Recombinant Single-chain Antibody Fragments Research Articles

Genome-scale metabolic model-based engineering of Escherichia coli enhances recombinant single-chain antibody fragment production.

Escherichia coli is an attractive and cost-effective cell factory for producing recombinant proteins such as single-chain variable fragments (scFvs). AntiEpEX-scFv is a small antibody fragment that has received considerable attention for its ability to target the epithelial cell adhesion molecule (EpCAM), a cancer-associated biomarker of solid tumors. Due to its metabolic burden, scFv recombinant expression causes a remarkable decrease in the maximum specific growth rate of the scFv-producing strain. In the present study, a genome-scale metabolic model (GEM)-guided engineering strategy is proposed to identify gene targets for improved antiEpEX-scFv production in E. coli. In this study, a genome-scale metabolic model of E. coli (iJO1366) and a metabolic modeling tool (FVSEOF) were employed to find appropriate genes to be amplified in order to improve the strain for incresed production of antiEpEX-scFv. To validate the model predictions, one target gene was overexpressed in the parent strain Escherichia coli BW25113 (DE3). For improving scFv production, we applied the FVSEOF method to identify a number of potential genetic engineering targets. These targets were found to be localized in the glucose uptake system and pentose phosphate pathway. From the predicted targets, the glk gene encoding glucokinase was chosen to be overexpressed in the parent strain Escherichia coli BW25113 (DE3). By overexpressing glk, the growth capacity of the recombinant E. coli strain was recovered. Moreover, the engineered strain with glk overexpression successfully led to increased scFv production. The genome-scale metabolic modeling can be considered for the improvement of the production of other recombinant proteins.

Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential.

Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30–35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody’s neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.

Isolation and Characterization of Cross-Neutralizing Human Anti-V3 Single-Chain Variable Fragments (scFvs) Against HIV-1 from an Antigen Preselected Phage Library.

Recently conducted human phase- I trials showed protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). The V3 region of the HIV-1 envelope is highly conserved as it is the co-receptor binding site, and is highly immunogenic. Recombinant single-chain antibody fragments (scFvs) can serve as potential tools for construction of chimeric/bispecific antibodies that can target different epitopes on the HIV-1 envelope. Previously, we have constructed a V3 specific human scFv phage recombinant library by a combinational approach of Epstein-Barr virus (EBV) transformation and antigen (V3) preselection, using peripheral blood mononuclear cells (PBMCs), from a subtype C HIV-1 infected antiretroviral naive donor. In the present study, by biopanning this recombinant scFv phage library with V3B (subtype B) and V3C (subtype C) peptides, we identified unique cross reactive anti-V3 scFv monoclonals. These scFvs demonstrated cross-neutralizing activity when tested against subtype A, subtype B, and subtype C viruses. Further, molecular modeling of the anti-V3 scFvs with V3C and V3B peptides predicted their sites of interaction with the scFvs, providing insights for future immunogen design studies. A large collection of such monoclonal antibody fragments with diverse epitope specificities can be useful immunotherapeutic reagents along with antiretroviral drugs to prevent HIV-1 infection and disease progression.

Generation of recombinant scFv antibody against Ochratoxin A (OTA)

Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in the blood and tissues after that consuming contaminated food. Recombinant single-chain antibody fragments (scFv) against OTA were selected from phage display libraries. After of one round of biopanning against BSA-conjugated OTA (OTA-BSA), 52 and 6 phage clones displaying scFv antibodies were isolated from human (Yamo I.3) and rabbit (Bozmix I.2) libraries. Two phage clones (one from each libraries, i.e., yOTA1e3 and bOTA2a9) showed binding to free toxin by competitive ELISA. The soluble scFv antibodies were produced by superinfecting phage clones into E. coli suppressor strain HB2151. The scFv genes from these two phage clones were sub-cloned into pKP300ΔIII vectors to generate scFv-AP fusions. The binding affinity (IC50) of antibody derived from human library was higher than those from rabbit library. The binding property of recombinant antibody in the form of scFv-AP was better than those of soluble scFv form. Cross-reactivity analysis indicated that the two recombinant antibodies did not cross-react with other soluble toxins, namely AFB1, DON, ZEN and FB. The ability to use the recombinant scFv-AP to detect contaminated toxins in agricultural product (corn) was demonstrated.

Polymer Cancerostatics Targeted with an Antibody Fragment Bound via a Coiled Coil Motif: In Vivo Therapeutic Efficacy against Murine BCL1 Leukemia.

A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate.

Full-length antibodies versus single-chain antibody fragments for a selective impedimetric lectin-based glycoprofiling of prostate specific antigen

The main aim of the research was to design a functional impedimetric biosensor able to glycoprofile prostate specific antigen (PSA), a biomarker for prostate cancer (PCa), with high specificity using lectins as glycan recognising proteins. Traditionally, full-length antibody is immobilised on the biosensor interface for specific capture of PSA with subsequent glycoprofiling of PSA by addition of lectins. Since full-length antibodies contain glycans in the Fc domain, particular attention has to be paid to suppress direct binding of lectins to immobilised full-length antibodies, which would compromise accurate glycoprofiling. This issue is addressed here using a recombinant single-chain antibody fragments (scAb), which do not contain any carbohydrate moiety. Surface plasmon resonance was applied to prove negligible interaction of lectins with immobilised scAb fragments, while substantial binding of lectins to full length antibodies was observed. Eight different biosensor designs were tested for their ability to detect PSA. The biosensor device based on scAb fragments covalently immobilised on the gold electrode surface, patterned by a mixed SAM using standard amine coupling chemistry, proved to be the most sensitive. The scAb fragment-based biosensor exhibited sensitivity of 15.9±0.8% decade−1 (R2=0.991 with an average RSD of 4.9%), while the full antibody-based biosensor offered sensitivity towards PSA of 4.2±0.1% decade−1 (R2=0.999 with an average RSD of 4.8%). Moreover, the selectivity of the scAb-based biosensor was tested using a kallikrein 2 protein, a protein structurally similar to PSA, and the results indicated high selectivity for PSA detection.

A novel toolbox for E. coli lysis monitoring

The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future.

Avidin-conjugated polymers with monobiotinylated antibody fragments: A new strategy for the noncovalent attachment of recombinant proteins for polymer therapeutics

The high affinity and specificity between avidin and biotin were employed to bind a recombinant single-chain antibody fragment to synthetic hydrophilic polymer drug carriers. Two semitelechelic polymers, based on poly(ethylene glycol) and poly[ N-(2-hydroxypropyl)methacrylamide], each containing a single thiol end group, were conjugated to dithiopyridyl-modified avidin. The biotinylated recombinant single-chain antibody fragment of the M75 antibody was then noncovalently bound to the polymer-avidin conjugates. The recombinant protein was chosen as a targeting ligand against carbonic anhydrase IX, a marker overexpressed by tumor cells of various human carcinomas. The antigen-binding affinity of the polymer–single-chain antibody fragment complex was confirmed by enzyme-linked immuno sorbent assay (ELISA). This approach provides an original, nondestructive way of preparing supramolecular systems intended for targeted delivery of therapeutics utilizing modern chemical procedures, including reversible addition–fragmentation chain-transfer polymerization and recombinant DNA techniques.

Diabody Mixture Providing Full Protection against Experimental Scorpion Envenoming with Crude Androctonus australis Venom

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD(50) s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom.

96 Recombinant SCFV Antibodies for IGE Epitope Mapping and Detection of Parvalbumins

BackgroundParvalbumin, the major fish allergen, is responsible for IgE cross-reactivity among different fish species. We aimed to generate recombinant single chain antibody fragments (scFv) binding to epitopes responsible for IgE cross-reactivity among parvalbumins.MethodsThe parvalbumin-specific scFvs were selected from the human synthetic scFv phage library ETH-2 by alternating the parvalbumin from cod (Gad m 1), carp (Cyp c 1) and trout (Onc m 1) during 3 rounds of sequential biopanning. Based on their reactivity to parvalbumins by ELISA, 2 clones were expressed in Escherichia coli. The ability of the 2 scFv antibodies to inhibit the binding of parvalbumin-specific IgE from fish allergic patients’ sera was showed by ELISA competition experiments and the rat basophilic leukemia mediator release assay.ResultsBased on ability to bind different parvalbumins and sequence analysis, phage clones scFv-gco9 and scFv-goo8 were selected for production of soluble scFv antibodies. We obtained 1 mg of scFv-gco9 and 1.3 mg of scFv-goo8 per litre of bacterial culture. The scFv-gco9 was able to detect all 3 parvalbumins at a concentration of 10 ng/mL. The scFv-goo8 bound to cod parvalbumin, but not to carp and trout parvalbumin. The detection limit for 1 μg/mL of the scFv-gco9 was 0.01 μg/mL of the Gad m 1 and 0.2 μg/mL of Onc m 1 or Cyp c 1. We found that scFv-gco9 dose-dependently blocked the binding of IgE to immobilized Gad m 1, Cyp c 1 and Onc m 1. At a concentration of 5 μg/mL of scFv-gco9 binding of IgE to the 3 parvalbumins was inhibited by approximately 40%, and at a concentration of 20 μg/mL the IgE binding was inhibited to ∼70%. In the case of the scFv-goo8, inhibition of IgE binding to Gad m 1 was about 15%. The inhibition of degranulation of basophils was 55% in the presence of 2 μg/mL scFv-gco9.ConclusionsThis work, supported by grant SFB-F01802, revealed that the scFv antibodies can be used for the standardization of protein extracts used for allergy diagnosis and for IgE epitope mapping. Epitope characterization enables the engineering of parvalbumin molecules with reduced IgE binding for allergen-specific immunotherapy.

Conversion of scFv peptide-binding specificity for crystal chaperone development.

In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 Å resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a ∼52 Å channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

Mannose phosphorylation in health and disease

Lysosomal hydrolases catalyze the degradation of a variety of macromolecules including proteins, carbohydrates, nucleic acids and lipids. The biogenesis of lysosomes or lysosome-related organelles requires a continuous substitution of soluble acid hydrolases and lysosomal membrane proteins. The targeting of lysosomal hydrolases depends on mannose 6-phosphate residues (M6P) that are recognized by specific receptors mediating their transport to an endosomal/prelysosomal compartment. The key role in the formation of M6P residues plays the GlcNAc-1-phosphotransferase localized in the Golgi apparatus. Two genes have been identified recently encoding the type III α/β-subunit precursor membrane protein and the soluble γ-subunit of GlcNAc-1-phosphotransferase. Mutations in these genes result in two severe diseases, mucolipidosis type II (MLII) and III (MLIII), biochemically characterized by the missorting of multiple lysosomal hydrolases due to impaired formation of the M6P recognition marker, and general lysosomal dysfunction. This review gives an update on structural properties, localization and functions of the GlcNAc-1-phosphotransferase subunits and improvements of pre- and postnatal diagnosis of ML patients. Further, the generation of recombinant single-chain antibody fragments against M6P residues and of new mouse models of MLII and MLIII will have considerable impact to provide deeper insight into the cell biology of lysosomal dysfunctions and the pathomechanisms underlying these lysosomal disorders.

The avß6 integrin - a novel target for antibody conjugated magnetic fluid hyperthermia for therapy in squamous cell carcinoma

Introduction: Magnetic fluid hyperthermia (MFH) is a novel technique whereby superparamagnetic iron oxide nanoparticles (SPIONs) are placed within and excited by an alternating magnetic field to generate heat.1 This approach for cancer therapy has previously been demonstrated in patients using direct intratumoural injection of SPIONs.2,3 We propose MFH can be delivered more effectively using antibody-targeted SPIONs. To test this hypothesis we have developed a recombinant single chain antibody fragment (scFv) to deliver SPIONs to the epithelial restricted avß6 integrin, an exciting new target in head and neck cancers.

Generation and application of a fluorescein-specific single chain antibody

A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K D of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.

Quantitative interpretation of gold nanoparticle-based bioassays designed for detection of immunocomplex formation

The authors present in this paper how the extended Mie theory can be used to translate not only end-point data but also temporal variations of extinction peak-position changes, Deltalambda(peak)(t), into absolute mass uptake, Gamma(t), upon biomacromolecule binding to localized surface plasmon resonance (SPR) active nanoparticles (NPs). The theoretical analysis is applied on a novel sensor template composed of a three-layer surface architecture based on (i) a self-assembled monolayer of HS(CH(2))(15)COOH, (ii) a 1:1 mixture of biotinylated and pure poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), and (iii) NeutrAvidin. Assisted by independent estimations of the thickness of the three-layer architecture using quartz crystal microbalance with dissipation (QCM-D) monitoring, excellent agreement with parallel mass-uptake estimations using planar SPR is obtained. Furthermore, unspecific binding of serum to PLL-g-PEG was shown to be below the detection limit, making the surface architecture ideally suited for label-free detection of immunoreactions. To ensure that the immunocomplex formation occurred within the limited sensing depth (approximately 10 nm) of the NPs, a compact model system composed of a biotinylated human recombinant single-chain antibody fragment (slashed circle approximately 2 nm) directed against cholera toxin was selected. By tracking changes in the centroid (center of mass) of the extinction peak, rather than the actual peak position, signal-to-noise levels and long-term stability upon cholera toxin detection are demonstrated to be competitive with results obtained using conventional SPR and state-of-the-art QCM-D data.

Refolding of a Recombinant Single-chain Antibody Fragment in the Presence of Ionic Liquids

Refolding of a Recombinant Single-chain Antibody Fragment in the Presence of Ionic Liquids

Targeting Tumor Angiogenesis with Adenovirus-Delivered Anti-Tie-2 Intrabody

Inhibition of tumor angiogenesis is a promising approach for cancer therapy. As an endothelial cell-specific receptor kinase expressed almost exclusively on the surface of vascular endothelium, Tie-2 has an important role in tumor angiogenesis. To explore the therapeutic potential of blocking Tie-2 receptor-interaction pathway, an adenoviral vector was used to deliver a recombinant single-chain antibody fragment rabbit intrabody (pAd-2S03) capable of inhibition of both mouse and human Tie-2 surface expression. pAd-2S03 was given to mice with well-established primary tumors, either a human Kaposi's sarcoma (SLK) or a human colon carcinoma (SW1222). The intrabody significantly inhibited growth of both tumors (75% and 63%, respectively) when compared with pAd-GFP control-treated tumors (P < 0.01). Histopathologic analysis of cryosections taken from mice treated with pAd-2S03 revealed a marked decrease in vessel density, which was reduced by >87% in both tumor models when compared with control-treated tumors (P < 0.01). In contrast, human Tie-2-monospecific pAd-1S05 intrabody did not affect the growth of tumors, indicating that the antitumor effect of pAd-2S03 was due to the inhibition of tumor angiogenesis in these murine models. Our results show that the Tie-2 receptor pathway is essential for both SLK sarcoma and SW1222 colon carcinoma xenograft growth. The present study shows the potential utility of antiangiogenic agents that target the endothelium-specific receptor Tie-2 for down-regulation or genetic deletion.

Role of an arbovirus nonstructural protein in cellular pathogenesis and virus release.

The insect-borne Bluetongue virus (BTV) is considered the prototypic Orbivirus, a member of the Reovirus family. One of the hallmarks of Orbivirus infection is the production of large numbers of intracellular tubular structures of unknown function. For BTV these structures are formed as the polymerization product of a single 64-kDa nonstructural protein, NS1, encoded by the viral double-stranded RNA genome segment 6. Although the NS1 protein is the most abundant viral protein synthesized in infected cells, its function has yet to be determined. One possibility is that NS1 tubules may be involved in the translocation of newly formed viral particles to the plasma membrane, and NS1-specific monoclonal antibodies have been shown to react with viral particles leaving infected cells. In the present study we generated a mammalian cell line that expresses a recombinant single-chain antibody fragment (scFv) derived from an NS1-specific monoclonal antibody (10B1) and analyzed the effect that this intracellular antibody has on BTV replication. Normally, BTV infection of mammalian cells in culture results in a severe cytopathic effect within 24 to 48 h postinfection manifested by cell rounding, apoptosis, and lytic release of virions into the culture medium. However, infection of scFv-expressing cells results in a marked reduction in the stability of NS1 and formation of NS1 tubules, a decrease in cytopathic effect, an increased release of infectious virus into the culture medium, and budding of virions from the plasma membrane. These results suggest that NS1 tubules play a direct role in the cellular pathogenesis and morphogenesis of BTV.

Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex

Human cytochrome P450 aromatase (P450arom) is responsible for biosynthesis of estrogens from androgens. Monoclonal antibody MAb3-2C2 to P450arom specifically binds to a conformational epitope and suppresses the enzyme activity in a dose-dependent manner. The crystal structure of the Fab fragment of MAb3-2C2 has been used to engineer a recombinant single chain antibody fragment (scFv) and a homodimeric variable domain of the light chain (VL 2). These recombinant antibody fragments have been expressed in Escherichia coli and purified. Here, we show that the recombinant scFv suppresses P450arom activity with an IC 50 value similar to that of natural MAb3-2C2 F(ab′) 2. The recombinant VL 2 also exhibits dose-dependent suppression of the P450arom activity, but at a reduced level, demonstrating that the homodimer is unable to fully mimic the complementarity determining region (CDR) of a variable heavy chain (VH)–VL heterodimer. We prepare and purify a stable complex of P450arom with MAb3-2C2 F(ab′) 2 and show that the complex migrates and precipitates as a single molecular assembly. Efforts to crystallize P450arom for structure-function studies have yielded small single crystals. Our results suggest that formation of stable complexes with fragments of the monoclonal antibody could provide an alternative method for crystallization of P450arom.

Phage-displayed recombinant single-chain antibody fragments with high affinity for cholesteryl ester transfer protein (CETP): cDNA cloning, characterization and CETP quantification.

Cholesteryl ester transfer protein (CETP) greatly affects the metabolism of all lipoprotein classes including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), both known to constitute powerful risk factors for coronary artery disease (CAD). We now report the successful first cloning and characterization of single-chain antibody fragments specific for CETP. A recombinant phage display library was generated using spleen mRNA isolated from BALB/c mice that had been immunized with highly purified CETP. Screening of the library yielded two single-chain antibody fragments with high affinity for CETP, termed 1CL8 and 1CL10, displaying respective KD values of 4.36 x 10(-9) M and 4.64 x 10(-9) M as determined by affinity sensor technology. Amino acid sequence comparison indicated the complementarity-determining regions of the respective heavy chains to be responsible for CETP high affinity binding. Fragment 1CL8 was successfully employed in clinical chemical quantification systems that uncovered an association in humans between plasma CETP concentration and total body fat mass (r=0.50, p<0.002). Because of the demonstrated superb CETP capturing capacity, combined with high binding affinity to CETP, ready access and unlimited supply, 1CL8 and 1CL10 are expected to prove powerful tools for studies on the role of CETP in atherogenesis.