Affinity separation using an Fv antibody fragment-"smart" polymer conjugate

• Published in: Biotechnology and bioengineering 2002-08, Vol.79 (3), p.271-276
• Main Authors: Fong, Robin B., Ding, Zhongli, Hoffman, Allan S., Stayton, Patrick S.
• Format: Article
• Language: English
• Subjects: Acrylic Resins - chemistry; Animals; Antibodies, Monoclonal; Antibody Specificity; Biological and medical sciences; Biotechnology; Chemical Precipitation; Chickens; Chromatography, Affinity - methods; Fluoroimmunoassay - methods; Fundamental and applied biological sciences. Psychology; Humans; Immunoglobulin Fragments - chemistry; LCST; lysozyme; Methods. Procedures. Technologies; Models, Molecular; Molecular Structure; Muramidase - analysis; Muramidase - chemistry; Others; poly(N-isopropylacrylamide); precipitation; Reproducibility of Results; Sensitivity and Specificity; Temperature; temperature-responsive; Various methods and equipments
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.10315

Poly(N‐isopropylacrylamide), or PNIPAAm, is considered a “smart” polymer because it sharply precipitates when heated above a critical temperature, about 32°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°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. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 271–276, 2002