Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: neutralization capacity versus thermodynamic stability

The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab?scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.     

A novel disulfide-stabilized single-chain variable antibody fragment against rabies virus G protein with enhanced in vivo neutralizing potency

Rabies is a fatal infectious disease requiring efficient protection provided by post-exposure prophylaxis (PEP) with rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv) is a small engineered antigen binding protein derived from antibody variable heavy (V(H)) and light (V(L)) chains. This novel antibody format may potentially replace the current application of RIG to detect and neutralize rabies virus (RV). However, the broad use of scFvs is confined by their generally low stability. In this study, a scFv (FV57) was constructed based on the monoclonal antibody, MAB57, against RV. To enhance its stability and neutralizing potency, a disulfide-stabilized scFv, ds-FV57, was also derived by introduction of cysteines at V(H)44 and V(L)100. Furthermore, the cysteine at V(L)85 of ds-FV57 was mutated to serine to construct ds-FV57(VL85Ser) in order to avoid potential mis-formed disulfide bonds which would alter the affinity of the scFv. The stability and activity of all three proteins expressed in Escherichia coli were evaluated. All of the constructed scFvs could provide efficient protection against RV infection both in vivo and in vitro. However, the stability of ds-FV57(VL85Ser) was notably improved, and its in vitro neutralizing potency against RV infection was enhanced. Our findings from these stabilization modifications support the feasibility of developing scFvs for PEP treatment of rabies.     

Microbial platform technology for recombinant antibody fragment production: A review

Recombinant antibody fragments are being used for the last few years as an important therapeutic protein to cure various critical and life threatening human diseases. Several expression platforms now days employed for the production of these recombinant fragments, out of which bacterial system has emerged a promising host for higher expression. Since, a small antibody fragment unlike full antibody does not require human-like post-translational modification therefore it is potentially expressed in prokaryotic production system. Recently, small antibody fragments such as scFvs (single-chain variable fragments) and Fabs (antibody fragments) which does not require glycosylation are successfully produced in bacteria and have commercially launched for therapeutic use as these fragments shows better tissue penetration and less immunogenic to human body compared to full-size antibody. Recently developed Wacker’s ESETEC secretion technology is an efficient technology for the expression and secretion of the antibody fragment (Fab) exceeded up to 4.0?g/L while scFv up to 3.5?g/L into the fermentation broth. The Pfenex system and pOP prokaryotic expression vector are another platform used for the considerably good amount of antibody fragment production successfully. In this review, we summarize the recent progress on various expression platforms and cloning approaches for the production of different forms of antibody fragments in E. coli.     

A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice

The variable region genes of a murine anti-coronavirus monoclonal antibody (mAb) were joined by assembly polymerase chain reaction and expressed in Escherichia coli in a single chain variable fragment (scFv) configuration. After induction of expression, the expected 32-kDa protein was identified by Western immunoblotting with specific rabbit anti-idiotype antibodies. The scFv fragments were purified from soluble cytoplasmic preparations by affinity chromatography on nickel agarose, which was possible with an N-terminal but not with a C-terminal histidine tag. Purified scFv fragments retained the antigen-binding properties of the parental antibody, could inhibit its binding to viral antigens with apparently higher efficiency than monovalent antigen-binding (Fab) fragments, but neutralized viral infectivity with lower efficiency (about sevenfold at a molar level). To evaluate the usefulness of these smaller and less immunogenic molecules in the treatment of viral diseases, mice were treated with purified recombinant scFv fragments and challenged with a lethal viral dose. A small delay in mortality was observed for the scFv-treated animals. Therefore, even though the scFv could neutralize viral infectivity in vitro, the same quantity of fragments that partially protected mice in the form of Fab only slightly delayed virus-induced lethality when injected as scFv fragments, probably because of a much faster in vivo clearance: the biologic half-life was estimated to be about 6 min. Since a scFv derived from a highly neutralizing and protective mAb is only marginally effective in the passive protection of mice from lethal viral infection, the use of such reagents for viral immunotherapy will require strategies to overcome stability limitations.     

In vivo biotinylated recombinant antibodies: high efficiency of labelling and application to the cloning of active anti-human IgG1 Fab fragments.

In vivo biotinylation of antibody fragments with a gene fusion approach is a realistic alternative to conventional in vitro chemical labelling. We have previously reported the construction of a vector system suitable for the bacterial expression of the binding fragment of antibody (Fab) genetically linked to the C-terminal domain of Escherichia Coli biotin carboxy carrier protein (BCCP*). A minor fraction of the expressed hybrids was biotinylated in vivo and therefore able to interact with streptavidin. We now show that the large majority of bacterially-expressed Fab-BCCP* fusions are labelled with biotin when plasmid-encoded biotin holoenzyme synthetase (BirA) is co-expressed. The yield of biotinylated Fab is maximal when overexpression of BirA is driven by a second compatible plasmid. We took advantage of this property to develop a novel filter assay for the rapid identification of recombinant Fab reacting with immunoglobulin. Starting with total RNA of two newly established murine hybridoma cell lines producing anti-human IgG1 antibodies, we selected in a single experiment the bacterial clones that expressed in vivo biotinylated anti-IgG1 Fab. Sequence analysis of the isolated Fabs showed that they did not derive from a single B clone. In addition, we found that these recombinant Fabs labelled with biotin in vivo are useful for the specific detection of human IgG1 by a solid-phase immunoassay.     

Chicken single-chain antibody fragments directed against recombinant VP7 of bluetongue virus

VP7, the major structural core protein of bluetongue virus, is conserved among the 24 bluetongue virus serotypes. The gene encoding VP7 of serotype 4 was expressed in Escherichia coli. A semi-synthetic chicken antibody library was screened with the resulting protein. Six single-chain antibody fragments (scFvs) were isolated. Immune sera blocked the binding of four of the six scFvs in enzyme-linked immunosorbent assays. These scFvs recognised recombinant VP7 coated directly onto a plastic surface. Their behaviour therefore differs from that of scFv F10 which was selected earlier on directly immobilised bluetongue virus and which binds to VP7 only when it is captured by an immobilised immunoglobulin directed against bluetongue virus.     

Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments.

Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.     

从噬菌体库中筛选抗SARS病毒S蛋白的Fab中和活性抗体研究

目的 从SARS病毒噬菌体抗体库中筛选出具有中和活性的抗S蛋白Fab片段抗体。方法ELISA方法对抗体库进行富集筛选及人源抗体克隆结合活性的测定，竞争ELISA初步筛选有中和活性的抗体，中和试验进一步确定中和活性，进而对其序列进行测定和分析。结果特异性富集了抗S蛋白噬菌体抗体，竞争ELISA筛选出两株抗体可部分阻断中和抗体2C5与S蛋白的结合，中和试验确定其具有一定巾和活性，编号为M1A和P8A，测序结果表明它们为两株不同的抗体克隆。结论筛选获得了两株抗体可部分中和SARS病毒活性，它们的获得为探索SARS病毒感染的被动免疫治疗打下了一定基础。     

Yeast display of antibody fragments: a discovery and characterization platform

Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single-chain Fv (scFv) antibodies for increased affinity and thermal stability and, more recently, for the display and screening of a non-immune scFv and immune Fab libraries. A major strength of yeast display as a novel antibody discovery platform is the ability to characterize the binding properties, i.e., the affinity and epitope binding characteristics, of a clone without the need for subcloning, expression and purification of the scFv. This review focuses on novel attributes of yeast display for antibody engineering endeavors.     

Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus

A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.     

Expression and detection strategies for an scFv fragment retaining the same high affinity than Fab and whole antibody: Implications for therapeutic use in prion diseases

Since antibodies currently constitute the most rapidly growing class of human therapeutics, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. Using as model a monoclonal antibody directed against the human prion protein that we prepared previously and tested for its therapeutic value, we describe here experimental conditions allowing the production of large quantities (up to 35 mg/l of bacterial culture) of correctly refolded and totally functional single chain fragment variable (scFv). These quantities were sufficient to characterize the binding properties of this small recombinant fragment through in vitro and ex vivo approaches. Interestingly, this scFv retains full binding capacity for its antigen, i.e. the human prion protein, when compared with the corresponding Fab or whole antibody, and recognizes soluble, solid-phase-adsorbed, and membrane-bound prion protein. This strongly suggests that from the mAb cloning step to the refolding of the recombinant fragment, each stage is well controlled, leading to almost 100% functional scFv. These results are of interest not only in view of possible immunotherapy for prion diseases, but also more generally in emphasizing the great promise of these small recombinant molecules in the context of targeted therapies.     

Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins

BaCKGROUND: Monoclonal antibodies are a valuable tool in the study of allergens, but the technology used in their generation can be slow and labour-intensive. Therefore, we have examined recombinant antibody development by phage-display against single allergens and protein mixtures. OBJECTIVE: We used the avian immunoglobulin system (generated from single V(H) and V(L) genes) to provide a rapid method for generating highly specific recombinant antibody fragments from a minimal number of animals. METHODS: A single-chain antibody fragment (scFv) library was generated from a single chicken immunized with model allergens. ScFvs were isolated by phage-display and their properties investigated by ELISA and Western blot. RESULTS: Mono-specific scFvs were generated against recombinant Fel d 1 and native Amb a 1. Pannings against yellow jacket venom extracts only yielded clones that reacted with multiple proteins in the venom extract. The scFvs from each panning type were effectively expressed in Escherichia coli and readily purified. Highly specific and sensitive recognition of Fel d 1 and Amb a 1 was demonstrated in ELISA, with scFvs displaying antibody-concentration-dependent absorbance curves down to picogram levels of antibody. The specificity of selected antibodies for their cognate antigen was further confirmed in Western blot analysis, with scFvs directed to either Fel d 1 or Amb a 1 showing no reactivity for the other antigens used in immunization. Anti-Amb a 1 scFvs also mapped Amb a 1-isoform location in Western blot of ragweed extracts separated by 2D SDS-PAGE. DNA sequence analysis of scFvs showed that multiple different clones had been generated against Fel d 1 and Amb a 1. Using two anti-Fel d 1 scFv for ELISA analysis of Fel d 1 content in crude cat pelt extracts, we could produce data which were highly similar (P=0.33 and 0.89 by paired t-test analysis) to those obtained using conventional assays (radial immunodiffusion). CONCLUSION: Phage-display technology may generate multiple allergen-specific recombinant antibody fragments from a single chicken, to allergens from mammalian, plant and insect sources. The resulting antibody fragments are of demonstrable use in allergen identification and quantification, in comparison with standard immunoassays.     

Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin

Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires. However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming. In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries. The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatography. Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv.     

Humanization of a highly stable single-chain antibody by structure-based antigen-binding site grafting

The murine single-chain variable fragment F8 (scFv(F8)) is endowed with high intrinsic thermodynamic stability and can be functionally expressed in the reducing environment of both prokaryotic and eukaryotic cytoplasm. The stability and intracellular functionality of this molecule can be ascribed mostly to its framework regions and are essentially independent of the specific sequence and structure of the supported antigen-binding site. Therefore, the scFv(F8) represents a suitable scaffold to construct stable scFv chimeric molecules against different antigens by in vitro evolution or antigen-binding site grafting. Thanks to the favourable pharmacokinetic properties associated to a high thermodynamic stability of antibody fragments, such scFv(F8) variants may be exploited for a wide range of biomedical applications, from in vivo diagnosis to therapy, as well as to interfere with the function of intracellular proteins and pathogens, and for functional genomics studies. However, the potential immunogenicity of the murine framework regions represents a limitation for their exploitation in therapeutic applications. To overcome this limitation, we humanized a derivative of the scFv(F8), the anti-lysozyme scFv(11E), which is endowed with even higher thermodynamic stability than the parent antibody. The humanization was carried out by substituting the framework residues differing from closely related V(H) and V(L) domains of human origin with their human counterparts. Site-directed mutagenesis generated the fully humanized product and four intermediate scFvs, which were analyzed for protein expression and antigen binding. We found that the substitution Tyr 90-->Phe in the V(H) domain dramatically reduced the bacterial expression of all mutants. The back-mutation of Phe H90 to Tyr led to the final humanized variant named scFv(H5)H90Tyr. This molecule comprises humanized V(H) and V(L) framework regions and is endowed with HEL-binding affinity, stability in human serum and functionality under reducing conditions comparable to the murine cognate antibody. Consequently, the humanized scFv(H5)H90Tyr represents a suitable scaffold onto which new specificities towards antigens of therapeutic interest can be engineered for biomedical applications.     

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.     

In vitro neutralization of equid herpesvirus 1 mediated by recombinant antibodies

A phage antibody display library of single chain fragment variables (scFv) was applied to develop anti-equid herpesvirus-1 (EHV-1) glycoprotein D (gD) neutralizing antibodies. To enrich for specific scFvs, the phage antibody library was panned against epitope derived from the N-terminal part of EHV-1 gD. Unique clones were differentiated by BstNI fingerprinting and further characterized by sequencing and immunoreactivity. The neutralizing effect of each clone was assessed by plaque reduction assay. Three clones with neutralizing effect were isolated.     

Selection of human antibodies against cell surface-associated oligomeric anthrax protective antigen

The protective antigen (PA(83)) of Bacillus anthracis is the dominant antigen in natural and vaccine-induced immunity to anthrax infection. Three human single-chain variable fragments (scFvs) against cell bound PA were isolated from an antibody phage display library. Specifically, the antibodies were evaluated for their ability to bind to cell bound heptameric PA and ultimately protect against the cytotoxicity of lethal toxin. In total, all three scFvs possessed neutralizing activity against the cytotoxic effects of lethal toxin in a macrophage lysis assay. The K(d) values of the Fabs were determined, interestingly their protective effects did not parallel their affinities; hence, a simple binding argument alone to PA(63) cannot be used as the distinguishing feature for the prediction of their neutralization abilities. Immunofluorescent microscopy experiments were conducted and provided strong evidence for Fab binding to oligomeric PA on the cell surface and thus a plausible mechanism for the toxin neutralization activity that was observed. The results of this study presented herein suggest that our antibodies compete with LF-PA cell surface interactions, and thus may provide potential application of human antibodies as passive immunization prophylactics in cases of B. anthracis exposure and infection.     

Sheep red blood cells armed with anti-CD20 single-chain variable fragments (scFvs) fused to a glycosylphosphatidylinositol (GPI) anchor: a strategy to target CD20-positive tumor cells

Single-chain variable fragment antibodies (scFv) retain antigen specificity and offer advantages over intact antibodies as therapeutic agents. We cloned the cDNA of the V(H) and V(kappa) regions from a mouse hybridoma (HB-9645) directed against human CD20. In addition to the basic scFv construct (V(kappa)-L-V(H)), we genetically engineered a secretory signal, six histidine residues, and a 'Flu' tag to facilitate secretion, purification, and detection. A glycosyl-phosphatidylinositol (GPI) modification signal was added at the C terminus. The GPI-tagged and the non-tagged scFvs were expressed in high yields on the surface of stably transfected insect cells. The CD20-binding properties of purified non-GPI tagged scFv were examined using flow cytometry and immunocytochemistry. The non-GPI-tagged scFv selectively recognizes CD20-positive cells in a concentration-dependent manner. Double-flow cytometry analysis using fresh peripheral blood lymphocytes and WSU-FSCCL cells revealed that our scFv resolves the B-cell population better than the intact antibody. The GPI-tagged scFv was loaded onto the surface of sheep erythrocytes to form rosettes with CD20-positive cells. The genetically engineered anti-CD20 scFv and GPI-tagged derivative have binding specificity for the CD20 antigen. The scFvs described here has potential uses as an in vivo tumor-imaging agent and as a carrier vehicle for targeted delivery of cytocidal agents to CD20-positive cancer cells.     

Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutralization potency and breadth

Previously we reported a broadly HIV-1 neutralizing mini-antibody (Fab 3674) of modest potency that was derived from a human non-immune phage library by panning against the chimeric gp41-derived construct N_CCG-gp41. This construct presents the N-heptad repeat of the gp41 ectodomain as a stable, helical, disulfide-linked trimer that extends in helical phase from the six-helix bundle of gp41. In this paper, Fab 3674 was subjected to affinity maturation against the N_CCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtype B and C HIV-1 reference panels. The parental Fab 3674 is 10-20-fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (∼ 50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (∼ 120 kDa) across the subtype B and C reference panels.     

Selection of Recombinant, Library-Derived Antibody Fragments against p24 for Human Immunodeficiency Virus Type 1 Diagnostics

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of V_H and V_κ regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.