Anti-free Prostate Specific Antigen (fPSA) scFv (B8H8) [B8H8scFv] recombinant antibody
Invented by Paul Conroy , James Whisstock , Caroline Murphy
Invented at Dublin City University
- Datasheet
- References (2)
- Inventor Info
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| Catalogue Number | 156519 |
| Applications | ELISA WB |
| Antigen/Gene or Protein Targets | Free Prostate Specific Antigen |
| Synonyms | fPSA |
| Reactivity | Human |
| Relevance | Prostate-specific antigen, or PSA, is a protein produced by normal, as well as malignant, cells of the prostate gland. The blood level of PSA is often elevated in men with prostate cancer. However, highly sensitive and label free detection of prostate specific antigen (PSA) still remains a challenge in prostate cancer diagnosis. |
| Host | Chicken |
| Immunogen | Prostate Specific Antigen (PSA) |
| Formulation |
Antibody Selection Single chain fragment variable (scFv) libraries were generated in the VL-VH orientation, as described by Andris-Widhopf et al. Clone B8 was selected from a PSA protein (human seminal fluid; SCIPAC)-immunized repertoire. Briefly, an adult leghorn was immunized with PSA, sacrificed, and the antibody repertoire was accessed from mRNA isolated from B-cells (femur bone marrow and spleen tissue) and displayed on the surface of filamentous phage. The antibody was isolated by iterative cycles phage display with increasing stringency exerted by serial limitation of adsorbed PSA. Antibody Expression and Purification ScFv antibodies were expressed within the periplasmic space of E. coli Top10F′ with the pComb3x vector. Single colonies were selected from LB-agar supplemented with 25 μg/ml carbenicillin and grown overnight in 5 ml of Superbroth supplemented with 25 μg/ml carbenicillin and 1% (w/v) glucose at 37 oC with shaking at 220rpm. This starter culture was used to inoculate 100 ml of Superbroth with 25 μg/ml carbenicillin and was grown to A600 = ∼0.6 before subculturing into 10 × 500 ml of Superbroth with 25 μg/ml carbenicillin (2-liter flasks). At A600 = ∼0.6, the cultures were induced with 0.2 mM isopropyl 1-thio-β-D-galactopyranoside at 30 oC with shaking at 230 rpm overnight (∼16 h). The bacteria were harvested by centrifugation (3220 × g) at 4 oC for 20 min. Soluble scFv were released from the periplasmic space by osmotic shock in a two-step process. The pellet was first thoroughly resuspended in 1× TBS (25 mM Tris, pH 8.0, 150 mM NaCl), and an equal volume of 2× shock buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 1 M sucrose, and 2 mM EDTA) was added before incubation at room temperature for 15 min. Shocked cells were recovered by centrifugation (12,400 × g at 4 oC for 20 min) followed by resuspension in ice-cold 5 mM MgSO4 and incubation on ice for 15 min. The periplasmic-stripped cells were collected by centrifugation (27,200 × g at 4 oC for 20 min), and 0.2 times the volume of 5× binding buffer (125 mM Tris, pH 8.0, 750 mM NaCl, 50 mM imidazole, 0.02% NaN3) was added to the supernatant. HisBind resin (1 ml equilibrated in 30 ml of 1× binding buffer) was added, and scFv was recovered by batch binding for 2 h at 4 oC on an end-over-end roller. The resin was collected by gravity flow and washed with 30 ml of binding buffer followed by a second wash with 30 ml of wash buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 20 mM imidazole, 0.05% (v/v) Polysorbate 20, 0.02% NaN3). Bound protein was eluted with 7–10 ml of elution buffer (1× running buffer, 300 mM imidazole) in 0.5-ml fractions, and protein-containing fractions were pooled and concentrated to 2 ml in a 3-kDa concentrator. The pooled fractions were resolved by size exclusion chromatography in 25 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3. The purified protein was analyzed by SDS-PAGE and Western blot. |
| Research Area | Cancer, Reproductive Biology |
| Immunogen UniProt ID | TBC |
| Notes |
Application working Concentration: ELISA (1:1,000 - 1:10,000) Storage: 1 x PBS -20C in 1 x PBS. Avoid repeated freeze and thaw cycles |
References: 2 entries
Spain et al. 2016. Biosens Bioelectron. 77:759-66. PMID: 26513282.
Conroy et al. 2014. J Biol Chem. 289(22):15384-92. PMID: 24737329.
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References: 2 entries
Spain et al. 2016. Biosens Bioelectron. 77:759-66. PMID: 26513282.
Conroy et al. 2014. J Biol Chem. 289(22):15384-92. PMID: 24737329.
Add a reference
Inventor Information
Inventors
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Paul Conroy |
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James Whisstock |
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Caroline Murphy |
