EGF-r detection kit and method

A kit operable for detecting the presence of epidermal growth factor receptor in cells comprising a tissue, the kit comprising:

• (a) Proteinase K proteolytic enzyme diluted in 0.05 M Tris-HCl, 0.015M sodium azide, pH 7.5.
• (b) Peroxidase Block comprising 0.03% hydrogen peroxide containing sodium azide.
• (c) Monoclonal Mouse Anti-human EGFR, Clone 2-18C9 supplied in concentrated form at 581 μg/mL as tissue culture supernatant (containing fetal calf serum) dialyzed against 0.05M Tris-HCl, pH 7.2, and 0.015M sodium azide.
• (d) Mouse IgG, Isotype Control comprising monoclonal mouse IgG1 antibody in concentrated form at 100 μg/mL as tissue culture supernatant (containing fetal calf serum) dialyzed against 0.05M Tris-HCl, pH 7.2, and 0.015M sodium azide.
• (e) Tris-based antibody diluent comprised of 0.05M Tris-HCl pH 7.6 containing stabilizing protein and 0.015M sodium azide.
• (f) a Visualization Reagent comprising peroxidase labeled polymer conjugated to affinity-isolated goat anti-mouse immunoglobulins in Tris-HCl buffer containing stabilizing protein and an antimicrobial agent.
• (g) DAB Substrate Buffer comprising Imidazole-HCl buffer pH 7.5, containing hydrogen peroxide, stabilizers, enhancers and an antimicrobial agent.
• (h) DAB Chromogen comprising 3,3′-diaminobenzidine in chromogen solution.
• (i) Tris Buffered Saline, pH 7.6.
• (j) Tween 20
• (k) At least one control slide containing three pelleted, formalin-fixed, paraffin-embedded cell lines, which represent different amounts of the EGFR protein expression.

A method for detecting the presence of epidermal growth factor in cells comprising a tissue specimen wherein the tissue specimen has been fixed by immersion in formalin and mounted on a slide comprising the steps of:

• a. covering the specimen with a solution containing Proteinase K;
• then rinsing the specimen;
• then
• b. covering the specimen with peroxidase block, then rinsing;
• and
• c. covering the specimen with primary antibody, then rinsing;
• then
• d. covering the specimen with a visualization reagent, then rinsing;
• then
• e. covering the specimen with a chromogen substrate, then rinsing;
• then
• f. covering the specimen with a counterstain, then rinsing;
• and
• g. viewing the specimen,
• wherein regions having a high concentration of reacted substrate indicate the presence of EGFR in the specimen.