Ionic compounds containing the cationic meriquinone of a benzidine

A process for visualizing an analyte selected from the group consisting of proteins, nucleic acids, carbohydrates and lipids, wherein the analyte contained in or on a test sample selected from the group consisting of a solid phase, a gel, a dissolved or suspended mixture containing complementary antibody and antigen, and a dissolved or suspended mixture containing single-stranded nucleic acids, which process comprises:

• (a) contacting the test sample with an oxidation catalyst attached to a detecting compound capable of specifically binding to the analyte;
• (b) incubating the test sample from step (a) under conditions whereby the detecting compound will analyte if it is present in the test sample;
• (c) washing the test sample from step (b) to remove unbound detecting compound;
• (d) adding to the washed test sample from step (c) a benzidine compound given by the structure ##STR8## in which R.sub.1 is H and R.sub.2 and R.sub.3 are independently selected from the group consisting of --H, --F, --Cl, --Br, --CH.sub.3, --OCH.sub.3, --(CH.sub.2).sub.n CH.sub.3, and --O--(CH.sub.2).sub.n CH.sub.3, where n is an integer of from 1 to 10;
• (e) subjecting the test sample to conditions under which the benzidine or substituted benzidine will oxidize to the meriquinone thereof in the presence of the oxidatively or peroxidatively active species, said conditions comprising a reaction temperature of 0.degree. to 60.degree. C. and an aqueous medium of pH 3 to 7;
• wherein an amount of an anion or polymeric anion effective to cause formation of a solid salt or immobilized complex of the anion or polymeric anion with the meriquinone is added during one or more of steps (a)-(e), and wherein the concentration of dissolved meriquinone in equilibrium with said salt or complex ion is less than about 10.sup.-5 M;
• and
• (f) detecting the formation of a solid salt or immobilized complex of said anion or polymeric anion and said cationic meriquinone, wherein said formation indicates the presence or characteristics of the analyte.

A process for visualizing an analyte selected from the group consisting of proteins, nucleic acids, carbohydrates and lipids, wherein the analyte is contained in or on a test sample selected from the group consisting of a solid phase, gel and liquid, which process comprises:

• (a) contacting the test sample with a detecting compound capable of specifically binding to the analyte;
• (b) contacting the test sample from step (a) with an oxidation catalyst attached to a moiety capable of specifically binding to the detecting compound;
• (c) incubating the test sample from step (b) under conditions whereby the detecting compound will bind to the catalyst and to the analyte if it is present in the test sample;
• (d) washing the test sample from step (c) to remove free detecting compound and catalyst;
• (e) adding to the washed test sample from step (d) a benzidine compound given by the structure ##STR9## in which R.sub.1 is H and R.sub.2 and R.sub.3 are independently selected from the group consisting of --H, --F, --Cl, --Br, --CH.sub.3, --OCH.sub.3, --(CH.sub.2).sub.n CH.sub.3, and --O--(CH.sub.2).sub.n CH.sub.3, where n is an integer of from 1 to 10;
• (f) subjecting the test sample to conditions under which the benzidine compound will oxidize to the meriquinone thereof in the presence of the analyte, said conditions comprising a reaction temperature of 0.degree. to 60.degree. C. and an aqueous medium of pH 3 to 7;
• wherein an amount of an anion or polymeric anion effective to cause formation of a solid salt or immobilized complex of the anion or polymeric anion with the meriquinone is added during one or more of steps (a)-(f), and wherein the concentration of dissolved meriquinone in equilibrium with said salt or complex ion is less than about 10.sup.-5 M;
• and
• (g) detecting the formation of a solid salt or immobilized complex of said anion or polymeric anion and said cationic meriquinone, wherein said formation indicates the presence or characteristics of the analyte.

A process for visualizing an analyte selected from the group consisting of an antigen, an antibody or a nucleic acid, contained in or on a solid phase using a Southern blot, a Northern blot, a DNA or RNA dot blot, a Western blot, an antigen dot blot or an antibody dot blot, which process comprises:

• (a) contacting the solid phase with a peroxidase attached to a detecting compound selected from the group consisting of (i) an antibody capable of binding to the antigen, (ii) an antigen or anti-antibody capable of binding to the antibody, or (iii) a nucleic acid hybridization probe containing a single-stranded nucleotide sequence which is complementary to a sequence contained in the nucleic acid;
• (b) incubating the solid phase from step (a) under conditions whereby the detecting compound will bind to the antigen, antibody or nucleic acid if it is present in the test sample;
• (c) washing the solid phase from step (b) to remove unbound detecting compound;
• (d) adding to the washed solid phase from step (c) a benzidine compound given by the structure ##STR10## in which R.sub.1 is H and R.sub.2 and R.sub.3 are independently selected from the group consisting of --H, --F, --Cl, --Br, --CH.sub.3, --OCH.sub.3, --(CH.sub.2).sub.n CH.sub.3, and --O--(CH.sub.2).sub.n CH.sub.3, where n is an integer of from 1 to 10;
• (e) subjecting the solid phase to conditions under which the benzidine compound will oxidize to the meriquinone thereof if the peroxidase is present, said conditions comprising a reaction temperature of 0.degree. to 60.degree. C. and an aqueous medium of pH 3 to 7 containing an amount of a hydroperoxide effective to oxidize the benzidine compound;
• wherein an amount of an anion or polymeric anion effective to cause formation of a solid salt or immobilized complex of the anion or polymeric anion with the meriquinone is added during one or more of steps (a)-(e), and wherein the concentration of dissolved meriquinone in equilibrium with said salt or complex ion is less than about 10.sup.-5 M;
• and
• (f) detecting the formation of a solid salt or immobilized complex of said anion or polymeric anion and said cationic meriquinone, wherein said formation indicates the presence of the material.

A process for visualizing an analyte selected from the group consisting of an antigen, an antibody and a nucleic acid, contained in or on a solid phase using a Southern blot, a Northern blot, a DNA or RNA dot blot, a Western blot, an antigen dot blot or an antibody dot blot, which process comprises:

• (a) contacting the solid phase with a detecting compound selected from the group consisting of (i) an antibody capable of binding to the antigen, (ii) an antigen or anti-antibody capable of binding to the antibody, or (iii) a nucleic acid hybridization probe containing a single-stranded nucleotide sequence which is complementary to a sequence contained in the nucleic acid;
• (b) contacting the solid phase from step (a) with a peroxidase attached to a moiety capable of binding to the detecting compound;
• (c) incubating the solid phase from step (b) under conditions whereby the detecting compound will bind to the peroxidase and to the antigen, antibody or nucleic acid if it is present in the solid phase;
• (d) washing the solid phase from step (c) to remove unbound detecting compound and peroxidase;
• (e) adding to the washed solid phase from step (d) a benzidine compound given by the structure ##STR11## in which R.sub.1 is H and R.sub.2 and R.sub.3 are independently selected from the group consisting of --H, --F, --Cl, --Br, --CH.sub.3, --OCH.sub.3, --(CH.sub.2).sub.n CH.sub.3, and --O--(CH.sub.2).sub.n CH.sub.3, where n is an integer of from 1 to 10, and wherein the concentration of dissolved meriquinone in equilibrium with said salt or complex ion is less than about 10.sup.-5 M;
• (f) subjecting the solid phase to conditions under which the benzidine compound will oxidize to the meriquinone thereof if the peroxidase is present, said conditions comprising a reaction temperature of 0.degree. to 60.degree. C. and an aqueous medium of pH 3 to 7 containing an amount of a hydroperoxide effective to oxidize the benzidine compound;
• wherein an amount of an anion or polymeric anion effective to cause formation of a solid salt or immobilized complex of the anion or polymeric anion with the meriquinone is added during one or more of steps (a)-(f);
• and
• (g) detecting the formation of a solid salt or immobilized complex of said anion or polymeric anion and said cationic meriquinone, wherein said formation indicates the presence of the antigen, antibody or nucleic acid.

A process for detecting the presence of an analyte comprising an antigen or an antibody in a liquid test sample, which process comprises:

• (a) incubating the test sample or an extract thereof with a surface to capture the antigen or antibody and with a peroxidase-labeled antibody or antigen which binds to the antigen or antibody to be detected, these incubations occurring separately, in either order, or together;
• (b) filtering the incubation mixture of step (a);
• (c) washing the filter of step (b);
• (d) incubating the washed product of step (c) with a hydroperoxide and a benzidine compound given by the structure ##STR12## in which R.sub.1 is H and R.sub.2 and R.sub.3 are independently selected from the group consisting of --H, --F, --Cl, --Br, --CH.sub.3, --OCH.sub.3, --(CH.sub.2).sub.n CH.sub.3, and --O--(CH.sub.2).sub.n CH.sub.3, where n is an integer of from 1 to 10, and an amount of an anion or polymeric anion effective to form a solid salt or immobilized complex with the meriquinone of said benzidine or substituted benzidine, and wherein the concentration of dissolved meriquinone in equilibrium with said salt or complex ion is less than about 10.sup.-5 M;
• (e) filtering and washing the incubate of step (d);
• and
• (f) detecting the formation of said solid salt or immobilized complex of said anion or polymeric anion and the meriquinone of said benzidine compound, wherein said formation indicates the presence of said antibody or antigen.