Biotechnol. Prog. 1991, 7, 173-177
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Antibody Immobilization onto Glow Discharge Treated Polymers Agneza Safranj,t David Kiaei, and Allan S. Hoffman* Center for Bioengineering, FL-20, University of Washington, Seattle, Washington 98195
Previous studies have shown that certain glow discharge treated polymers strongly retain adsorbed albumin and fibrinogen. On the basis of this phenomenon, we have investigated the possibility of immobilizing antibodies on glow discharge treated surfaces for diagnostic immunoassay applications. As a model for antibody immobilization, bovine IgG was immobilized on the following polymers: polyethylene (PE),tetrafluoroethylene glow discharge treated PE (TFE/PE), poly(ethy1ene terephthalate) (PET), TFE/PET, poly(tetrafluoroethy1ene) (PTFE), ethylene glow discharge treated PET (E/PET) and hexamethyldisiloxane glow discharge treated PET (HMDS/PET). IgG and immobilized by either of the following two methods: (a) was radiolabeled with 1251 physical adsorption of IgG on untreated and glow discharge treated polymers or (b) physical adsorption of albumin followed by chemical coupling of IgG to albumin by glutaraldehyde. IgG concentration as well as adsorption times were varied in order both to optimize the immobilization conditions and to investigate the adsorption and retention mechanisms. T o evaluate the efficiency of the immobilization techniques, blood plasma, Tween-20, and sodium dodecyl sulfate (SDS) were used to elute the adsorbed IgG layer. We found that IgG was successfully immobilized on the fluorocarbon glow discharge treated surfaces by using either the physical adsorption or the glutaraldehyde coupling method, although the former is more efficient than the latter method. Only 15% of the IgG adsorbed on TFE/PE for 4 h was eluted by SDS. In contrast, 75% of the IgG was eluted from untreated P E by SDS. Both Tween-20 and blood plasma were less effective than SDS in eluting the adsorbed IgG. Preliminary results indicate that antibodies immobilized by the first method retain their antigen binding capability.
Introduction Immunoassays are widely used clinicallyin the diagnosis of various illnesses. A common step in most of these assays is the physical adsorption of an antibody that is specific for the antigen of interest onto a solid surface, e.g., the wells of microtiter plates. Although this technique is very popular, it suffers from several drawbacks ( I ) . These problems are (a) loss of immunological activity of the antibody upon adsorption, (b) desorption of the antibody during assay, and (c) nonspecific binding of the antigen or a second, labeled antibody to the surface of the microtiter plate, giving rise to false positive results. Recent availability of monoclonal antibodies that recognize the circulating antigens of diseases such as schistosomiasis, malaria, tuberculosis, and leprosy has allowed the development of highly sensitive and specific immunoassays for these diseases. In addition to the need for a quantitative assay, there is also a great need for a diagnostic dipstick assay, yielding a rapid, on-site qualitative “yes/no” answer for the presence or absence of these antigens. Such a dipstick should retain the solidphase-immobilized antibody in a condition that maintains its activity over a long shelf life under extreme conditions of heat and humidity and be easy to handle. We have previously shown that albumin and fibrinogen tenaciously adsorb onto surfaces treated with a tetraflu+ Present address: Boris Kidric Institute of Nuclear Sciences, Beograd, Yugoslavia. 8756-7938/91/3007-0173$02.50/0
oroethylene (TFE)radio frequency glow discharge (RFGD) (2, 3 ) . Therefore, we propose here to immobilize monoclonal antibodies onto surfaces RFGD-treated with TFE for use in an immunodiagnostic dipstick assay. It is our hypothesis that a major fraction of the antibodies adsorbed on such surfaces will not be easily desorbed and will retain sufficient activity over a long shelf life under extreme conditions of heat and humidity to be useful for a diagnostic dipstick. This paper reports on the deposition of three different RFGD polymers on two polymer supports, followed by the immobilization of bovine IgG as a model for a monoclonal antibody. We have evaluated two routes of IgG immobilization by the RFGD treatment technique. The first route, referred to as method A, simply consists of physical adsorption of IgG onto RFGD-treated surfaces. In the second route (method B), IgG is coupled (by glutaraldehyde) to albumin that has been preadsorbed onto the RFGD-treated surfaces. Method B takes advantage of the previously observed tight binding of albumin on fluorocarbon RFGD-treated surfaces. It also avoids direct contact between IgG and the polymer surface (method A), which could lead to some unfolding of antibody and concomitant loss in activity. Also, the albumin layer should act to reduce the nonspecific adsorption of antigen or a second, labeled antibody.
Materials and Methods Base Polymers and Monomers. Poly(ethy1eneterephthalate) (PET) Thermanox cover slips (Nunc Inc., Na-
0 199 1 American Chemical Society and American Institute of Chemical Engineers
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perville, IL) and low-density polyethylene (PE) (Penn Fibre, Greenwood, DE) were used as substrates for the RFGD treatments. Poly(tetrafluoroethy1ene) (PTFE) was purchased from Berghof/America Inc. (Raymond, NH). Poly(dimethylsi1oxane) (PDMS) was a product of Dow Corning Corp. (Midland, MI). All polymers were cut into 11 X 16 mm rectangles, cleaned by 15-min ultrasonic treatments in each of the three solvents methylene chloride, acetone, and distilled water, and dried in a vacuum desiccator. Argon (Ar) was obtained from Air Products (Allentown, PA). Ethylene (E) was purchased from Byrne Specialty Gases (Seattle, WA). TFE was a product of PCR Inc. (Gainesville, FL). Hexamethyldisiloxane was received from Alfa Products (Danvers, MA). RFGD Treatments. The cleaned polymers were placed horizontally in the middle of a 135-cm-long, 16-mm i.d. Pyrex reactor. This arrangement allows uniform treatment of both sides of the samples. The radio frequency power, supplied by an EN1 Power Systems generator (Model HF-300, Rochester, NY) was capacitively coupled to the RFGD reactor by two external copper electrodes. The electrodes were positioned 1in. apart and were moved across the length of the reactor at a constant speed of 3.3 mm/s. The reactor was evacuated by a Stokes vane pump (Model 009-2,Pennwalt Corp., Philadelphia, PA) and the pressure was monitored by a Hastings thermocouple vacuum gauge (ModelVT-6), connected downstream from the reactor. The flow rate of the gas introduced into the reactor was measured by an ULTRAFLO mass flow sensor (Vacuum General Inc., San Diego, CAI. The samples were first treated with argon (2.5 W, 3 cm3/min, and 0.1 Torr) to further clean their surface and then treated with the selected monomer gas or vapor under the same conditions. HMDS was degassed prior to the RFGD polymerization by immersing the monomer in liquid nitrogen and allowing it to thaw under vacuum. Flow controllers were not used to measure the flow rate of this monomer. Surface Characterization. The elemental compositions of the surfaces were determined by electron spectroscopy for chemical analysis (ESCA), with a Surface Science Laboratories SSX-100 ESCA spectrometer at the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO) at the University of Washington. Preparation of Radiolabeled IgG. Bovine IgG (Sigma Chemical Co., St. Louis, MO) was labeled with 1251 by the iodine monochloride method of MacFarlane ( 4 ) ,as modified by Helmkamp (5) and Horbett (6). Briefly, 1 mCi of NalZ5I(Amersham, Arlington Heights, IL) was added to 0.5 mL of 0.4 M borate and 0.32 M sodium chloride at pH 7.75. This solution was mixed with 0.5 mL of cold iodine monochloride (2-fold molar excess of iodine over IgG) in 2 M sodium chloride. This solution was then added to 0.5 mL of IgG in borate buffer and mixed by gentle repipetting. After 20-30 min of incubation at 0 "C, the free lZ5Iwas separated from the labeled IgG by gel chromatography (Bio-Gel P-4, Bio-Rad, Richmond, CA) at room temperature with CPBSz (0.01 M citric acid, 0.01 M sodium monobasic phosphate, 0.12 M sodium chloride, and 0.02% sodium azide, pH = 7.4) as the mobile phase. The labeled protein was collected, stored at -70 "C and used within 2 weeks of preparation. IgG Immobilization Methods. (I)Method A . Surfaces were hydrated in CPBSzI (0.01 M citric acid, 0.01 M sodium monobasic phosphate, 0.11 M sodium chloride, 0.01 M sodium iodide, and 0.02 % sodium azide, pH = 7.4)
Biotechnol. Prog., 1991, Vol. 7, No. 2
buffer overnight at 4 "C. The next day, the buffer was replaced with 2 mL of fresh, degassed CPBSzI, and the samples were equilibrated at 37 "C for about 2 h. Adsorption was initiated by adding 1 mL of the IgG solution at 3 times the desired final concentration. Adsorption was allowed to take place for a predetermined length of time at 37 "C, after which the samples were rinsed by the dilution-displacement method, which washes away the unbound antibody while avoiding exposure of the film to air. Samples were placed into the surfactant solution (0.01 M Tris, 0.01 M phosphoric acid, and 1%(w/v) SDS or 0.2% (w/v) Tween-20, pH = 7.0) and the radioactivity of each sample was measured with a y counter (T.M. Analytic, Model 1185R, Elk Grove Village, IL). The amount of adsorbed IgG was calculated by dividing the radioactivity of each sample by the specific activity of the IgG solution and the planar surface area of the sample. To determine the amount of antibody retained, the samples were rinsed following overnight incubation at room temperature in the surfactant solution, and their radioactivity was measured again. The amount retained was calculated similarly to the amount adsorbed. (11)Method B. After overnight hydration at 4 "C in CPBSzI and 1-2 h of equilibration in freshly degassed buffer at 37 "C, the samples were incubated with 0.2 mg/ mL albumin solution in CPBSzI for 2 h. The adsorption was terminated by dilution-displacement rinsing. Then the samples were placed in new vials, containing glutaraldehyde solution (2.5 % glutaraldehyde, 0.05 M sodium monobasic phosphate, and 0.05 M sodium chloride, pH 7.0) for 1 h at 37 "C. The IgG solution was then added and the adsorption was allowed to proceed for 2 h at 37 OC. The samples were rinsed by the dilution-displacement method and placed in the 1% SDS solution overnight, and the radioactivity of each sample was measured in a y counter. After the overnight incubation, the samples were rinsed with CPBSzI and their radioactivity was recounted to determine the amount of IgG immobilized.
Results and Discussion Physical Adsorption of IgG (Method A). We have previously shown that albumin and fibrinogen tenaciously bind to TFE glow discharge deposited polymers (2, 3). One purpose of this study is to determine whether IgG also binds tightly to TFE-treated surfaces. We have investigated the physical adsorption and elutability of IgG for several untreated and RFGD-treated polymers. RFGD polymers of ethylene and hexamethyldisiloxane were deposited on poly(ethy1ene terephthalate) (PET). TFE glow discharge polymer was deposited on both PET and polyethylene (PE). All RFGD treatments resulted in uniform and continuous coatings on the base polymer, as revealed by SEM and ESCA. In Figures 1-3, highresolution carbon Is (ClJ spectra of RFGD-treated polymers are compared to those of the untreated control polymers. We can see from the C1, spectra and the elemental compositions that ethylene-treated PET (E/ PET) is similar to PE (Figure 1)and hexamethyldisiloxanetreated PET (HMDS/PET) is similar to poly(dimethy1siloxane) (PDMS) (Figure 2). In the case of TFE, we have a different situation. In Figure 3, glow discharge deposited polymer of TFE on PET (TFE/PET) is compared to PTFE. TFE/PET shows a very complex spectrum, in contrast to PTFE, which exhibits a single peak at 292 eV, corresponding to the CFZrepeating unit. The complexity of the spectrum for the TFE glow discharge treated surface indicates considerable molecular fragmentation and rearrangement of the TFE monomer during the glow
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Figure 3. ESCA high-resolution carbon 1s spectra of PTFE and TFE/PET. discharge, resulting in a polymer containing CFs, CFz, and CF groups. The elemental compositions of PTFE and the glow discharge deposited TFE are also different. TFE/ PET and TFE/PE (which exhibit similar spectra) have approximately 43 % carbon, 56 5% fluorine, and 196 oxygen, whereas PTFE is composed of 33% carbon and 67% fluorine. The source of oxygen in the glow discharge deposited polymers of TFE is believed to be post-RFGD reaction of the free radicals trapped in the deposited film with atmospheric oxygen. The oxidation is a rather slow process since no oxygen was detected when surfaces were analyzed on the same day that they received the RFGD treatment. Lack of oxygen on the freshly prepared TFE/PET also indicates that the CIS spectrum of the TFE/PET is exclusively due to the deposited polymer and not the underlying PET substrate. PET is composed of carbon, hydrogen, and ca. 16% oxygen. If any of the peaks in the CI, spectrum of TFE/PET were due to PET, one would also expect to detect a significant amount of oxygen on the surface. Further evidence in support of this is the fact that the C1, spectra of the TFE/PE and TFE/PET are
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Figure 4. IgG, 0.2 mg/mL, was adsorbed for 2 h at 37 "C. Overnight incubation with 1%SDS was utilized to elute the adsorbed IgG. identical despite the large difference in the chemical composition of the substrates (data not shown). Bovine IgG was adsorbed for 2 h onto the RFGD-treated and untreated polymer surfaces. The amounts of IgG adsorbed and retained on various surfaces after overnight elution with 1%SDS are compared in Figure 4. While the amount of adsorbed IgG varied within a small range, between ca. 400 and 460 ng/cm2,the retention values varied widely. Consistent with our earlier studies of albumin and fibrinogen retention, the TFE-treated surfaces (TFE/ PET and TFE/PE) retained the highest amount of IgG following elution with SDS. The control (untreated) polymers did not tightly bind the adsorbed IgG, and the same can be said for HMDS/PET. E/PET retained slightly more IgG than untreated PE, but it is still much less than the amount retained by either TFE/PET or TFE/PE. On the basis of these results, only TFE/PE and P E were selected for further studies. In order to maximize the amount of IgG adsorbed and retained by the treated surfaces, as well as to investigate the mechanism of adsorption and retention, various adsorption times and IgG concentrations were studied. IgG adsorption onto PE and TFE/PE from a 0.1 mg/mL solution increases with time and reaches a maximum at ca. 2 h (Figures 5 and 6). For adsorption times longer than 2 h, the amount of IgG on TFE/PE did not change, although a decrease was observed for the untreated PE. The amounts of IgG retained on TFE/PE and the control PE surfaces both increase with the adsorption time (Figures 5 and 6). The TFE/PE surface retains about 50% of the adsorbed IgG after only 5 min of adsorption, with the retention gradually increasing with longer adsorption times to almost 90% after 4 h of adsorption. On the control surface, untreated PE, the percentage of retained IgG increases almost linearly with the adsorption time but reaches only 23 5% after 4 h. The gradual increase in percent retention with time, for both PE and TFE/PE, suggests that the IgG may be changing its conformation on the surface to permit stronger intermolecular interactions. IgG concentration was varied from 0.005 to 0.2 mg/mL, while keeping the adsorption time constant at 2 h, which was deemed suitable from the data in Figures 5 and 6. The IgG adsorption isotherms (Figure 7) show typical Langmuir isotherm behavior: an initial steep rise followed by a plateau. The plateau value for IgG adsorption on TFE/ PE is ca. 360 ng/cmZ. The amount of IgG retained on TFE/PE increases with the IgG concentration and reaches a plateau at ca. 250 ng/cm2. If the IgG were adsorbed as a side-on close-packed monolayer, the amount would be 270 ng/cm2, assuming a prolate ellipsoid with the dimen-
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Biotechnol. Prog.., 1991, Vol. 7, No. 2 500 1
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sions of 235 X 44 X 44 A for IgG (7). Although the amount of IgG adsorbed on TFE/PE exceedsthe monolayer range (Figure 7), only an amount corresponding to a monolayer is retained by that surface. On the control P E surface, however, the amount of IgG retained never reaches the monolayer range and remains below 100 ng/cm2 for all concentrations studied (Figure 7). It is interesting to note that both retention curves show a decline a t higher concentrations (Figure 7). This decline in percent retention at higher IgG concentrations suggests that either (a) IgG binds strongly at certain preferred adsorption sites and less strongly at others and/or (b) the surface crowding at higher IgG concentrations prevents conformational changes that lead to larger retention values due to stronger intermolecular interactions. All the retention values presented so far were obtained after one overnight elution with SDS. Retention values did not change significantly even after repeating the
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overnightelution (Figure 8). Tween-20 and baboon plasma were not as effective as SDS in eluting the adsorbed IgG (Figures 9 and 10). In fact, no IgG was displaced by plasma proteins. Other researchers have also found that SDS is more effective than Tween-20in eluting proteins adsorbed on polymers (8). Physical adsorption is the simplest method of immobilization of bioactive materials. Depending on the nature of aqueous/solid interfaces, biomolecules generally interact with surfaces by a combination of ionic and van der Waals forces (polar plus hydrophobic interactions). The tight binding of IgG on the RFGD-deposited polymer of TFE is most likely due to strong hydrophobic interactions. Other possibilities include polar (C-F) interactions and reaction with reactive sites (e.g., radicals) formed during the RFGD treatment. The mechanism of this tight binding is not yet fully understood and is presently under investigation. Covalent Immobilization (Method B). A major disadvantage of protein immobilization by physical adsorption is the potential for conformationalchangesleading to denaturation and loss of bioactivity. We have shown that the physically immobilized IgG on the TFE-treated surface cannot be removed easily, even with the aid of a strong surfactant. Therefore, denaturation of a significant fraction of the IgG bound on the surface may be occurring. To overcomethis potential problem, IgG was immobilized covalently on albuminated surfacs. This route should prevent direct contact between IgG and the surface and, therefore, presumably avoids conformational changes and denaturation. Albumin can also act to resist nonspecific adsorption in a diagnostic assay. The amount of IgG immobilized by the albumin-glutaraldehyde coupling method is lower than by physical adsorption of IgG (Figure 11). This may be due to the difficulty of getting the IgG close enough to the albumin layer to be coupled to the glutaraldehyde coupling site. In addition, it is possible that higher IgG levels may be achieved with more concentrated solutions of albumin or IgG. The retention data in Figure 12show that a large fraction of the albumin-coupled IgG is retained by TFE/PE. This retention is comparable to that seen when the physical adsorption method was used. In addition, there is no significant difference in the amount of IgG retained by P E and TFE/PE when the albumin-glutaraldehyde coupling method is used. Cross-linking of the adsorbed albumin is most likely responsible for the inability of SDS to elute the albumin-IgG complex. This technique remains interesting and needs to be optimized. Activity of Immobilized Antibodies. The activity of monoclonal antibodies of schistosomiasis, malaria, and
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Figure 9. Elution of adsorbed IgG by SDS and Tween-20. IgG, 0.2 mg/mL, was adsorbed for 2 h and eluted overnight with a 1% solution of SDS or a 0.2% solution of Tween-20. Adsorbed
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Figure 11. Comparison of IgG immobilization by the two methods. Method A: IgG, 0.2 mg/mL, was adsorbed for 2 hand eluted with 1% SDS overnight. Method B: IgG, 0.2 mg/mL, was coupled to surfaces preadsorbed with 0.2 mg/mL albumin and activated with glutaraldehyde. IgG was eluted with 1% SDS overnight.
tuberculosis immobilized on PE by both routes are being investigated and will be published in a subsequent article. Preliminary results are encouraging and suggest that the antibodies adsorbed on the treated surfaces (method A) are still active and capable of recognizing their specific antigen (9). Conclusions. We can conclude that it is feasible to enhance IgG/surface interactions and prevent desorption of surface-bound IgG by TFE glow discharge treatment of the surface. The strength of the IgG/surface interac-
TFEIPE
Figure 12. Percent IgG immobilized by the physical adsorption and chemical coupling methods. Method A: IgG, 0.2 mg/mL, was adsorbed for 2 h and eluted with 1%SDS overnight. Method B: IgG, 0.2 mg/mL, was coupled to surfaces preadsorbed with 0.2 mg/mL albumin and activated with glutaraldehyde. IgG was eluted with 1%SDS overnight.
tions as determined by the reduced SDS elutability of the IgG, rapidly increases as the adsorption time increases. Covalent couplingof IgG to a preadsorbed layer of albumin, which is tenaciously bound onto TFE-treated surfaces, offers the option of immobilizing the IgG while avoiding direct contact between the surface and the IgG. The albumin layer may also act to resist nonspecific adsorption during the assay.
Acknowledgment We thank the NESAC/BIO (NIH grant RR01296) for the use of its ESCA instrument and the Washington Technology Centers a t the University of Washington and the International Atomic Energy Agency (TC 302-El-USA5280) for their financial support. Literature Cited (1) Kemeny, D. M.; Chantler, S. An introduction to ELISA. In ELISA and other solid phase immunoassays; Kemeny, D. M., Challacombe, S. J., Eds.; John Wiley and Sons Ltd.: New York, 1988; p 22. (2) Kiaei, D.; Hoffman, A. S.; Ratner, B. D.; Horbett, T. A.; Reynolds, L. 0. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1988, 42, 269. (3) Kiaei, D.; Hoffman, A. S.; Horbett, T. A. Transactions of the 15th Annual Meeting of the Society for Biomaterials, April 28-May 2, 1989, Lake Buena Vista, FL; Society for Biomaterials: Birmingham, AL, 1989; p 110. (4) MacFarlane, A. Nature 1959, 182, 53. (5) Helmkamp, R. W.; Goodland, R. L.; Bale, W. F.; Spar, I. L.; Mutschler, L. E. Cancer Res. 1960, 20, 1495. (6) Horbett, T. A. J. Biomed. Muter. Res. 1981, 15, 673. (7) Colander, C.-G.; Kiss, E. J. Colloid Interface Sci. 1988,121, 1. (8) Rapoza, R. J.; Horbett, T. A. J . Colloid Interface Sci. 1990, 136, 480. (9) Report of the Research Coordination Meeting on Development of Diagnostic Reagents for Communicable Diseases using Radiation Processing Techniques, Directed by J. B. Castelino and V. Markovic, International Atomic Energy Agency, Vienna, Austria, September 11-14, 1989.
Accepted January 15, 1991. Registry No. SDS, 151-21-3;PE, 9002-88-4; TFE, 116-14-3; PET, 25038-59-9; PTFE, 9002-84-0; E, 74-85-1;Tween-20,900564-5; hexamethyldisiloxane, 107-46-0.
