Luminex Multiplex Assays are widely used for supporting several stages of the drug development process. Complexities of such assays present the clinicians with several unique challenges involving adjustments for their validations. This validation often requires a compromise in decision-making for the selection of assay conditions and criteria for its acceptance. The critical parameters involved in these Luminex Multiplex assay validation processes include minimum required dilution, quantitative ranges for specific drug targets, quality control samples, freeze-thaw stability, and cross-talk across assays.
Multiplex assays suffer enormously from the analytical challenges throughout the process of drug development, validation, and the maintenance of these Luminex assay. Luminex multiplex ligand binding assays (LBAs) are routinely employed for measuring all the relevant clinical analytes such as biomarkers and biological drugs. Most LBAs rely on the use of antibodies, such as critical reagents, for capturing and detecting the analytes of interest within a biological matrix.
There are varied challenges associated with the Luminex multiplex assays. These include the selection of the biomarker, its characterization, and validation. Detection ranges, matrix interferences, complex specificity issues, and reagent cross-reactivity are other specific challenges associated with the multiplex assay development. All these challenges fall within the definition of fit-for-purpose Luminex multiplex assay.
Validation parameters for Luminex Multiplex Assays –
The basic parameters for validation of the Luminex Multiplex Assays are as follows –
Method precision and relative accuracy are two key performance characteristics used for describing the magnitude of both the systemic errors and random errors associated with repeated measurements.
At the start of your assay itself, the clinician needs to regulate and establish assay parameters such as intra-batch precision, method accuracy, and inter-batch precision. These parameters should preliminarily be developed during the drug development process and later validated through pre-study validation.
Selectivity is the Luminex Multiplex assay’s ability to measure the analyte of interest accurately, even in the presence of other interferences. Samples derived from multiple individuals of both normal and targeted populations should be evaluated. This provides assistance in assessing the endogenous values of the target biomarker present within each biological sample. Recovery of the reference standard (analyte) spiked into each sample should be evaluated both at high and low levels. This recovery can then be theoretically calculated by subtracting the value obtained from the basal value. Here, the total assay error may be used as an acceptance criterion for determining the spiked values of the respective samples.
Specificity is defined as the ability of a measuring protocol for determining the extent to which the multiplex assay generates a response. The exact target analyte structure for such small biomarkers and their metabolites is known if the biomarkers are small. The acceptance criteria of the small biomarker molecules should be set in such a way that it should resemble the acceptance PK criteria as they obey a similar experimental setup.
The presence of degrading enzymes, time, humidity, temperature, biomarker half-life, matrix, container system, and the storage conditions directly or indirectly affect the stability of biomarkers.