The Complete Guide to Antibody Detection by Immunoenzyme Technique: Antibody
Capturing ELISA, ABC-ELISA, Dot-ELISA and Immunohistochemistry Stainig
The immunoenzyme technique is an immunological technique that combines the specificity of the
antigen-antibody reaction and the efficient catalytic action of the enzyme on the substrate's color
reaction. Because of its specificity and high sensitivity, it has been widely used for screening and
identification of monoclonal antibody.
Antibody Capturing ELISA
a. Coat the enzyme plate with purified anti-mouse Ig antibodies of appropriate concentration.
Add 100ul into each hole, and maintain at 37℃ for 2 hours or at 4℃ overnight.
b. After washing and patting dry the plate from the last step, add McAb samples remained
untested. Maintain it at 37℃ for 1-2 hours.
c. After washing the plate from the last step, add appropriate amount of antigens, and maintain it
at 37 ℃ for 1-2 hours.
d. Wash the plate from the last step, add the enzyme-labeled polyclonal antibodies, and maintain
at 37 ℃ for 1-2 hours. After washing, add the substrate to show color. Analyze the result
according to the color.
ABC-ELISA
ABC-ELISA is based on the conventional ELISA principle. It increases the amplification function
between biotin and avidin. Avidin consisting of four subunits has high affinity to biotin. Biotin is
easy to covalently bind to protein. Thus, avidin binding with enzymes reacting to biotin binding
with antibodies can lead to multipolar amplification. The steps are as follows.
a. Coat the known antigens and add untested McAb samples, which is the same as indirect
ELISA.
b. Add 100ul biotinylated anti-mouse Ig antibodies to each hole. Maintain at 7 ℃ for 1 hour and
then wash.
c. Add 100ul enzyme-labeled avidin into each hole. Wash at 37℃ for 30 minutes. Add the
substrate to show color, and analyze the result according to the color.
Dot-ELISA
Dot-ELISA is an immune detection means which uses nitrocellulose membrane or cellulose
acetate membrane as a solid carrier to lead antigen-antibody reaction. This method uses insoluble
substrates (such as DAB, 4-chloronaphthol or AgNO3, etc.) to react with corresponding markers
