Multiple simultaneous antigen detection by immunohistochemistry

A method of simultaneously determining the presence of an antigen and a disease marker in a tissue sample suspected of containing the antigen and the disease marker, said method comprising:

• incubating an enzyme-conjugated antibody mix with said tissue sample, said enzyme-conjugated antibody mix including a first antibody against said antigen, said first antibody conjugated to a first enzyme, and a second antibody against said disease marker, said second antibody conjugated to a second enzyme;
• washing said tissue sample;
• adding a first substrate specific for said first enzyme and sequentially adding a second substrate specific for said second enzyme;
• and
• determining the presence of said antigen and said disease marker in said tissue sample.

A kit for the simultaneous identification of a first target molecule and a second target molecule in a pathological tissue sample, said kit comprising:

• an antibody solution including a first antibody or fragment thereof which immunologically recognizes said first target molecule, said first antibody or fragment thereof conjugated to a first means of detection, and a second antibody or fragment thereof which immunologically recognizes said second target molecule, said second antibody or fragment thereof conjugated to a second means of detection;
• a first reagent reactive with said first means of detection to produce a detectable reaction product;
• and
• a second reagent reactive with said second means of detection to produce a detectable reaction product.

A method of making a kit for use in simultaneously identifying at least two target molecules in a sample, said method comprising:

• providing an antibody solution including a first antibody or fragment thereof which immunologically recognizes a first target molecule, said first antibody or fragment thereof conjugated to a first means of detection, and a second antibody or fragment thereof which immunologically recognizes a second target molecule, said second antibody or fragment thereof conjugated to a second means of detection;
• providing a first reagent reactive with said first means of detection to produce a detectable reaction product;
• and
• providing a second reagent reactive with said second means of detection to produce a detectable reaction product.

A method of using a kit to simultaneously diagnose the presence of a disease, said method comprising:

• providing a kit capable of simultaneously identifying at least two markers for a disease, said kit comprising an antibody solution including a first antibody or fragment thereof which immunologically recognizes a first marker of said at least two markers, said first antibody or fragment thereof conjugated to a first enzyme, and a second antibody or fragment thereof which immunologically recognizes a second marker of said at least two markers, said second antibody or fragment thereof conjugated to a second enzyme;
• introducing a sample suspected of including said at least two markers to said antibody solution;
• and
• performing an assay to simultaneously detect the presence of said at least two markers for said disease.