scFv-alkaline Phosphatase Fusion Research Articles

Erratum: Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence.

An erratum was issued for:Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence. The Authors section was updated from: Cassandra Warden1 Alan J. Simmons2 Lejla Pasic3 Sean S. Davies4 Justin H. Layer5 Raymond L. Mernaugh3 Annet Kirabo4,6 1Vanderbilt Eye Institute, Vanderbilt University Medical Center 2Department of Cell and Developmental Biology, Vanderbilt University 3Department of Biochemistry, Vanderbilt University 4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center 5Division of Hematology and Oncology, Indiana University School of Medicine 6Department of Molecular Physiology and Biophysics, Vanderbilt University to: Cassandra Warden1 Alan J. Simmons2 Lejla Pasic3 Ashley Pitzer4,6 Sean S. Davies4 Justin H. Layer5 Raymond L. Mernaugh3 Annet Kirabo4,6 1Vanderbilt Eye Institute, Vanderbilt University Medical Center 2Department of Cell and Developmental Biology, Vanderbilt University 3Department of Biochemistry, Vanderbilt University 4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center 5Division of Hematology and Oncology, Indiana University School of Medicine 6Department of Molecular Physiology and Biophysics, Vanderbilt University.

Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence.

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins.

Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and—most importantly—it enables high-throughput procedures.

Cloning of scFv from hybridomas using a rational strategy: Application as a receptor to sensitive detection microcystin-LR in water

Single chain variable fragment (scFv), containing of heavy and light chains (VH and VL) joined by a short peptide linker, has been used widely for immunodetection. Nevertheless, cloning functional variable genes is still a bottle neck for the scFv generation technology. Here, a rational strategy for cloning and selecting variable region genes from an anti-microcystin-LR hybridoma was devised, then the functional VH and VL genes were recloned and assembled to scFv using splicing overlap extension PCR. The resulting scFv gene was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein (scFv−AP) by vector PLIP6/GN. Then an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for detection of microcystin-LR was developed. The half-maximum inhibition concentrations (IC50) and limits of detection (LODs, IC15) were 0.81 ± 0.04 μgL−1 and 0.13 ± 0.03 μgL−1, respectively. With the mean coefficient of variation lowing 8%, the mean recovery in intra-assay and inter-assay were 100.06% and 96.46%, The proposed strategy should be useful for generation scFv in a rapid and simple way.

Immunodetection of the “brown” spider (Loxosceles intermedia) dermonecrotoxin with an scFv-alkaline phosphatase fusion protein

Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32–35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39–20000ng/mL and a detection limit of 39ng/mL without any cross-reaction.

A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10−2μgmL−1, superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10−3mgg−1 of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food.

Selection and characterization of scFv antibodies against the Sin Nombre hantavirus nucleocapsid protein

Rodent-borne hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in the old world and hantavirus cardio-pulmonary syndrome (HCPS) in the new. Most cases of HCPS in North America are caused by Sin Nombre Virus (SNV). Current viral detection technologies depend upon the identification of anti-viral antibodies in patient serum. Detection of viral antigen may facilitate earlier detection of the pathogen. We describe here the characterization of two single-chain Fv antibodies (scFvs), selected from a large naïve phage antibody library, which are capable of identifying the Sin Nombre Virus nucleocapsid protein (SNV-N), with no cross reactivity with the nucleocapsid protein from other hantaviruses. The utility of such selected scFvs was increased by the creation of an scFv-alkaline phosphatase fusion protein which was able to directly detect virally produced material without the need for additional reagents.

A simple vector system to improve performance and utilisation of recombinant antibodies.

BackgroundIsolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression.ResultsWe have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs). Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3). The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3)) was investigated and found to be inferior to periplasmic expression in BL21 (DE3) cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner), bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule), or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and immunohistochemistry.ConclusionNine scFv expression vectors have been generated and tested. Three vectors showed utility for expression of functional scFv fragments. One vector, pSANG14-3F, produces scFv-alkaline phosphatase fusion molecules which offers a simple, convenient and sensitive way of determining the reactivity of recombinant antibody fragments in a variety of common assay systems.

Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries

Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c- myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC L for expression as fusion proteins with human kappa constant region (scFv-C L). Expression of these constructs in Escherichia coli yielded 0.2–1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c- myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.

PSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins.

The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSKAP/S allowed the scFv genes to be easily transferred from pUC119-derived phagemid vectors that are used frequently in phage display antibody library technology. Strong transcriptional control of expression was achieved using the tetracycline promoter, and induction of different individual clones with anhydrotetracycline resulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion proteins that were retained in the periplasm, these proteins could be isolated with a simple extraction procedure. Increased amounts of a scFv-alkaline phosphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testing for binding of the scFv-alkaline phosphatase fusion proteins to antigen was possible in an ELISA without the need for additional enzyme-conjugated antibodies. The pSKAP/S vector was successfully used to obtain scFv fragments from a preparation of phage-antibody clones after subcloning and expression of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones (and clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. Therefore, the pSKAP/S vector was shown to be useful in extending the range of scFv obtained from phage display libraries.

Selection of Single-Chain Variable Fragment Antibodies to Black Currant Reversion Associated Virus from a Synthetic Phage Display Library

ABSTRACT Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.

Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions.

Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore method, Western blots and enzyme-linked immunosorbent assay (ELISA). Western blots and ELISAs can be developed directly by using crude extracts of E.coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent. Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose. The dissociation constants, KD of the peptide KGGHHHHH (KD = 4 x 10(-7) M) and of imidazole (KD = 4 x 10(-4) M) were determined. Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.

A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA

A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V H3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-β- d-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.