COMPOUND CROSS-REACTION IN THE ACMIA/EVRO ASSAYS
The following tables present assay data on the cross-reactivity of sirolimus and metabolites versus the anti- SRL antibody used in the ACMIA/EVRO assay.
As shown in the tables, some data are missing because ACMIA/EVRO cannot provide reliable
quantification. This is caused by the fact that the assay is programmed to test the same specimen twice when the first determination gives a value of everolimus >10 ng/mL. In the second test, the sample is treated with no displacing agent (FK-506 succinate) in the lysis buffer, which is used to release everolimus from its bonds with proteins.
In fact, as the assay uses no protein precipitation, the solubility of the substrates available for antibody recognition depends on their displacement from the binding sites operated by the FK-506 succinate. Thus, in the absence of this compound in the lysis buffer, there should be no reaction signal. This means that a signal produced without a displacing agent is due to compounds that do not bind to proteins and thus do not compete with FK-506 succinate.
Therefore, the original table has been divided according to the following criteria:
Group “A” compounds: produce signal only with a displacing agent
Group “B” compounds: produce signal with a displacing agent only when the sample is not spiked with EVL
Group “ C” compounds: produce signal regardless of the use of a displacing agent and spiking of the sample with EVL
This peculiar behavior suggests that cross-reactivity data for ACMIA/EVRO are highly dependent on the type of group they belong to. For instance, the cross-reactivity of RAD PSA above 20 ng/mL is affected by the spiking of everolimus, as shown by the presence of signal in the absence of FK-506. In contrast, the sample spiked with the same concentration of RAD SA produces unspecific signal regardless of everolimus and FK-506 succinate.
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GROUP “A”
CROSS-REACTING COMPOUNDS THAT PRODUCE NO UNSPECIFIC SIGNAL IN THE ASSAY
Spiked compound Spiked EVL Cross-reactivity
Compound ng/mL ng/mL %
Sirolimus 5.0 0.0 57.0
5.0 5.2 91.0
20.0 0.0 69.0
20.0 5.2 90.0
40-Phosphatidylcholine EVL 5.0 0.0 64.0
RAD PC 5.0 5.2 86.0
20.0 0.0 96.0
20.0 5.2 98.0
45/46-OH EVL 5.0 0.0 14.0
5.0 5.2 14.0
20.0 0.0 17.0
20.0 5.2 12.0
24-OH EVL 5.0 0.0 26.0
5.0 5.2 24.0
20.0 0.0 21.0
20.0 5.2 17.0
16-O-DESMETIL EVL 5.0 0.0 22.0
5.0 5.2 12.0
20.0 0.0 17.0
20.0 5.2 16.0
11-OH EVL 5.0 0.0 27.0
5.0 5.2 24.0
20.0 0.0 26.0
20.0 5.2 24.0
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GROUP “B”
CROSS-REACTING COMPOUNDS THAT PRODUCE UNSPECIFIC SIGNAL IN THE ASSAY ONLY WHEN BOTH EVEROLIUS AND FK-506-SUCCINATE ARE PRESENT
Spiked compound Spiked EVL Cross-reactivity
Compound ng/mL ng/mL %
Precursor of Seco Acid of EVL 5.0 0.0 54.0
RAD PSA 5.0 5.2 39.0
20.0 0.0 49.0
20.0 5.2 *
OH-PIPERIDINE EVL 5.0 0.0 30.0
5.0 5.2 23.0
20.0 0.0 27.0
20.0 5.2 *
27-O DESMETIL EVL 5.0 0.0 40.0
5.0 5.2 46.0
20.0 0.0 41.0
20.0 5.2 *
*: A signal is produced in the second assay, where there is no FK-506 succinate 27
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GROUP “C”
CROSS-REACTING COMPOUNDS THAT PRODUCE UNSPECIFIC SIGNAL IN THE ASSAY REGARDLESS OF THE ADDITION OF EVEROLIUS OR FK-506-SUCCINATE
Spiked compound Spiked EVL Cross-reactivity
Compound ng/mL ng/mL %
Seco Acid of EVL 5.0 0.0 52.0
(RAD SA) 5.0 5.2 44.0
20.0 0.0 *
20.0 5.2 *
25-OH EVL 5.0 0.0 74.0
5.0 5.2 69.0
20.0 0.0 *
20.0 5.2 *
12-OH EVL 5.0 0.0 54.0
5.0 5.2 62.0
20.0 0.0 *
20.0 5.2 *
*: A signal is produced in the second assay, where there is no FK-506 succinate 30
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