Biomolecular Interaction Analysis: Strategies for Antibody and Antigen Characterization

Roder, Uwe W.; Markey, Francis

doi:10.1385/0-89603-485-2:531

Cited by 3 publications

(2 citation statements)

References 6 publications

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“…The scFv genes were subcloned into expression vector pETFlag, expressed and purified to homogeneity (38). Dissociation constants (K d ) were calculated from the measured association (k on ) and dissociation (k off ) rate constants by using the BIAcore instrumentation and software (Amersham Pharmacia) (39)(40)(41)(42). For BIAcore experiments, protein antigens were immobilized on CM5 chips.…”

mentioning

confidence: 99%

A method for the generation of combinatorial antibody libraries using pIX phage display

Gao

Mao

Kaufmann

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

117

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For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and select a wide variety of antibodies. We previously reported that the phage coat proteins pVII and pIX could be used to display the heterodimeric structure of the antibody Fv region. Herein, aspects of this technology were invoked and extended to construct a large, human single-chain Fv (scFv) library of 4.5 ؋ 10 9 members displayed on pIX of filamentous bacteriophage. Furthermore, the diversity, quality, and utility of the library were demonstrated by the selection of scFv clones against six different protein antigens. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning. Analyzed scFvs were also found to be of high affinity. For example, kinetic analysis (BIAcore) revealed that scFvs against staphylococcal enterotoxin B and cholera toxin B subunit had a nanomolar and subnanomolar dissociation constant, respectively, affording affinities comparable to, or exceeding that, of mAbs obtained from immunization. High specificity was also attained, not only between very distinct proteins, but also in the case of the Ricinus communis (''ricin'') agglutinins (RCA60 and RCA120), despite >80% sequence homology between the two. The results suggested that the performance of pIX-display libraries can potentially exceed that of the pIII-display format and make it ideally suited for panning a wide variety of target antigens.

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confidence: 99%

A method for the generation of combinatorial antibody libraries using pIX phage display

Gao

Mao

Kaufmann

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

117

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“…BIAcore Analysis. Binding kinetics was determined by surface plasmon resonance using a BIAcore 2000 instrument (Pharmacia) …”

Section: Methodsmentioning

confidence: 99%

Phage-Display Selection of a Human Single-Chain Fv Antibody Highly Specific for Melanoma and Breast Cancer Cells Using a Chemoenzymatically Synthesized G_M3−Carbohydrate Antigen

et al. 2002

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Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 10(-5) M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.

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Field Detection of Eastern Equine Encephalitis Virus in the Amazon Basin Region of Peru Using Reverse Transcription-Polymerase Chain Reaction Adapted for Field Identification of Arthropod-Borne Pathogens

O’Guinn

Lee²,

Kondig³

et al. 2004

Am J Trop Med Hyg

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In support of efforts to develop rapid diagnostic assays for use in the field, reverse transcription-polymerase chain reaction (RT-PCR) assays were developed to detect arboviruses circulating in the Amazon Basin region of Peru. Previous knowledge of arthropod/pathogen relationships allowed a focused evaluation to be conducted in November 2000 that assessed the feasibility and reliability of a mobile, rapid, field-expedient RT-PCR diagnostic system aimed at detecting eastern equine encephalitis virus (EEEV) in Culex (Melanoconion) pedroi mosquitoes. Modifications were made to a commercially available mobile molecular laboratory kit and assay procedures were tailored for use under harsh environmental conditions with field-collected and field-processed mosquitoes. From CO2 baited mosquito light traps, 3,227 Cx. (Mel.) pedroi mosquitoes were collected and sorted into 117 pools. The pools were processed and assayed in the field by RT-PCR and five of those pools were found positive for EEEV. Laboratory sequence analysis confirmed the presence of two distinct subtypes of EEEV.

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Biomolecular Interaction Analysis: Strategies for Antibody and Antigen Characterization

Cited by 3 publications

References 6 publications

A method for the generation of combinatorial antibody libraries using pIX phage display

A method for the generation of combinatorial antibody libraries using pIX phage display

Phage-Display Selection of a Human Single-Chain Fv Antibody Highly Specific for Melanoma and Breast Cancer Cells Using a Chemoenzymatically Synthesized G_M3−Carbohydrate Antigen

Field Detection of Eastern Equine Encephalitis Virus in the Amazon Basin Region of Peru Using Reverse Transcription-Polymerase Chain Reaction Adapted for Field Identification of Arthropod-Borne Pathogens

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Biomolecular Interaction Analysis: Strategies for Antibody and Antigen Characterization

Cited by 3 publications

References 6 publications

A method for the generation of combinatorial antibody libraries using pIX phage display

A method for the generation of combinatorial antibody libraries using pIX phage display

Phage-Display Selection of a Human Single-Chain Fv Antibody Highly Specific for Melanoma and Breast Cancer Cells Using a Chemoenzymatically Synthesized GM3−Carbohydrate Antigen

Field Detection of Eastern Equine Encephalitis Virus in the Amazon Basin Region of Peru Using Reverse Transcription-Polymerase Chain Reaction Adapted for Field Identification of Arthropod-Borne Pathogens

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Product

Resources

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Phage-Display Selection of a Human Single-Chain Fv Antibody Highly Specific for Melanoma and Breast Cancer Cells Using a Chemoenzymatically Synthesized G_M3−Carbohydrate Antigen