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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 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

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 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
(HRP, AP and colloidal gold), forming insoluble products which show as spot-like coloring and
help us to analyze the result easily. According to different markers, they can be classified into HRP
Dot-ELISA, AP Dot-ELISA, Dot-IGSS and so on. The steps are as follows.

a. Add 2-5ul antigen solution on the cellulose membrane, and dry at 37 .

b. Soak the cellulose membrane in blocking solution, and store at 37 C for 30 minutes.

c. Wash with washing solution for 2 times, add untested McAb samples, and then store at 37
for 1 hour. Wash in vibro washer for 3 times, and each time lasts for 5 minutes. Add HRP or
AP or colloidal gold labeled anti-mouse Ig antibodies, and maintain at 37 for 30 minutes.

d. Wash in the same way and sip up. Color with the fresh corresponding substrate solution, wash
to terminate the reaction, and then analyze the result.

Immunohistochemistry Stainig

This method is mainly used to detect McAb against cell antigen components. Commonly used
approaches include indirect immunoperoxidase and APAAP technology. The results can be
checked by light microscope or inverted microscope.

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Creative Biolabs is specialized in custom biotechnology and pharmaceutical services that cover
the full scope of biotechnology needs of early drug discovery and development, such as next gen
antibody sequencing, antibody manufacturing, phage display library construction, and so on.