OBJECTIVETo isolate single chain antibody fragments (scFv) against cell surface molecules by pathfinder selection from an anti-KG1a cell scFv phage library.

METHODSThe anti-KG1a scFv library was enriched by KGla cell panning for three rounds, or unenriched, then processed for pathfinder selection respectively using anti-CD34 monoclonal antibody as pathfinder molecule. ScFv phage clones were randomly picked and identified by binding KG1a cells using immunofluorescein and flow cytometry. The KG1a+ clones were further identified by KG1a, HL60, U937, and CEM cell lines and ELISA. Their antigenic molecules on cell surface were digested by chymopapain and analyzed by flow cytometry. DNAs from ten positive clones were sequenced. The scFv clones with different primary structure were used to analyze the molecular weight of their antigens by Western blot.

RESULTSOne hundred and two KG1a+ scFv phage clones were isolated from 144 enriched and 96 unenriched scFv phage library respectively, among which 47 bound KG1a, HL60, U937, and CEM cells, 55 bound KG1a cells exclusively. None of 28 KG1a+, HL60-, U937-, and CEM- scFv clones bound to the CD34 antigen, as confirmed by ELISA, although most of their antigens were sensitive to chymopapain digestion. DNA sequences from ten positive clones showed that they were from four different clones. They bound antigens with different molecular weight.

CONCLUSIONSOne hundred and two scFv phage clones specific for hematopoietic stem and progenitor cells have been isolated from an anti-KG1a cell scFv phage library. The pathfinder selection has showed advantages to improve the screening efficacy of scFv phage clones against antigens, which present at very low densities on the cell surface.

Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Monoclonal ; genetics ; Antibody Specificity ; Bacteriophages ; genetics ; Cloning, Molecular ; Hematopoietic Stem Cells ; immunology ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.
Animals ; Immunoglobulin Variable Region/genetics* ; Immunoglobulin Fragments/metabolism* ; Single-Chain Antibodies/metabolism* ; Peptide Library ; Mammals/genetics*

OBJECTIVETo screen and characterize the variable region gene about prostate specific membrane antigen (PSMA) of the Chinese Fab fragment, and to establish a new approach to researches on PSMA and prostate gene therapy.

METHODSWe used purified PSMA protein as antigen, stuck it on the ELISA plate and scanned the phage Fab fragment antibody library by phage display technology. After five cycles of "absorbing-elution-amplification", we got the Fab fragment phage antibody of PSMA with high antigen binding ability and specificity, and tested it with immunodetection and sequencing.

RESULTSThe sequence of Fd fragment was 696 base pairs encoding 232 amino-acid residues, with 98% homological similarity to the human immunoglobulin gamma chain, while the light chain was constructed by 630 base pairs encoding 210 amino-acid residues, with 93% homological similarity to kappa chain.

CONCLUSIONUsing phage display technology, we obtained the gene sequence of Fab antibody fragment specific to PSMA, and the antibody gene has the classic structural features of immunoglobulin light chain and heavy chain. The coding output of the antibody gene has the specificity and immunological competence to PSMA.

Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Genetic Code ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Variable Region ; genetics ; Male ; Peptide Library ; Prostate-Specific Antigen ; immunology

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.
Animals ; Complementarity Determining Regions ; genetics ; immunology ; Cytokines ; immunology ; Humans ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Heavy Chains ; genetics ; immunology ; Mice ; Peptide Library ; Protein Binding ; Receptors, Immunologic ; immunology ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification

OBJECTIVETo screen anti-c-Met Fab from a phage antibody library and identify its binding activity.

METHODSThe expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines.

RESULTSc-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence.

CONCLUSIONThe anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.

Antibodies ; immunology ; isolation & purification ; Cell Line, Tumor ; Cloning, Molecular ; Gene Library ; Humans ; Immunoglobulin Fab Fragments ; immunology ; Immunoglobulin Variable Region ; immunology ; Peptide Library ; Proto-Oncogene Proteins c-met ; immunology ; Recombinant Fusion Proteins ; immunology

OBJECTIVETo construct a human phage display antibody library, which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic. This can provide a platform for human antibody preparation and diagnosis, prophylaxis and therapy of RSV infection in children.

METHODSPeripheral blood lymphocytes were isolated from 52 children with RSV infection. cDNA was synthesized from the total RNA of lymphocytes. The light and heavy chain Fd (VH-CH1) fragments of immunoglobulin gene were amplified by RT-PCR. The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue. The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs. The plasmids extracted from amplified E.coli were digested with restriction endonucleases Sac I, Xba I, Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes. RSV virions were utilized as antigens to screen Fab antibodies.

RESULTSBy recombination of light and heavy chain genes, an immune Fab phage display antibody library against RSV containing 2.08x10(7) different clones was constructed, in which 70% clones had light chains and heavy chain Fd genes. The capacity of Fab phage antibody gene library was 1.46x10(7) and the titre of the original Fab antibody library was about 1.06x10(12) pfu/mL. The antibody library gained an enrichment in different degrees after the preliminary panning.

CONCLUSIONSUtilizing the technology of phage display, an immune Fab phage display antibody library against RSV was successfully constructed in this study, which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis, therapy and prophylaxis of RSV infection in children.

Antibodies, Viral ; genetics ; Bacteriophages ; genetics ; Child ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Peptide Library ; Respiratory Syncytial Viruses ; immunology

BACKGROUNDCloning recombinant human Fab fragment against HCMV for the purpose of prophylaxis and control of HCMV infection.

METHODSThe authors constructed a HCMV phage display library with 2 x 10(6) clones, then used purified HCMV viral lysates to pan the library, then screened by ELISA.

RESULTSThree clones showed positive responses in ELISA, they also showed high specificity in IFA, two of them could neutralize HCMV in neutralizing assays.

CONCLUSIONThe specific binding of Fab antibodies to HCMV was demonstrated by ELISA, IFA and neutralizing activities. These results provide us the basis for further research of neutralizing recombinant human whole IgG molecule.

Antibodies, Viral ; genetics ; immunology ; Cytomegalovirus ; genetics ; immunology ; Cytomegalovirus Infections ; immunology ; virology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Neutralization Tests ; Peptide Library

OBJECTIVESTo obtain a single-chain antibody with high affinity against hepatocellular carcinoma (HCC).

METHODSA second single-chain antibody mutant library was established using an error-prone PCR and a phage display. Single-chain antibodies with high affinity for hepatocellular carcinoma were selected using ELISA.

RESULTSThe content of the second single-chain antibody mutant library was about 4.5 x 10(7). Two selected mutants, M90 and M116, were obtained after 3 rounds of panning and ELISA. Immunoassay showed that both M90 and M116 could bind to human HCC cells. The relative affinity of M90 was 1.7-fold higher than that of the original antibody, and M116 was 2-fold higher than that of the original antibody.

CONCLUSIONError-prone PCR is an effective and simple method for affinity maturation of antibodies isolated from a phage antibody library.

Antibodies, Neoplasm ; immunology ; Antibody Affinity ; Antibody Specificity ; Carcinoma, Hepatocellular ; immunology ; pathology ; Humans ; Immunoglobulin Fragments ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Liver Neoplasms ; immunology ; pathology ; Mutation ; Peptide Library

BACKGROUNDTo acquire the recombinant human monoclonal antibodies and IgG to adeno-associated virus type 2 (AAVs-2).

METHODSConstruct and pan human Fab antibody library to AAVs-2 was established from normal volunteer donors by using phage display technology and secreted expression in E.Coli system. The positive Fab clones were selected and characterized through ELISA and immunofluorescent assay, and then the heavy and light chain were sequenced. The gene of light chain and heavy chain Fd fragment of recombinant mAb were inserted into baculovirus expression vector pAC-L-Fc and construct expression vectors of intact IgG, then transfected insert sf-9 cell secreted expression in Baculovirus/Insert system. Immunoprecipitation test was used to detect its recognizing region.

RESULTSOne clone named AAVs-31 showed positive responses in ELISA and IFA, the Fab was composed of gamma chain and kappa chain IgG was positive in ELISA and IFA. The IgG failed to detect nonassembled or denatured capsid proteins, but recognized the AAVs-2 stock from immunoprecipitation test.

CONCLUSIONThe authors isolated a clone of Fab and IgG to adeno-associated virus type 2 by phage display technology, they perhaps recognize an epitope which is formed during capsid assembly.

Amino Acid Sequence ; Animals ; Antibodies, Viral ; genetics ; immunology ; Cell Line ; Dependovirus ; genetics ; immunology ; Gene Expression ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin G ; genetics ; immunology ; Molecular Sequence Data ; Peptide Library ; Recombinant Proteins ; genetics ; immunology ; Spodoptera

BACKGROUNDTo investigate preparation of human-derived anti-HBs IgG antibody.

METHODSmRNA was extracted from lymphocytes of a volunteer with high level antibody to HbsAg. Fab phage display library was constructed. Purified HBsAg was used to screen the Fab. Eukaryotic expression vector was constructed and transfected into sf9 cells, then the supernatant was purified. The expression of anti-HBs human IgG was tested with blocking ELISA and immunofluorescent test.

RESULTSA whole human anti-HBs IgG molecule was obtained, the antibody reacts with HBsAg "a" determinant.

CONCLUSIONIt can be concluded preliminarily that this antibody has neutralizing characteristic against HbsAg.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antigen-Antibody Reactions ; Cell Line ; Hepatitis B Antibodies ; biosynthesis ; immunology ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin G ; biosynthesis ; immunology ; Peptide Library ; Recombinant Proteins ; biosynthesis ; immunology ; Spodoptera