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Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting
Author(s) -
Krizkova Sona,
Adam Vojtech,
Eckschlager Tomas,
Kizek Rene
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200900201
Subject(s) - polyclonal antibodies , electroblotting , chemistry , membrane , primary and secondary antibodies , horseradish peroxidase , monoclonal antibody , antibody , detection limit , metallothionein , differential pulse voltammetry , cadmium , microbiology and biotechnology , chromatography , biochemistry , biology , cyclic voltammetry , immunology , electrochemistry , enzyme , organic chemistry , electrode , nitrocellulose
Metallothionein (MT) is a low‐molecular mass protein playing an essential role in homeostasis of heavy metal ions. Its relation with formation and progression of a tumour disease is discussed in this article. Here, we propose a new methodological approach for visualization of MT on PVDF membranes after dot‐ and electroblotting by using a commercial mouse monoclonal antibody E9 and polyclonal chicken antibodies. The optimized procedure was as follows. We dotted 1 μL sample volume on PVDF membrane and let it to dry. Then, we blocked the membrane surface with 2% BSA in PBS for 30 min. After that, the membrane was incubated in chicken primary antibody (diluted 1:500), washed, and incubated in rabbit‐anti‐chicken secondary antibody conjugated with horseradish peroxidase. To visualize the interaction, we used 3‐aminoethyl‐9‐carbazole. Under these conditions, we estimated detection limit as 3 pg of MT per 1 μL. The optimal approach was further utilized for detection of MT level in two human fibroblast cell lines and in blood serum obtained from children with medulloblastoma. The results were in good agreement with differential pulse voltammetry‐Brdicka reaction.