Proprietary AnySource® Sample Dilution

Optimized sample dilution buffers and procedures

Sample dilution is a powerful tool in the optimization of immunoassays. Developed in parallel to its ultra sensitive Imperacer® platform, Chimera advanced AnySource® sample dilution technology to take full advantage of Imperacer’s® strong tolerance for sample dilution. The minimum required dilution (MRD) effect - optimal immunoassay performance in a diluted sample - is well known regarding immunoassays in general. Ultra sensitive platforms providing a broad assay range such as Imperacer or Simoa, however, allow for greater sample dilution. This gives rise to rare matrix or microsampling study support with extremely reduced sample consumption at sensitivities still better than conventional immunoassays. In this regard, Imperacer might be favorable due to technical factors which lead to less required sample per well.

In a design of experiment (DOE) approach, buffer conditions are optimized by running dilution experiments in five standard AnySource® diluents. Each standard buffer represents a family of AnySource® sample dilution buffers (SDB). Based on the experimental outcome, a customized AnySource® SDB buffer for the particular assay is identified from our library or developed according to the buffer attributes which performed best. AnySource® is also employed for fine tuning or converting an assay, e.g. from being ultra sensitive to microsampling support with minimal sample consumption.

The handling and storage of low volumes of biological matrices is challenging. Pipetting of volumes below 10 µl can lead to higher variability, strong evaporation effects, and sample loss in frozen storage. Even though at Chimera we are highly trained and well equipped to handle sample volumes down to 0.5 µl, we acknowledge the basic requirements and limitations in preclinical and clinical trials.

To analyze a trial’s biological matrix and required parameters, a mutual AnySource® storage buffer is developed. This diluent is designed to be compatible with stability requirements for long-term frozen storage of the analyte in the diluted specimen, as well as with downstream assay requirements. Pipetting volumes can be adjusted in regard to available sample volume and minimal pipetting volume that can be adjusted to study requirements.

  1. Taking backup volume for potential re-testing into account, dead volume is avoided and only the sample material needed for the bioanalytical assays is shipped to Chimera. Residual material can be used for additional procedures or be stored with the sponsor or central lab.
  2. Upon receipt of the pre-diluted sample at Chimera’s GLP/GCP compliant analytical facility, working aliquots are prepared. Once more, the amounts pipetted are typically in manageable volumes of more than 10 µl.
  3. Each working aliquot can be diluted a second time in assay specific AnySource® sample dilution buffer (SDB), converting the buffer conditions from storage prerequisite to optimal analyte binding for the particular antibody pair used in the actual assay.
  4. Finally, quantification data are generated from parallel assays run in duplicate wells. In contrast to typical multiplexing immunoassays, this Polyplexing® approach ensures the highest possible, antibody intrinsic specificity as antibody cross talk is completely avoided  

Depending on sensitivity requirements, typically 10 analytes (e.g. drug plus 9 biomarkers) can be tested from 25 µl of sample. Taking backup volume into account, 50 µl of neat sample volume is sufficient for testing of 10 analytes in parallel, duplicate well assays with single digit pg/ml sensitivities each.

Please read further in this rare matrix case study from Ophthalmology drug development support.