ing RIA of T3 in the dialysate standardized with the same antibody and by a ... serum. To validate this assay, we developed an equilibrium dialysis method ...
CLIN. CHEM. 33/3, 372-376 (1987)
A Two-Step Radioimmunoassayfor Free Triiodothyroninein Serum M. G. R. Rajan
and A. M. Samuel
Using a high-affinity solid-phase-bound
antibody (I = 1.2 x 1011 LJmol), we have standardized a two-step radioimmunoassay for free triiodothyronine (FT3) in serum, based
on immunoextraction. The method was validated by comparison with an equilibrium-dialysis procedure (r = 0.96) involving RIA of T3 in the dialysate standardized with the same antibody and by a commercial (Liso-Phase, InternationalCIS) method. The two-step RIA could detect as little as 0.2 pg per milliliter. The mean CVs within and between assays were 9% and 12%, respectively. FT3 values measured in 30 normal adults ranged from 1.77 to 4.77 ng/L. Comparison with ratios of total T3 to thyroxin-binding globulin showed good agreement in normal subjects, pregnant women, and hypothyroid and hyperthyroid patients. AdditIonal
Keyphrases: thyroid status
dism pregnancy “kit’Smethods
hypo- and hyperthyroiequilibrium-dialysis procedure compared
The free moiety is believed to be responsible for the physiological action of thyroid hormones, and measurements of free triiodothyromne (FF3) are used to detect hyperthyroidism and monitor treatment by thyroid hormone replacement (1-4).’ Traditional methods of measuring free thyroid hormones are tedious and subject to artefactual errors; this is especially true for FF3 because of its lower concentrations. Recent reports on alternative methods for FF4 show that two-step radioimmunoassay (RIA) with immunoextraction (5) and the procedure of the Liso-Phase kit method (6) give results that correlate very well with those by equilibrium dialysis method for a variety of clinical conditions, whereas analogbased methods for FF4 have been severely criticized (7). The theory of two-step RIA has been amply substantiated by Ekins (8). We describe here a two-step RIA for measuring FF3 in serum. To validate this assay, we developed an equilibrium dialysis method involving RIA of T3 in the dialysate (EDIRIA). Intra- and interassay variation, effect of sample volume, and the effect of the amount of T3 extracted by the antibody from the sample were also studied. Preliminary results for normal subjects, pregnant women, and for hypothyroid, hyperthyroid, and nonthyroidally ill patients were compared with the T3/TBG ratios for these groups. We also measured FF3 with a commercial kit and report the comparison of results. Radiation Medicine Centre, Bhabha Atomic Research Centre, Tate Memorial Centre Annexe, Parel, Bombay-400 012. India. ‘Nonstandard abbreviations: T3, total triiodothyronine; FF3, free triiodothyronine; FF4, free thyroxin; TBG, thyroxin-binding globulin; ED, equilibrium dialysis; HEPES, 4-(2-hydroxy-ethyl)-1piperazineethanesulfonic acid; HarEs-gel, HEPEs buffer containing gelatin; HEPEs-GT, REPES buffer containing gelatin and Tween 20 (see text); cal, cellulose; and T3-ab-cel, solid-phase-conjugated antibody. Received September 24, 1986; accepted November 19, 1986. 372
CLINICALCHEMISTRY, Vol. 33, No. 3, 1987
MaterIals and Methods Materials Reagents. 1,1 ‘-Carbonyldiimidazole, HEPES, microcrystalline cellulose (Sigmacell 20), IrP3 (free acid), gentamicin sulfate, activated charcoal, and Tween 20 [polyoxyethylene (20) sorbitan monolaurate] were all from Sigma Chemical Co., St. Louis, MO. All other reagents were of analytical grade and were obtained locally. Polystyrene tubes, 11 mm (i.d.) x 75 mm, with a round bottom were used. Dialysis tubing (6 mm o.d., Mr 6000-8000 cutoff) was from Thomas Scientific, Swedesboro, NJ. Buffers. We prepared HEPES buffer to contain, per liter, 10 mmol of HEPES, 106 mmol of NaCl, 15 mg of gentamicin sulfate, and 60 mg of NaN3. HEPEs-gel buffer contained 1 g of gelatin per liter of HEPES buffer. HEPES-GT buffer had Pwen-20 (0.5 mLIL) in HEPE5-gel buffer. Antiserum to T3. Antiserum to T3, raised in sheep, was from Guildhay, London (batch no. HP/S/60 WA). We found it to have an affinity of 1.2 (± 0.1) x lOll L/mol. Its cross reactivity with L-T4 (50% displacement) was 0.2%. [1251]T3. We obtained label with high specific activity (>3000 Ci/g) from Radiopharmaceutical Division, B.A.R.C., Bombay, India. The exact specific activity, determined with each batch, was used to calculate the amount of [1mJT3 to use in experiments. We used the label within four weeks of its production. RIA kits. We used T3 and T4 kits from B.A.R.C. and RIA kits for TBG and FF3 (Liso-Phase) from International CIS, St. Quentin-Yvelines Cedex, France. We followed the protocols supplied with the kits and found their intra- and interassay CVs to be within the limit specified by the supplier. Control sera. Thyroid control sera 1, 2, and 3 were from Amersham International, Amersham, Bucks., U.K. (code no. QAS 101, lot no. 10). These contained thyroid hormones (total and free) calibrated at three concentrations and were used for studying intra- and interassay variations. Hormone-free sera. Pooled serum from normal volunteers was treated with activated charcoal to remove all hormones. The concentration of total protein was 68 gIL. FT3 serum standards. Hormone-free serum containing r.T3 in various concentrations was prepared by serially diluting a 6.0 mg/L solution of i..-P3 with the serum. We determined the FF3 concentration at each dilution by ED/RIA (n = 5 each), correcting for statistical variations as suggested by Ekins (8). Thus, a set of six standards for FF3 with concentrations in the range 0-18.4 ng/L were prepared. These were aliquoted and stored at -20 #{176}C; each aliquot was thawed and used once. Specimens. Serum samples were obtained from normal healthy volunteers, pregnant women in the second and third trimesters of pregnancy, patients admitted to hospital for various nonthyroidal illnesses, and patients attending the thyroid clinics at our institute. Serum was stored at -20 #{176}C until analysis. All samples were assayed for FF3 (by twostep RIA), T4, T3, TBG, and (by the bromcresol green dye-
binding method) albumin. For the purpose of correlation, we also analyzed some samples from each group by ED/RIA and Liso-Phase. Procedures Conjugation of antibodies to cellulose. We activated microcellulose with 1,1’-carbonyldiimidazole and couimmunoglobulin fraction of T3 antisera, with subsequent washing, exactly as directed by Chapman and Ratcliffe (10). We suspended 200 mg of the solid-phase conjugated antibody (T3-ab-cel) in 10 mL of HEPES-gel buffer (the stock suspension). We observed no change in the antiserum’s afiInity for T3 after coupling to cellulose. Stored at 4#{176}C in HEPES-gel buffer, the T3-ab-cel was stable for at least a year. Equilibrium dialysis/RIA. We followed the principle of Ellis and Ekins (11) but used a different set up. Dialysis tubing, 125 mm long, was washed several times in hot (50 #{176}C) de-ionized water and rinsed in HES buffer. Wearing polythene gloves, we folded each strip in two and carefully introduced 250 iL of serum into the tubing at the bend. After placing this in a lO-mL Corning glass scintillation vial (washed with de-ionized water) containing 5 mL of HEPES buffer, with the open ends of the dialysis tubing projecting out of the vial, we stapled the open ends together. The dialysis assembly was placed in a waterbath at 37#{176}C and left for 18 h. The T3 concentration in the dialysate was estimated by RIA as follows. To 1.0 mL of the dialysate in a polystyrene tube we added 1.5 pg of [1mIfl3 in 50 uL of HEPES-gel buffer, 50 L of 20 g/L gelatin solution, 100 ML of suitably diluted T3-ab-cel stock suspension, and 0.5 mg of plain cellulose, to give a visible bulk to the solid phase. Concurrently, we set up seven standards (0-27 ng/L), prepared in 1.0 mL of dialysate from hormone-free serum, to use in constructing a standard curve. The standards and samples (in triplicate) were incubated overnight on an orbital shaker at 25#{176}C, centrifuged, and supernates were aspirated, and the solid phases were washed once with 2.0 mL of ice-cold HEPES-GT buffer. The radioactivity remaining in the tubes was counted. Samples with FF3 values exceeding 27 ng/L were re-assayed to ascertain that the concentrations were not artefactually crystalline pled the
increased by serum, which could have leaked out of the dialysis tubing. Two-step RIA for FT3. This method resembles the one suggested by Ekins for FF4 (8). Incubate, in duplicate, for 3 h at 37 #{176}C, 200 pL of FF3 serum standards, as prepared above, or unknowns, with 500 L of HEPE5-gel buffer containing suitably diluted T3-ab-cel stock plus 0.5 mg of plain cellulose for bulk. The T3-ab-cel dilution is determined in separate experiments as that at which about 3% of the total T3 in 200 L of Amersham thyroid control serum 2 is extracted. Pellet the solid phase by centrifugation at 1500 x g for 5 mm, then wash the pellet twice with 2-mL portions of ice-cold HEPES-GT buffer. Add to the tubes 10 to 12 pg of [‘I]T3 in 1.0 mL of cold HEPE5-gel buffer and incubate at 4#{176}C for 2 h. After centrifuging and washing as before, count the radioactivity remaining in the tubes. T3 extraction. To control sera 1,2, and 3 we added known quantities of [I]T3 to yield about 106 counts/mm in 1 mL and incubated for 1 h at 20#{176}C. We used this tracercontaining sera in the first step of the two-step RIA and the Liso-Phase procedure to determine the amount of T3 extracted. The radioactivity in the solid phase (in the two-step RIA) or in the Sephadex LH2O column (in the Liso-Phase assay), after the prescribed washing steps, was counted and used to determine the percentage extracted. Effect of sample volume. T3-ab-cel in 500 A.L of HEPES-gel buffer as standardized for the two-step RIA, was incubated with 50, 100, 150,200, or 250 L of control serum 2 to which [‘I]T3 tracer had been added. The percentage and absolute amount extracted in each case was thus determined. Results Calibration curves. Figure 1 depicts typical standard curves obtained with the two-step RIA, ED/RIA, and LisoPhase methods. In all three cases, 50 to 60% of the label was bound in the zero standard tube (B0). The detection limits (n =6) of the assays with 95% confidence at B0 was 0.2 ngfL for two-step RIA, 0.8 ng/L for ED/RIA, and 0.4 ng/L for LisoPhase. The precision proffles for the assays shown in Figure 1 represent the intra-assay variation. The interassay variations (CV) for control sera 1, 2, and 3 were respectively 12%,
ED/RIA --TSRIA 80 -
ED/RI A -TSRIA e-L iso-Phase
60
40
20 /
,
A
0.1
10
Fl3 pglml. FIg. 1. Standard curves(left) and intra-assay precision profiles (nght) for two-step AlA, ED/RIA,and Uso-Phasemethodsof FT3
50 estimation
CLINICALCHEMISTRY,Vol. 33, No. 3,
1987
373
8%, and 10% for the two-step RIA and 25%, 16%, and 14% for the ED/RIA. Nonspecific binding, determined by using plain cellulose instead of T3-ab-cel, was
