Enhanced sensitivity of immunoblotting with peroxidase-conjugated antibodies using an adsorbed substrate method.
Immunoblots probed with immunoglobulin E (IgE)-containing sera from allergic patients are frequently used in allergy research. Current techniques for detection of specific IgE include radiolabeled and enzyme-linked methods. Although radiolabeled methods are very sensitive, many research groups prefer non-radioactive procedures with equal or greater sensitivity. Alkaline phosphatase (AP) and horse radish peroxidase (HRP) are the most frequently used conjugating enzymes for immunoblotting with the former generally recognized as more sensitive. We describe a method of immunoblot detection using HRP-conjugated immunochemicals with sensitivity equal to and for some systems greater than that of AP conjugates. An adsorbed substrate method for developing immunoblots probed with HRP immunochemical conjugates is compared with traditional AP and HRP methods. The adsorbed substrate system, when used to detect IgE binding to allergic proteins, gives high resolution and delineates bands not otherwise seen. The system has advantages of high sensitivity, rapid development and conservation of immunochemicals. Problems of fading, sensitivity to heat and light, and high background can be solved with increased washing, prompt photography and computer scanning.
Barnes
C S
CS
Section of Allergy/Immunology, Children's Mercy Hospital, Kansas City, MO 64108.
Upadrashta
B
B
Pacheco
F
F
Portnoy
J
J
D000327:Adsorption; D000469:Alkaline Phosphatase / Q000032:analysis / Q000276:immunology; D000528:Alternaria / Q000737:chemistry / Q000254:growth & development; D000906:Antibodies / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000032:analysis / Q000276:immunology; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007124:Immunoenzyme Techniques; D007073:Immunoglobulin E / Q000032:analysis / Q000276:immunology; D007202:Indicators and Reagents
D000906:Antibodies; D007202:Indicators and Reagents; D007073:Immunoglobulin E; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase