antibodies are appropriate for the customer's specific needs. This can typically be completed within 2 weeks, depending on the availability of the antibodies desired.
Upon reception of the antibodies to be included in the study, each antibody is conjugated twice, to both the HTRF donor and acceptor dyes, so that both orientations of each antibody pair can be tested. This process is typically completed in less than 1 week.
Antibody pair testing consists of a comprehensive matrix testing approach that includes:
At the end of the project, the best antibody pairs are delivered to the customer for further assay optimization, along with a detailed report on all data generated.
This project was undertaken for the identification of the best possible antibody pair for a known biomarker. The initial assay was a sandwich ELISA, time-consuming and non-amenable to miniaturization as a format. The conversion to a "mix & measure" HTRF format was requested in order to run the test in a more cost-effective way.
As in most cases, it is critical to test multiple combinations, given that the best antibody pair for an HTRF assay is determined empirically.
The figure on the left illustrates some results of this Best Pair analysis (only 25 pairs out of the 100 pairs that were tested for this project are shown). Five antibodies (Ab1 to AB5) were labeled with both Europium cryptate and d2 acceptor. All the possible donor/acceptor combinations were then tested. AB5-K (cryptate) / AB4-d2 was found to be the antibody pair generating the highest assay signal. This was therefore the project's recommendation for further assay optimization.
|Custom lab. 2 antibodies + best pair test||64CUS2AY|
|Custom lab. 3 antibodies + best pair test||64CUS3AY|
|Custom lab. 4 antibodies + best pair test||64CUS4AY|
|Custom lab. N antibodies + best pair test||64BPP000|
|Best pair additional optimization||63BPP000|