GB1571197A

GB1571197A - Polymeric materials for use in immunoassay

Info

Publication number: GB1571197A
Application number: GB1169979A
Authority: GB
Original assignee: Cordis Corp
Current assignee: Cordis Corp
Priority date: 1975-09-29
Filing date: 1976-09-29
Publication date: 1980-07-09
Also published as: GB1571198A

Description

(54) IMPROVEMENTS IN OR RELATING TO POLYMERIC MATERIALS FOR USE IN IMMUNOASSAY (71) We, CORDIS CORPORATION, a Corporation organised and existing under the laws of the State of Florida, United States of America, residing at 3901 Biscayne Blvd., Miami, Florida 33137, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention is concerned with improvements in or relating to polymeric materials for use in immunoassay, particularly but not exclusively hepatitis immunoassay.

The present application is divided out of our copending application No. 40376/76, (Serial No. 1,571,196) which discloses inter alia a process for detecting the presence of antigens associated with hepatitis, in which there is used an insoluble polymeric matrix having reactive groups grafted onto its surface and hepatitis antibody bonded to the reactive groups so that the hepatitis antibody is immobilised. The matrix has a surface configuration such that when placed in a fiat-bottomed vial, it will be substantially in contact with any solution in the vial while minimising the surface-to-surface contact between the matrix and the bottom of the vial.

The polymeric matrix may not only be used to immobilise hepatitis antibody but also other suitable proteins, for example antibodies to drugs, e.g. digoxin, opiates and steroids; antibodies to natural products e.g.

insulin and other hormones; and specific enzymes to metabolites found in blood and other body fluids.

For further explanation of the background to and advantages of the present invention attention is directed to said copending application No. 40376/76. (Serial No.

1,571,196).

The present invention provides a material adapted for use in immunoassay comprising a water-insoluble shaped polymeric matrix having a layer of reactive groups grafted onto its surface, said reactive groups being capable of covalently bonding to protein to enable the structure to be used in immunoassay wherein waffle-like surfaces are provided on said polymeric matrix which, when placed in a flat-bottomed vial, will be substantially in contact with any solution in the vial while minimising the surface-to-surface contact between the matrix and the bottom of the vial.

The invention also provides a material adapted for use in immunoassay comprising a water-insoluble shaped polymeric matrix having (i) reactive groups grafted onto its surface said reactive groups being capable of bonding to protein to enable the structure to be used in immunoassay and (ii) surfaces configured to comprise a field of high and low points to provide a matrix which, when placed in a flat-bottomed vial, will be substantially in contact with any solution in the vial while minimising the surface-to-surface contact between the matrix and the bottom of the vial.

The invention also provides a method of producing a material useful in immunoassay comprising the steps of: (a) providing a water-insoluble disc shaped polymeric matrix which has reactive groups grafted onto its opposed surfaces which groups are capable of covalently bonding to protein; (b) shaping the opposed surfaces on said matrix to provide a field of high and low points so that when said matrix is placed in the bottom of a vial its surface will be substantially in complete contact with any solution in the vial and there will be a minimum of surface-to-surface contact between the matrix and the bottom of the vial; (c) covalently bonding a protein onto the reactive groups to enable the material to be utilised in assaying the presence of material which reacts with the bound protein.

The invention also provides a material produced by a method according to the invention.

There now follows a description, to be read with reference to the accompanying drawing of an embodiment of the invention.

This description, which is illustrative of product and method aspects of the invention is given by way of example only, and not by way of limitation of the invention.

In the accompanying drawing the single figure shows a perspective view of a preferred water-insoluble member embodying the invention.

The water-insoluble member embodying the invention has a plurality of groups reactive with proteins grafted uniformly to its surface layer. Purified hepatitis antibody is covalently bonded to the reactive groups to provide an exterior layer of hepatitis antibody.

Thus the member has a high concentration of purified, hepatitis associated antibody distributed uniformly over its entire surface, and such that the antibody will not be dislodged by mechanical or chemical forces to which it may be subjected during use. In this regard, the attachment is effected such that the antibody remains functional, i.e. capable of participating in its immunological reaction and immunochemically unaltered by its attachment.

The insoluble antibody coated solid has an improved shape which is easy to handle during immunoassay and which is designed to expose a constant and a greater portion of its coated surface to solutions used during incubations.

A step in the preparation of the reagents for the immunoassay process of said copending application No. 40376/76 (Serial No.

1,571,196) is to covalently bond a portion of purified antibody to an insoluble member.

To effect this bonding, the insoluble member used is provided with reactive groups or sites capable of reacting with the specific antibody used in the bioassay.

U.S. Patent No. 3,700,609 entitled Graft Copolymers, to G. W. Tregear et al., discloses an insoluble continuous polymeric substance comprising a polymeric backbone onto which side chains of another polymer or copolymer are grafted. By suitable choice of the grafted polymer, it is possible to chemically link biological substances to the insoluble matrix.

A product which is disclosed in the above patent is commercially available in a disc form under the trade name PROTAPOL DI/i from Imperial Chemical Industries of Australia and New Zealand (ICIANZ).

The PROTAPOL DI/1 comprises a polytetrafluoroethylene backbone having isothiocyanatopolystyrene groups grafted uniformly over its surface and is designed for use in radioimmunoassay. The discs, as presently available, are approximately 0 01 inches thick and 0.5 inch in diameter.

In accordance with one important embodiment of the matrix as shown in the drawing each disc 10 is provided with a waffle-like pair of surfaces 11 comprising a first series of linear ridges 12 and a second series of linear ridges 14 which form grids. Ridges 12 and 14 are preferably perpendicular to each other and hence define a plurality of square depressions 16. The sides of each ridge 12 and 14 taper upwardly from adjacent pairs of depressions 16 to form a line defining the top of the ridge.

It should be noted that in order to facilitate description the ridges 12 and 14 are greatly exaggerated in the drawing.

The desired configuration of the disc is achieved by passing the disc between the surfaces of two rollers having projections on the surface of the rollers designed to impart the desired configuration on the disc. As is obvious, the rollers are designed to provide a sufficient amount of pressure to disfigure the -polymeric material in the disc without actually puncturing the disc. This is important because the disc has a reactive layer on its surface. Thus, penetration of the disc would expose interior portions to which no antibody can be bonded. Exposure of the polytetrafluoroethylene layer would actually result in a disc which would have a lower bonding capacity.

The main consideration is to provide a disc matrix with surfaces which, when placed in a flat bottomed vial 15, will be substantially in complete contact with the test sample, i.e., there should be a minimum of surface-tosurface contact between the matrix and the bottom of the vial; and it will be realized that the disc's surfaces are configured to have a field of high and low points.

Hepatitis antibody, produced as disclosed in the application No. 40376/76 (Serial No.

1,571,196) in the lyophilized form, is recc stituted by adding 100 ml of 0.1M Na -CO3 (pH 9'6) for each 5.0 mg of antibody. In general, the procedure for attachment involves contacting the waffled dise with the dilute solution at 2-80C for 8 to 16 hours, with agitation. Afterwards, the antibody solution is discarded and the discs are washed twice with successive volumes of 0.1M NaHCO3, pH 9-6, phosphate buffered saline, and cold (28 C) 0 3 % bovine serum albumin in phosphate buffered saline with 0 5 % TWEEN (Registered Trade Mark) 20.

After an additional washing with crystalline bovine serum albumin, and freezing over dry ice, lyophilization is carried out and the discs are stored at 2-80C until ready for use.

Although this description relates to the preparation of discs having hepatitis antibody bonded thereto, it will be realized that discs embodying the present invention are useful to immobilise any suitable protein. For example, the increased contact between the test sample and the disc enables the disc to be used in tests which involve the bonding of the following proteins thereto; antibodies to drugs such as digoxin, opiates, and steroids; antibodies to natural products, for example insulin and other hormones; and specific enzymes to metabolites found in blood and other body fluids.

Example The following procedure was used to prepare 8,000 discs, each of which were first treated with the press to produce the desired configuration as described. A batch of 8,000 discs requires 40 mg of hepatitis antibody, i.e., 5 mg per 1,000 discs. The protein content of the reconstituted hepatitis antibody solution is adjusted to .05 mglml in the final volume of 800 ml in 0 IM NaHCO3 (pH 9.6). The entire 800 ml of buffered antibody is then added to a 1,000 ml screw-cap bottle provided with a leak proof liner containing the 8,000 discs, the bottle is rotated for 16 hours, e.g., overnight, at 2-80C to slowly tumble the discs through each rotation cycle. Afterwards, the liquid is poured from the bottle and discarded and the discs are transferred to a wide-mouth 2 liter flask.

The discs are washed twice with successive 1 liter volumes of cold (28OC) 0-1M NaRCO3, pH 9 6, following which the buffer is removed. The discs are then washed again, this time using two successive 1 liter volumes of cold buffer (0OIM sodium phosphate, 0 15M NaCI, pH 7.4). After removing residual buffer, the discs are washed for a third time, using two successive one liter volumes of cold bovine serum albumin solution (0.3 %).

The discs are finally washed with two successive 1 liter volumes of a solution of cold crystalline bovine serum albumin (pH 8) at a concentration of 2 mg/ml. This step is performed to provide a protein environment for the protein on the disc. The discs, after removing the residual wash, are then transferred to dishes or trays (9 x 9"), each of which is lined with a sheet of filter paper and each of which contains 200 ml of the crystalline bovine serum albumin solution. When the transfer is complete, a sheet of filter paper is used to cover them. Buffer is thoroughly removed. The discs are then quick frozen for 30 minutes on dry ice.

The contents of the tray are then lyophilized and the dry discs are removed and stored in stoppered containers.

For further details attention is again directed to copending application No.

40376176, (Serial No. 1,571,196) and also to our copending application No. 7920659 (Serial No. 1,571,198) which is also divided out of application No. 40376/76. (Serial No.

1,571,196).

WHAT WE CLAIM IS: 1. A material adapted for use in immunoassay comprising a water-insoluble shaped polymeric matrix having a layer of reactive groups grafted onto its surface, said reactive groups being capable of covalently bonding to protein to enable the structure to be used in immunoassay wherein waffle-like surfaces are provided on said polymeric matrix which, when placed in a flat-bottomed vial, will be substantially in contact with any solution in the vial while minimising the surface-tosurface contact between the matrix and the bottom of the vial.

2. A material adapted for use in immunoassay comprising a water insoluble shaped polymeric matrix having (i) reactive groups grafted onto its surface said reactive groups being capable of bonding to protein to enable the structure to be used in immunoassay and (ii) surfaces configured to comprise a field of high and low points to provide a matrix which, when placed in a flat-bottomed vial, will be substantially in contact with any solution in the vial while minimising the surface-to-surface contact between the matrix and the bottom of the vial.

3. A material according to claim 1 or claim 2, having a protein bonded thereto.

4. A material according to claim 1 or claim 2, having an antibody bonded thereto.

5. A material according to claim 1, wherein an antibody reactive with antigens associated with hepatitis is covalently bonded to the reactive groups on said polymeric matrix.

6. A material according to claim 2, having a hepatitis antibody bonded thereto, 7. A method of producing a material useful in immunoassay comprising the steps of: (a) providing a water-insoluble dise shaped polymeric matrix which has reactive groups grafted onto its opposed surfaces which groups are capable of covalently binding to protein; (b) shaping the opposed surfaces on said matrix to provide a field of high and low points so that when said matrix is placed in the bottom of a vial its surface will be substantially in complete contact with any solution in the vial and there will be a minimum of surface-to-surface contact between the matrix and the bottom of the vial; (c) covalently bonding a protein onto the reactive groups to enable the material to be utilized in assaying the presence of material which reacts with the bound protein.

8. A method according to claim 7, wherein said surfaces are shaped to have a wafflelike configuration.

9. A method according to claim 8, wherein said surfaces are shaped by passing the matrix between the surfaces of two

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. to immobilise any suitable protein. For example, the increased contact between the test sample and the disc enables the disc to be used in tests which involve the bonding of the following proteins thereto; antibodies to drugs such as digoxin, opiates, and steroids; antibodies to natural products, for example insulin and other hormones; and specific enzymes to metabolites found in blood and other body fluids. Example The following procedure was used to prepare 8,000 discs, each of which were first treated with the press to produce the desired configuration as described. A batch of 8,000 discs requires 40 mg of hepatitis antibody, i.e., 5 mg per 1,000 discs. The protein content of the reconstituted hepatitis antibody solution is adjusted to .05 mglml in the final volume of 800 ml in 0 IM NaHCO3 (pH 9.6). The entire 800 ml of buffered antibody is then added to a 1,000 ml screw-cap bottle provided with a leak proof liner containing the 8,000 discs, the bottle is rotated for 16 hours, e.g., overnight, at 2-80C to slowly tumble the discs through each rotation cycle. Afterwards, the liquid is poured from the bottle and discarded and the discs are transferred to a wide-mouth 2 liter flask. The discs are washed twice with successive 1 liter volumes of cold (28OC) 0-1M NaRCO3, pH 9 6, following which the buffer is removed. The discs are then washed again, this time using two successive 1 liter volumes of cold buffer (0OIM sodium phosphate, 0 15M NaCI, pH 7.4). After removing residual buffer, the discs are washed for a third time, using two successive one liter volumes of cold bovine serum albumin solution (0.3 %). The discs are finally washed with two successive 1 liter volumes of a solution of cold crystalline bovine serum albumin (pH 8) at a concentration of 2 mg/ml. This step is performed to provide a protein environment for the protein on the disc. The discs, after removing the residual wash, are then transferred to dishes or trays (9 x 9"), each of which is lined with a sheet of filter paper and each of which contains 200 ml of the crystalline bovine serum albumin solution. When the transfer is complete, a sheet of filter paper is used to cover them. Buffer is thoroughly removed. The discs are then quick frozen for 30 minutes on dry ice. The contents of the tray are then lyophilized and the dry discs are removed and stored in stoppered containers. For further details attention is again directed to copending application No. 40376176, (Serial No. 1,571,196) and also to our copending application No. 7920659 (Serial No. 1,571,198) which is also divided out of application No. 40376/76. (Serial No. 1,571,196). WHAT WE CLAIM IS:

1. A material adapted for use in immunoassay comprising a water-insoluble shaped polymeric matrix having a layer of reactive groups grafted onto its surface, said reactive groups being capable of covalently bonding to protein to enable the structure to be used in immunoassay wherein waffle-like surfaces are provided on said polymeric matrix which, when placed in a flat-bottomed vial, will be substantially in contact with any solution in the vial while minimising the surface-tosurface contact between the matrix and the bottom of the vial.

3. A material according to claim 1 or claim 2, having a protein bonded thereto.

6. A material according to claim 2, having a hepatitis antibody bonded thereto,

7. A method of producing a material useful in immunoassay comprising the steps of: (a) providing a water-insoluble dise shaped polymeric matrix which has reactive groups grafted onto its opposed surfaces which groups are capable of covalently binding to protein; (b) shaping the opposed surfaces on said matrix to provide a field of high and low points so that when said matrix is placed in the bottom of a vial its surface will be substantially in complete contact with any solution in the vial and there will be a minimum of surface-to-surface contact between the matrix and the bottom of the vial; (c) covalently bonding a protein onto the reactive groups to enable the material to be utilized in assaying the presence of material which reacts with the bound protein.

rollers to press the waflile-like configuration into the polymeric matrix.

10. A method according to any one of claims 7, 8 and 9, wherein an antibody reactive with antigens associated with hepatitis is covalently bonded to the reactive groups on said polymeric matrix.

Il. A method of producing a polymeric matrix substantially as hereinbefore described with reference to the Example.

12. A material produced by a method according to any one of claims 7 to 11.

13. A polymeric matrix substantially as hereinbefore described with reference to the accompanying drawing.