SimplyDilutingSerumbefore RadloimmunoassayDoes Not PreventBindingof Prednisoloneto Protein

Simple dilution of serum is commonly thought to prevent prednisolone binding to plasma protein. Colburn and Buller (1) reported that 50-fold diluted normal serum could not be distinguished from the buffer control, and in their assay control serum and unknown serum were used after 50-fold dilution, without any extraction or denaturing step. The following describes an investigation to determine whether plasma protein still binds prednisolone after simple dilution. The double-antibody technique was used for the assay. The first antibody (anti-prednisolone rabbit serum) was prepared as described below. Prednisolone-21-hemisuccinate was conjugated to bovine serum albumin by the mixed anhydride method of Erlanger et al. (2): 25 mg of prednisolone-21-hemisuccinate in a mixture (pH 7.5) consisting of 0.6 mL of dioxane, 20 iL of tributylamine, and 10 mL of isobutylchlorocarbonate was mixed and stirred for 30 mm at 4 #{176}C. This mixture was then added to a stirred solution of 90 mg of bovine serum albumin in 5 mL of an equivolume mixture of water and dioxane, and 70 iL of 1 mol/L NaOH, increasing the pH to 8.5. Stirring and cooling at 4 #{176}C were continued for 4 h. The solution was dialyzed overnight against running water, brought to pH 4.5 by adding 50 . tL of 1 moliL HC1, and allowed to stand for 2 h at 4 #{176}C. The resulting precipitate, collected by centrifugation, was dissolved by bringingthepHto5.5with30. iLofa 1 moll L solution of NaHCO3. Again, this solution was dialyzed against running water for 4 h at 4 #{176}C. The remaining solution was lyophilized and stored at -20 #{176}C. A 1 mg/mL solution of prednisolone hapten in saline (NaC1, 9 gIL) was emulsified with an equal volume of Freund’s adjuvant. Three albino rabbits were injected with 0.5 mL of this emulsion, intracutaneously on the back, once a week for four weeks. Complete adjuvant was used for the first three weeks, and incomplete adjuvant for the fourth week. Serum from blood collected at the fifth week was stored in small aliquots at -20 #{176}C. This firstantibody antiserum was used in 100fold dilution. Second antibody (anti-rabbit gamma-globulin goat serum) was prepared as follows. Rabbit gamma-globulin, 2 mg/mL in saline, was emulsified with an equal volume of complete Freund’s adjuvant. A goat was injected subcutaneously with 5 mL of this emulsion once each week for four weeks. Serum from blood collected at the fifth week was stored in small aliquots at -20 #{176}C. This second-antibody antiserum was used in eightfold dilution. [6,7-3H(N)]Prednisolone (Amersham, no. TRK-425) with specific activity 50 kCilmol was purified by chromatography on a column of LH 20 (0.8 x 20 cm) in benzene/methanol (85/15 by vol), and the concentration of radioactivity was adjusted to 25 000 cpm/mL with Tris buffer (pH 8.0, 50 mmolIL). For the assay we prepared three different samples. Sample A, 50 mL of control serum diluted to one-, 10-, 50-, or 100-fold with Tris buffer, was added to 350 p.L of Tris buffer. Sample B, 50 . tL of undiluted control serum, was added to 350 jL of Tris buffer and warmed in a 70#{176}C water bath for 30 mm. Sample C was 400 . mL of Tris buffer only. All samples were similarly assayed, according to the following protocol: Add 100 iL of first antibody and 200 . mL of [3Hlprednisolone to all samples. After incubation for 2 h at room temperature, separate free and bound prednisolone by adding 100 . mLof second antibody. After incubation overnight at 4 #{176}C, centrifuge at 2000 rpm for 20 mm, and count the radioactivity in 500 iL of the supernate in 10 mL of Aquasol (New England Nuclear, Boston, MA 02118), using a liquid scintillation counter. The percentage of free prednisolone found was influenced only by protein binding of prednisolone. As a result, the percent of free counts for the heattreated control serum (sample B) and buffer only (sample C) was similar, 13.4% and 13.6%, respectively, which shows that the heat treatment of serum obviates protein binding. On the other hand, the percent of free counts for the diluted control serum (sample A) was respectively 59.8, 25.0, 19.5, and 18.4% in one-, 10-, 50-, and 100fold dilutions. Even in 100-fold dilution the value was greater than that of buffer only (p < 0.001). Evidently the protein still binds the prednisolone and affects radioimmunoassay results. Thus, simple dilution of serum does not suffice to prevent prednisolone binding to serum protein, so either heat-treatment or extraction of serum before assay is essential.