Aliphatic polyamides are reacted on their surfaces with sulphuryl chloride, either neat or in solution in paraffins or cycloparaffins to give reactive intermediate whcih may convert into a support capable of reaction with antibodies or enzymes by providing covalent chemical links to which the antibodies or enzymes may be bound.

FIELD OF THE INVENTION 
This invention relates to the preparation of sulphuryl chloride treated 
aliphatic polyamides which may be covered to provide supports for use in 
affinity chromatography and for immobilizing enzymes. 
BACKGROUND ART 
Affinity chromatography is a separation technique based on the reversible, 
specific interaction of a biological substance in solution or suspension 
when passed through a subdivided filling holding a complementary 
biological substance. 
Most prior work on affinity chromatography has used a filling comprised of 
small beads of agarose. Extensive literature exists on means of activating 
the agarose with suitable chemicals to provide a covalent chemical link 
for the binding of an antibody so that harvesting of the desired antigenic 
protein is possible. After the desired antigenic protein has been bound to 
the filling containing the covalently bonded antibody impurities are 
washed away and the desired pure antigenic protein is eluted by a solution 
from which it is easily separated. The eluting solution should leave the 
filling containing the covalently bonded antibody ready for reuse. 
Immobilized enzymes are enzymes suitably held on an extensive surface of 
the filling and have reached industrial scale use. The support matrix used 
is often a membrane of suitable shape and porosity. Steric requirements 
usually dictate that a suitable spacer arm be provided between the support 
matrix and the covalent chemical link to the enzyme to give high 
efficiency and selectivity. Suitable spacer arms and the activating 
chemicals required are described by O.R. Zaborsky in "Immobilized 
Enzymes", C.R.C. Press, Cleveland, Ohio, 1974. 
Canadian Pat. No. 1,083,057 deals with the prior art of forming some 
suitable porous membranes, their activation and a method of reacting the 
enzyme whilst forcing it through the membrane. 
Although nylons are physically very suitable as a support matrix because of 
tolerance of sterilising temperatures, stiffness, hydrophilicity and ease 
of forming, they have been little used since they are difficult to 
activate, particularly if isothiocyanate end groups are needed. 
The reaction of sulphuryl chloride on the polyamide fibre Nylon 6 has been 
investigated by D.S. Varma and Thomas Eapen. As reported in the Indian 
Journal of Textile Research, Vol. 1, March 1976, pp. 26-28 their findings 
were: 
"Our results thus indicate that the reaction of Nylon 6 with sulphuryl 
chloride is accompanied by predominant chain scission reactions. 
Cross-bond formation is indicated by the appearance of an additional peak 
in the DTA thermogram. As a result of hydrolysis, a deterioration in the 
properties is observed." 
These unpromising results confirm the earlier work of S.A.M. El-Garf and Y. 
Abou-Street in Faserforschung Textiletechnik Vol. 25(6), pp. 248-51, 1974 
where it was reported that degradation increased as the temperature 
increased. Furthermore, the rates of reaction and of degradation were 
higher in carbon tetrachloride than in toluene. 
The Varma and Eapen paper contains no suggestion of useful, reactive 
intermediates being prepared by the reaction of sulphuryl chloride on 
Nylon 6. 
American CHemical Abstracts record no reactions of sulphuryl chloride with 
monomeric aliphatic secondary amides, thus giving no expectation of useful 
intermediates being formed by the reaction of sulphuryl chloride on 
aliphatic polyamides. 
DISCLOSURE OF THE INVENTION 
We have surprisingly found that sulphuryl chloride may be used as a 
convenient, economical reactant to convert the surface of aliphatic 
polyamides into suitable intermediates which may then be converted into 
stable ready-to-use supports capable of direct reaction with antibodies or 
enzymes. 
According to one aspect of the invention aliphatic polyamides are reacted 
on their surfaces in anhydrous conditions with sulphuryl chloride, either 
neat or in solution in paraffins or cycloparaffins, to give reactive 
intermediates which were suitable for preparing derivatives for affinity 
chromatography. These derivatives were found to be stable and could be 
designed to contain ready-to-use spacer arms and a variety of terminal 
functional groups able to combine covalently with antibodies or enzymes. 
The invention thus provides a process for preparing supports capable of 
reaction with antibodies or enzymes comprising the steps of: 
(i) reacting an aliphatic polyamide with sulphuryl chloride to form an 
intermediate, and, 
(ii) converting the intermediate into the support by providing thereon a 
covalent chemical link to which the antibodies or enzymes may be bound 
The invention also provides a support capable of reaction with antibodies 
or enzymes comprising an aliphatic polyamide reacted with sulphuryl 
chloride and having thereon a plurality of covalent chemical links to 
which the antibodies or enzymes may be bound. 
The supports may be supplied in sterile form, in suitable shape and 
porosity and require no special activation procedure to provide spacer 
arms ending in chemical groups capable of reacting covalently with the 
terminal groups of enzyme or antibody proteins. 
The spacer arms may be provided by reacting the intermediate with 1,6 - 
diaminohexane and the covalent chemical link by further reacting with 
glutaraldehyde. In one form of the invention, the spacer arm and covalent 
chemical link are formed by reacting the intermediate with 4 - 
hydroxybenzaldehyde. 
DESCRIPTION OF PREFERRED EMBODIMENTS 
The preferred methods of the invention to provide the reactive 
intermediates and to convert the intermediates to a useful range of 
protein-binding fillings will now be illustrated by the following 
examples. All the examples were performed on a woven Nylon 6 membrane of 
pore size around 5 microns. In all cases after staining to confirm 
functionality, the membrane was washed with water, boiled in ethanol and 
then washed again in water. Only then was a final assessment made of the 
stainability of the membrane.

EXAMPLE 1 
The membrane was soaked in a solution of 25% sulphuryl chloride in 
petroleum spirit (b.p. 40 .degree.-60.degree. C.) at 45.degree. C. for 5 
minutes. It was then washed in petroleum spirit and dried at 65.degree. C. 
for 10 minutes. The product was tacky to touch and stuck readily to 
itself. A small portion of the material stained blue in an aqueous 
solution of methylene Blue stain, indicating the presence of sulphonyl 
chloride groups. This membrane changed its properties after several hours 
in air, presumably due to hydrolysis of the pendant sulphonyl chloride 
groups. It was thus convenient to store it under petroleum spirit until 
used as an intermediate. 
EXAMPLE 2 
The membrane obtained as described in Example 1 was soaked in a molten mass 
of 1,6 - diaminohexane at 65.degree. C. for 1 hour. It was then washed 
thoroughly in water. The presence of pendant amino groups (as spacer arms) 
was demonstrated by staining with an aqueous solution of 
4-nitrobenzenediazonium tetrafluoroborate. 
EXAMPLE 3 
The membrane obtained as described in Example 1 was soaked in an aqueous 
solution of potassium hydroxide (0.5%) and 4-hydroxybenzaldehyde (1.12%) 
for 15 minutes at room temperature. The membrane was then washed 
thoroughly in water. The presence of aldehyde groups in the membrane was 
confirmed by staining with 2,4 - dinitrophenylhydrazine reagent. The 
stained membrane darkened on immersion in aqueous sodium hydroxide 
solution (10%), confirming the initial presence of aldehyde groups. 
EXAMPLE 4 
The membrane obtained as described in Example 2 was soaked for 8 hours in 
hexamethylene diisocyanate at room temperature. The membrane was then 
washed thoroughly in ether. A small sample of this membrane failed to 
stain with 4 - nitrobenzenediazonium tetrafluoroborate solution. This 
confirmed the expected conversion of all original amino groups into a urea 
spacer arm, terminated by an isocyanate group. 
EXAMPLE 5 
The membrane obtained as described in Example 2 was soaked for 15 hours at 
room temperature in 2,3 - epoxy - 1 -chloropropane (epichlorhydrin). It 
was then washed thoroughly in ether. The sequence of staining treatment 
changes described in Example 4 confirmed the presence of epoxide groups on 
the membrane. 
EXAMPLE 6 
The membrane of Example 2 was reacted with 25% glutaraldehyde for 15 hours 
at 20.degree. C. at pH7 and then well washed with water. The presence of 
aldehyde groups in the membrane was confirmed by staining with 2,4 
-dinitrophenylhydrazine reagent. The stained membrane darkened on 
immersion in aqueous sodium hydroxide solution (10%), confirming the 
initial presence of aldehyde groups. 
EXAMPLE 7 
The membrane of Example 2 was dried in an oven at 60.degree. and reacted at 
20.degree. with a solution of 5% sebacoylchloride in dry ether. The 
product was immersed in water at 20.degree. C. for 10 hours to hydrolyse 
the resulting acid chloride to the carboxylic acid. The presence of the 
acid groups was confirmed by titrating a washed portion with dilute alkali 
to a phenolphthalein end-point as well as by staining with methylene blue 
solution. 
The acidic membrane was freed of water by washing three times for thirty 
minutes in dry dioxane, followed by draining. Five grams of the dry acidic 
membrane were treated in 25ml of dry dioxane with 0.6g. of 
N-hydroxysuccinimide and shaken until this dissolved. Then lg. of 
dicyclohexylcarbodiimide were added and shaken for 1/2 hour. The resulting 
membrane was washed four times with dry dioxane, then four times with dry 
methanol, then twice again with dioxane. Before use as a 
N-hydroxy-succinimide ester activated support the membrane was drained of 
free dioxane. Dry tetrahydrofuran can be used in place of the more toxic 
dioxane. 
EVALUATION OF EXAMPLES 
The membranes of Examples 3, 4, 5, 6 and 7 were then evaluated as protein 
immobilization supports. These attachment studies were performed using a 
monoclonal antibody (IgG class) directed against a medium molecular weight 
protein antigen. 5/16" discs were prepared from each membrane. 
Triplicate samples were incubated together in 0.5ml solution containing 50, 
200, or 300 micrograms per millilitre monoclonal antibody (MAb) and a 
known amount of 125.sub.I-MAb. After agitating for 24 hours the discs were 
washed to remove non-covalently bound MAb, then treated with ethanolamine 
to fill vacant active sites. Measurement of the radioactivity of each disc 
allowed the uptake of MAb to be quantified. 
Samples of unactivated nylon were similarly treated to account for 
non-specific adsorption to the polymer. 
Two of the three discs from each of the previous experiments were incubated 
in 0.5ml solution containing antigen and .sup.125 I-labeled antigen. The 
third disc was placed in 0.5ml buffer to observe MAb desorption. After 
overnight incubation, the level of radioactivity bound to each disc was 
determined and the difference between counts prior to antigen attachment 
was used as a measure of antigen adsorption. 
As a test of the strength of the covalent bond between polymer and MAb, the 
discs were incubated in 4 Molar NaSCN, a commonly-used chaotropic elutant. 
Satisfactory levels of MAb remaining after this treatment should indicate 
a bond of acceptable strength. 
The results of these tests are shown in Table I. The supports made in 
accordance with Examples 3, 4, 5, 6 and 7 compared favourably with the 
agarose gel beads commonly used, especially on activity. The main 
advantages are the resistance to compression which limits the depth of 
present gels, the high flow rates allowable and the ability to adapt the 
shape and the porosity to meet the engineering needs of large scale use. 
TABLE I 
__________________________________________________________________________ 
Initial Covalently Bound 
Antibody 
Antibody Activity 
% Antibody Remaining 
Concentration 
Microgram/cm.sup.2 
Microgram/gram 
Percent 
After 4 Molar NaSCN 
EXAMPLE 
Microgram/ml 
*(n = 3) 
(n = 3) (n = 2) 
(n = 1) 
__________________________________________________________________________ 
3 200 1.6 .+-. 0.2 
290 .+-. 25 
12 .+-. 1 
83 
4 200 6.5 .+-. 0.5 
1150 .+-. 90 
8 .+-. 1 
88 
5 200 4.2 .+-. 0.4 
740 .+-. 65 
6 .+-. 1 
92 
6 50 4.0 .+-. 0.5 
710 .+-. 85 
16 .+-. 0.5 
95 
6 200 10.0 .+-. 1.3 
1770 .+-. 233 
4 .+-. 1 
96 
6 300 13.7 .+-. 3.4 
2400 .+-. 600 
4 .+-. 1 
96 
7 200 0.5 .+-. 0.2 
91 .+-. 25 
not done 
98 
__________________________________________________________________________ 
*NOTE: 
Amount of antibody per unit planar surface area (single side) of mesh 
EXAMPLE 8 
An aromatic analogue of the secondary intermediate with a sulfur spacer arm 
described in Example 2 was prepared by soaking the membrane of Example 1 
in an ethanolic solution of p - phenylene diamine (5.4%) for 15 minutes at 
room temperature. It was then washed with water. The presence of amine 
groups was confirmed by staining with an aqueous solution of 4 - 
nitrobenzenediazonium tetrafluoroborate. 
Although the structure of the intermediate has not been proven, the 
presence of sulphonyl groups has been positively established (see Example 
1). Therefore, the intermediate contains one or both of the following 
structures: 
##STR1## 
which represent tautomeric structures. 
Various other modification of the processes and products of the invention 
may be made without departing from the scope and ambit of the invention.