Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting

Abstract

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.

Evaluation ofalpha-methylacyl-CoA racemase, metallothionein and prostate specific antigen as prostate cancer prognostic markers

“…Moreover, this marker has been found elevated even in other types of cancers such as breast carcinoma [66], head and neck cancer [67], medulloblastoma [35], melanoma [68,69] and other [70][71][72][73][74]. Nevertheless, combination of MT and PSA levels could be of prognostic significance due to possible revealing of false positive results, but this assumption needs to be investigated in greater details.…”

“…In addition, metallothioneins regulate level, activity and cellular localisation of transcriptional factor NF-κB, which activates antiapoptotic genes Bcl-2, c-myc and TRAF-1 that belong to the group of prooncogenes. This antiapoptotic cascade can be efficient as the protective mechanism of prostate carcinoma cells against apoptotic signals at their proliferation [35,36].…”

Isolation of metallothionein from cells derived from aggressive form of high‐grade prostate carcinoma using paramagnetic antibody‐modified microbeads off‐line coupled with electrochemical and electrophoretic analysis

“…1). Advantages of both steps are discussed in the previously published articles [7,51]. The MT antibody is immobilized with high affinity on protein G-conjugated paramagnetic particles via its Fc fragment and then MT is selectively isolated.…”

“…In the previous experiments, we have shown that chicken egg yolk IgY anti-MT antibody (HENA, Czech Republic) led to significantly higher signal in Western blot assay as compared with other anti-MT antibodies [7]. However, according to the paramagnetic beads' manual, protein G or protein A beads do not bind to the chicken IgY antibodies.…”

Microfluidic tool based on the antibody‐modified paramagnetic particles for detection of 8‐hydroxy‐2′‐deoxyguanosine in urine of prostate cancer patients

“…Then, the membrane was incubated with a chromogenic substrate for horseradish peroxidase (0.4 mg/mL À1 3-aminoethyl-9-carbazole in 0.5 M acetate buffer with 0.1% H 2 O 2 , pH 5.5), after the adequate development the reaction was stopped by rinsing with water. After drying the membranes were scanned according to the protocol mentioned in Krizkova et al [77], in which function optical density (OD) of Biolight software (Vilber-Lourmat, France) was used for the calculation of dot volumes by contour recognition.…”