Affinity separation using an Fv antibody fragment-"smart" polymer conjugate.
Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.
Department of Bioengineering, University of Washington, Seattle 98195, USA.
Robin B
RB
Fong
Zhongli
Z
Ding
Allan S
AS
Hoffman
Patrick S
PS
Stayton
D000180:Acrylic Resins / Q000737:chemistry*; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D011232:Chemical Precipitation; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D009113:Muramidase / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
D000180:Acrylic Resins; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv; C052970:poly-N-isopropylacrylamide; C421864:hen egg lysozyme; D009113:Muramidase