Solid-phase immunoradiometric assay for serum amyloid a protein using magnetisable cellulose particles

An immunoradiometric assay for human serum amyloid A protein (SAA) was developed using magnetisable cellulose particles as the solid phase. Rabbit antiserum to SAA was raised by immunization with SAA isolated from acute-phase serum by gel filtration in formic acid. The antiserum was rendered monospecific for SAA by solid-phase immunoabsorption with normal human serum, which contains only trases of SAA, and some was coupled covalently to the cellulose particles. Immunopurified anti-SAA antibodies were isolated from the monospecific anti-SAA serum by binding to, and elution from insolubilized acute-phase serum and were radiolabelled with ¹²⁵I. The assay was calibrated with an acute phase serum which contained 6000 times more SAA than normal sera with the lowest detectable level of SAA, and an arbitrary value of 6000 U/1 was assigned to this standard. Sera were tested in the native, undenatured state and there was no increase in SAA immunoreactivity following alkali treatment or heating. The assay range was from 1-2000 U/1 so that all SAA levels above 6 U/1 could be measured on a single (1:6) dilution of serum. The intra- and interassay coefficients of variation were 11.7 and 15.0% respectively. Among 100 healthy normal subjects (50 male, 50 female) the median SAA level was 9 U/1, range <1-100, with 93% below 20 U/1 and only 2% below the lower limit of sensitivity of the assay (1 U/1).