E!114334 C-LITE - Improved human free light chain diagnostic immunoassay reagents

Serum FLC measurements are increasingly relevant in the screening for monoclonal gammopathies, prognostic stratification and monitoring of therapy responses. The techniques of protein electrophoresis are gradually being replaced by turbidimetric platforms. Analytical issues are documented for commercially available sFLC immunoturbidimetric assays related to specificity, sensitivity, linearity, and lot to lot and inter-analyzer variation. Our aim was to develop, validate, and launch new reagents and calibrators for a turbidimetric test (PETIA) of free light chains in serum (sFLC) that can be analyzed on open clinical chemistry instruments. Reagents necessary for the final PETIA product have been developed during the project: high affinity IgY antibodies specific for sFLC kappa and lambda have been produced and verified by proteomics. The IgYs have been conjugated with optimized nanoparticles and paired with a suitable assay buffer. Calibrator and control materials have been produced and buffers have been optimized. The new PETIA is being evaluated at two independent clinical sites for validation of sensitivity and reproducibility, method comparison, and sFLC reference range determination using the new sFLC PETIA. The long-term potential outcomes of the C-LITE project are improved diagnostics of plasma cell disorders, which is likely to have a profound effect on patient management and prognosis.

The aim of the C-LITE project was to develop novel, specific, and sensitive turbidimetric reagents for sFLC that can be run on open clinical chemistry platforms. This was to be achieved by addressing the following sub-goals: 1. Produce and purify high-affinity IgY antibodies that are specific for sFLC kappa and lambda and are verified by proteomics 2. Optimize nanoparticle size and surface chemistry for use with the IgY in the assays 3. Develop reagents and buffers for a turbidimetric immunoassay for rapid detection of sFLC 4. Produce proteomics-verified FLC calibrator and a control-reagents set to assist standardisation of FLC immunoassays 5. After project completion, deliver validated reagents for the quantification of sFLC using open turbidimetric platforms Goals achieved: 1. Stabilized FLC kappa and lambda antigens that give a good immune response were identified and prepared. Procedures to produce large quantities of high-affinity, mono-specific FLC kappa and FLC lambda IgY were optimised and verified by proteomics. Results demonstrated that the antibodies bind their target FLC with low cross-reactivity and high specificity. Goal complete. 2. Antibody-coated nanoparticles giving best signal were prepared using optimal size and surface chemistry. Goal complete. 3. Assay reagents and buffers were developed tor optimal stability and measurement range to be used in a rapid sFLC turbidimetric immunoassay (PETIA) using the antibody-coated nanoparticles. Assay procedures (application notes) were established and successfully tested on three open clinical chemistry platforms. Goal complete. 4. The final formulation and calibration range of the proteomics-verified calibrator material was documented and meets the design specifications. Testing was performed on three open clinical chemistry platforms. Stability of the calibrator and control materials was demonstrated, and storage conditions defined. Goal complete. 5. The new PETIAs for sFLC kappa and lambda are being evaluated at two independent clinical sites for validation of sensitivity and reproducibility, method comparison, and sFLC reference range determination using the new sFLC PETIA. A further 3 sites are eager to test these sFLC PETIAs. Conclusions The C-LITE project developed novel, specific and sensitive rapid PETIAs for sFLC kappa and lambda for use on open clinical platforms. During the project, we identified a major reason for the documented analytical issues for commercially available sFLC immunoturbidimetric assays related to specificity and linearity. These findings have been communicated to clinical partners and will be disseminated to the greater clinical audience via a manuscript in a peer-reviewed clinical journal. The long-term potential outcomes of the C-LITE project are improved diagnostics of plasma cell disorders, which is likely to have a profound effect on patient management and prognosis.

Serum FLC measurements are increasingly relevant in the screening for monoclonal gammopathies, prognostic stratification and monitoring of therapy responses. The techniques of gel precipitation are gradually being replaced by turbidimetric platforms. Analytical issues are documented for commercially available sFLC immunoturbidimetric assays related to specificity, sensitivity, linearity, and lot-to-lot and interanalyzer variation. We will address these issues through the astute selection of the antibody and antigens used. The C-LITE project will develop novel specific, and sensitive turbidimetric reagents for sFLC that can be run on open clinical platforms.