Open sandwich ELISA: A novel immunoassay based on the interchain interaction of antibody variable region

We describe an immunoassay that is based on the interchain interaction of separated VL and VH chains from a single chain antibody variable region. In the presence of antigen, the chains reassociate. VL fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 were immobilized on microtiter plates. Samples were coincubated with an M13-displayed VH chain, and assayed with peroxidase-labeled anti-M13 antibody. Signal was detected in direct proportion to the amount of HEL in the sample. Wide dynamic range with <15 ng/ml sensitivity was attained.

[1]  L. Riechmann,et al.  Antibody VH Domains as Small Recognition Units , 1995, Bio/Technology.

[2]  K. D. Hardman,et al.  Single-chain antigen-binding proteins. , 1988, Science.

[3]  S. Smith‐Gill,et al.  Contributions of immunoglobulin heavy and light chains to antibody specificity for lysozyme and two haptens. , 1987, Journal of immunology.

[4]  M. Klein,et al.  Relative noncovalent association constant between immunoglobulin H and L chains is unrelated to their expression or antigen-binding activity. , 1987, Journal of immunology.

[5]  César Milstein,et al.  Man-made antibodies , 1991, Nature.

[6]  M. Polymenis,et al.  Domain interactions and antigen binding of recombinant anti-Z-DNA antibody variable domains. The role of heavy and light chains measured by surface plasmon resonance. , 1995, Journal of immunology.

[7]  Y. Satow,et al.  Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A. , 1985, Journal of molecular biology.

[8]  S. Smith‐Gill,et al.  Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme. , 1992, Journal of immunology.

[9]  S. Muyldermans,et al.  Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. , 1994, Protein engineering.

[10]  G. Cohen,et al.  Structure of an antibody-antigen complex: crystal structure of the HyHEL-10 Fab-lysozyme complex. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[11]  R. R. Robinson,et al.  Escherichia coli secretion of an active chimeric antibody fragment. , 1988, Science.

[12]  S. Paul,et al.  Selection of functional human immunoglobulin light chains from a phage-display library , 1994, Applied biochemistry and biotechnology.

[13]  P. T. Jones,et al.  Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli , 1989, Nature.

[14]  M. Klein,et al.  The role of the VL- and VH-segments in the preferential reassociation of immunoglobulin subunits. , 1986, Molecular immunology.

[15]  A. Davies.  “Baculophage:” A new tool for protein display , 1995, Bio/Technology.

[16]  M. Wittekind,et al.  Production of stable anti-digoxin Fv in Escherichia coli. , 1992, Molecular immunology.

[17]  S. Eklund,et al.  Vasoactive intestinal peptide hydrolysis by antibody light chains. , 1991, The Journal of biological chemistry.

[18]  L. Riechmann,et al.  ‘Camelising’ human antibody fragments: NMR studies on VH domains , 1994, FEBS letters.

[19]  R. Bruccoleri,et al.  Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[20]  S. Paul,et al.  Antigen recognition by an antibody light chain. , 1994, The Journal of biological chemistry.

[21]  K. Tsumoto,et al.  Effect of the order of antibody variable regions on the expression of the single-chain HyHEL10 Fv fragment in E. coli and the thermodynamic analysis of its antigen-binding properties. , 1994, Biochemical and biophysical research communications.

[22]  G. Winter,et al.  Engineering bispecific antibodies. , 1993, Current opinion in biotechnology.

[23]  M. Gavish,et al.  Folding and interaction of subunits at the antibody combining site. , 1976, Biochemistry.

[24]  K Watanabe,et al.  Contribution to antibody-antigen interaction of structurally perturbed antigenic residues upon antibody binding. , 1994, The Journal of biological chemistry.

[25]  G. Winter,et al.  In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation. , 1994, Journal of molecular biology.

[26]  G. Winter,et al.  Expression of an antibody Fv fragment in myeloma cells. , 1988, Journal of molecular biology.

[27]  K Watanabe,et al.  Role of Tyr Residues in the Contact Region of Anti-lysozyme Monoclonal Antibody HyHEL10 for Antigen Binding (*) , 1995, The Journal of Biological Chemistry.

[28]  M. Klein,et al.  Structural basis for the preferential association of autologous immunoglobulin subunits: role of the J region of the light chain. , 1984, Molecular immunology.

[29]  R. Glockshuber,et al.  A comparison of strategies to stabilize immunoglobulin Fv-fragments. , 1990, Biochemistry.

[30]  Kumagai Izumi,et al.  Synthesis and expression of a DNA encoding the Fv domain of an anti-lysozyme monoclonal antibody, HyHEL10, in Streptomyces lividans , 1993 .

[31]  M. Klein,et al.  Noncovalent association of heavy and light chains of human immunoglobulins. III. Specific interactions between VH and VL. , 1982, Journal of immunology.

[32]  K. J. Dorrington,et al.  Equilibrium and kinetic aspects of the interaction of isolated variable and constant domains of light chain with the Fd' fragment of immunoglobulin G. , 1979, Biochemistry.