Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

Immunoassays for IF analysis in biological samples

Simplified methods for the assessment of O-desmethylangolensin (O-DMA) and equol in human biological fluids have been developed. Currently, expensive and cumbersome GC-MS methodologies have to be used to ensure adequate sensitivity.

These methods are based on time-resolved fluoroimmunoassay (TR FIA) using a europium chelate as a label. Immunoassays have the advantage of allowing a high thruput at relatively low costs with equipment that most laboratories will be able to afford. The use of these methods has a validity that goes beyond the present project. Indeed, the presence of equol rather than O-DMA in urine is considered a marker of decreased cancer risk.

In the first method, after the synthesis of 4-O-carboxymethyl-O-DMA, this compound is coupled to bovine serum albumin, and then used as antigen in immunization of rabbits. The tracers with the europium chelate are synthesized using the same 4-O-derivative of the α-methyldeoxybenzoin. After enzymatic hydrolysis and ether extraction the immunoassay is carried out by time resolved fluoroimmunoassay (TR FIA).

Cross-reactivity was tested with angolensin, dihydrogenistein, dihydrodaidzein, equol, 6-OH-angolensin, trans-4-OH-equol, 6-OH-O-DMA, cis-4-OH-equol and 5-OH-equol. The antiserum cross-reacted only with angolensin. This cross-reactivity seems not to influence the results, which were highly specific. The correlation coefficient between the plasma TR FIA results and the GC-MS results was high; r value was 0.985. The intra-assay coefficients of variation (CVs) for plasma O-DMA concentrations and for urine O-DMA concentrations at three different concentrations varied 2.8-7.7 and 3.0-6.0%, respectively and the inter-assay CVs varied 3.8 - 8.9 and 4.4-6.6%, respectively.

The working range of the plasma and urine O-DMA assays was 0.5-512nmol/l. In the second method, after synthesis of 4-O-carboxymethylequol the compound is coupled to BSA, and then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4-O-derivative of the isoflavan. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids.

For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a GC-MS method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula is used to correct the urine values making them comparable to the GC-MS results.

The correlation coefficients between the TR-FIA methods and GC-MS methods were high; r values, respectively for the plasma and urine method, were 0.98 and 0.91. The intra-assay coefficient of variation (CV %) for the TR-FIA plasma and urine method at three different concentrations vary between 5.5-6.5 and 3.4-6.9, respectively. The inter-assay CV % vary between 5.4-9.7 and 7.4-7.7, respectively. The working ranges of the plasma and urine assay are 1.27-512 nmol/l and 1.9-512 nmol/l, respectively. The development of these methods has great potential for commercial exploitation and allows simplifying the implementation of studies of phytoestrogen effects in humans.

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