Production of Nanobodies against Prostate-Specific Membrane Antigen (PSMA) Recognizing LnCaP Cells

2014 ◽  
Vol 29 (2) ◽  
pp. 169-179 ◽  
Author(s):  
Hamed Zare ◽  
Masoumeh Rajabibazl ◽  
Iraj Rasooli ◽  
Walead Ebrahimizadeh ◽  
Hamid Bakherad ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Protein A ◽  
High Specificity ◽  
Prostate Specific Membrane Antigen ◽  
Lncap Cells ◽  
E Coli ◽  
Dna Fragment ◽  
Specific Membrane ◽  
Cell Elisa ◽  
Selection Of

Prostate cancer is the most common type of cancer in men. The antibody-mediated therapy for cancer treatment depends on the identification of selected molecular targets. The prostate-specific membrane antigen (PSMA) is a potential molecular target in prostate cancer and is abundantly expressed in this type of cancer. This study is aimed at designing and producing a recombinant PSMA epitope and a monoclonal nanobody with a high affinity toward the PSMA protein. A DNA fragment encoding the dominant epitopes of PSMA was designed, synthesized, and expressed in E. coli BL21 (DE3). A camel was immunized with the purified recombinant PSMA (rPSMA). Following mRNA isolation and cDNA synthesis, the variable fragment of heavy-chain antibodies (VHH) fragments were cloned and displayed on the surface of an M13 phage and used in sequential panning rounds. After phage ELISA and selection of colonies with the highest affinity, soluble nanobodies were produced and evaluated. Affinity of the nanobodies to rPSMA was estimated to be 3.5 × 10−7. Adherence of the purified anti-PSMA VHH was tested in cell-ELISA in the LnCaP and PC3 cell lines. VHH efficiently bound to LnCaP cells. The high specificity and affinity of this nanobody suggests its possible application as an effective tool in the diagnosis and treatment of prostate cancer.

scholarly journals Surface Modification of Liposomes by a Lipopolymer Targeting Prostate Specific Membrane Antigen for Theranostic Delivery in Prostate Cancer

Materials ◽  
2019 ◽  
Vol 12 (5) ◽  
pp. 756 ◽  
Author(s):  
Hooman Yari ◽  
Gregory Nkepang ◽  
Vibhudutta Awasthi
Keyword(s):  
Prostate Cancer ◽  
Surface Modification ◽  
Surface Functionalization ◽  
Targeted Delivery ◽  
Prostate Specific Membrane Antigen ◽  
Lncap Cells ◽  
Binding Motifs ◽  
Ic50 Value ◽  
Pc3 Cells ◽  
Specific Membrane

Prostate specific membrane antigen (PSMA) is a marker for diagnosis and targeted delivery of therapeutics to advanced/metastasized prostate cancer. We report a liposome-based system for theranostic delivery to PSMA-expressing (PSMA+) LNCaP cells. A lipopolymer (P3) comprising of PSMA ligand (PSMAL), polyethylene glycol (PEG2000), and palmitate was synthesized and post-inserted into the surface of preformed liposomes. These P3-liposomes were loaded with doxorubicin and radiolabeled with 99mTc radionuclide to study their theranostic characteristics. Differential expression of PSMA on LNCaP and PC3 cells was confirmed by immunoblotting as well as by uptake of PSMAL labeled with 18F radionuclide. We found that the uptake of 99mTc-labeled P3-liposomes by LNCaP cells was >3-fold higher than 99mTc-labeled Plain-liposomes; the amount of doxorubicin delivered to LNCaP cells was also found to be >3-fold higher by P3-liposomes. Cell-based cytotoxicity assay results showed that doxorubicin-loaded P3-liposomes were significantly more toxic to LNCaP cells (p < 0.05), but not to PSMA-negative PC3 cells. Compared to doxorubicin-loaded Plain-liposomes, the IC50 value of doxorubicin-loaded P3-liposomes was reduced by ~5-fold in LNCaP cells. Together, these results suggest that surface functionalization of liposomes with small PSMA-binding motifs, such as PSMAL, can provide a viable platform for specific delivery of theranostics to PSMA+ prostate cancer.

scholarly journals Positive Selection of Specific Antibodies Produced against Fusion Proteins

2020 ◽  
Vol 3 (2) ◽  
pp. 37
Author(s):  
Lukas Kramberger-Kaplan ◽  
Tina Austerlitz ◽  
Holger Bohlmann
Keyword(s):  
Fusion Protein ◽  
Affinity Chromatography ◽  
Positive Selection ◽  
Fusion Proteins ◽  
Protein A ◽  
High Specificity ◽  
Western Blots ◽  
Specific Antibodies ◽  
E Coli ◽  
Selection Of

A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in Escherichia coli, purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of E. coli and purified. GFP without tags was also expressed in E. coli and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different E. coli compartments for antibody production and affinity chromatography.

Preparation of QDs@SiO2/Polystyrene Composite Particles for Cancer Cells Detection

Nano LIFE ◽  
2018 ◽  
Vol 08 (04) ◽  
pp. 1841006 ◽  
Author(s):  
Xiao Han ◽  
Yilong Wang ◽  
Donglu Shi
Keyword(s):  
Prostate Cancer ◽  
Cancer Cells ◽  
Polymer Nanocomposites ◽  
Polyclonal Antibodies ◽  
Composite Particles ◽  
Prostate Specific Membrane Antigen ◽  
Lncap Cells ◽  
Enhanced Fluorescence ◽  
Magnetic Probes ◽  
Specific Membrane

We report a sandwich immunoassay strategy for specific isolation and detection of cancer cells. Prostate cancer LNCaP cells overexpressing PSMA (Prostate-specific membrane antigen) were used as the model target to evaluate the performance of immunoassay. Inorganic/polymer nanocomposites with Janus-like structure and enhanced fluorescence properties were synthesized and applied for immobilization of QDs and the antibodies. PSMA antibodies were covalently conjugated to the QDs@SiO2/PS Janus-like composite nanoparticles to construct the fluorescent probes. Magnetic probes conjugated with polyclonal antibodies were used to capture and isolate the cancer cells. Results indicated high potential of the unique nanoprobes in specific isolation and detection of cancer cells.

479: Prostate-Specific Membrane Antigen (PSMA) Expression as a Predictor of Prostate Cancer Progression

2006 ◽  
Vol 175 (4S) ◽  
pp. 155-156
Author(s):  
Matthias D. Hofer ◽  
Sven Perner ◽  
Haojie Li ◽  
Rainer Kuefer ◽  
Richard E. Hautmann ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Progression ◽  
Prostate Specific Membrane Antigen ◽  
Prostate Cancer Progression ◽  
Specific Membrane ◽  
Psma Expression
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