Immunoassay with membrane immobilized antibody

Claim 1

A method for detecting the presence of a diagnostic antigen in an unfractionated sample of feces or colon aspirate dispersed in a liquid, comprising: immersing in the sample first and second microporous membranes each of which has a network of voids permitting liquid flow therethrough, the first membrane being a test membrane and having immobilized to its external and internal surfaces a first antibody which is specifically immunochemically reactive with said antigen, the second membrane being a control membrane having immobilized on its external and internal surfaces a second antibody which is immunochemically non-reactive with said antigen, said membranes having capillary pore sizes in the range from about 25 nanometers to 25 micrometers and having a thin, water-insoluble coating of an inert proteinaceous material on their surfaces, said antibodies being immobilized to said coatings, said first and second membranes being immersed together in the sample and being treated essentially identically in all steps: incubating the membranes in said sample to permit immunochemical reaction between the immobilized antibody of the first membrane and the antigen to be detected; washing the membranes to remove non-specifically bound materials; immersing the membranes in a serum diluted solution of a conjugate of an enzyme and an antibody to the antigen to be detected, in order to immunochemically bind the conjugate to any of said diagnostic antigen bound to said first immobilized antibody on the test membrane; again washing the membranes to remove non-specifically bound materials; thereafter testing the membranes for any activity of the enzyme moiety of the conjugate bound thereto; and comparing the first and second membranes for evidence of enzyme activity, any substantial difference being indicative of the presence of the diagnostic antigen in the sample.

Claim 2

A method for detecting the presence of a diagnostic antigen in an unfractionated sample of feces or colon aspirate dispersed in a liquid, comprising: immersing in the sample first and second microporous membranes each of which has a network of voids permitting liquid flow therethrough, the first membrane being a test membrane and having immobilized to its external and internal surfaces a first antibody which is specifically immunochemically reactive with said antigen, the second membrane being a control membrane having immobilized on its external and internal surfaces a second antibody which is immunochemically non-reactive with said antigen, said membranes having capillary pore sizes in the range from about 25 nanometers to 25 micrometers and having a thin, water-insoluble coating of an inert proteinaceous material on their surfaces, said antibodies being immobilized to said coatings, said first and second membranes being immersed together in the sample and being treated essentially identically in all steps: incubating the membranes in said sample to permit immunochemical reaction between the immobilized antibody of the first membrane and the antigen to be detected; washing the membranes to remove non-specifically bound materials; immersing the membranes in a serum diluted solution of a conjugate of an enzyme and an antibody to the antigen to be detected, in order to immunochemically bind the conjugate to any of said diagnostic antigen bound to said first immobilized antibody on the test membrane; again washing the membranes to remove non-specifically bound materials; thereafter testing the membranes for any activity of the enzyme moiety of the conjugate bound thereto; and comparing the first and second membranes for evidence of enzyme activity, any substantial difference being indicative of the presence of the diagnostic antigen in the sample.

Claim 8

A device for use in detecting a diagnostic antigen in an unfractionated sample of feces or colon aspirate in a liquid, said device comprising a support member having first and second microporous membranes attached thereto in an arrangement so that both membranes can be immersed together in said unfractionated sample, the membranes having a network of capillary pores therethrough so that upon immersion in the sample, the sample contacts the membranes and the liquid penetrates the membranes and makes contact with the surfaces defined by the network of capillary pores therein, said membranes having capillary pore sizes in the range from about 25 nanometers to 25 micrometers and having a thin, water-insoluble coating of an inert proteinaceous material on their external and internal surfaces, the first membrane being a test membrane having immobilized on the proteinaceous coating on the external and internal surfaces thereof an antibody which is specifically immunochemically reactive with the diagnostic antigen in the unfractionated feces or colon aspirate, the second membrane being a control having immobilized on the protein aceous coating on the external and internal surfaces thereof a second antibody which is immunochemically non-reactive with the diagnostic antigen in the unfractionated feces or colon aspirate, whereby upon immersion of the membranes in the sample, the diagnostic antigen becomes preferentially bound to the test membrane by immunochemical binding, and nonspecifically bound materials may be washed from the membranes.

Claim 9

A device for use in detecting a diagnostic antigen in an unfractionated sample of feces or colon aspirate in a liquid, said device comprising a support member having first and second microporous membranes attached thereto in an arrangement so that both membranes can be immersed together in said unfractionated sample, the membranes having a network of capillary pores therethrough so that upon immersion in the sample, the sample contacts the membranes and the liquid penetrates the membranes and makes contact with the surfaces defined by the network of capillary pores therein, said membranes having capillary pore sizes in the range from about 25 nanometers to 25 micrometers and having a thin, water-insoluble coating of an inert proteinaceous material on their external and internal surfaces, the first membrane being a test membrane having immobilized on the proteinaceous coating on the external and internal surfaces thereof an antibody which is specifically immunochemically reactive with the diagnostic antigen in the unfractionated feces or colon aspirate, the second membrane being a control having immobilized on the protein aceous coating on the external and internal surfaces thereof a second antibody which is immunochemically non-reactive with the diagnostic antigen in the unfractionated feces or colon aspirate, whereby upon immersion of the membranes in the sample, the diagnostic antigen becomes preferentially bound to the test membrane by immunochemical binding, and nonspecifically bound materials may be washed from the membranes.