Bonding proteins to inorganic supports

Proteins such as enzymes or antibodies can be immobilized in a biologically active state on various inorganic supports via an intermediate residue of o-dianisidine.

BACKGROUND OF THE INVENTION 
1. Field 
This invention is concerned generally with the field of immobilized 
proteins and specifically with enzymes and immune bodies which have been 
immobilized on inorganic support materials. 
2. Prior Art 
The desirability of immobilizing enzymes, antibodies, and other proteins 
for repetitive use and/or ease of handling is well known. Proteins have 
been immobilized or fixed on a wide variety of materials, both organic 
(e.g. U.S. Pat. No. 3,705,084 to Reynolds) and inorganic (e.g. U.S. Pat. 
No. 3,519,538 to Messing et al. disclosing enzymes bound via silanes and 
U.S. Pat. No. 3,652,761, to Weetall disclosing antibodies bound via 
silanes). See also, U.S. Pat. No. 3,850,751, to Messing disclosing enzymes 
bonded via adsorption to certain inorganics and U.S. Pat. No. 3,930,951 to 
Messing disclosing the use of another intermediate, 4,4'-bi 
(methoxybenzenediazonium chloride) or BMBD, to bond enzymes to various 
inorganics. 
Although bonding by adsorption or via covalent bonds both have advantages 
and disadvantages relative to each other, it can be appreciated that, in 
general, bonding via chemical coupling means provides a stronger bond 
which is not subject to such factors as pH change. Hence, considerable 
attention has been directed toward finding relatively inexpensive 
"coupling agents" which can be used as intermediate links between proteins 
and various inorganic supports. Although substances known as silane 
coupling agents have been used as coupling agents for some time now, such 
materials are relatively expensive and must often be chemically modified 
after attachment to the inorgainic, but before reaction with the protein, 
thus adding an undesirable processing step and added cost. The BMBD 
compound disclosed recently in U.S. Pat. No. 3,930,951 offers some 
advantages over silane coupling agents but it is still rather expensive. 
We have now found there exists an intermediate coupling agent which is 
relatively inexpensive and requires little or no modification prior to 
protein bonding. Details of our method of using such coupling agent to 
bond a variety of proteins are disclosed herein. 
SUMMARY OF INVENTION 
Our method of bonding proteins to inorganic support materials to form 
composities of immobilized biologically active proteins comprises the 
steps of reacting a high surface area, essentially water insoluble 
inorganic support material having available oxide or hydroxide surface 
groups with a solution of o-dianisidine under conditions sufficient to 
form a support coated with residues of o-dianisidine, and then reacting 
the coated support with an aqueous solution of the proteins to be bonded. 
In preferred embodiments, the inorganic support has a high surface area 
(&gt;0.2 m.sup.2 /g) and is siliceous. 
SPECIFIC EMBODIMENTS 
The carriers which can be reacted with the o-dianisidine solution include 
any essentially water-insoluble inorganics having available surface oxide 
or hydroxyl groups capable of reacting with the o-dianisidine. The exact 
mechanism whereby the surface inorganic reacts with the organic compound 
is not fully understood although the bond formed was found to be quite 
strong. For example, the bond could not be washed off with 0.5 M sodium 
chloride, boiling water, or urea. 
For reasons of economy, convenience, and control of surface area, and, 
where desired, porosity, we prefer siliceous support materials, consisting 
mainly of silica. Silica per se, glass, and porous glass or fritted glass 
are all useful supports and can be readily surface activated and coated 
with o-dianisidine from either an aqueous or organic solvent. 
After the inorganic support has been reacted with the o-dianisidine 
solution to form a surface coating of o-dianisidine residues, the coated 
support can be reacted directly with a protein solution or, where desired 
to introduce certain functional groups, indirectly with the solution after 
some further modification. For example, in some of the examples below, 
glutaraldehyde was found to be an excellent modifier between the 
o-dianisidine residue and the proteins of the protein solution, thus 
permitting a relatively mild protein bonding step. Where it is desirable 
to space the protein away from the support a "coupling arm" of 
o-dianisidine and modifiers can be extended by simply alternating the 
reaction of o-dianisidine and modifiers such as glutaraldehyde. 
In general, the reactions using the o-dianisidine are relatively rapid, an 
obvious advantage. The o-dianisidine molecule, somewhat similar to the 
BMBD of U.S. Pat. No. 3,930,951, cited above, has the following structure: 
##SPC1## 
The compound can be applied in an aqueous solution or in such solvents as 
methanol. In either case, the concentration used can be fairly small, 
preferably about 1% by weight. The reaction can be completed in about 10 
minutes and can be done at room temperature. Once an o-dianisidine residue 
has been formed on the inorganic surface, the treated support may be 
reacted directly with the aqueous protein solution in which case it is 
thought that the available amino group of the o-dianisidine residue reacts 
with available reactive groups (e.g. --COOH) on all proteins. 
Alternatively, the o-dianisidine residue is modified with a modifier such 
as glutaradlehyde to tailor-make a surface for subsequent reaction with 
the protein. For example, in some cases, to protect an active site on an 
enzyme or complexing site on an antibody, it may be desirable to modify 
the available amino groups of the o-dianisidine to form a non-interfering 
group capable of bonding with the protein. 
The proteins which can be usefully immobilized according to this disclosure 
include any polypeptides which, when immobilized according to the 
teachings herein, will retain a useful function (e.g. biological 
activity). In the case of antibodies, complexing ability or affinity for 
antigenic substances or haptens must be preserved. Examples of the wide 
variety of both enzymes and antibodies and proteinaceous antigenic 
substances which can be bonded according to this disclosure can be found 
in U.S. Pat. No. 3,519,538, (enzymes) and U.S. Pat. No. 3,652,761, 
(antibodies and antigenic substances). 
In the examples below, we describe the bonding of a variety of enzymes and 
one antibody to a variety of inorganic carriers and then demonstrate 
retention of the respective biological activities. In some preliminary 
experiments, o-dianisidine was attached to the surfaces of porous silica, 
controlled pore porous glass (CPG) and fine fritted glass using a 1% 
o-dianisidine aqueous solution containing 0.7 ml HC1 per 100 ml of 
solution. 
In some cases, the surface modified siliceous materials were washed and 
then converted to materials having surface aldehyde groups by the addition 
of glutaraldehyde. The aldehyde derivative was then washed. To various 
samples of these derivatives enzymes such as papain, lactase and catalase 
were attached. The papain composite was then shown to have enzymatic 
activity with casein. Catalase activity was demonstrated with peroxide 
solution. Alkaline Bacillus subtilis protease was attached directly to the 
o-dianisidine derivative of the porous silica and then shown to have 
retained enzymatic activity by use of casein. 
PREATION OF SURFACE DERIVATIVES 
The inorganic surfaces can be readily derivatized via both aqueous and 
organic solvents. 
METHOD 1 
Aqueous Solution Preparation 
For this method, the carrier material consisted of porous silica particles 
(45 to 80 mesh) having a surface area of 40 m.sup.2 /g and an average pore 
diameter of 425A. The minimum pore diameter was 270A and the maximum 
diameter was 475A. The o-dianisidine solution consisted of 5 grams of 
o-dianisidine plus 4 ml HCl diluted to 500 ml with water. 
A 100 gram sample of the silica was transferred to a coarse, 350 ml fritted 
glass funnel. To this sample, 200 ml of the o-dianisidine solution was 
delivered at a rate of 1000 ml per hour. The o-dianisidine was trapped at 
the top surface of the silica and the solution came through clear. After 
delivery of the 200 ml was completed, the silica cake was drained. An 
additional 200 ml of the o-dianisidine was delivered to the silica. Again, 
after passage through the silica, the solution was colorless. After the 
second delivery, isopropanol was delivered to the derivative to wash away 
excess o-dianisidine. The first 100 ml of alcohol delivered was intensly 
colored brownish-red; however, after a second 100 ml volume of the alcohol 
was delivered, the wash solution began to lose color. The support was then 
washed with 300 ml of water followed by another 100 ml of isopropanol and, 
finally, washed with 100 ml of acetone and then air dried with aspiration 
for 1 hour. The carrier derivative was transferred to a glass bottle and 
used subsequently for preparing the immobilized proteins. 
METHOD 2 
Organic Solution Preparation 
For this procedure, the inorganic support consisted of silica particles 
having a surface area of 370 m.sup.2 /g and an average particle size of 
about 4 microns (Syloid 72, Grace Div., Davidson Chem. Co.). The 
o-dianisidine solution consisted of one gram of o-dianisidine diluted to 
100 ml with methanol. The carrier was prepared by mixing 20 grams of the 
silica with 100 ml of the o-dianisidine solution in a 150 ml beaker for 10 
minutes at room temperature. The slurry was transferred to a fine frit 600 
ml funnel and filtered with aspiration on a filter flask. The solution 
came through the funnel with about 50% of the color of the original 
solution. The silica was then washed on the funnel with aspiration with 
100 ml of methanol. The filtrate was clear. The carrier derivative was 
then washed with 50 ml of acetone and air dried. Samples of this carrier 
were used for the immobilization of antibodies, described below. 
Another sample of the same carrier was derivatized further for aldehyde 
function as follows: To 3.64 grams of the above-described treated carrier, 
18.2 ml of a 2.5% aqueous glutaraldehyde solution was added in a 30 ml 
beaker and stirred for about 10 minutes. This derivative was then 
immediately filtered with aspiration on a Buchner funnel containing S and 
S576 filter paper. The derivative was then washed with 100 ml of distilled 
water and finally washed with 50 ml of acetone and air dried.

The immobilization of enzymes is described in Examples I-III below. Example 
IV illustrates the immobilization of an antibody. 
EXAMPLE I 
Immobilized Glucose Oxidase and Catalase 
A 10 mm fritted glass disc was placed in a 5 ml beaker. One ml of the 1% 
o-dianisidine solution containing HCl (described above) was added to the 
beaker which was then placed on a hot plate for one minute. As the 
solution came to a boil, the disc was removed from the beaker and held 
under a distilled water faucet for one minute. The disc was then placed in 
a 5 ml beaker to which 3 ml of water and 0.1 ml of glutaraldehyde was 
added and allowed to react with hand shaking for 1 minute. The disc was 
then washed by holding it under a distilled water faucet for one minute. 
The disc was replaced in a beaker and 3 ml of a glucose oxidase-catalase 
solution (Miles, DeeO) was added and reacted for 1 minute with mixing. The 
disc was removed from the beaker and held under the distilled water for 
one minute. The total time used for this preparation was about 11 minutes 
with an actual operation time of 6 minutes and a bonding time of about 3 
minutes. This represents a significant improvement over many past bonding 
methods requiring longer preparation times. The above preparation was 
assayed for catalase activity by merely observing bubbles of O.sub.2 
produced from a 1% H.sub.2 O.sub.2 solution. The O.sub.2 bubbles were 
generated from the pores in a fine stream. The preparation was visually 
observed with 4 different aliquots of H.sub.2 O.sub.2 with no diminution 
in the evaluation of O.sub.2. Further information describing the 
advantages of immobilizing both glucose oxidase and catalase on the same 
carrier can be found in U.S. Pat. No. 3,841,971 to Messing wherein it is 
pointed out how the two enzymes act synergistically. 
EXAMPLE II 
Immobilized Lactase 
Lactase derived from Escherichia coli was immobilized on the derivatized 
carrier of Method 1 by placing about 1.0 g of the carrier in a column and 
circulating through the column at 400 ml/hr about 12.5 ml of an aqueous 
slurry of the enzyme containing about 417 lactase activity units. The 
reaction was carried out at 20.degree.C. with circulation for about 22 
hours. It was found that the amount of enzyme immobilized yielded between 
92 and 98 lactase units per gram. It is significant to note that in this 
preparation no further functionalization of the surface o-dianisidine 
residue was required. In the past, this level of activity per gram 
commonly required added processing steps -- e.g. silanization of an 
inorganic followed by further functionalization with glutaraldehyde. In a 
subsequent experiment where the same carrier of Method 1 was further 
functionalized with glutaraldehyde, an enzyme loading of 261 lactase units 
per gram was achieved. When the carrier of Method 1 having the 
o-dianisidine residue was further functionalized to form a reactive 
diazonium group, the lactase loadings were about 197 lactase activity 
units per gram. 
EXAMPLE III 
Immobilized Lactase From Aspergillis Niger (Column) 
Two grams of o-dianisidine silica carrier described in Method 1 was 
transferred to a 9 .times. 150 mm water jacketed column. 30 ml of a 2.5% 
glutaraldehyde in water solution was circulated at 530 ml per hour in a 
downward flow through the column for 10 minutes. The flow was then 
reversed upward for an additional 10 minutes and reversed again downward 
for another 10 minutes. The glutaraldehyde was then removed from the 
column and 1 liter of water was pumped through the column at 530 ml per 
hour to wash the derivative. The derivative was then ready for coupling. 
30 ml of a lactase solution (500ml lactase diluted to 30 ml with water and 
containing 5619 lactase units was circulated in a downward flow through 
the column at 530 ml per hour at room temperature. This circulation was 
continued for approximately 17 hours, after which the enzyme was removed 
from circulation and the column was washed with 100 ml of water at 
approximately 70 ml per hour flow rate. The enzyme was then washed on the 
column with 50 ml of 0.5 M sodium chloride followed by an additional 20 ml 
of water. The total volume of washes and enzyme solution was approximately 
200 ml. This wash plus enzyme solution was assayed and found to contain 
1570 units of lactase activity in a 200 ml sample. 
The immobilized enzyme was evaluated in the same column without removal 
from the column as was used for preparing the enzyme. The substrate 
solution contained 200 gms of lactose plus 3800 ml of water plus 1.3 ml of 
2 M HCl and the pH of this solution was 3.5. The column was maintained 
continually at 50.degree.C. with a circulating water bath. The feed rate 
at which the substrate was supplied to the column was between 49 and 71 ml 
per hour. This column was fed continuously over a 56 day period. 
Approximately twice a week, the column and pump were washed with 100 ml of 
0.17 M acetic acid to remove microbial growth within the pump and on the 
column. The results were as follows: 
TABLE I 
______________________________________ 
DAYS ACTIVITY (U/g) 
______________________________________ 
0 620 
1 619 
2 650 
6 535 
7 565 
8 488 
14 465 
16 553 
23 357 
27 451 
28 369 
29 484 
34 421 
35 413 
36 428 
41 347 
42 383 
51 316 
55 285 
56 273 
______________________________________ 
It should be noted in the table above, that the low results achieved were 
on those days where the column had not been washed for considerable 
lengths of time and substantial growth was noted both in the column and in 
the substrate feed. On the days when the feed was clear within 4 days 
after washing the column, the activities were rather substantial. The 
results of the activities were plotted, and the half-life of this column 
was found to be 52 days with an LCL (95%) of 43 days and UCL (95%) of 65 
days. 
The activity recovery for the preparation of this enzyme is as follows: 
Enzyme recovery = [(2 .times. 620) + 1570].div. 5619 .times. 100 = 50%. 
The coupling efficiency for this preparation is as follows: Coupling 
efficiency = (620 .times. 2) .div. (5619 - 1570) .times. 100 = 31%. 
EXAMPLE IV 
Immobilized Antibodies (anti-thyroxine) 
A representative antibody (anti-thyroxine or anti-T.sub.4) useful in the 
radioimmunoassay (RIA) of T.sub.4 was immobilized and found to retain its 
immunochemical complexing properties. 
Preparation of the Immobilized Antibody (IMA) 
Silica particles were derivatized with o-dianisidine by Method No. 2. After 
drying, 1.0 gm of the carrier was diazotized with sodium nitrite. After 
diazotization the derivative was washed several times with borate (0.03M) 
buffered saline (0.15M) pH 8.0. The derivative was then reacted with 1.5 
ml of T.sub.4 antisera (titer 1:10000) at 4.degree.C. with stirring. The 
pH was maintained between 8.0 - 8.5 with 0.1N NaOH. After 30 minutes the 
pH stabilized at 8.2 and the reaction was stirred slowly at 4.degree.C. 
overnight. After this time the composite gave a negative .beta.-napthol 
test indicating no more remaining functional diazo groups. The preparation 
was then washed (4X) with phosphate-buffer (0.03M) saline (0.15M) 
containing 0.1% bovine serum albumin (PBS-BSA). 
Titering of the Immobilized Antibody 
The derivative was diluted in PBS-BSA to contain 10 mgs. of IMA per ml. The 
IMA (0.1 ml) was serially diluted with PBS-BSA in 12 .times. 75 
polystyrene tubes. Each tube then would contain 0.1 ml of PBS-BSA 
containing 100 ug of 8-anilino-1-napthalene sulfonic acid (ANS) and 
finally 0.1 ml of labeled T.sub.4 (.about.25,000 c.p.m.). The tubes were 
vortexed and incubated at 37.degree.C. for 2.0 hours. Appropriate controls 
were run to account for non-specific binding. At this time the tubes were 
centrifuged (15 minutes at 5000 r.p.m.) and the supernatant decanted into 
duplicated tubes and both tubes counted in the gamma spectrometer. The per 
cent of label bound was calculated. A typical titer curve was made where 
per cent bound was expressed as a function of final dilution. As a control 
1.0 gm of silica was stirred at pH 8.0 overnight with antisera. Titering 
of this control preparation indicated little or no antibody attached to 
the particles. 
Assay of T.sub.4 Using the IMA 
A dilution of IMA was chosen so that 0.1 ml bound between 50-60% of the 
labeled material added. This quantity was added to duplicate 12 .times. 75 
tubes. To this was added 0.1 ml of T.sub.4 free serum, 0.1 ml of T.sub.4 
standards (12.5-400 ng/ml), 0.6 ml of PBS-BSA containing 200 ng of ANS and 
finally 0.1 ml of labeled T.sub.4. The tubes were vortexed and incubated 
at 37.degree.C. for 12 hours. At this time, the tubes were centrifuged, 
decanted into duplicate tubes and counted for 1.0 minutes on the gamma 
spectrometer. A typical dose response curve was prepared. 
Three commercially available analyzed control sera, (A), (B), and (C) were 
assayed and Table II shows the good correlation of results. 
TABLE II 
______________________________________ 
Control Calculated 
Assayed (Found) 
______________________________________ 
(A) 121 126 
(B) 72 71 
(C) 80 92 
______________________________________ 
Standard curves were prepared for measuring T.sub.4 in a clinically 
significant concentration range and these curves were compared with 
standard curves obtained using immobilized anti-T.sub.4 coupled via a 
silane coupling agent to glass particles. The curves were substantially 
the same within that concentration range. 
From the above experiments, it is clear that biologically active proteins 
such as enzymes and antibodies can be successfully immobilized according 
to the novel method disclosed herein. Since the disclosed method is 
subject to modifications, it is intended that the scope of the disclosed 
invention should be limited only by the following claims.