Dataset Information

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.

ABSTRACT: 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 VH and Vkappa 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.

SUBMITTER: de Haard HJ

PROVIDER: S-EPMC95634 | biostudies-literature | 1998 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.

de Haard H J HJ Kazemier B B Koolen M J MJ Nijholt L J LJ Meloen R H RH van Gemen B B Hoogenboom H R HR Arends J W JW

Clinical and diagnostic laboratory immunology 19980901 5

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 ant ...[more]

PMID: 9729530

Similar Datasets

Project description:Solubility of recombinant proteins (i.e., the extent of soluble versus insoluble expression in heterogeneous hosts) is the first checkpoint criterion for determining recombinant protein quality. However, even soluble proteins often fail to represent functional activity because of the involvement of non-functional, misfolded, soluble aggregates, which compromise recombinant protein quality. Therefore, screening of solubility and folding competence is crucial for improving the quality of recombinant proteins, especially for therapeutic applications. The issue is often highlighted especially in bacterial recombinant hosts, since bacterial cytoplasm does not provide an optimal environment for the folding of target proteins of mammalian origin. Antibody fragments, such as single-chain variable fragment (scFv), single-chain antibody (scAb), and fragment antigen binding (Fab), have been utilized for numerous applications such as diagnostics, research reagents, or therapeutics. Antibody fragments can be efficiently expressed in microorganisms so that they offer several advantages for diagnostic applications such as low cost and high yield. However, scFv and scAb fragments have generally lower stability to thermal stress than full-length antibodies, necessitating a judicious combination of designer antibodies, and bacterial hosts harnessed with robust chaperone function. In this review, we discuss efforts on not only the production of antibodies or antibody fragments in microorganisms but also scFv stabilization via (i) directed evolution of variants with increased stability using display systems, (ii) stabilization of the interface between variable regions of heavy (V H ) and light (V L ) chains through the introduction of a non-native covalent bond between the two chains, (iii) rational engineering of V H -V L pair, based on the structure, and (iv) computational approaches. We also review recent advances in stability design, increase in avidity by multimerization, and maintaining the functional competence of chimeric proteins prompted by various types of chaperones.

Project description:Human babesiosis is an emerging tick-borne parasitic disease and blood transfusion-transmitted infection primarily caused by the apicomplexan parasite, Babesia microti. There is no licensed vaccine for B. microti and the development of a reliable serological screening test would contribute to ensuring the safety of the donated blood supply. The recent sequencing of the B. microti genome has revealed many novel genes encoding proteins that can now be tested for their suitability as subunit vaccine candidates and diagnostic serological markers. Extracellular proteins are considered excellent vaccine candidates and serological markers because they are directly exposed to the host humoral immune system, but can be challenging to express as soluble recombinant proteins. We have recently developed an approach based on a mammalian expression system that can produce large panels of functional recombinant cell surface and secreted parasite proteins. Here, we use the B. microti genome sequence to identify 54 genes that are predicted to encode surface-displayed and secreted proteins expressed during the blood stages, and show that 41 (76%) are expressed using our method at detectable levels. We demonstrate that the proteins contain conformational, heat-labile, epitopes and use them to serologically profile the kinetics of the humoral immune responses to two strains of B. microti in a murine infection model. Using sera from validated human infections, we show a concordance in the host antibody responses to B. microti infections in mouse and human hosts. Finally, we show that BmSA1 expressed in mammalian cells can elicit high antibody titres in vaccinated mice using a human-compatible adjuvant but these antibodies did not affect the pathology of infection in vivo. Our library of recombinant B. microti cell surface and secreted antigens constitutes a valuable resource that could contribute to the development of a serological diagnostic test, vaccines, and elucidate the molecular basis of host-parasite interactions.

Dataset Information

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.

Publications

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure