Bioconjugate Chem. 1003, 4, 166-171
168
New Application of Silane Coupling Agents for Covalently Binding Antibodies to Glass and Cellulose Solid Supports Niedre M. Pope, David L. Kulcinski, Alan Hardwick, and Yu-An Chang' Baxter Healthcare Corporation, Biotech Group-Immunotherapy Division, 3015 South Daimler Street, Santa Ana, California 92705. Received October 2, 1992
Bifunctional silane reagents (3-iodopropy1)trimethoxysilane(I), (y-glycidoxypropy1)trimethoxysilane (2), and [l-(trimethoxysilyl)-2-(m-(or p-)chloromethyl)phenyllethane (3) were used to covalently link goat anti-mouse (GAM) antibodies (Ab) to glass microbeads and cuprammonium rayon hollow-fiber dialyzers. An average of 0.79 and 0.83 bg of GAM Ab/ cm2was immobilized on the hollow-fiber dialyzers and the glass beads, respectively. The antibodies immobilized on glass microbeads or on hollow-fiber dialyzers were then used to selectively deplete CD34+ cells or CD4+ peripheral blood mononuclear cells (PBMC), respectively. Glass microbeads depleted 80% CD34+ cells with good selectivity, and the hollow-fiber dialyzers depleted an average of 81% CD4+ PBMC.
INTRODUCTION
Scheme I. Bifunctional Silane Reagents
Immobilization of biologicals had its beginning in 1916 with work done by Nelson and Griffin (1) when they immobilized invertase onto charcoal and alumina. Since then, the immobilization of biologicalsto a variety of solid supports has become a widely used technology. Bifunctional silanes of the structure (Me0)3Si(CHdnX (Scheme I) were used extensively in the 1970s as coupling agents to link enzymes to inorganic solid supports (21, such as minerals, including sand, diatomaceous earth, and hornblende, or fabricated materials, such as controlledpore glass and ceramics, nickel oxides, and iron oxides (3-6). Since that time, technology has shifted to the method of directly coupling biologicals to chemically modified organic solid supports, such as derivatized vinyl polymers, polyamides, and polysaccharides (agarose, cellulose, Sepharose, Sephadex, etc.) (7). This shift to the use of organic solid supports occurred because these supports can be derivatized under mild conditions, and because a wide variety of functional groups can be put on organic solid supports (8). Until recently, reaction conditions used to silanize inorganic solid supports were relatively harsh. For example, Weetall (9) activated glass by refluxing in a 10% solution of (y-aminopropy1)triethoxysilanein toluene for 24 h. Toluene, with a boiling point of 110.6 "C, would destroy many organic solid supports. Frequently after silanization, further derivatization of the organic functionality on the silane was necessary prior to immobilization of a biological. Derivatization methods for silaneactivated inorganic solid supports were limited and equally as harsh as those for silanization. Greenfield and Lawrence (10) used thiophosgene in chloroform to form a thiocyanate derivative which could then be coupled with the enzyme. Weetall and Filbert (11) used p-nitrobenzyl chloride in chloroform to form an aminoacyl derivative. The nitro group was then reduced with dithionite. After forming a diazo group with nitrous acid, the enzyme could be coupled to the solid supports. In this study, we have used bifunctional silane coupling agents to immobilize antibodies onto low-density-glass microbeads and onto acetylated cellulose hollow-fiber dialyzers. Reaction conditions for silanizing the solid supports were mild and silane reagents were chosen such that further derivatization was not necessary. The im-
0
(Me0)3SiCH2CH2CH21
(Me0)3SiCH&H2CH20CH2A (2)
(1)
(MeO),SiCH,CH,
H C ZJT'
(3)
mobilized antibodies were then tested for their ability to selectively deplete target cells. EXPERIMENTAL PROCEDURES Reagents. (3-Iodopropyl)trimethoxysilane, (y-glycidoxypropyl)trimethoxysilane, and [l-(trimethoxysilyl)-2(m-(orp-)chloromethyl)phenyllethanewere all purchased from Hiils American Corp., Bristol, PA. Cupraphan hollow fiber dialyzers were obtained from Baxter Healthcare Corp., Membrane and Materials Technology Group, Round Lake, IL. Low density floating glass Scotchlite microbeads (catalog no. H50/1000) were obtained from 3M, St. Paul, MN, boric acid and ethanol reagent alcohol were purchased from Baxter Healthcare Corporation, Scientific Products Division, McGaw Park, IL, and goat anti-mouse (cat. no. 115-005-071)and mouse IgG/HRPO were purchased from Jackson ImmunoResearch Labs, Inc., West Grove, PA. Anti-CD4 FITC, GAM FITC, GAM PE, Simultest control, mouse anti-Leu 3a (anti-CD4) Ab, and mouse anti-HPCA-1 (anti-CD34) Ab were purchased from Becton-Dickinson, Mountain View, CA; ABTS substrates A and B, from Kirkegaard and Perry; and Ficoll Histopaque 1077,Dulbecco's phosphate-buffered saline (dPBS), Hank's balanced salt saline (HBSS), Tween 20, RPMI, and fetal bovine serum (FBS), from Sigma Chemical Co., St. Louis, MO. lZ5I-labeledGAM Ab was prepared using Boulton-Hunter Reagent from Du Pont. Paraformaldehyde fixative was prepared by dissolving cacodylic acid (10.7 g) into 900 mL of deionized water. The pH was adjusted to 7 with 1M HCI. The solution was heated to 70 "C with a water bath. Then paraformaldehyde (10 g) was added and the solution was returned to the 70 "C water bath with occasional stirring until all the paraformaldehyde was dissolved. The solution was then cooled to ambient temperature and the pH adjusted to 7.2 using 1 N NaOH. Next, the volume was adjusted to 1 L
1043-1802/93/2904-0166$04.00/0@ 1993 American Chemical Society
Bioconjugate Chem., Vol. 4, No. 2, 1993
Silane Reagent for Coupling Abs to Solid Supports
Scheme 11. Reagents
Activation of Glass Beads by Silane
(Me0)3Si-X
x
+
H20
FUNCTIONAL GROUPS SUCH AS IODIDE, BENZYL CHLORIDE AND EPOXIDE.
: ALKYL or ARYL SIDE ARMS
95% EtOHIH20
OH
+
(H0)3S-i
X
Dehydration at 5OoC, 6 hours
or high vacuum (1 mmHg) at r.t. overnight.
t I I I
0 I OSi I
x
F
0 I I
I
and the solution filtered through a Corning 0.22-pm cellulose acetate membrane filter unit. The paraformaldehyde fixative was stored a t 4 "C. Preparation of the Microbeads. Floating glass microbeadswere washed in concentrated HC1for 2 h followed by concentrated nitric acid a t ambient temperature overnight to remove any coatings or contaminates on the microbeads. The microbeads were washed with deionized water until all traces of acid were removed. The microbeads were then centrifuged in 100 mL of deionized water for 5 min at 1000 rpm to remove any cracked or broken microbeads, which tended to sediment. This was done until no broken microbeads sedimented. Then the microbeads were washed with 100 mL of ethanol, filtered, and dried under reduced pressure. Silane Activation of Microbeads. Silane solutions (40 mL, 10% solution in 95% ethanol/deionized water) were prepared from the three silanes (Scheme 11). Each of them was added to 4 g of microbeads. The mixture was rotated end-over-end a t ambient temperature for 1h. The excess silane solution was filtered off and the microbeads were dried a t 50 "C for 6 h, or under high vacuum (1mmHg) overnight. Excess silane and aggregated microbeads were washed away by filtering microbeads through a metal screen (100 mesh) with ethanol. The microbeads were filtered and dried a t 50 "C. These microbeads can be stored up to 6 months without loss of activity. Coupling of GAM Ab to Silane-Activated Microbeads. Goat anti-mouse antibody was added to the microbeads in borate buffer (0.05 M, pH 9.6). The mixture was rotated end-over-end at ambient temperature overnight. The borate buffer was filtered off under reduced pressure, and the microbeads were washed with dPBS. Microbeads were then weighed out and stored in borate
167
buffer for use in either functional protein binding or cell binding studies (Scheme 111). Analysis of Functional GAM Ab Content on Microbeads. HRPO-conjugated mouse IgG (mouse IgG/ HRPO) was used to determine the quantity of functional GAM Ab bound to the microbeads. The ELISA was performed using 2 mg of silane-activated, GAM Ab coupled microbeads, and 2 mL of a 50X dilution of mouse IgG/ HRPO (1 pg/mL) in 0.05% Tween 20/dPBS. The microbeads and mouse IgG/HRPO were incubated by endover-end rotation a t ambient temperature for 30 min. A set of standards was prepared (50 pL of diluted mouse IgG/HRPO + 950 pL dPBS) by serial dilution with dPBS. Three to five washes were done just prior to, and after, incubation using 1%Tween 2O/PBS (2 mL), to remove nonbound antibody. Microbeads were filtered, weighed, and placed in dry test tubes. ABTS substrates A and B were mixed in a 1:lratio and then added to the microbeads (2 mL) in 5-9intervals and incubated a t room temperature for 20 min. The reaction was quenched with oxalic acid (1 mL, 0.25 M). Concentrations of mouse IgG/HRPO bound were determined by comparison with a standard curve read a t 405 nm (Scheme IV). Preparation of KGla Cells for Cell-DepletionExperiments. A flask of about 1X 108fluorochromed (100 pL) KGla cells, which possess CD34 antigen on their surface, was spun down for 7 min at 1400 rpm and 4 "C. Cells were resuspended in a solution of 2% FBS/RPMI (40 mL) and were centrifuged as above. This procedure was repeated three times. Mouse anti-CD34Ab was added to the cells (1pg/l X lo6 cells), after which the cells were incubated at 4 "C for 30 min. Cells were washed three times in 2% FBS/RPMI, centrifuged as above between washes and counted using a hemocytometer. Cells were resuspended in 5 mL 2% FBS/RPMI and stored on ice until added to the glass microbeads. Depletion of KGla Cells by the Glass Microbeads. KGla cells were added to the glass microbeads in the ratio of 1X lo4 cells/mg bead. The volume was adjusted to 20 mL, and the microbeads and cells were incubated endover-end at 10 rpm for 1 h at 4 "C. After incubation, microbeads were allowed to float to the top of the suspension and the underlying fluid (effluent) was drawn off. Cells in the effluent were counted with a hemocytometer (Scheme V). Silane Activation of Hollow-Fiber Dialyzers. Activation solution [10% (yglycidoxypropy1)trimethoxysilane in 95% ethanol/water] was pumped via a peristaltic pump through a flow loop containing a small-scale hollowfiber dialyzer for 20-22 h a t a flow rate of 0.4 mL/min. The hollow-fiber dialyzer was dried under reduced pressure a t ambient temperature. Hollow-fiber dialyzers were used within 3 days to a week (Figure 1). Coupling of GAM Ab to Hollow-FiberDialyzers. A solution of 1.67 mg GAM Ab/mL dPBS was pumped and recycled through the loop and hollow-fiber dialyzer for 20-22 h at a flow rate of 0.4 mL/min. Hollow-fiber dialyzers were then used for either lZ5I-labeledGAM Ab binding or for cell binding studies. Analysis of Antibody Content on Hollow-Fiber Dialyzers. lZ5I-labeledGAM Ab buffer (3 mg GAM Ab + 0.16 mg lZ5I-GAMAb in 5 mL of dPBS) at a radiation level of 1 x lo6 counts per minute (cpm) was recycled through the flow loop and hollow-fiber dialyzer for 20-22 h a t a flow rate of 0.4 mL/min. After recycling, the hollowfiber dialyzer was washed by flushing 35 mL of dPBS through the flow loop, followed by washing with 25 mL of 2 % Tween 2O/dPBS, to remove nonbound antibody. The
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168 Bioconjugafe Chem., Vol. 4, No. 2, 1993
Scheme 111. GAM Ab Coupling to the Activated Glass Beads
x
0 I
osi
)(
I
: FUNCTIONAL GROUPS SUCH AS IODIDE,
BENZYL CHLORIDE AND EPOXIDE. :ALKYLorARYLSlDEARMS
0
I I I
0 0.05 M Borate Buffer
+
I
w
OSi
pH 9.6
I
0
v
-i N~
I
I I I
NU = NH2 01 SH
Scheme IV. HRPO Assay for Quantification of Functional GAM Ab I
I I
0
GAM Ab
0
I
I
I
I
I
I
Incubate 30 min. at r t
+
*
I
0 I
I I
HRPO
Scheme V. KGla Cell Depletion by GAM Ab Immobilized Glass Beads I
I I
0
0 I I I
I
GAMAb
I I
KGla Cell Depletlonln 2% FBSlRPMl buffer
+
0 I
OSi I
0 I
I
I I
HPCA-1 MOU-
Ab
CD 34 Antlgen
hollow-fiber dialyzer was immediately taken out of the flow loop, and the fibers were cut into 1-cm pieces with a razor blade. The fiber bundles were placed in sequentially numbered test tubes and assayed in a y-counter for radioactivity. The amount of GAM Ab bound to a fiber bundle was calculated as follows: bound GAM Ab (mg) = measured cpm X (3.16 mg GAM Ab/l X lo6 cpm).
Preparation of Cells for PBMC Depletion Experiments. Fresh human blood (approximately50 mL) was collected and heparinized. The blood was aliquoted such that 15 mL was added to a 50-mL centrifuge tube containing Ficoll Histopaque 1077 (10 mL). Each centrifuge tube was then filled to 40 mL with Hank's bufferedsaline solution (HBSS). The tubes were centrifuged a t
Bioconjugate Chem., Vol. 4, No. 2, 1993
Silane Reagent for Coupling Abs to Solid Supports
160
Y
Reservoir
Figure 1. Flow loop for hollow-fiber dialyzer. Table I. Preparation of Cell Samples for FACScan Analysis sample sample prep. number 1 after 1st pass 2 after 1st pass 3 after 2nd pass 4 after 2nd pass 5 after 3rd pass 6 after3rdpass
first antibody anti-CDC anti-CD4 anti-CD4 antLCD4 anti-CD4 anti-CD4
second antibody Simultest control GAM-PE Simultest control GAM-PE Simultest control GAM-PE
third antibody none anti-CD4-FITC none anti-CD4-FITC none anti-CD4-FITC
2000 rpm for 15 min a t room temperature. The resulting layer of peripheral blood mononuclear cells (PBMC), some of which possess the CD4 surface antigen, was removed from each tube and pooled. The PBMC were washed twice in HBSS (centrifuged a t 1000 rpm for 10 min) and were then resuspended in 2% FBS/RPMI (10 mL), yielding approximately 6 X lo7PBMC as determined by a Coulter counter. Anti-CD4 antibody was then added to 4 X lo7 PBMC in 2 % FBS/RPMI (5 mL) and mixed for 30 min a t 4 OC. Unbound antibody was removed by washing the cells in 2 % FBS/RPMI twice (centrifuged a t lo00 rpm for 10min). The PBMC were then resuspended in 5 mL 2% FBS/ RPMI and kept on ice until needed. Depletion of PBMC Cells by the Hollow-Fiber Dialyzers. The starting PBMC suspension, which consisted of approximately 3 X lo7 PBMC in 5 mL, was pumped once through the GAM Ab-coupled hollow-fiber dialyzer a t a flow rate of 0.4 mL/min. Fresh 2 % FBS/ RPMI (2 mL) was then pumped through the hollow-fiber dialyzer to flush out unbound cells. All effluent was collected into a test tube. An effluent aliquot was removed for counting cells with a Coulter counter and for later FACScan analysis. The remaining effluent was then pumped through the hollow-fiber dialyzer a second time a t a flow rate of 0.4 mL/min, followed by a 2-mL flush with 2% FBS/RPMI. Effluent from this second pass through the hollow-fiber dialyzer was also collected in a test tube, and aliquots were taken for cell counts and FACScan analysis. After a third and final pass of effluent through the hollow-fiber dialyzer, samples were prepared for FACScan analysis. Preparation of Cell Samples for FACScan Analysis. After passage through the hollow-fiber dialyzer, cell samples were washed in 2 % FBS/O.O2 % NaN3/PBS buffer, and a second antibody, GAM Ab/FITC or -/PE, was added (Table I). Samples were incubated with a second antibody (20 pL) for 30 min a t 4 "C. Samples to be incubated with
1.25
2.5
5
10
ug GAM Ab Offeredlmg Beads
Figure 2. Optimum GAM Ab to bead ratio. a third antibody, anti-CD4 AWFITC, were first washed with 2% FBS/O.O2% NaNdPBS buffer and then were incubated with the antibody (20 pL) for 30 min a t 4 OC. All samples were washed once more and then resuspended in 500 pL of paraformaldehyde fixative solution. Samples were stored in the dark at 4 "C overnight until analyzed. RESULTS
Functional GAM Ab Binding Assay. ELISA was used to determine the optimum functional GAM Ab coupled to glass microbeads activated with the benzyl chloride silane by varying the amount of GAM Ab offered per milligram of microbeads (Figure 2). The optimum GAM Ab to bead ratio was found to be 2.5 pg GAM Ab/mg of microbeads for this particular system. Next, the ability of microbeads activated with the three silane reagents to bind GAM Ab functionality a t a 2.5 pg GAM Ab/mg microbead ratio (Figure 3) was compared. The iodide (1) and benzyl chloride (3)silanes performed better than the epoxide silane (21, perhaps due to instability of the epoxide under the basic (pH 9.6) conditions used to couple GAM Ab onto the silane-activated beads. KGla Cell Depletion. Control experiments were done using KGla cells to compare the effects that the following conditions have on KGla cell depletion: (A) nonactivated microbeads with and without GAM Ab incubation, (B) silane-activated microbeads with and without GAM Ab incubation, or (C) the use of KGla cells which have not been sensitized by mouse IgG anti-CD34 Ab with silaneactivated, GAM Ab-coupled microbeads (Figure 4). There
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170 Bioconjugate Chem., Vol. 4, No. 2, 1993 100
4
v
e
m
-
..
@ 3
2 B
8 2
a
$P
P 0
0
Epoxide
iodide
Benzyl Chloride
Figure 3. Comparison of three silane reagents.
'
With
HLG
hllr
K G l a Deoktion Wilhout Hi66 C.lie
Figure 5. KGla cell depletion with and without the presence of nontarget HL60 cells. Table 11. Location of lZ6I-LabeledGAM Ab
--6
80.0%
80-
L
dialyzer description
P
60-
% %in % %in %in bound recycle PBS Tween recovto fibers solution wash wash ery
untreated dialyzer silane-activated dialyzer #l silane-activated dialyzer #2
2 3
7
75 84 25
10 10 58
6 5 1
93 102 91
Table 111. Amount of GAM Ab Bound to the Hollow-Fiber Dialyzers 0.8%
1
2
3
4
5
Figure 4. KGla cell-depletioncontrol experiments: unactivated beads (1) without GAM Ab and (2) with GAM Ab; activated beads (3)without GAMAband (4) with GAMAb;(5)unsensitized KGla cells using activated, GAM Ab-coupled beads. was essentially no cell depletion observed for microbeads which were not incubated with GAM Ab. In addition, no cell depletion was obtained using nonsensitized KGla cells. Silane-activated, GAM Ab-coupled microbeads showed significant cell depletion, although a small amount of depletion was seen with nonactivated microbeads which had been incubated with GAM Ab. These results indicate that the silane-activated glass bead system can efficiently deplete target cells without nonspecific trapping of the cells. Additionally, 2 % fluorochromed KGla cells were mixed with nontarget cells (HL60 cells). These nontarget cells do not contain the CD34 antigen and were not fluorochromed. The GAM Ab-immobilized glass microbeads were incubated with this cell mixture. Percent KGla cell depletion was essentially the same as those experiments done without HL60 cells. Further, no nonfluorochromed cells were depleted by the microbeads. This indicates that GAM Ab-immobilized glass microbeads can selectively deplete target cells (KGla cells) from a large quantity of nontarget cells (98% HL60 cells) without sacrificing depletion purity and efficiency (Figure 5). lZ5I-GAMAb Binding Assay. GAM Ab was offered to the hollow-fiber dialyzers a t a concentration of 1.66 mg/mL. Table 11, which is a mass balance for the radiolabeled Ab, shows the final location of the radiolabeled antibody. Most of the 1251-labeledGAM Ab ended up in the recycle solution or in the PBS wash. A Tween 20 detergent wash was done to remove any noncovalently bound protein. However, since the hollow-fiber dialyzers were saturated with GAM Ab for maximum immobilization, it is possible that even with a 2% Tween 2O/PBS
dialyzer description
untreated dialyzer silane-activated dialyzer #I silane-activated dialyzer #2 GAM Ab-saturated glass beads GAM Ab-optimized glass beads
bound Ab (rg)
bound Ab/surface area (pg/cm2)
69 104 217 171 33
0.34a 0.51 1.07 0.83b 0.16c
a The surface area of the hollow fiber dialyzers is 203.5 om2. The surface area of the glass microspheres is 1.029 m2/g. The micrograms of GAM Ab bound was determined indirectly by a Pierce protein assay of the GAM Ab left in the supernatent after incubation with 20 mg of silane-activated glass beads. The micrograms of GAM Ab bound under optimized conditions (0.0025 mg of GAM Ab/mg glass microsphere) was also determined by Pierce protein assay.
Table IV. Results of the CD4+ Cell Depletions
CD4+ cells non-CD4+ cella CD4+ cells non-CD4+ cells CD4+ cells non-CD4+ cells
cummulative 7% cell depletion after 1st uass after 2nd uass after 3rd uass Control Experiment 13O 30 Experiment 1 39 50 63 18 20 36 Experiment 2 99.70 99.90 99.90 49 50 53
Represent cells nonspecifically trapped in the hollow-fiber dialyzers.
wash, some noncovalently bound GAM Ab remained (Table 111). The radiolabeled Ab binding studies also enabled the determination of the absolute amount of antibody bound to the hollow-fiber dialyzers, as shown in Table 111. CD4+ Cell Depletion. Three CD4+ cell depletions from peripheral blood mononuclear cells were carried out. Results from the Coulter counter and FACScan analysis are shown in Table IV. The control experiment differed from experiments 1and 2 in that the hollow-fiber dialyzer was neither silane-activated, nor GAM Ab-coupled. Of
BloconJugate Chem., Vol. 4, No. 2, 1993
Silane Reagent for Coupling Abs to Solid Supports
the 1.37 X lo8 starting PBMC, 60% were CD4+. In experiment 1, the starting cell population consisted of 2 X 107 PBMC, of which 59% were CD4+ cells. In experiment 2, the starting cell population consisted of 3.4 X lo7PBMC, of which 29 % were CD4+ cells. After passing these PBMC through the hollow-fiber dialyzers, the CD4 positive cells were depleted 13% (control), 63 % (experiment l), and 99% (experiment 2), respectively. In addition to the CD4+ cells that were depleted, 3 5% ,36 % , and 53 % of the CD4- cell population was also depleted. This indicates that there was a certain degree of nonspecific trapping of PBMC by the hollow-fiber dialyzers. DISCUSSION
In this study we have developed a simple and versatile method to activate both organic and inorganic solid supports, under mild conditions, for subsequent immobilization of biologicals using bifunctional silane-coupling reagents. We have shown that silane-activated glass microbeads and acetylated cellulose hollow-fiber solid supports will indeed bind biological agents without loss of biological activity, so that specific target cells can then be captured. The solid supports used in this work were chosen because they are inexpensive relative to many now on the market, and they provide a large surface area for bioselective separation. One interesting observation made of the hollow fibers requires discussion. For covalent attachment of silane to take place with acetylated cellulose, the following reaction takes place: 0 S H O H
+
H O - SI i w X
I 0'
-H20
53 +HzO
0'
I C H O - 7
w x
0'
thus forming a C-0-Si bond, which has been generally observed to be labile. In the liquid phase, lability is expected due to rapid hydrolysis of the C-0-Si bond. In the case of hollow-fiber solid supports, the C-0-Si linkages
171
were formed under mild dehydration conditions. In terms of kinetics, two-phase reactions are usually slow. Since the C-0-Si linkages were made on the solid phase, when the activated hollow-fiber dialyzer was subjected to an aqueous environment, hydrolysis of these bonds was relatively slow. The 1261-labeled-GAMAb study and the CD4+ cell depletion data both indicate that the C-0-Si bonds on the solid support were stable during the bioassay time frame. ACKNOWLEDGMENT
We wish to thank Ms. Virginia H. Mansour for providing KGla cells, Mr. Dan Boggs for providing the hollow-fiber dialyzers, Dr. Bonnie Mills for aid with FASCan analysis, and Dr. William Lake for useful advice. LITERATURE CITED (1) Nelson, J. M.,and Griffin, E. G. (1916) J . Am. Chem. SOC. 38, 1109. (2) Plueddeman, E. P. (1982) Silane Coupling Agents, Chapter 8, pp 207-232, Plenum Press, New York. (3) Cabral, J. M. S., and Kennedy, J. F. (1991) Covalent and Coordination Immobilization of Proteins. In Protein Immobilization Fundamentals and Applications (R. F . Taylor, Ed.) p p 73-138, Marcel Dekkar, New York. (4) Kent, C., Rosevear, A., and Thomson, A. R. (1978) Enzymes Immobilized on Inorganic Supports. In Topics in Enzyme
and Fermentation Biotechnology (5) Scouten, W. H. (1981) Chemical Analysis. Affinity Chromatography (P. J. Elving, and J. D. Winefordner, Eds.) Vol. 59, John Wiley & Sons, New York. (6) Scouten, W. H.(1983) Chemical Analysis. Solid Phase Biochemistry (P. J. Elving, and J. D. Winefordner, Eds.) Vol. 66, John Wiley & Sons, New York. (7) Kinner, K. J. (1975) Chem. Eng. News Aug 8, 22-41. (8) Wingard,L. B.,Katchalski-Katzir,E.,andGoldstein,L. (1976) Applied Biochemistry and Bioengineering. Immobilized Enzyme Principles, Vol. 1, Academic Press, New York. (9) Weetall, H. H.(1969) Nature 223,959. (10) Greenfield, P. F., and Laurence, R. L. (1975) J . Food Sci. 40, 906. (11) Weetal1,H. H., andFilbert,A. M. (1974) Methods Enzymol. 34, 59-72.
