Polymer-protein conjugates. II. Affinity precipitation separation of human immunogammaglobulin by a poly(N-isopropylacrylamide)-protein A conjugate.

The conjugate of protein A with poly(N-isopropylacrylamide) was synthesized and utilized in the separation of human immunogammaglobulin. In the separation process, poly(N-isopropylacrylamide)-protein A conjugate binds to the immunoglobulin with high specificity to form the poly(N-isopropylacrylamide)-protein A/immunoglobulin complex. The complex can be conveniently separated by precipitation upon heating above the lower critical solution temperature of the poly(N-isopropylacrylamide)-protein A/immunogammaglobulin complex. The separation capacity of poly(N-isopropylacrylamide)-protein A conjugate for human immunogammaglobulin was studied and it was demonstrated that approximately one out of every four protein A molecules binds to human immunogammaglobulin with a dissociation constant (Ks) of 3 x 10(-6) M. The affinity precipitation separation of human immunogammaglobulin is a rapid process which avoids the need for chromatographic columns. It can also be designed to run in a continuous mode.

[1]  A S Hoffman,et al.  A novel immunoassay system and bioseparation process based on thermal phase separating polymers , 1987, Applied biochemistry and biotechnology.

[2]  J. Sjöquist,et al.  Characterization of the soluble complex formed by reacting rabbit IgG with protein A of S. aureus. , 1978, Immunochemistry.

[3]  Langone Jj.  Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci. , 1982 .

[4]  G. Scatchard,et al.  THE ATTRACTIONS OF PROTEINS FOR SMALL MOLECULES AND IONS , 1949 .

[5]  H. Snyder,et al.  Selective removal of antigen-complexed IgG from cat plasma by adsorption onto a protein A-silica matrix. , 1987, Journal of immunological methods.

[6]  B. Meloun,et al.  Protein A isolated from Staphylococcus aureus after digestion with lysostaphin. , 1972, European journal of biochemistry.

[7]  J. Sjödahl.  Repetitive Sequences in Protein A from Staphylococcus aureus , 1977 .

[8]  J. J. Langone.  [125I]protein A: a tracer for general use in immunoassay. , 1978, Journal of immunological methods.

[9]  G. Kronvall,et al.  Immunochemical aspects of Fc-medicated binding of human IgG subclasses to group A, C and G streptococci. , 1980, Molecular immunology.

[10]  J. Sjödahl,et al.  Structural studies on the four repetitive Fc-binding regions in protein A from Staphylococcus aureus. , 1977, European journal of biochemistry.

[11]  S. Ohlson,et al.  Profiling of circulating immune complexes by high performance liquid affinity chromatography (HPLAC) on a protein A-silica matrix. , 1988, Journal of immunological methods.

[12]  R. Lindmark,et al.  Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera. , 1983, Journal of immunological methods.

[13]  J. Deisenhofer.  Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. , 1981, Biochemistry.

[14]  S. Ohlson.  HIGH PERFORMANCE LIQUID AFFINITY CHROMATOGRAPHY (HPLAC) WITH PROTEIN A-SILICA , 1983 .

[15]  A. Hoffman,et al.  N-isopropylacrylamide and N-acryloxysucccinimide copolymer: a thermally reversible, water-soluble, activated polymer for protein conjugation , 1987 .

[16]  T. Borsós,et al.  Studies on the interaction between protein A and immunoglobulin G. I. Effect of protein A on the functional activity of IgG. , 1978, Journal of immunology.

[17]  T. Borsós,et al.  Studies on the interaction between protein A and immunoglobulin G. II. Composition and activity of complexes formed between protein A and IgG. , 1978, Journal of immunology.

[18]  J. Goding.  Conjugation of antibodies with fluorochromes: modifications to the standard methods. , 1976, Journal of immunological methods.

[19]  A. Hoffman,et al.  Polymer-protein conjugates. I. Effect of protein conjugation on the cloud point of poly (N-isopropylacrylamide). , 1990, Biomaterials.

[20]  T. Borsós,et al.  125I protein A: applications to the quantitative determination of fluid phase and cell-bound IgG. , 1977, Journal of immunological methods.

[21]  M. Heskins,et al.  Solution Properties of Poly(N-isopropylacrylamide) , 1968 .

[22]  J. Marchalonis,et al.  Studies on immune responses to larval cestodes in mice. Immunoglobulins associated with the larvae of Mesocestoides corti. , 1977, The Australian journal of experimental biology and medical science.

[23]  A. Forsgren,et al.  “Protein A” from S. Aureus I. Pseudo-Immune Reaction with Human γ-Globulin , 1966 .

[24]  S. Ohlson,et al.  High-performance liquid affinity chromatographic separation of mouse monoclonal antibodies with protein A silica. , 1987, Journal of chromatography.

[25]  P. Quie,et al.  Quantitation of staphylococcal protein A: Determination of equilibrium constant and number of protein A residues on bacteria. , 1970, Journal of immunology.

[26]  J Carlsson,et al.  Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. , 1978, The Biochemical journal.

[27]  D. Lancet,et al.  Interactions between staphylococcal protein A and immunoglobulin domains. , 1978, Biochemical and biophysical research communications.

[28]  A. Forsgren,et al.  “Protein A” from Staphylococcus Aureus III. Reaction with Rabbit γ-Globulin , 1967 .