Phage‐derived monoclonal anti‐Lua
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Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing reagents. Many blood group specificities, however, are not amenable to classic hybridoma technology. The phage display technology, aimed at isolating peptides or antibody fragments, offers an alternative strategy. Recombinant antibodies derived from this technology would greatly facilitate phenotyping and decrease analysis cost.A human single-chain Fv (scFv) phage-displayed library was panned on red blood cells (RBCs) in an attempt to isolate clones recognizing human RBC specificities. Three rounds of biopanning were performed. Enrichment was monitored by phage titration, and selected phage populations were analyzed further.Three major clones were identified by clone diversity analysis. One of them showed a specificity for Lua. This scFv was reconstructed into a human IgG1 by recombinant DNA methods. The reactivity of the reconstructed human IgG1 toward Lua is indistinguishable from its parent scFv. Moreover, the specificity of the antibody was confirmed by serologic assays, flow cytometry, and biochemical analysis with RBCs of different Lu phenotypes and a recombinant cell line expressing Lu glycoproteins.With phage display and standard recombinant DNA methods, isolation of a scFv of Lua specificity was successful, from which a complete human IgG1 MoAb of equivalent reactivity was reconstructed. To our knowledge, this is the first MoAb specific for Lua.Keywords:
Biopanning
Polyclonal antibodies
clone (Java method)
Hybridoma technology
Objective To screen and identify peptides with high affinity to lipopolysaccharide (LPS) from phage display random peptide library. Methods Using LPS as a ligand, phage clones that bond to LPS were screened from phage display random 12-mer peptide library through 4 rounds of biopanning. After binding activity was measured by ELISA and plaque reduction testing, the phage clones presenting high affinity to LPS was selected and amplified. Their DNA were extracted for sequencing, and the coding peptide sequences were deduced from the results and analyzed by bioinformatics including homology analysis through blasting. Results After 4 rounds of effective screen, 86 eluted clones were obtained and amino acid sequences of 12 positive clones with high affinity to LPS were selected and identified. The core peptide sequence of phage clones was deduced as HWQWPHWSPPP (named P11) through comparing with the results from phage display random 7-mer peptide library, which was considered to contain the motif of the common amino acid sequence for LPS affinity. There was no patent declared based on P11 sequences retrieving from patent database. There were 908 proteins matching partly to the peptide found by homology retrieval. Conclusion Peptides with high affinity to LPS are obtained from phage display random peptide library, which provide us structural foundations on further study of direct-evolution using these peptides as primers.
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Biopanning
Phagemid
Epitope mapping
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Combinatorial phage display technology may be applied to decipher the molecular diversity of peptide binding specificity to isolated proteins, purified antibodies, cell surfaces, intracellular/cyto-domains, and blood vessels in vivo. The application of such a strategy ranges from identifying receptor-ligand pairs and antigen binding sites to understanding the progression of diseases by their differential expression patterns and developing therapeutic targeting strategies. Different strategies can be used to isolate peptides from diverse libraries displayed on the surface of bacteriophage by exposing the library to a target molecule or organ, washing away nonbinding phage, eluting and amplifying the bound phage for multiple round use, and then analyzing the peptide sequences of the enriched phage. The following methods first outline the construction of a phage library and then delineate various in vitro and in vivo biopanning applications to probe isolated integrins, purified antibodies, cell surface molecules, and vascular endothelial cells.
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DECIPHER
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Peptide phage display libraries have been successfully applied in areas of mapping antibody epitope, finding ligands for enzymes, receptors, and many other molecules. But it has been demonstrated to be very difficult to select cytokine-binders from peptide phage display libraries probably because cytokine is not so sticky as antibody that there are rare chances of capturing peptide phages during biopanning. A pVIII-based peptide phage display library was panned with the cytokine GM-CSF and some GM-CSF binding clones were selected based on high throughput screening (HTS) method and confirmed by ELISA and micropanning assays. These cytokine-binders may be utilized in affinity chromatography in cytokine downstream processing and even act as potential antagonists of GM-CSF if their affinity are further improved through secondary library strategy.
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Mimotope
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Biopanning
Binding selectivity
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Phage display has been widely used to develop high‐affinity peptide ligands against proteins and cell lines. Such peptides are typically selected using biopanning to exhibit significant binding affinity and specificity against a molecular target. Rarely are the same peptides identified from separate phage display selections, since the presence of these ligands in a selection typically signify specific binding to the target. However, here, we report the identification of the same peptide (TG1) from two different phage display selections against the hemagglutinin (HA) epitope and human pancreatic cancer (Mia Paca‐2) cells. Phage display selections were conducted to identify a peptide with high affinity and specificity for the HA epitope. In brief, a pre‐cleared 15‐mer fUSE5 phage library was subjected to four rounds of selections against 0.04–4 μg HA. Following the last round of selection, the phage DNA was amplified using PCR and submitted to next‐generation sequencing. These results identified peptide TG1 (RNVPPIFNDVYWIAF) as the most prevalent ligand in the selection. Modified enzyme‐linked immunosorbent assay (ELISA) results showed that 109 nM TG1 bound significantly higher (p<0.05) to 5 μg immobilized HA compared to a non‐relevant peptide J18 known to bind ovarian adenocarcinoma (SKOV‐3) cells. In a separate, previously reported phage display selection, the same 15‐mer fUSE5 phage library was subjected to selections against human pancreatic cancer (Mia‐Paca‐2) cells. Again, next‐generation sequencing results identified TG1 as the third most prevalent peptide. Modified ELISA results using 10 μM TG1, and a known pancreatic cancer‐targeting peptide MCA2, showed that both ligands bound significantly (p<0.01) to Mia‐Paca‐2 cells compared to dimethyl sulfoxide (DMSO; vehicle). A literature search revealed that TG1 was identified by two separate research groups conducting phage display selections against caveolin protein and mouse embryos. These results indicate that the peptide is an indiscriminate binder. Thus, we sought to elucidate the binding properties of peptide TG1 and phage TG1 (pTG1). Modified ELISA with 10 μM TG1, MCA2, J18, or DMSO were carried out against SKOV‐3 and human normal pancreatic (hTERT‐HPNE) cells. Results showed that TG1 bound significantly higher (p<0.01) to both SKOV‐3 and hTERT‐HPNE cells compared to DMSO. As expected MCA2 showed no binding to either cell line. Peptide J18 showed significant binding (p<0.05) to SKOV‐3 cells. Phage qPCR analysis was performed to further elucidate the binding properties of the pTG1. 10 10 V/mL pTG1, pancreatic cancer‐targeting pMCA1, or WT phage was incubated with Mia‐Paca‐2, SKOV‐3, or hTERT‐HPNE cells. After washing with Tris‐buffered saline (TBS), bound phage were eluted with 2.5% 3‐cholamidopropyl dimethylammonio 1‐propanesulfonate (CHAPS). Collected phage virions (5 μL) were quantified by qPCR using Fast SYBR Green Master Mix on an Applied Biosystems StepOnePlus Real‐Time qPCR System (Applied Biosystems, CA). The results revealed that pTG1 demonstrated significantly higher binding (p<0.01) compared to WT for all cell lines. As expected, pMCA1 showed binding to Mia‐Paca‐2, and pJ18 exhibited binding to SKOV‐3 cells. Taken together, these results indicate that TG1 indiscriminately binds multiple targets.
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Phagemid
Polystyrene
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