{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,23]],"date-time":"2025-12-23T19:30:38Z","timestamp":1766518238873,"version":"3.48.0"},"reference-count":0,"publisher":"Maad Rayan Publishing Company","content-domain":{"domain":["apb.tbzmed.ac.ir"],"crossmark-restriction":false},"short-container-title":["Adv Pharm Bull"],"abstract":"<jats:p>Purpose: Nanobodies possess unique properties that make them promising as tumor targeting agents. Prostate-specific membrane antigen (PSMA), overexpressed in prostate cancer, can be an excellent target for prostate cancer diagnosis. This study aimed to express and purify an anti-PSMA nanobody (PSMA-Nb) and assess its potential for detecting PSMA antigen in prostate cancer through immunohistochemistry (IHC). Methods: The PSMA-Nb gene was subcloned into pET-28a and expressed in E. coli Rosetta (DE3) and Rosetta-gami2 under varying IPTG\/temperature\/time conditions (0.5\/1.0\/1.5 mM; 16\/30\/37 \u00b0C; 4\/16 h). The soluble and inclusion-body fractions were analyzed, and PSMA-Nb was purified via native Ni-NTA, confirmed by SDS-PAGE and anti-c-Myc Western blot. Binding was validated by ELISA against recombinant PSMA and by flow cytometry on LNCaP (PSMA+) and DU145 (PSMA-). For tissue studies, FFPE IHC quantified staining as fractional fluorescent area per mm\u00b2. Results: PSMA-Nb (~27 kDa) was enriched in E. coli inclusion bodies. A factorial screen identified Rosetta (DE3) with 1 mM IPTG at 37 \u00b0C for 16 h as the highest-expressing condition; resulting in a yield of approximately 84 mg\/L. ELISA showed dose-dependent binding to recombinant PSMA, and flow cytometry confirmed antigen selectivity (LNCaP 68.5% vs. DU145 2.35%). IHC showed higher PSMA levels in tumor vs. normal tissue with both reagents (PSMA-Nb: 21.61 \u00b1 2.89 vs. 5.82 \u00b1 1.80; commercial antibody: 20.55 \u00b1 3.80 vs. 5.50 \u00b1 2.14; P&lt;0.0001), with no difference between reagents (P=0.9624), supporting analytical validity. Conclusion: The superior features of nanobodies support antibody-based diagnostics in solid tumors. Purified PSMA-Nb detected PSMA on cancer cells and FFPE tissues, indicating a promising tool for prostate cancer diagnosis.<\/jats:p>","DOI":"10.34172\/apb.025.45447","type":"journal-article","created":{"date-parts":[[2025,12,23]],"date-time":"2025-12-23T19:27:11Z","timestamp":1766518031000},"update-policy":"https:\/\/doi.org\/10.34172\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimizing Expression of a Llama-Based Anti-PSMA Nanobody in Escherichia coli and Its Application in Immunohistochemistry of Prostate Cancer Tissues"],"prefix":"10.34172","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-5723-2789","authenticated-orcid":true,"given":"Seyedeh Sheila","family":"Seyed-Motahari","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shahriyar","family":"Abdoli","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohammad Ali","family":"Shokrgozar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shiva","family":"Irani","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4419-0315","authenticated-orcid":true,"given":"zahra","family":"Sharifzadeh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"20123","published-online":{"date-parts":[[2025,12,23]]},"container-title":["Advanced Pharmaceutical Bulletin"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/apb.tbzmed.ac.ir\/Inpress\/apb-45447.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/apb.tbzmed.ac.ir\/Inpress\/apb-45447.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,23]],"date-time":"2025-12-23T19:27:11Z","timestamp":1766518031000},"score":1,"resource":{"primary":{"URL":"https:\/\/apb.tbzmed.ac.ir\/Inpress\/apb-45447"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,23]]},"references-count":0,"URL":"https:\/\/doi.org\/10.34172\/apb.025.45447","relation":{},"ISSN":["2228-5881","2251-7308"],"issn-type":[{"value":"2228-5881","type":"print"},{"value":"2251-7308","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,23]]},"assertion":[{"value":"Tabriz University of Medical Sciences","name":"journal_owner","label":"Journal Owner"},{"value":"Tabriz University of Medical Sciences","name":"journal_publisher","label":"Journal Publisher"},{"value":"2025-04-11","order":1,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-12-20","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-12-23","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}