Anti-metatype antibodies stabilize the fluorescein single-chain antibody 4-4-20 complex against dissociation by hydrostatic pressure

Hydrostatic pressure was used to promote dissociation of fluorescein (Fl) from single-chain antibody 4-4-20 (SCA 4-4-20). Fl fluorescence intensity was quenched by 97% upon binding to SCA 4-4-20. Increasing pressure to 2.4 kbar enhanced Fl fluorescence from the remaining 3% to 14-17%. The capacity o...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 1994-03, Vol.269 (11), p.8146-8152
Main Authors: T Coelho-Sampaio, E W Voss, Jr
Format: Article
Language:English
Subjects:
Affinity Labels
Animals
Antibodies - chemistry
Antibodies, Monoclonal - chemistry
Antigen-antibody reactions, antigen-antibody complexes, antibody-complement and others. Study of affinity. Antigen presentation
Biological and medical sciences
Cloning, Molecular
Escherichia coli
Fluorescein
Fluoresceins
Fluorescence Polarization
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Hydrostatic Pressure
Kinetics
Mice
Molecular immunology
Protein Binding
Recombinant Proteins - chemistry
Spectrometry, Fluorescence
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Hydrostatic pressure was used to promote dissociation of fluorescein (Fl) from single-chain antibody 4-4-20 (SCA 4-4-20). Fl fluorescence intensity was quenched by 97% upon binding to SCA 4-4-20. Increasing pressure to 2.4 kbar enhanced Fl fluorescence from the remaining 3% to 14-17%. The capacity of anti-metatype antibodies (anti-Met), which specifically recognize liganded anti-Fl antibodies, to protect against pressure-induced Fl dissociation was tested. Both polyclonal and monoclonal anti-Met antibodies protected against Fl dissociation, reducing the fluorescence intensity at 2.4 kbar from 14-17% to 6-8%. Additive effects of anti-Met antibodies in protection against pressure-induced Fl dissociation were suggested by the fact that a 2-fold molar excess polyclonal anti-Met reagent promoted additional protection relative to an equimolar amount. On the other hand, combination of different monoclonal anti-Met antibodies did not promote additive protection, suggesting recognition of overlapping metatopes by these monoclonals. The complex formed by SCA 4-4-20 and the Fl analog HPF was more sensitive to pressure than the Fl-SCA 4-4-20 complex. Addition of both polyclonal and monoclonal anti-Met antibodies reduced the Fl fluorescence recovery at 2.4 kbar from 75% to 40-55%. In order to directly study binding of anti-Met antibodies to mAb 4-4-20, monoclonal anti-Met antibody 3A5-1 was labeled with 2-dimethylaminonaphthalene-5-sulfonyl chloride (2,5-Dns-Cl) and Dns fluorescence anisotropy measured. Unliganded mAb 4-4-20 did not bind to 2,5-Dns-3A5-1 as indicated by the absence of measurable changes in Dns fluorescence anisotropy upon increasing mAb concentration. Addition of mAb 4-4-20 bound to Fl produced a sigmoidal increase in Dns anisotropy, compatible with association of the primary immune complex and 3A5-1. An affinity constant, K0.5, of 1.5 x 10(-7) M and a cooperativity coefficient (n) of 3.1 were calculated for formation of the Fl-mAb 4-4-20 complex. The HPF-mAb 4-4-20 complex was also recognized by 2,5-Dns-3A5-1 but with lower affinity, indicating that the monoclonal anti-Met 3A5-1 distinguished between mAb 4-4-20 liganded to different haptens.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)37172-7