A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis.
Expression of carbonic anhydrase IX (CAIX) significantly increases under hypoxic conditions in tumor cells. CAIX activity is executed by the catalytic domain (CA) located on the extracellular part of the enzyme. Neutralization of CAIX enzymatic activity reduces malignancy and survival of tumor cells. To inhibit the enzymatic activity, a VHH nanobody was developed against the CA domain of CAIX using phage display technology. Following immunization of a camel with the recombinant CAIX, VHH fragments were isolated by nested PCR on lymphocyte cDNA. Binding affinity of isolated nanobodies was tested by ELISA. A clone (K24) with the highest binding affinity was expressed in a soluble form. Affinity of K24 nanobody was determined to be approx. 2.3 × 10(-5). K24 nanobody recognized the expressed CAIX in the HeLa cell lines with high selectivity and specificity. These findings thus have usefulness for the diagnosis and treatment of cancers.
Department of Biology, Faculty of Basic Science, Shahed University, Opposite of Imam Khomeini's Shrine, Tehran-Qom, Express Way, 3319118651, Tehran, Iran.
Fatemeh
F
Araste
Walead
W
Ebrahimizadeh
Iraj
I
Rasooli
Masoumeh
M
Rajabibazl
Seyed Latif
SL
Mousavi Gargari
D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D002162:Camelus; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D020134:Catalytic Domain; D061505:Cell Surface Display Techniques; D002846:Chromatography, Affinity; D006367:HeLa Cells; D006801:Humans; D008297:Male; D061905:Single-Domain Antibodies / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D061905:Single-Domain Antibodies; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases