Page 343 - Vitamin D and Cancer

330 B.W. Hollis

especially vulnerable to the matrix effects of any PBA. Anything present in the
sample assay vessel that is not present in the calibrator assay vessel can cause matrix
effects. These matrix effect substances are usually lipid but in the newer direct
assays, they could be anything contained in the serum or plasma sample. These
matrix factors change the ability of the binding agent, antibody or binding protein to
associate with 25(OH)D in the sample or standard in an equal fashion. When this
occurs, it markedly diminishes the assay’s validity. Experience has demonstrated
that the DBP is more susceptible to these matrix effects than antibodies [8]. The
original Haddad procedure overcame the matrix problem by using chromatographic
sample purification before CPBA [1].
Researchers had a strong desire to simplify this cumbersome CPBA for 25(OH)
D, so Belsey and colleagues developed a streamlined CPBA in 1974 [9]. The goal
of this second-generation CPBA was to eliminate chromatographic sample purifi-
3
cation as well as individual sample recovery using H-25(OH)D . However, after
3
several years of trying, researchers were unable to validate the Belsey assay due to
matrix problems originating from ethanolic sample extraction [10].
The 25(OH)D CPBA’s did have the advantage of being co-specific for 25(OH)
D and 25(OH)D and thus provided a “total” 25(OH)D value if the assay was
2 3
valid. The DBP’s binding co-specificity for 25(OH)D and 25(OH)D , as well as
2 3
its stability, made it an attractive candidate for incorporation into automated
direct chemiluminescent assays. In fact, Nichols Institute Diagnostics used this
approach when its researchers developed the Advantage® 25(OH)D Assay. The
U.S. Food and Drug Administration (FDA) approved this assay for clinical use
but Nichols ultimately withdrew it from the market place due to its propensity
to overestimate total circulating 25(OH)D concentrations and its surprising
inability to detect circulating 25(OH)D [11, 12]. Although never described,
2
these problems were probably linked to the DBP’s inability to resolve the matrix
problems associated with direct sample assay. Currently, the CPBA for 25(OH)
D is rarely used. Also, one cannot accurately compare most CPBA results for
circulating 25(OH)D concentrations from the past with values from current
methods because many of the matrix interferences were not linear in the old
CPBA’s.

15.3.2 Radioimmunoassay

In the early 1980s, my group decided that a non-chromatographic RIA for circulat-
ing 25(OH)D would be the best approach to measuring the substance. We therefore
designed an antigen that would generate an antibody that was co-specific for
25(OH)D and 25(OH)D [13]. In addition, we designed a simple extraction method
2 3
that allowed simple non-chromatographic quantification of circulating 25(OH)D.
In 1985 Immunonuclear Corp., now known as DiaSorin, introduced this H-based
3
RIA as a kit on a commercial basis. This RIA was further modified in 1993 to
incorporate a I-labeled reporter and calibrators (standards) in a serum matrix [14].
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