Optimization and Validation Neutralizing Antibody Assays

In either approach, if the goal is to compare close titer values (such as eightfold difference between titers of two ADA samples), it is important to apply the interpolation method to determine the titers, or if a noninterpolation method is used, the serial dilutions should be narrowly spaced (ideally twofold or less). In addition, an objective method is needed for determining the ability of the assay to differentiate titers (titer precision). This titer precision is defined by the minimum significant ratio (MSR), which is the smallest fold change between the titers of any two ADA-positive samples that can be considered as significant. For example, MSR = 5 means that at least a fivefold difference in the titer results between any two positive samples will be required to conclude that they are statistically different. Evaluation of MSR is mostly relevant when comparison of samples with close titers is of interest. 

The determination of MSR for titer precision requires dilution profile data of at least five high-positive mock samples, diluted past the screening cut point, from at least three independent runs by two or more analysts (if relevant). The mock positive samples are drug-naive samples from individual donors spiked with a high concentration of the ADA-positive control. The titer results of all these mock positive samples from different runs should be log transformed and analyzed to obtain the overall standard deviation. This estimate of overall SD is then used to determine the MSR of the titer results, where MSR = 10A[2 sqrt(2) SD], assuming that base 10 was used in the log transformation of the titer results. In addition to the practical usefulness of the MSR concept in this application, an attractive feature is that it applies to any range of titer results. This is because the variability in log scale tends to be quite similar across the entire range of titer results. 

The following points should be noted in the evaluation of MSR (1) because the mock positive samples will generally have different characteristics from the real ADApositive patient samples, the MSR determined from this approach may not be a fully accurate reflection of the variability of patient titer results and (2) the use of mock positive samples rather than a pooled positive control is stressed here due to the relevance of both the biological and analytical variability in the interpretation of MSR. 

Refer to Table 8.1 for solutions to common problems observed with development of ADA assays using LB As. 

High background response Poor blocking of solid Evaluate alternate 

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