Thorough analytical verification of an assay is of great importance in the qualification of biomarkers for clinical research and therapeutic development. The Conferma™ Sandwich ELISAs are created with a focus on their critical components. The monoclonal capture and detection antibodies and accompanying recombinant protein standard are rigorously evaluated from lot-to-lot using physiochemical characterization techniques. Combined with finished kit quality control (QC) analysis, the start to finish Conferma™ ELISA validation provides a high degree of consistency with each lot to give you confidence in your research.
Figure 1.Three lots of IL-6 ELISA run independently. The curves show excellent similarity.
A sandwich ELISA is a common enzyme-linked immunosorbent assay (ELISA) procedure that measures the antigen level of a sample. The antigen is “sandwiched” between two antibodies, the capture antibody and the detection antibody. These antibodies can be either monoclonal (mAb) or polyclonal antibodies, however our Conferma™ ELISAs utilize mAbs. Sandwich ELISAs are considered to be one of the more sensitive and specific types of ELISAs as two unique epitopes are required to determine that the correct protein has been identified.
Figure 2.Illustration of how sandwich ELISAs work.
Learn about troubleshooting common ELISA problems in our ELISA Troubleshooting Guide.
The immunoassay has undergone many re-inventions over the last few decades, moving from single to multiple analyte detection with increasing levels of sensitivity and specificity. Over time there has been an influx of commercially available kits by numerous vendors. The majority of these kits fall within the Research Use Only (RUO) category and are required to be labeled as such by the U.S. Food and Drug Administration (FDA). However, unlike In Vitro Diagnostic (IVD) assays, there are no regulatory requirements for the validation and confirmation of RUO assays. With no requirement for validation, the burden of responsibility falls to the end-user to evaluate and accept the manufacturer’s claims. This has led to a range of commentary by both academic and industrial assay users, reflected in the 2015 paper by Khan et al., who are looking to provide a framework for commercial assay validation in the hands of a researcher.1
A key aspect of an assay’s analytical validation is the evaluation of lot-to-lot reproducibility, which has often been traced back to the variability within the assay’s components. Papers published by King et al. in 2014 and O’Hara et al. in 2012 indicate strategies that may be used to generate and monitor critical reagents used in ligand binding assays, which could be applied to both in-house and commercial vendors.2,3
To address these concerns, the Conferma™ ELISAs are built with the following key principles:
Developing the critical reagents in-house ensures control of both the supply and the manufacturing. It also provides the ability to obtain the samples necessary to send to our corporate Center of Excellence for Mass Spectrometry in St. Louis, MO, who utilize 1D Gel Analysis, Amino Acid Analysis (AAA), Liquid Chromatography–Mass Spectrometry (LC-MS), Reverse Phase – LC-MS (RP-LC-UV-MS), and Surface Plasmon Resonance (SPR, Biacore™ T-200 system), where appropriate, to characterize and compare the reagents (pre- and post-biotinylation in the case of the detection mAbs) lot-to-lot.
The characterization of the critical reagents provides for an extremely granular evaluation of the materials lot-to-lot. The assessment (using AAA, Mass-Spec, and 1D-Gel Analysis) of the recombinant protein calibrator (used in the standards and QCs) allows us to confirm that there is no change in mass or sequence coverage. Additionally, the work done on the mAbs confirms both the consistency of the mass and an assessment of the biotin bound to the detection antibody, which can be reflected in the optical density (OD) of the standard curve.
An important test within the process is SPR carried out on a Biacore™ T-200 platform. This test allows us to understand the relationship between the mAbs and the calibrator. We are looking for the mAbs to have, and maintain, a high affinity against the calibrator with an equilibrium dissociation constant (Kd) <0.5 nM. The activity of each lot of mAb should also be consistent lot-to-lot. This technique is carried out pre- and post-biotinylation in the case of a detection mAb to judge the effect of the preparation. The purpose of the SPR testing is to verify that the biotinylation did not impact the affinity of the mAb and to confirm post-biotinylation consistency from batch to batch.
When each batch of raw materials is confirmed, they are then utilized by the manufacturing group to create individual lots of assays. While the mAbs and recombinant proteins can be stable and stored for a long time, careful QC procedures are required to ensure that the new lots produced from the raw materials are consistent with previous lots across all QC parameters (e.g. component relative potency and curve mathematical parallelism). The Conferma™ assays add finished kit biological sample testing, with kits from the new and prior lot run against a library of samples to ensure that they are effectively bridged.
The details and data from the process outlined here are made available for evaluation in two forms, firstly a Verification Report is produced which details the work done in establishing the assays reagents, feasibility, and testing. Secondly, each lot of assays has a Certificate of Analysis consisting of the component lot details, the expected OD of the standard curves, and details of the sample testing.
Discover more details on this whole process with our Conferma™ IL-6 ELISA application note.
By establishing this process, we are looking to address a key need within the industry -- establishing assays with a high level of lot-to-lot consistency, anchored by consistent critical reagents and evaluated sample bridging. As we develop assays based on this process you will find them, and their accompanying documentation listed on this page in the table below.