Using of chicken antibodies for metallothionein detection in human blood serum and cadmium-treated tumour cell lines after dot- and electroblotting.
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 microL 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 microL. 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.
Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Czech Republic.
Sona
S
Krizkova
Vojtech
V
Adam
Tomas
T
Eckschlager
Rene
R
Kizek
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods*; D002104:Cadmium / Q000494:pharmacology*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor / Q000187:drug effects / Q000378:metabolism; D002645:Chickens / Q000276:immunology; D002648:Child; D003101:Collodion; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008527:Medulloblastoma; D008668:Metallothionein / Q000032:analysis / Q000097:blood* / Q000378:metabolism; D011145:Polyvinyls; D005465:Fluorocarbon Polymers
D000906:Antibodies; D002104:Cadmium; D003101:Collodion; D008668:Metallothionein; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers