Screening Panels of Monoclonal Antibodies Using Phage-Displayed Antigen

A procedure is described to screen panels of hybridomas or purified monoclonal antibodies using antigen displayed on the surface of filamentous bacteriophage. In this system, samples containing murine monoclonal antibodies are incubated with phage-displayed antigen in microtiter plates coated with r...

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Bibliographic Details
Published in:Analytical biochemistry 1997-06, Vol.248 (2), p.211-215
Main Authors: Lijnen, H.R., Lasters, I., Verstreken, M., Collen, D., Jespers, L.
Format: Article
Language:English
Subjects:
Animals
Antibodies, Monoclonal
Antibody Affinity
Antibody Specificity
Antigens
Bacteriophages - immunology
Humans
Hybridomas - immunology
Immunologic Techniques - statistics & numerical data
In Vitro Techniques
Kinetics
Mice
Plasminogen - immunology
Rabbits
Sensitivity and Specificity
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Description
Summary:A procedure is described to screen panels of hybridomas or purified monoclonal antibodies using antigen displayed on the surface of filamentous bacteriophage. In this system, samples containing murine monoclonal antibodies are incubated with phage-displayed antigen in microtiter plates coated with rabbit anti-mouse IgG, and bound antibody–phage complex is detected with horseradish peroxidase–sheep anti-phage M13 conjugate. The assay has been validated with a panel of 16 monoclonal antibodies directed against human plasminogen, using phage-displayed miniplasmin(ogen) (amino acids Ala444through Asn791comprising kringle 5 and the proteinase domain of plasminogen) or microplasminogen (amino acids Ala543through Asn791comprising the proteinase domain). Six monoclonal antibodies were identified directed against miniplasminogen and miniplasmin; this was confirmed using a microtiter plate coated with antigens. One of these monoclonal antibodies (MA-42B12) did not react with microplasminogen, suggesting that its epitope is comprised within the kringle 5 domain. This test is rapid and sensitive (detecting 10–20 ng/ml of monoclonal antibody), and screening can be performed using phage-displayed zymogens or active enzymes or selected domains thereof. The procedure eliminates the need for large amounts of purified antigen for screening. Furthermore, immunization can be performed with partially purified antigen because only antibodies raised against the antigen of interest will be identified with the use of phage-displayed antigen. Therefore, this test may offer distinct advantages over the classical one-site enzyme-linked immunosorbent assay using antigen-coated microtiter plates.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1997.2131