Chromogenic substrate with a pH indicator dye

A method for detecting an analyte using 3,3′,5,5′-tetramethylbenzidine (TMB) as a chromogenic substrate in an enzyme-linked immunosorbent assay (ELISA) and distinguishing a negative reaction from a false negative reaction in the ELISA, which comprises:

• (a) providing a composition comprising a mixture of the TMB which is oxidizable by peroxide in a reaction for the ELISA catalyzed by the peroxidase of the ELISA to form a first color with an absorbance at a first wavelength, a pH indicator dye which is colorless or has a color which is essentially not detectable by eye when the composition is applied to a reaction vessel for the ELISA but which forms a second color which is detectable by eye and has an absorbance at a second wavelength when a stop solution is added to the composition in the reaction vessel, and a peroxide;
• (b) adding an aliquot of the composition to each of the reaction vessels for the ELISA to form a reaction mixture;
• (c) incubating the reaction mixture for a time sufficient to oxidize the TMB to the first color;
• (d) adding the stop solution to the reaction mixture to stop the reaction, which produces the second color and further oxidizes the TMB to the first color;
• and
• (e) measuring spectrographically absorbances at the first and second wavelengths, wherein absorbance at the first wavelength indicates the detection of the analyte, absorbance at only the second wavelength indicates the negative reaction, and absence of absorbance at the first and second wavelengths indicates the false negative reaction or detecting the second color by eye wherein the second color indicates that the reaction mixture contains the composition and the stop solution.

An enzyme immunoassay for colorimetric detection of an analyte of a type wherein a quantity of a first antibody is adsorbed to a solid support in a reaction vessel;

• a conjugate is formed between an immunologic reagent and a peroxidase;
• the conjugate is admixed with a sample to be tested for the analyte, the analyte binds to the first antibody and to the conjugate to form an immunologic complex in solid phase;
• and the quantity of the analyte is determined by measuring the reaction of the immunologic complex with a chromogenic substrate oxidizable by peroxide and the peroxidase, wherein the improvement comprises:
• (a) providing a liquid solution comprising the chromogenic substrate, peroxide, and a pH indicator dye, wherein the chromogenic substrate is oxidizable to a first color with an absorbance at a first wavelength and the pH indicator dye produces a second color which is detectable by eye and which has an absorbance at a second wavelength when a stop solution is added to the reaction vessel containing the liquid solution;
• (b) mixing the liquid solution with the immunologic complex in the reaction vessel to form a reaction mixture which oxidizes the chromogenic substrate;
• (c) adding the stop solution to the reaction mixture in the reaction vessel to produce the first and second colors;
• and,
• (d) measuring spectrographically the absorbances at the first and second wavelengths, wherein absorbance at the first wavelength indicates detection of the analyte, absorbance at the second wavelength indicates a negative reaction, and an absence of absorbance at the first or second wavelength indicates a false negative reaction or detecting the second color by eye wherein the second color indicates that the reaction mixture contains the liquid solution and the stop solution.

An enzyme immunoassay for colorimetric detection of an antibody of a type wherein a quantity of an analyte is adsorbed to a solid support in a reaction vessel;

• a conjugate is formed between an immunologic reagent and a peroxidase;
• the conjugate is admixed with a sample to be tested for the antibody, the antibody binds to the analyte and to the conjugate to form an immunologic complex in solid phase;
• and the quantity of the antibody is determined by measuring the reaction of the immunologic complex with a chromogenic substrate oxidizable by peroxide and the peroxidase, wherein the improvement comprises:
• (a) providing a liquid solution comprising the chromogenic substrate, peroxide, and a pH indicator dye, wherein the chromogenic substrate is oxidizable to a first color with an absorbance at a first wavelength and the pH indicator dye produces a second color which is detectable by eye and which has an absorbance at a second wavelength when a stop solution is added to the reaction vessel containing the liquid solution;
• (b) mixing the liquid solution with the immunologic complex in the reaction vessel to form a reaction mixture which oxidizes the chromogenic substrate;
• (c) adding the stop solution to the reaction mixture in the reaction vessel to produce the first and second colors;
• and,
• (d) measuring spectrographically the absorbances at the first and second wavelengths, wherein absorbance at the first wavelength indicates detection of the analyte, absorbance at the second wavelength indicates a negative reaction, and an absence of absorbance at the first or second wavelength indicates a false negative reaction or detecting the second color by eye wherein the second color indicates that the reaction mixture contains the liquid solution and the stop solution.

An enzyme immunoassay for colorimetric detection of an analyte of a type wherein a quantity of a first antibody is adsorbed to a solid support in a reaction vessel;

• a conjugate is formed between an immunologic reagent and a peroxidase;
• the conjugate is admixed with a sample to be tested for the analyte, the analyte binds to the first antibody and to the conjugate to form an immunologic complex in solid phase;
• and the quantity of the analyte is determined by measuring the reaction of the immunologic complex with 3,3′,5,5′-tetramethylbenzidine (TMB) which is oxidizable by peroxide and the peroxidase, wherein the improvement comprises:
• (a) providing a liquid solution comprising the TMB, peroxide, and a pH indicator dye, wherein the TMB is oxidizable to a first color with an absorbance at a first wavelength and the pH indicator dye is colorless or has a color which is essentially not detectable by eye when the liquid solution is applied to the reaction vessel for the immunoassay but which produces a second color which is detectable by eye and which has an absorbance at a second wavelength when a stop solution is added to the reaction vessel containing the liquid solution;
• (b) mixing the liquid solution with the immunologic complex in the reaction vessel to form a reaction mixture which oxidizes the TMB;
• (c) adding the stop solution to the reaction mixture in the reaction vessel to produce the first and second colors;
• and,
• (d) measuring spectrographically the absorbances at the first and second wavelengths, wherein absorbance at the first wavelength indicates detection of the analyte, absorbance at the second wavelength indicates a negative reaction, and an absence of absorbance at the first or second wavelength indicates a false negative reaction or detecting the second color by eye wherein the second color indicates that the reaction mixture contains the liquid solution and the stop solution.

An enzyme immunoassay for colorimetric detection of an antibody of a type wherein a quantity of an analyte is adsorbed to a solid support in a reaction vessel;

• a conjugate is formed between an immunologic reagent such as an antibody, Fab fragment, Fv fragment, single-chain Fv polypeptide, or other antibody derivative and a peroxidase;
• the conjugate is admixed with a sample to be tested for the antibody, the antibody binds to the analyte and to the conjugate to form an immunologic complex in solid phase;
• and the quantity of the antibody is determined by measuring the reaction of the immunologic complex with 3,3′,5,5′-tetramethylbenzidine (TMB) which is oxidizable by peroxide and the peroxidase, wherein the improvement comprises:
• (a) providing a liquid solution comprising the TMB, peroxide, and a pH indicator dye, wherein the TMB is oxidizable to a first color with an absorbance at a first wavelength and the pH indicator dye is colorless or has a color which is essentially not detectable by eye when the liquid solution is applied to the reaction vessel for the immunoassay but which produces a second color which is detectable by eye and which has an absorbance at a second wavelength when a stop solution is added to the reaction vessel containing the liquid solution;
• (b) mixing the liquid solution with the immunologic complex in the reaction vessel to form a reaction mixture which oxidizes the TMB;
• (c) adding the stop solution to the reaction mixture in the reaction vessel to produce the first and second colors;
• and,
• (d) measuring spectrographically the absorbances at the first and second wavelengths, wherein absorbance at the first wavelength indicates detection of the analyte, absorbance at the second wavelength indicates a negative reaction, and an absence of absorbance at the first or second wavelength indicates a false negative reaction or detecting the second color by eye wherein the second color indicates that the reaction mixture contains the liquid solution and the stop solution.

A method for detecting an analyte in a enzyme-linked assay and distinguishing a negative reaction from a false negative reaction in the assay, which comprises:

• (a) providing a composition comprising a mixture of a chromogenic substrate selected from the group consisting of ortho-phenylenediamine (OPD), 2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), diaminobenzidine (DAB), and 3,3′dimethyloxybenzidine (ortho-dianisidine or ODN) which is converted by an enzyme in a reaction vessel for the assay to a first color which has an absorbance at a first wavelength and a pH indicator dye which produces a second color detectable by eye and which has an absorbance at a second wavelength when an acid or base stop solution is added to the composition in the reaction vessel;
• (b) adding an aliquot of the composition to the reaction vessel for the enzyme-linked assay to form a reaction mixture;
• (c) incubating the reaction mixture for a time sufficient to produce the first color;
• (d) adding the stop solution to the reaction mixture to stop the reaction and to generate the second color with the second wavelength;
• and
• (e) measuring spectrographically absorbances at the first and second wavelengths, wherein absorbance at the first wavelength indicates the detection of the analyte, absorbance at only the second wavelength indicates the negative reaction, and absence of absorbance at the first and second wavelengths indicates the false negative reaction or detecting the second color by eye wherein the second color indicates that the reaction mixture contains the composition and the stop solution.