Bad Coefficients of Variation
The reasons for interference differ, but most problems have one thing in common—they are derived from low-to-medium affinity binding. Even HAMAs rarely show high affinity, although they are often described as high-affinity binders.
Assay antibodies and analyte should bind to each other with high affinity. Thus a solution to all interference problems would suppress the low-and-medium binding affinities. At the same time such a solution would keep the highest affinities untouched. This binding affinity discriminator would work similar to a wavelength filter in optical physics.
Candor Bioscience’s (www.candor-bioscience.de) LowCross-Buffer® meets these criteria. It is an assay buffer and antibody diluent that enhances the reliability of immunoassays. When antibodies and analytes are incubated in LowCross-Buffer, high-affinity binding is not negatively affected, but low-to-medium affinity binding is depressed. LowCross-Buffer has been shown to be effective in many biomarker assays.
Its ease of use, general applicability in assay formats like ELISA, Western blotting, immunohistochemistry, protein arrays, immuno-PCR, and others, and its applicability with many different matrices like blood, serum, plasma, urine, CSF, tissue specimen, milk, meat, food extracts, and environmental samples make it a useful new tool.
As a result of the specific characteristics of any analyte or matrix, the LowCross-Buffer has to be tested to be effective in any assay with a new validation. Testing is simple because one has only to exchange LowCross-Buffer for the currently used assay buffer or assay diluent. In competitive assays, however, the concentrations of tracer and assay antibodies have to be titered again exactly to the new assay conditions.
LowCross-Buffer is effective. Figure 1 shows avoidance of false-positives in an ELISA. This ELISA against IgG from guinea pigs is used for immunotoxicological studies. In this assay false positives in the specificity control (row A1–A12) as well as at the blank value (H1–H12) spoiled the interpretation and evaluation.
The use of LowCross-Buffer, on the other hand, prevented the false positives and made the detection of concentrations possible in more rows. More often one has assays that don’t show such significant false-positives, but the precision profiles are not in accordance to the acceptance criteria.
Figure 2a shows the precision profile of a biomarker assay. Standard assay buffers could not ensure reliability over the complete concentration range. The interference in this assay is not easy to understand, because the precision profile does not show an expected graph. Such unexpected precision profiles are well known, though. Due to FDA guidance, these assays are validated in a strict manner showing all the erroneous results.
Switching to LowCross-Buffer results in a better precision profile where acceptance criteria are met. Figure 2b shows the CV over the whole measurement range.