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 in tensity was quenched by 97% upon binding to SCA 4-4-20. Increasing pre ssure to 2.4 kbar enhanced Fl fluorescence from the remaining 3% to 14 -17%. The capacity of anti-metatype antibodies (anti-Met), which speci fically recognize liganded anti-Fl antibodies, to protect against pres sure-induced Fl dissociation was tested. Both polyclonal and monoclona l anti-Met antibodies protected against Fl dissociation, reducing the fluorescence intensity at 2.4 kbar from 14-17% to 6-8%. Additive effec ts of anti-Met antibodies in protection against pressure-induced Fl di ssociation were suggested by the fact that a 2-fold molar excess polyc lonal anti-Met reagent promoted additional protection relative to an e quimolar amount. On the other hand, combination of different monoclona l 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 pres sure than the Fl-SCA 4-4-20 complex. Addition of both polyclonal and m onoclonal anti Met antibodies reduced the Fl fluorescence recovery at 2.4 kbar from 75% to 40-55%. In order to directly study binding of ant i-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 di d not bind to 2,5-Dns-3A5-1 as indicated by the absence of measurable changes in Dns fluorescence anisotropy upon increasing mAb concentrati on. Addition of mAb 4-4-20 bound to Fl produced a sigmoidal increase i n Dns anisotropy, compatible with association of the primary im immune complex and 3A5-1. An affinity constant, K-0.5, 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 reco gnized by 2,5-Dns-3A5-1 but with lower affinity, indicating that the m onoclonal anti-Met 3A5-1 distinguished between mAb 4-4-20 liganded to different haptens.