antisera to purified human ferritin were prepared in two animal species ( rabbits and guinea pigs ). anti ( rabbit igg ) serum was prepared in guinea pigs . guinea pig anti ( ferritin ) diluted 1 : 2000 was used to coat polystyrene tubes . 125 i - anti ( rabbit igg ) was prepared in the same way as previous radioactive antibodies as is known in the literature . ferritin standards were made up in normal rabbit serum diluted 1 : 20 with 0 . 05 m veronal buffer ph 8 . 0 , containing 4 . 5 gm / l nacl , 200 mg / l sodium azide , 1 gm / l bovine serum albumin ( hereafter referred to as bsa - buffer ). ferritin protein concentration was determined using the method of lowry with bovine serum albumin standards . the assay was carried out as follows : 0 . 2 ml of human ferritin standard was dispensed into the bottom of the antibody coated tube . the tubes were left at 4 ° c . for 24 hours ( reaction 1 ), aspirated and washed once with bsa - buffer . 0 . 2 ml of rabbit anti ( ferritin ) diluted 1 : 2000 with bsa - buffer , was added and the tubes left at 4 ° c . for a further 48 ( reaction 2 ). the tubes were then aspirated and washed twice with 0 . 04 m phosphate buffer ph 7 . 4 , before adding 0 . 2 ml ( 20 , 000 cpm ) of purified 125 i - labelled guinea pig anti ( rabbit igg ). the tubes were left at 4 ° c . for 48 hours ( reaction 3 ), and then again aspirated , washed twice with the phosphate buffer to remove unbound labelled antibody and counted for 1 minute in an automatic gamma counter . as the concentration of antigen increases , the amount of radioactivity in the solid - phase increases . a standard curve was derived from quadruplicate determinations on ferritin standards containing 0 , 0 . 33 , 1 . 33 , 5 . 31 , 21 . 25 , 85 and 340 ng of ferritin protein per ml . the data was analyzed using a version of the weighted , four - parameter logistic curvefitting method of rodbard and hutt , adapted for use on the ibm - 360 and with 2 - site irma . the mean zero - dose radioactivity in this assay was 1772 cpm as compared with 2302 cpm for the lowest concentration of ferritin measured ( 0 . 33 ng / ml ). the minimal detectable dose ( two standard deviations from zero - dose ) was 34 picograms and occurred when the tube radioactivity increased from 1772 to 2031 cpm ( an increase from 7 . 7 % to 8 . 83 % of the total radioactivity added to the tube ) ( 23 , 000 cpm ). a 4 . 8 fold increase in tube radioactivity ( 2031 to 9781 cpm ) was seen throughout the working range of the standard curve ( 0 . 17 to 50 ng / ml ). in order to demonstrate the versatility of this system , a &# 34 ; mirror image &# 34 ; assay was established . in this assay the tubes were coated with rabbit - anti ( ferritin ); guinea - pig - anti ( ferritin ) was used in reaction 2 ; and 125 i - rabbit anti ( guinea - pig igg ) was used in reaction 3 . the results showed a good assay standard curve , but the zero - dose radioactivity is higher and the slope of the dose response curve not as steep as in example i , probably due to the lower affinity of the commercial anti ( guinea - pig igg ). this example demonstrates the application of this same assay system ( using the same labelled antibody ) to the measurement of several different antigens . antisera to glial fibrillary acidic protein ( gfap ) and bovine parathyroid hormone ( pthh ) were prepared in both guinea pigs and rabbits . the assays were carried out as was done in the ferritin assay ( example i ) with the guinea pig antiserum in the solid phase and the rabbit antiserum in reaction 2 . reaction 1 was carried out using various concentrations of gfap or pthh . reaction 3 used the same preparation of 125 i anti ( rabbit igg ) used in example i ( total counts per minute = 45 , 000 ). results showed that in the gfap assay 5829 cpm were bound into the solid phase in the absence of added gfap , and the radioactivity increased steadily with increasing concentrations of added gfap ( 11370 cpm at 1 . 1 μg / ml and 13470 cpm at 11 μg / ml ). in the pthh assay 4224 cpm were bound into the solid phase at zero dose , and the radioactivity increased to 5565 cpm ( 7 μg crude extract / ml ) and 7601 ( 70 μg / ml ).