Fluorometric assay of total IgE level and reagents therefor

Claim 1

A method for identifying and quantifying total IgE levels in patient serum comprising a) contacting an insoluble support having anti-IgE antibody adhering thereto with patient serum for a sufficient time to permit conjugation and removing the patient serum therefrom; b) contacting the insoluble support with anti-IgE antibody labeled with a fluorogenic enzyme in an aqueous solution containing polyethylene glycol and a non-ionic surfactant for a sufficient time to permit conjugation of anti-IgE antibody to any allergen specific IgE conjugated to the support and removing unconjugated enzyme labeled anti-IgE antibody therefrom; c) contacting the insoluble support with a solution of a substrate which undergoes reaction in the presence of the fluorogenic enzyme to yield fluorescent product; and d) measuring the fluorescence level in the solution, wherein the anti-IgE antibody on the insoluble support and labeled anti-IgE antibody are individually selected from the group consisting of affinity purified polyclonal antibody or a mixture of monoclonal antibodies derived from a plurality of different clones.

Claim 2

A method for identifying and quantifying total IgE levels in patient serum comprising a) contacting an insoluble support having anti-IgE antibody adhering thereto with patient serum for a sufficient time to permit conjugation and removing the patient serum therefrom; b) contacting the insoluble support with anti-IgE antibody labeled with a fluorogenic enzyme in an aqueous solution containing polyethylene glycol and a non-ionic surfactant for a sufficient time to permit conjugation of anti-IgE antibody to any allergen specific IgE conjugated to the support and removing unconjugated enzyme labeled anti-IgE antibody therefrom; c) contacting the insoluble support with a solution of a substrate which undergoes reaction in the presence of the fluorogenic enzyme to yield fluorescent product; and d) measuring the fluorescence level in the solution, wherein the anti-IgE antibody on the insoluble support and labeled anti-IgE antibody are individually selected from the group consisting of affinity purified polyclonal antibody or a mixture of monoclonal antibodies derived from a plurality of different clones.

Claim 5

The method of any of claims 1 to 3 wherein the anti-IgE antibody is labeled with alkaline phosphatase.

Claim 6

The method of any of claims 1 to 4 wherein the insoluble! support is a microtiter plate of polystyrene or a styrene-(vinyl monomer) copolymer and said support is contacted in step (b) with enzyme labeled anti-IgE antibody in an aqueous solution containing from 1 to 8 wt.% polyethylene glycol having a molecular weight within the range of from 1000 to 10,000.

Claim 8

The method of any of claims 1 to 6 wherein the insoluble support is prerinsed with an aqueous buffered solution containing from 0.0001 to 0.5 wt.% of soluble animal protein before being contacted with patient serum.

Claim 10

The method of any of claims 1 to 8 wherein the insoluble support having anti-IgE antibody adhering thereto has been treated with an aqueous buffer solution containing water-soluble saccharide and water-soluble animal protein,

Claim 16

A method for stabilizing protein adhering to an insoluble support by contacting the insoluble support with the protein adherent thereto with a 0.01 to 0.5 M phosphate buffer solution having a pH of from 6 to 8, containing from 0.05 to 1.0 wt.% water-soluble animal protein and from 0.1 to 5.0 wt.% water-soluble saccharide.

Claim 17

A method for stabilizing protein adhering to an insoluble support by contacting the insoluble support with the protein adherent thereto with a 0.01 to 0.5 M phosphate buffer solution having a pH of from 6 to 8, containing from 0.05 to 1.0 wt.% water-soluble animal protein and from 0.1 to 5.0 wt.% water-soluble saccharide.