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 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.
Coelho-Sampaio
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Department of Microbiology, University of Illinois, Urbana 61801.
Voss
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D000345:Affinity Labels; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003001:Cloning, Molecular; D004926:Escherichia coli; D019793:Fluorescein; D005452:Fluoresceins; D005454:Fluorescence Polarization; D006874:Hydrostatic Pressure; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D000906:Antibodies; D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D011994:Recombinant Proteins; D019793:Fluorescein