In vivo clearing of idiotypic antibodies with antiidiotypic antibodies and their derivatives

At immunolocalization of experimental tumors, idiotypic monoclonal antibodies, such as TS1 against cytokeratin 8, can be used to carry and deposit in vivo terapeutics in the tumor. These carriers also remain in the circulation and may cause negative side-effects in other tissues. In this report, several derivatives of the antiidiotypic antibody alphaTS1 were produced and tested for their clearing capacity of the idiotypic carrier antibody TS1. Intact monoclonal alphaTS1, scFv of a alphaTS1 and alphaTS1 Fab'2 and fragments were produced by recombinant technology or by cleavage with Ficin. The scFv was tailored by use of the variable domain genes of the light and heavy chain from the hybridoma clone in combination with a (Gly4Ser)3-linker, followed by expression in E. coli. When tested for clearing capacity, the intact divalent antiidiotypic IgG was found to be the most efficient. The divalent and the monovalent Fab fragment also demonstrated significant clearing, but lower than the intact antiidiotypic IgG. The alphaTS1 scFv antibody when injected separately was not found to clear the idiotype, but could do so when preincubated with the idiotype. Rapid excretion and in vivo instability of this low molecular weight antibody fragment may be the major reasons. Similar results were obtained when the system was reversed and the 131I-labeled antiidiotype IgG was cleared with the idiotype fragment. It is concluded that both intact antiidiotypic IgG, and Fab'2 fragments are able to clear the idiotypic antibodies. The experimental data support the conclusion that the Fc parts from both the idiotype and the antiidiotype may contribute to this elimination.     

Carrier-specificity of a phosphorylcholine-binding antibody requires the presence of the constant domains and is not dependent on the unique VH49 glycine or VH30 threonine residues

Bacterially-produced antibody fragments, such as single-chain Fv (scFv) which comprises the variable regions of the light (VL) and heavy (VH) chains joined together by a short flexible linker, are useful as diagnostic and therapeutic agents. We previously constructed a scFv fragment from a hybridoma antibody (Mab2) but it unexpectedly lacked the unique carrier specificity of the native antibody. Thus, it bound indiscriminately to various phosphorylcholine (PC)-associated antigens, whereas the hybridoma antibody recognized the PC epitope only in the context of the immunizing antigen. Here, we investigated whether the problem was linker-related by changing the linker composition or by deleting it, but these attempts proved futile. Instead, we have constructed a recombinant Fab fragment of the antibody in bacteria that was carrier-specific. This suggests that constant regions are required for the carrier specificity, which presumably helps to mould the fine structure of the antibody combining site or in stabilizing such a structure. Consistent with this global effect is the finding that replacing specific residues in VH with germ-line residues, namely, VH49 glycine and VH30 threonine, both thought previously to be important for the carrier specificity, had no effect on the carrier specificity of the recombinant Fab.     

Expression of a functional single-chain variable-fragment antibody against complement receptor 1 in Streptococcus gordonii

Streptococcus gordonii, an oral commensal organism, is a candidate vector for oral-vaccine development. Previous studies have shown that recombinant S. gordonii expressing heterologous antigens was weakly immunogenic when delivered intranasally. In this study, antigen was specifically targeted to antigen-presenting cells (APC) in order to potentiate antigen-APC interactions and increase the humoral immune response to the antigen. To achieve this goal, a single-chain variable-fragment (scFv) antibody against complement receptor 1 (CR1) was constructed. Anti-CR1 scFv purified from Escherichia coli was able to bind to mouse mixed lymphocytes and bone marrow-derived dendritic cells. The in vivo function of the anti-CR1 scFv protein was assessed by immunizing mice intranasally with soluble scFv and determining the immune response against the hemagglutinin (HA) peptide located on the carboxy terminus of the scFv. The serum anti-HA immunoglobulin G (IgG) immune response was dose dependent; as little as 100 ng of anti-CR1 scFv induced a significant IgG immune response, while such a response was minimal when the animals were given an unrelated scFv. The anti-CR1 scFv was expressed in S. gordonii as a secreted protein, which was functional, as it bound to dendritic cells. Mice orally colonized by the anti-CR1-secreting S. gordonii produced an anti-HA IgG immune response, indicating that such an approach can be used to increase the immune response to antigens produced by this bacterium.     

Expression of a humanized single-chain variable fragment antibody targeting chronic myeloid leukemia cells in Escherichia coli and its characterization

Chronic myeloid leukemia (CML) is a malignant blood disease originating from hematopoietic stem cells. Drug resistance and tumor recurrence have become major problems for the treatment of this disease. Therefore, new therapeutic methods need to be developed. Antigens expressed on the surface of cancer cells are potential targets for antibody-mediated drug delivery. In our study, an anti-CML cell single-chain variable fragment (scFv) antibody has been produced and characterized because it is the first step towards the construction of a novel cancer-targeted agent for cancer diagnosis and treatment. Here, a 46?kDa antibody derivative was produced by genetic fusion of a humanized scFv antibody against a CML cell surface antigen with the 6xHis-tag, which can specifically bind to CML cells. The recombinant scFv against CML cells was expressed as a fusion protein containing the 6xHis-tag at its N-termini, and purified by Ni2+-NTA column chromatography. The recombinant scFv, which was soluble, was expressed and produced in bacteria, and was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays. Finally, its cell-binding activity and immunoactivity were demonstrated by enzyme-linked immunosorbent assay (ELISA). Furthermore, flow cytometry analysis demonstrated that this scFv specifically targeted CML cells expressing the associated antigen (47.9 and 34.4%) other than non-expressing tumor cells (1.25%) in?vitro. The results presented in this study illustrate that the humanized anti-CML cell scFv antibody may function as a novel therapeutic tool for CML.     

The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies

The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.     

Characterization of a unique human single-chain antibody isolated by phage-display selection on membrane-bound mosquito midgut antigens

The insect midgut is the primary site for food digestion, as well as for vector-borne pathogen infection into the invertebrate host. Accordingly, antigens of this critical insect organ are targets for anti-vector vaccines, insecticidal toxins, and transmission-blocking vaccines. We used midgut proteins of the African malaria vector mosquito Anopheles gambiae to select single-chain human antibody fragments (scFv) from a high-diversity, phage-displayed library. Using a phage-display selection method on western-blotted antigens, we selected an unusual truncated scFv clone, consisting of a heavy-chain only, which binds to An. gambiae midgut tissue. This clone binds a spectrum of mosquito antigens from the midgut and other mosquito tissues, as well as various mammalian glycoproteins, but binding was reduced when these glycoproteins were enzymatically deglycosylated. We also observed that this clone preferentially binds the lumenal midgut surface. Furthermore, antigen binding by our selected scFv was limited by competition with increasing concentrations of certain soluble carbohydrates, most dramatically by galactose and N-acetyl glucosamine. Our results show that the cognate epitope of this scFv is a carbohydrate moiety. This paper describes a phage-display selection of antibody fragments on mosquito midgut tissue and it also describes a method for phage-display selection on membrane-immobilized heterogeneous antigens. These selection methods resulted in the isolation of a novel, truncated, carbohydrate-binding human antibody fragment from a naive phage-display library.     

抗阿尔茨海默病Aβ人源单链抗体基因的筛选和表达

目的 从人源噬菌体单链抗体库中筛选与阿尔茨海默病发病中起关键作用的β淀粉样多肽(Aβ)1-42特异性结合的单链可变区抗体(scFv)基因,利用原核生物大肠杆菌进行可溶性表达,以获得抗AB1-42人源抗体.方法 利用噬菌体展示技术对人源噬菌体单链抗体库进行多轮富集,以Aβ1-42为抗原经酶联免疫吸附(ELISA)检测,筛选与Aβ1-42特异性结合的阳性噬菌体克隆,再用阳性噬菌体感染E.coli HB2151进行可溶性表达,经SDS-PAGE、Western blot及ELISA法检测scFv单抗的可溶性表达水平及抗原结合活性.并对阳性scFv抗体基因进行基因测序鉴定.结果 获得了7个特异性的抗Aβ1-42 scFv阳性噬菌体克隆,其中4个克隆ELISA检测吸光度值(A值)高于阴性对照5倍以上;其中1株(A 10)获得可溶性单链抗体的成功表达,表达产物主要位于菌体中,ELISA效价显示在全菌蛋白中A值高于对照5倍以上.其相对分子质量约为33 000.DNA测序表明所获抗体的可变区基因属于scFv抗体基因,推导得到的氨基酸序列具有典型的抗体可变区结构.结论 用人源噬菌体单链抗体库筛选出抗Aβ1-42的人源抗体单链可变区基因,并成功表达了具有抗原结合活性的可溶性单链抗体,为进一步研究阿尔茨海默病的发病机制和治疗奠定了基础.     

Testing the anti-fibrotic potential of the single-chain Fv antibody against the α2 C-terminal telopeptide of collagen I

This study focuses on the single-chain fragment variable (scFv) variant of the original IgA-type antibody, recognizing the α2 C-terminal telopeptide (α2Ct) of human collagen I, designed to inhibit post-traumatic localized fibrosis via blocking the formation of collagen-rich deposits. We have demonstrated that the scFv construct expressed in yeast cells was able to fold into an immunoglobulin-like conformation, but it was prone to forming soluble aggregates. Functional assays, however, indicate that the scFv construct specifically binds to the α2Ct epitope and inhibits collagen fibril formation both in vitro and in a cell culture model representing tissues that undergo post-traumatic fibrosis. Thus, the presented study demonstrates the potential of the scFv variant to serve as an inhibitor of the excessive formation of collagen-rich fibrotic deposits, and it reveals certain limitations associated with the current stage of development of this antibody construct.     

Highly specific inhibition of C1q globular-head binding to human IgG: a novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment

We sought to specifically regulate the binding of human C1q, and thus the activation of the first complement component, via the construction of a single chain antibody variable binding region fragment (scFv) targeting the C1q globular heads. Here we describe details of the construction, expression and evaluation of this scFv, which was derived from a high-affinity hybridoma (Qu) specific for the C1q globular heads. The scFv was comprised of the Qu variable heavy chain domain (VH) linked to the Qu variable light chain domain (VL) and was termed scFv-QuVHVL. When mixed with either purified C1q or with human serum as a source of C1, scFv-QuVHVL bound to C1q and competitively restricted the interaction of C1q or C1 with immobilized IgG or with IgG1 antibody-coated cells, and prevented the activation of native C1 in human serum as determined by analyses of C1-mediated C4 deposition and fluid-phase C4 conversion. However scFv-QuVHVL could be manipulated to become a C1 activator when it was irreversibly immobilized onto microtiter ELISA plates, prior to contact with human serum complement. This functional dichotomy can be a useful tool in selectively elucidating, differentiating, inducing or inhibiting specific roles of human C1q and the classical complement pathway in complement-mediated physiological processes. We project that once fully humanized, fluid-phase scFv-QuVHVL could become a useful therapeutic in limiting inadvertent host tissue damage elicited by the classical complement pathway.     

Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against the targeted epitope

We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the “knob” region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity.     

An ELISA for detection of infectious bursal disease virus and differentiation of very virulent strains based on single chain recombinant chicken antibodies.

Two chicken single-chain variable antibody fragments (scFv) designated scFv154 and scFv88, previously shown to react with either all or very virulent (vv) infectious bursal disease virus (IBDV) strains, respectively, were evaluated for use in an enzyme-linked immunosorbent assay (ELISA) for differentiation of vvIBDV. Specificity and sensitivity of the vvIBDV ELISA was assessed when scFv154 and scFv88 were expressed as soluble antibodies (sAb), phage antibodies (pAb) or hyper-phage antibodies (hpAb). The highest test sensitivity and specificity was obtained using hpAb154 to detect all IBDV and pAb88 to differentiate vvIBDV strains. Such an ELISA was eight to 16 times more sensitive for IBDV antigen detection than the mouse monoclonal antibody ELISA. Using field samples, the scFv ELISA was able to differentiate between flocks infected with vvIBDV and those infected with classical or variant IBDV. In one instance IBDV was detected in a flock found to be negative by the monoclonal antibody ELISA. The results showed that scFv can be utilized as highly specific and sensitive ELISA reagents for the detection and discrimination of avian pathogens.     

The use of intracellular single-chain antibody fragments to inhibit specifically the expression of cell surface molecules

One possible method to inhibit specifically the function of a protein inside a cell is to express an intracellular antibody combining site that can block function or prevent expression of the targeted molecule. In this report the parameters involved in the production and expression of functional, endoplasmic reticulum-retained, single chain Fv antibody fragments (scFv) were investigated. These intracellular scFv constructs were tested for their ability to inhibit specifically the expression of a CHO cell line pretransfected with the relevant cell surface antigen CD2. No scFv was detected in the cell supernatant although functional scFv, as assayed by ELISA, was detected in an NP-40 soluble fraction if an N-linked glycosylation site had been introduced into the antibody construct. This demonstrates that functional antibody combining sites can be produced even though they are not secreted. Inhibition of CD2 was obtained but was not complete and differed between clones. Levels of scFv could be increased by gene amplification but the level of functional binding activity remained constant and no further inhibition of CD2 expression was obtained. Immunofluorescence analysis at the single-cell level of the permeabilised transfected cell lines showed that less than 8% of cells expressed detectable levels of intracellular scFv, indicating selection against cells producing high levels of single-chain antibody. This selection was not seen when comparable single-chain TCR constructs, known to be retained intracellularly, were used. Thus, production of scFv with binding activity is not sufficient for good inhibition of gene expression although introduction of an N-linked glycosylation site is beneficial. The best strategy is probably to screen a panel of scFv constructs and use those that are secreted rather than those that are retained intracellularly     

Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigatus membrane protein

Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.     

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A1 with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A1 scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the preferred selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv.     

Characterization of human variable domain antibody fragments against the U1 RNA-associated A protein,selected from a synthetic and a patient-derived combinatorial V gene library

This is the first study describing recombinant human antibody fragments directed to the U1 RNA-associated A protein (U1A). Three anti-U1A antibody fragments (Fab) were isolated from a semi-synthetic human Fab library and one anti-U1A single-chain variable fragment (scFv) was isolated from a library which was derived from the IgG-positive splenic lymphocytes of an autoimmune patient. Competition studies with autoantibodies against the U1 small nuclear ribonucleoprotein (snRNP) particle from patients with systemic lupus erythematosus (SLE) and SLE-overlap syndromes revealed that U1A binding of these antibody fragments can be inhibited by about 40% of the patient sera. All antibody fragments recognized the native U1 snRNP in immunoprecipitation assays. Two of three Fab clones as well as the scFv clone derived from the repertoire of an autoimmune patient use the same heavy chain germ-line gene DP-65. Epitope mapping revealed that these three clones appear to recognize an identical epitope domain present on the C-terminal RNP motif of the U1A protein. The DP-65 heavy chain gene is used in less than 1% of the B cells in healthy individuals, while three out of four anti-U1A antibody fragments use this gene. This points to a restricted V_H gene usage in the case of U1A, suggesting that the DP-65 heavy chain has a natural shape complementarity to the U1A protein.     

Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins.

The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen binding affinity; internalization rate, intracellular processing and intrinsic toxin domain potency. This study examines the influence of antigen binding affinity on potency in various immunotoxin fusion proteins where target antigen binding is mediated by single chain antibody variable region fragments (scFv). Firstly, the relationship between affinity and potency was examined in a panel of four scFv immunotoxins generated from different anti-CD3 monoclonal antibodies fused to the 38 kDa fragment of Pseudomonas aeruginosa exotoxin A (PE38). Of these four scFv-PE38 immunotoxins, the one derived from the anti-CD3 monoclonal antibody UCHT1 has highest cell killing potency. Analysis of these four scFv-PE38 immunotoxins indicated a correlation between antigen binding affinity and immunotoxin potency in the cell killing assay with the exception of the scFvPE38 immunotoxin derived from the antibody BC3. However this scFv appeared to suffer a greater drop in affinity ( approximately 100x), relative to the parent Mab than did the other three scFvs used in this study (2-10x). Secondly, the scFv(UCHT1)-PE38 immunotoxin was then compared with a further panel of scFv(UCHT1)-derived immunotoxins including a divalent PE38 version and both monovalent and divalent Corynebacterium diphtheriae toxin (DT389) fusion proteins. When the scFv-UCHT1 domain was amino-terminally positioned relative to the toxin, as in the scFv(UCHT1)-PE38, an approximately 10-fold higher antigen-binding affinity was observed than with the C-terminal fusion, used in the DT389-scFv(UCHT1) molecule. Despite this lower antigen-binding activity, the DT389-scFv immunotoxin had a 60-fold higher potency in the T-cell-killing assay. Thirdly, a divalent form of the DT389-scFv construct, containing tandem scFv domains, had a 10-fold higher binding activity, which was exactly reflected in a 10-fold increase in potency. Therefore, when comparing immunotoxins in which scFvs from different antibodies are fused to the same toxin domain (DT or PE) a broad correlation appears to exist between binding affinity and immunotoxin potency. However, no correlation between affinity and potency appears to exist when different toxin domains are combined with the same scFv antibody domain.     

Enhancement of scFv fragment reactivity with target antigens in binding assays following mixing with anti-tag monoclonal antibodies

The phage display Ab library technology has been found to be a useful method to isolate antigen-specific Ab fragments, since the repertoire of antibody specificities is broad and since it bypasses the need of immunization. However, when screening clones isolated from a phage display Ab library, the yield of isolating antigen-specific Ab fragments is low and the rate of false negative results is high. This limitation reflects the low affinity/avidity of Ab fragments and/or the low density of the target antigen. To facilitate the isolation of Ab fragments with a broad range of affinities to antigens of interest from phage display Ab libraries, we have developed a simple method to increase the sensitivity of binding assays to detect the reactivity of single-chain fragments of antibody variable regions (scFv) with target antigens. This method involves the mixing of scFv fragments, expressing a c-myc epitope tag, with anti-tag mAb 9E10 prior to their use in binding assays in order to form stable dimeric Ab fragment-anti-tag mAb complexes. The increase in the reactivity of scFv fragments with the corresponding antigen is observed over a broad range of scFv fragment (6-800 microg/ml) and mAb 9E10 (0.5-30 microg/ml) concentrations, thereby facilitating the testing of scFv fragment preparations with unknown scFv fragment concentrations. Use of this method in binding assays resulted in a twofold increase in the reactivity of low-affinity purified scFv fragments with the corresponding antigen. Moreover, application of this method to screen clones isolated from phage display scFv libraries resulted in a reproducible increase in both the yield of antigen-specific scFv clones and the titer of scFv fragment preparations by a factor of 5 and 2- to 32-fold, respectively. Lastly, this method can be applied in both ELISA and flow cytometry and is independent of the characteristics of the antigen (i.e. whole cells, carbohydrates and purified protein) and/or of the library (synthetic scFv Library (#1), a large semi-synthetic phage display scFv library and the human synthetic VH+VL scFv library (Griffin.1 library)) used. Therefore, the method we have described represents a sensitive, simple and reproducible technique that will facilitate the isolation and use of scFv fragments.     

Human autoimmune anti-proteinase 3 scFv from a phage display library

This is the first study describing recombinant human antibody fragments directed to the autoantigen proteinase 3 (PR3) from an immune B cell source. Detection of these autoantibodies has proven valid for the diagnosis and monitoring of Wegener's granulomatosis. The described antibody fragment (scFv) was isolated from a phage display library prepared from the IgG-positive splenic lymphocytes of a patient with systemic autoimmunity. The cloning strategy was designed to maintain the diversity of the antibody variable gene repertoire, and sequencing of several variable genes demonstrated that all major heavy and light chain families were represented. We found an over-representation of particular heavy chain variable domains in splenic lymphocytes which differ from the ones frequently found in peripheral blood lymphocytes. It was possible to obtain specific scFv to PR3 after a single round of selection and the binding could be inhibited by the patients' sera. Although the antibody fragments in the splenic repertoire were found to be highly mutated, it was interesting to find that the selected scFv showed only limited somatic mutation. Furthermore, we could demonstrate that the removal of the mutations had no effect on binding specificity.     

Construction and characterization of a novel recombinant single-chain variable fragment antibody against Western equine encephalitis virus

A novel recombinant single-chain fragment variable (scFv) antibody against Western equine encephalitis virus (WEE) was constructed and characterized. Using antibody phage display technology, a scFv was generated from the WEE specific hybridoma, 10B5 E7E2. The scFv was fused to a human heavy chain IgG1 constant region (CH1-CH3) and contained an intact 6 His tag and enterokinase recognition site (RS10B5huFc). The RS10B5huFc antibody was expressed in E. coli and purified by affinity chromatography as a 70-kDa protein. The RS10B5huFc antibody was functional in binding to WEE antigen in indirect enzyme-linked immunosorbent assays (ELISAs). Furthermore, the RS10B5huFc antibody was purified in proper conformation and formed multimers. The addition of the human heavy chain to the scFv replaced effector functions of the mouse antibody. The Fc domain was capable of binding to protein G and human complement. The above properties of the RS10B5huFc antibody make it an excellent candidate for immunodetection and immunotherapy studies.