Identification of single-chain antibody fragments specific against SARS-associated coronavirus from phage-displayed antibody library

To develop early diagnostic reagents, effective vaccines, and even drugs against SARS-associated coronavirus (SARS-CoV), the human single fold single-chain antibody fragments, (scFv) libraries I+J (Tomlinson I+J) were used to identify novel scFvs, which can specifically bind to SARS-CoV. Interestingly, two scFvs (B5 and B9) exhibited higher binding specificity to SARS-CoV with the OD(450) value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of V(H) gene (351bp) and V(L) gene (327bp), so the two scFvs were uniformly named as SA59B and chosen for further analysis. SA59B scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The soluble 30kDa SA59B scFv-antibody was verified in SDS-PAGE and Western-blot. The purified SA59B scFv-antibody was labeled with HRP by the glutaraldehyde method, and the concentration of HRP and SA59B scFv-antibody in the SA59B-HRP solution reached 2.4 and 2.28mg/ml, respectively. Then, the binding ability of SA59B-HRP to SARS-CoV was evaluated by ELISA with S/N of 11.6, indicating higher binding specificity between them. Finally, both the SA59B sequence specificity and its application for diagnosis, prophylaxis or therapy of SARS were discussed.     

Chaperone-assisted folding of a single-chain antibody in a reconstituted translation system

A protein-synthesizing system based on a minimal set of purified components was used to investigate the roles molecular chaperones play in the folding of newly synthesized polypeptides. After we ascertained that this system lacks intrinsic chaperones, the effect of adding chaperones in a co-translational or post-translational manner was directly evaluated. An aggregation-prone single-chain antibody was used as the model nascent chain. The participation of the trigger factor or the DnaK system during translation efficiently increased the level of functional protein that was generated. In addition, both systems also acted as chaperones after translation had been stopped. In contrast, the GroEL/ES system showed little or no co- or post-translational assistance in folding.     

An immunoassay method for quantitative detection of proteins using single antibodies

A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose-response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assay (ELISA) and better than that of the antigen direct labeling method. In addition, multiple proteins can be measured simultaneously by SALRA. Using the SALRA method, the detection limit for most of the cytokines tested was approximately 0.01 ng/ml. Further SALRA tests on interleukin 6 (IL-6) showed the linear dose-response was 3.3 to 0.01 ng/ml, the accuracy of the test was 71 to 91%, the intraassay variation was 3.6 to 7.4%, and the interassay variation was 3.8 to 10.0%. The applications of SALRA include quantitatively measuring proteins for which there are no ELISA tools available and providing a new platform for protein microarrays.     

Lectin immunoassays using antibody fragments to detect glycoforms of human chorionic gonadotropin secreted by choriocarcinoma cells

Immobilized antibodies are commonly used to recognize and bind proteins of interest from heterogeneous samples; however, subsequent probing of the glycan(s) of captured glycoproteins with lectins is limited by interference due to the competing oligosaccharides inherently present on antibodies. To prepare capture antibodies with significantly reduced binding of any lectin, the glycosylated protein domains (F(c)) of two anti-human chorionic gonadotropin antibodies were proteolytically removed. Depending on the individual antibody, usable fragments were generated either directly or effectively separated after cleavage through partial reduction and thiol coupling to an appropriate matrix. Importantly, neither method required additional purification of the antibody fragments before immobilization. Binding of a variety of lectins to the functional fragments was reduced by approximately 90% compared with intact immunoglobulin G in both an enzyme-linked immunosorbent assay and a biosensor format. These carbohydrate-free antibody fragments were used to bind the glycoprotein hormone, human chorionic gonadotropin, produced during normal pregnancy and that secreted by three human choriocarcinoma cell lines. Lectins bound to the unpurified gonadotropin glycoforms in distinct patterns consistent with glycan structures previously elucidated by others on hormone samples purified from the urine of pregnant women and of patients with choriocarcinoma. The methods described in this article are applicable for generating capture reagents universally suitable for lectin immunoassays of glycoproteins.     

Genetically engineered intracellular single-chain antibodies in gene therapy

The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins.     

Applications of single-chain variable fragment antibodies in therapeutics and diagnostics

Antibodies (Abs) are some of the most powerful tools in therapy and diagnostics and are currently one of the fastest growing classes of therapeutic molecules. Recombinant antibody (rAb) fragments are becoming popular therapeutic alternatives to full length monoclonal Abs since they are smaller, possess different properties that are advantageous in certain medical applications, can be produced more economically and are easily amendable to genetic manipulation. Single-chain variable fragment (scFv) Abs are one of the most popular rAb format as they have been engineered into larger, multivalent, bi-specific and conjugated forms for many clinical applications. This review will show the tremendous versatility and importance of scFv fragments as they provide the basic antigen binding unit for a multitude of engineered Abs for use as human therapeutics and diagnostics.     

Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of botulinum neurotoxin using high-affinity antibodies

A fluorescence sandwich immunoassay using high-affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum neurotoxin serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-H_C-fragment) as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. Detection to 31 pM with a total incubation time of 3 h was demonstrated. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the reactions were carried out in microcentrifuge tubes with an incubation time of 1 h. The beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell equipped with a fiber optic system for fluorescence measurements. In PBS buffer, the BoNT/A-H_C-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.     

Expression of a single-chain Fv antibody fragment specific for the Hepatitis B surface antigen in transgenic tobacco plants

An anti-Hepatitis B virus surface antigen (HBsAg) single chain Fv (scFv) antibody fragment was expressed in Nicotiana tabacum transgenic plants. The 6-histidine tagged scFv was targeted to either the cytosol, apoplast, and vacuole, or for retention in the endoplasmic reticulum. Expression of active scFv was detected by ELISA in fresh leaf material from F1 transgenic plant lines representative of the genetic constructs targeting the antibody fragment to the apoplastic fluid (AF-12, 0.031% of the total soluble protein), vacuole (V-20, 0.032% of the total soluble protein), and endoplasmic reticulum (ER-52, 0.22% of the total soluble protein). No scFv was detected by ELISA or western blot in the plants transformed with the cytosol construct. The biologically active scFv was easily purified (to 94–95% purity) from ER-52 and AF-12 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the ER-52 plant line indicates that 15–20g of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.     

Expression and long-term stability of a recombinant single-chain Fv antibody fragment in transgenic Nicotiana tabacum seeds

A functionally active anti-hepatitis B surface antigen single-chain Fv antibody fragment (scFv) was expressed in seeds of transgenic tobacco plants using genetic constructs for expression in the vacuole or the apoplastic fluid. Antibody levels close to 0.2% of the total soluble protein were found. After storage of the transgenic tobacco seeds for one year and a half a year at room temperature, the scFv maintained its antigen-binding activity in full.     

Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants

We constructed a recombinant antibody fragment—single chain fragment-variable (scFv) antibody—derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.Communicated by F. Sato     

Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5-8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 degrees C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.     

Functional improvement of antibody fragments using a novel phage coat protein III fusion system

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.     

Establishment of a miniaturized enzyme-linked immunosorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate

A miniaturized enzyme-linked immunosorbent assay (ELISA) with a reaction volume of 5 μl for human transferrin quantification has successfully been developed using an intelligent multifunctional analytical plate (IMAPlate 5RC96), the first miniature analytical platform capable of manually performing parallel liquid transfer, reaction, and analysis. This is the first article to validate the platform for the ELISA application. The data obtained from the standards in this miniaturized ELISA can well be fitted by a one-site binding reaction mode, the coefficient of variation (CV) of the whole plate for an artificial sample (spiking a known concentration of human transferrin into the assay diluent) is 7.0%, and the mean recovery is between 94 and 114% (n = 96), comparable to the values from conventional ELISA in a 96-well format plate. The IMAPlate 5RC96-based miniaturized ELISA not only can reduce sample and reagent consumption to 5% of the conventional ELISA but also can shorten the reaction time. Combined with the advantages brought by miniaturization, the easy-to-handle, parallel, and simultaneous liquid transfer features of the IMAPlate 5RC96 provide a completely new lab tool for manually performing high-throughput ELISA. Our results demonstrate that the IMAPlate 5RC96 is a convenient, robust, high-throughput lab device feasible for miniaturized ELISA in an ordinary laboratory.     

Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli

The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30°C or 37°C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic \-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30°C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A ₆₆₀ at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding.     

Recent developments in colorimetric immunoassays using nanozymes and plasmonic nanoparticles

Enzyme-linked immunosorbent assay (ELISA) is a popular detection technique for the screening and diagnosis of diseases. The sensitivity of ELISA can be increased by the incorporation of nanoparticles. Through this article, we discuss the utilization of nanoparticles in ELISA. Nanoparticles possess an intrinsic biological peroxidase-like activity which allows it to act as an enzyme mimic for the development of an improved analysis method. Different nanoparticles (gold nanoparticles, silver nanoparticles, etc.) carry different peroxidase-mimic characteristics. Besides this, nanoparticles can also perform as a colorimetric substrate in ELISA where it gives a more prominent color change compared to the commonly used colorimetric substrate TMB. This article also focuses on the mechanisms behind this color change including aggregation, in situ nanoparticle growth, seeding, and etching.     

Antibody-mediated improved resistance to ClYVV and PVY infections in transgenic tobacco plants expressing a single-chain variable region antibody

Transgenic Nicotiana tabacum plants expressing a single-chain variable region antibody fragment derived from a broad-spectrum monoclonal antibody 3-17 showed suppression of virus infection following challenge by two distinct potyviruses: potato virus Y strain D, and clover yellow vein virus strain 300. Monoclonal antibody 3-17, which was raised against the potyvirus Johnsongrass mosaic virus, was shown to react strongly with 14 potyvirus species. Two different single-chain antibody constructs were used to produce chimeric genes encoding recombinant proteins designed to be targeted either to the apoplasm or to the cytoplasm. Transgenic plant lines showed reduced numbers of local lesions and systemic symptoms when challenged with potato virus Y, strain D and reduced local lesions following challenge with clover yellow vein virus, strain 300. The level of suppression conferred by the transgene when plants were challenged under laboratory conditions with high concentrations of virus, together with the ability of the transgene to partially protect plants against distinct viruses suggest that one single-chain gene construct might be used to protect plants from distinct potyviruses.     

Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays

The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Prnp knockdown in transgenic mice using RNA interference

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.     

RNA interference in immune cells by use of osmotic delivery of siRNA

Delivery of siRNA to immune cells has been one of the major obstacles to widespread application of RNAi in the immunology field. Here, we report that osmotic delivery of siRNA can be used to silence genes in macrophage RAW264.7 without incurring either cytotoxic or immunomodulatory activity. We also showed usefulness of the osmotic delivery in other types of cells including T cell DO11.10. By repeated osmotic delivery of siRNA, long-term gene silencing was readily achieved. When TLR4 was disrupted in RAW264.7 cells for 48 h and the cells were stimulated with the TLR4 ligand LPS, a significant decrease in TNFalpha production was observed. DNA microarray-based gene expression profile analysis showed that gene silencing by osmotic delivery of siRNA was target-specific and the delivery method itself had little influence on overall gene expression.     

An application of protein microarray in the screening of monoclonal antibodies against the oyster mushroom spherical virus

Protein detection is a common yet time-intensive task in many laboratories. Here we report a protocol that makes use of cold microwave technology to reduce the total processing time to less than 1 h with dot and Western blot applications while yielding lower background noise at similar signal strength when compared with conventional protocols. With dot blots, the time savings was accompanied by a decrease in reagent use. With Western blots, the visibility of prestained markers was maintained, in stark contrast to conventional procedures. Experiments kept at a constant temperature of 21 degrees C support the existence of a microwave radiation effect, whereas an additional thermal effect is noted when the temperature is increased to 37 degrees C from ambient. Microwave-assisted dot blotting is suggested as an effective way of facilitating large-scale screening of expressed proteins.