Scintillation proximity radioimmunoassay with microporous membranes

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Appl. Radiat. lsot. Vol. 47, No. 3, pp. 323-328, 1996 Copyright © 1996ElsevierScienceLtd Printed in Great Britain.All rights reserved 0969-8043/96 $15.00+ 0.00

Scintillation Proximity Radioimmunoassay With Microporous Membranes R O B E R T K. M A N S F I E L D * , D I B A K A R B H A T T A C H A R Y Y A , N E l L G. H A R T M A N t a n d M I C H A E L JAY:~ Division of Medicinal Chemistry and Pharmaceutics, Department of Chemical Engineering and Center of Membrane Science, University of Kentucky, Lexington, KY 40536, U.S.A. (Received 18 October 1995) Fluors were incorporated into the matrix of microporous polymeric membranes and antibodies were subsequently bound to their surfaces. These membranes were used in competitive binding assays (secondary RIAs) for cyclic-AMP in which the bound and unbound radioligands did not require separation. Membrane-based scintillation proximity assays have utility for the detection of a variety of radioanalytes and can be configured in a number of sizes and shapes.

Introduction Radioimmunoassay (RIA) is a competitive binding assay in which a radiolabeled antigen competes with an unlabeled antigen for a limited number of specific antibody binding sites (Yalow and Berson, 1959; Berson and Yalow, 1968). In standard RIAs, it is necessary to separate the radiolabeled antigen bound to the antibody from unbound radiolabeled antigen prior to assay. Recently, immunoassays in which the separation step can be avoided have been developed (Witherspoon, 1989). One such method involves fluorescence polarization immunoassay (FPIA), which employs fluorescein molecules as the "reporter" (Jolley et al., 1981). However, this method is not easily adaptable to all antigens. A technique known as scintillation proximity assay (SPA), initially developed by Hart and Greenwald, has been applied toward RIAs in which the separation step can be avoided (Bosworth and Towers, 1989; Hart and Greenwald, 1979; Nelson, 1987; Udenfriend and Gerber, 1987). SPAs detect only those radiolabeled antigens that are bound to fluor-containing matrices. The radioisotopes used in SPA (3H and ~-'5I)emit low energy electrons which are absorbed within a very short distance of aqueous solutions (4/zm for ~H negatrons and 35/~m for ~-'~I Auger electrons)

(Bosworth and Towers, 1989). Therefore, only those radiolabeled antigens bound to the fluor-containing matrix are in close enough proximity to excite the fluors and induce scintillations. The emitted light can be conveniently measured in a liquid scintillation counter. Unbound radiolabeled antigens are too far removed from the solid phase fluor to be detected. As long as the solution is adequately dilute, only a small amount of unbound radiolabeled antigen is in close enough proximity to the solid phase scintillant to be detected. The RIAs based on SPA technology (SPRIAs) currently in use are based on a microbead design. The beads either contain or are composed of scintillant materials. Antibodies are bound to the surface of the beads which are subsequently suspended in solutions containing mixtures of labeled and unlabeled antigens. In this paper, we describe a membranebased SPRIA in which antibodies were bound to the pore surface of fluor-containing microporous membranes. Organic and inorganic fluors were entrapped within the matrix of polymeric membranes composed of polyvinyl chloride (PVC) or hydroxyethylcellulosecoated polysulfone.

Experimental Materials

*Current address: Allergen Inc., Irvine, CA 92713-9534, U.S.A. tCurrent address: Departement de medecine nucleaire, Hopital Notre-Dame, Montreal, Quebec, Canada H2L 4MI. :~To whom all correspondence should be addressed.

Polysulfone (MW = 752,000) was obtained from Union Carbide (Marietta, OH). 2,5-Diphenyloxazole (PPO), 2-(4-tert-butylphenyl)-5-(4-biphenyl)-l,3,4oxadiazole (butyl-PBD), and p-bis-(o-methylstyryl)benzene (bis-MSB) (all scintillation grade) were

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purchased from Research Products International (Elk Grove Village, IL). Cerium-activated yttrium silicate (Type 158 regular) was purchased from Sylvania Chemicals/Metals (Towanda, PA). Affinitypurified goat anti-rabbit immunoglobulin was purchased from Zymed Laboratories (South San Francisco, CA) and standard radioimmunoassay kits were purchased from Amersham International (Melville, NY).

Chloromethylation of polysulfone Polysulfone was chloromethylated as previously described (Radovanovic et al., 1992) and involved dissolution of 7 g (0.016 mol) of polysulfone (PSF) in 120 mL of freshly distilled tetrachloroethane. After heating to 50-55°C (Radovanovic et al., 1992), 5 g (0.036 mol) of zinc chloride and 3 mL of chloromethyl methyl ether (0.037 mol) were then added, and the reaction flask was purged with dry nitrogen, sealed and allowed to stir for 4 h. The reaction mixture was diluted with 100 mL of chloroform and the chloromethylated polymer precipitated in 1400 mL of acetone:2-propanol (50:50) under vigorous agitation. After stirring for 5 min, the precipitate, which had a rubbery consistency, was allowed to settle and the excess supernatant was removed and replaced with 500 mL of fresh 2-propanol. The off-white colored precipitate was collected via suction filtration using a sintered glass funnel. The polymer was allowed to air dry for 15-20 min, dissolved in 250 mL of tetrahydofuran and then re-precipitated. Analytical samples were further purified via Soxhlet extraction using 2-propanol for 96 h. All polymer samples were dried under vacuum for 24h at 80-90°C. The degree of chloromethylation (number of PSF repeating units chloromethylated) was determined by elemental analysis and ~H N M R spectroscopy to be approx. 50%.

Membrane preparation Membranes composed of either PVC or chloromethylated PSF in which organic or inorganic fluors were trapped were prepared using a phase inversion technique. Chloromethylated PSF membranes containing the cerium-activated yttrium silicate (CASY) particles (3 mm dia) were prepared by dissolving 2 g of dry chloromethylated PSF and 0.3 g polyvinyl pyrrolidinone (PVP) in a mixture containing 17 mL of N-methyl pyrrolidinone (NMP) and 8 mL triethylene glycol. Once all the components had dissolved, 2.5 g of CASY were suspended in the solution. This suspension was heated to 50°C and a small sample (4 mL) was poured onto a pre-heated (50°C) glass plate with two strips of standard masking tape bordering two edges of the plate (see Figure 1). A glass stirring rod was used to evenly distribute the casting solution over the plate, and the plate was immediately submerged in an aqueous gelling bath heated to 75°C. The membrane was allowed to gel for 1 h and the water-swollen mem-

brane was cut into 25 mm discs. Chloromethylated PSF membranes containing organic scintillators were prepared using a similar procedure where the desired amount of organic scintiUant (PPO, butyl-PBD and bis-MSB) was homogeneously dissolved in the casting solution. PVC membranes were prepared by dissolving 2.4 g of poly(vinylchloride), 0.3 g PVP and the desired amount of organic fluor in an NMP/trietheylene glycol solution. The membranes were cast and the gels formed in a manner similar to that described above for the chloromethylated PSF membranes.

Coating of polysulfone membranes In order to decrease the surface hydrophobicity and provide attachment points for antibodies, chloromethylated PSF membranes were coated with the hydroxyethyl cellulose (HEC) hydrophilic polymer. Twenty-two circular chloromethylated PSF membranes (25 mm dia) were placed in a 1 L Erlenmeyer flask containing 500 mL of I M NaOH. The solution was heated to 80-85°C and agitated on an orbital shaker (150-200 rev/min) for 20h. This procedure converted the surface-available chloromethyl groups to the corresponding alcohol. After thorough washing with cold water, the membranes were placed in Erlenmeyer flasks containing 50mL of 0.1 N NaOH and 20 mL of acetonitrile. Seven mL (6.3 g) of ethylene glycol diglycidyl ether (EGDGE) were added where acetonitrile served as a co-solvent to completely solubilize the EGDGE. The membranes were mixed in this solution using an orbital shaker (200 rev/min) for 4 h at room temperature after which the solutions were decanted and the membranes washed thoroughly with cold water. 80 mL of a 0.6 N NaOH solution containing 2% (w/v) HEC were added to the membranes and this mixture was heated to 60-65°C and returned to the orbital shaker (200 rev/min) for 20 h. The supernatant was decanted Casting solution containing polymer, solvent and fluor

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and the membranes were washed with 1 L of 60-70°C water. The membranes were treated with E G D G E and HEC repeatedly for a total of 5-7 cycles (Sepracore, 1989).

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HEC-coated PSF membranes containing inorganic fluor (CASY). Thirty HEC coated PSF membranes were washed four times with dry acetonitrile to remove trapped water. Dry acetonitrile (15 mL) was used to dissolve 0.6g (0.002mol) of 2-fluoro-1methylpyridinium toluene-4-sulfonate (FMP) and combined with 500 mL (0.38 g, 0.004mol) of dry triethylamine. After the membranes were added, the solution was purged with dry nitrogen, sealed with parafilm and shaken vigorously for 1 h. The membranes were washed three times with 10 mL of dry acetonitrile and four times with 2 mM HCI, and then transferred to 10mL polypropylene tubes containing 3 m L of a 100mg/mL solution of goat anti-rabbit IgG in bicarbonate buffer (0.03 M, pH 8.1). These solutions were vigorously shaken on an orbital shaker (400 rev/min) for 6 h (Ngo, 1986). Thirty control HEC coated membranes which were not treated with F M P were exposed to antibody in the same manner described above. After 6 h, the membranes were thoroughly washed with acetate buffer (0.05 M, pH 5.8). The amount of antibody bound to the membrane was determined by measuring the difference in protein concentration between the initial and spent antibody solutions using the Lowry method (Lowry, 1951). PVC membranes containing organic fluor (butylPBD). Thirty PVC membranes containing 30% (w/w) butyl-PBD were placed in 2 mL of phosphate buffer (0.01 M, pH 7.4) containing 150pg of goat anti-rabbit IgG (75 pg/mL) in a glass scintillation vial. The vial was shaken at 200 rev/min in an orbital shaker for 3 h. To promote noncovalent attachment of the antibody to the membranes. Three 100 mL samples were removed from the original antibody stock solution for subsequent protein analysis using the Lowry assay where bovine serum albumin (BSA) served as standards. After 3 h, the membranes were removed and the supernatant was analyzed for protein content. The amount of antibody bound to the membranes was determined by the difference in protein concentration between the stock and supernatant protein solutions. The membranes were thoroughly washed with phosphate buffer and placed in 5 mL of 3% (w/v) BSA in phosphate buffer to block the remaining nonspecific binding sites. Three control membranes that were not exposed to antibody were treated in the same manner with BSA. Control membranes using normal goat IgG, i.e. not anti-rabbit IgG, were also employed.

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Scintillation proximity radioimmunoassay The procedures that accompanied a commercially scintillation proximity assay kit available for cyclic

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Number of HEC coatings Fig. 2. The effect of the number of hydroxyethylcellulose coating cycles on the nonspecific absorption of ['25I]bovine serum albumin on polysulfone membranes. These studies were carried out by placing 7 mm membrane discs in polypropylene tubes containing 500 mL of phosphate buffer (pH 7.4, 0.01 M) and 20mL (8nCi) of ['-'~I]albumin (l.9nCi/mg). The tubes were shaken at 350rev/min for 18h, washed with acetate buffer, 0.15 M NaCI, acetate buffer again and distilled water and counted in a ),-scintillation counter. adenosine monophosphate (cAMP) were followed to prepare the assay buffer (acetate buffer, 0.05 M, pH 5.8), rabbit anti-cAMP antiserum, cAMP standards and [~25I]cAMP tracer. Samples were prepared in triplicate by adding the following to appropriately labeled 2 mL polypropylene tubes: ['25I]cAMP tracer, cAMP standards (0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 pmol/tube prepared via 1/2 dilutions from a 256 pmol/mL stock cAMP solution), rabbit anticAMP antiserum and acetate buffer in 100pL additions (for a total volume of 400 #L). The rabbit anti-cAMP antiserum was reconstituted from a commercially available kit by the addition of distilled water to the reagent so that the final antiserum solution contained 0.5% bovine serum albumin and 0.01% thimerosal in 0.05 M acetate buffer. Fluorcontaining membranes cut into 7 mm discs were then added to each tube. Nonspecific binding of t25IcAMP was determined by adding 200 pL of acetate buffer, 100 p L of ['25I]cAMP tracer and 100/aL of 3% bovine serum albumin in acetate buffer to a 2 m L polypropylene tube containing a scintillation proximity membrane (fluor-containing membranes with goat anti-rabbit IgG, or control membranes). Zero standards were prepared by adding 100 pL of each of the following tracer to a 2 mL polypropylene tube containing a scintillation proximity membrane: [~2SI]cAMP tracer, acetate buffer, 3% bovine serum albumin in acetate buffer and rabbit anti-cAMP anti-serum. After all samples were prepared, the tubes were sealed, vortexed and rotated at 25°C for 20 h. This time period was sufficient for equilibrium to be

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achieved as samples incubated for longer than 20 h showed no increase in binding. The tubes were then placed in a 20 mL glass scintillation vial and counted in a liquid scintillation counter for 2 min.

Results and Discussions Two types of membranes were prepared for use in scintillation proximity radioimmunoassays: polysulfone membranes containing the inorganic fluor CASY that were coated with hydroxyethylcellulose, and polyvinyl chloride membranes that contained the organic fluor butyl-PBD. The PSF membranes were subjected to several coating cycles with HEC. The HEC coating on PSF membranes served two functions: (1) the hydrophilic surface created by HEC reduced nonspecific binding of proteins; and (2) the hydroxyl groups of HEC were used to couple the secondary antibody (goat anti-rabbit IgG) to the membrane surface. The effect of the number of coating cycles on nonspecific binding (determined by

the binding of [~SI]albumin) is depicted in Figure 2. The secondary antibody was coupled to the PVC membrane hydrophobically in a noncovalent manner. These membranes were added to a mixture containing fixed amounts of primary antibody (rabbit antiserum directed against cAMP) and [~25I]cAMP tracer, as well as variable amounts of unlabeled cAMP, as illustrated in Figure 3. Similar curves for the two membranes were obtained when the percentage of [~2SI]cAMP bound to the membrane (determined by analyzing the membranes in a liquid scintillation counter) was plotted against the amount of unlabeled cAMP present in the tube (Figure 4 and Figure 5). Because the fluor was entrapped within the matrix of the membrane, there was no need to add a liquid scintillation cocktail to the tubes before counting. These results indicate that the two membranes yielded similar results since the curves were nearly identical to those obtained using the standard radioimmunoassay kit for cAMP. However,

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cAMP (pmol/tube) Fig. 4. Percentage [~2~I]cAMPactivity bound and detected by hydroxyethylcellulose-coated fluor-containing polysulfone membranes as a function of unlabeled cAMP in a scintillation proximity radioimmunoassay. Samples were prepared in triplicate by adding [~25I]cAMPtracer, cAMP standards (0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 pmol/tube), rabbit anti-cAMP antiserum and acetate buffer in 100 pL additions to polypropylene tubes. Fluor-containing membranes cut into 25 mm discs were then added to each tube which were subsequently shaken for 20h, placed in scintillation vials and counted for 2 min. the standard RIA for cAMP involves a separation step which was avoided with the scintillation proximity membranes. The solid phase scintillant plays a crucial role in scintillation proximity assays. An effective solid phase must contain sufficient amounts of fluor to efficiently detect electrons released during the decay of 3H and '2sI, and it must serve as a support for the receptors (immunoglobulins in the case of scintillation proximity radioimmunoassay). Fluor molecules are easily entrapped within the matrix of a polymeric membrane which can be functionalized to couple a wide variety of receptors, including antibodies. In scintillation proximity assays, only bound radio100

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ligands are detected due to their proximity to the entrapped fluor molecules. The fluor must remain in the solid support to ensure that only bound radioligands are detected. If the tluors leach from the support, the assay may be subject to significant error due to the detection of unbound radioligands. Organic fluors commonly used in liquid scintillation counting (PPO, butyl-PBD and bis-MSB) were observed to leach from the polysulfone membranes, even after the membranes were thoroughly washed with buffer. Since CASY is insoluble in both aqueous and organic solvents (Ross et al., 1991), this problem was avoided by adding the inorganic fluor CASY in the matrix of the polysulfone membranes during the casting procedure. The PVC membranes were easier to prepare than the HEC-coated PSF membrane since the native polymer was not chemically modified and the PVC membranes were not subjected to any time consuming coating procedures. Thus, the time required to prepare PVC membranes for SPRIA is only a fraction of the time required to prepare HEC coated PSF membranes. Membrane-based systems offer several advantages over microbeads for scintillation proximity assays. Because receptors can be bound to the porous surface of the membrane, the bound radiolabeled analyte can be surrounded by the fluors entrapped within the matrix of the membrane. This results in a nearly 100% geometric detection efficiency, whereas the maximum geometric detection efficiency of a microbead system is 50%. In addition, membranes can potentially be used with higher energy electron emitters such as ~4C because the membrane is not homogeneously spread throughout the analyte solution. Thus, emission of more energetic electrons emitted from ~4C-labeled analytes that are not bound to the membrane may be further from the membrane than the range of the electrons. In microbead assays where the beads are homogeneously suspended in the analyte solution, the range of the ~4C-negatrons is sufficiently long so that unbound ~4C-analytes may excite the fluor within the beads. Membranes can also be employed in convective flow systems which are not diffusion limited; in the microbead assays, the diffusion of the analyte to the antibodies bound on the microbeads is rate limiting. Membranes can easily be configured in a variety of sizes and shapes, which can be particularly useful in applications such as sensors or fiber optic devices. Acknowledgements--This work was supported in part by NSF Grant EHR-9108764 and the Kentucky EPSCoR Program.

cAMP (pmol/tube) Fig. 5. Percentage [~25I]cAMPactivity bound and detected by polyvinyl chloride membranes as a function of unlabelled cAMP in a scintillation proximity radioimmunoassay. Assay conditions were identical to those desribed in the legend for Figure 3.

References

Berson S. and Yalow R. (1968) General principles of radioimmunoassay. Clin. Chim. Acta. 22, 51. Bosworth N. and Towers P. (1989) Scintillation proximity assay. Nature 341, 1444.

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Hart H. and Greenwald E. (1979) Scintillation proximity assay (SPA): a new method of immunoassay. Mol. Immunol. 16, 265. Jolley M., Stroup¢ S., Wang C., Panas H., Keegan C., Schrnidt R. and Schwenzer K. (1981) Fluorescence polarization immunoassay I. Monitoring aminoglycoside antibody in serum and plasma. Clin. Chem. 27, 1190. Lowry O. H. (1951) J. Biol. Chem. 193, 265. Nelson N. (1987) A novel method for the detection of receptors and membrane proteins by scintillation proximity assay. Analyt. Chem. 165, 287. Ngo T. (1986) Facile activation of sepharose hydroxyl groups by 2-fluor-l-methyl-pyridinium toluene 4-sulfonate: preparation of affinity and covalent chromatographic matrices. BioTechnology 4, 134. Radovanovic P., Thiel S. and Hwang S. (1992) Formation of asymmetric polysulfone membranes by immersion

precipitation. Part II. The effects of casting solution and gelation bath compositions on membrane structure and skin formation. J. Membrane. Sci. 65, 231. Ross H., Noakes J. and Spaulding J. (1991) Liquid Scintillation Counting and Organic Scintillators. Lewis, Chelsea, MI. Sepracore (1989) International Patent No. PCT/US89/ 84620. Udenfriend S. and Gerber L. (1987) Scintillation proximity assay: a sensitive and continuous isotopic method for monitoring ligand/receptor and antigen/antibody interactions. Analyt. Biochem. 161, 494. Witherspoon L. (1989) Radioimmunoassayists must embrace new technology. J. Nucl. Med. 30, 1571. Yalow R. and Berson S. (1959) Radiobiology. Nature 184, 1648.