Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity. 2010

Jian-Xin Zhao, and Lian Yang, and Zhen-Nan Gu, and Hai-Qin Chen, and Feng-Wei Tian, and Yong-Quan Chen, and Hao Zhang, and Wei Chen
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China; E-Mails: jxzhao@jiangnan.edu.cn (J.-X.Z.); yanglian27@yahoo.com.cn (L.Y.); haiqinchen@jiangnan.edu.cn (H.-Q.C.); fwtian@jiangnan.edu.cn (F.-W.T.).

The interdomain instability of single-chain fragment variable (scFv) might result in intermolecular aggregation and loss of function. In the present study, we stabilized H4-an anti-aflatoxin B(1) (AFB(1)) scFv-with an interdomain disulfide bond and studied the effect of the disulfide bond on antibody affinity. With homology modeling and molecular docking, we designed a scFv containing an interdomain disulfide bond between the residues H44 and L100. The stability of scFv (H4) increased from a GdnHCl(50) of 2.4 M to 4.2 M after addition of the H44-L100 disulfide bond. Size exclusion chromatography revealed that the scFv (H44-L100) mutant existed primarily as a monomer, and no aggregates were detected. An affinity assay indicated that scFv (H4) and the scFv (H44-L100) mutant had similar IC(50) values and affinity to AFB(1). Our results indicate that interdomain disulfide bonds could stabilize scFv without affecting affinity.

UI MeSH Term Description Entries
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D004220 Disulfides Chemical groups containing the covalent disulfide bonds -S-S-. The sulfur atoms can be bound to inorganic or organic moieties. Disulfide
D004797 Enzyme-Linked Immunosorbent Assay An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. ELISA,Assay, Enzyme-Linked Immunosorbent,Assays, Enzyme-Linked Immunosorbent,Enzyme Linked Immunosorbent Assay,Enzyme-Linked Immunosorbent Assays,Immunosorbent Assay, Enzyme-Linked,Immunosorbent Assays, Enzyme-Linked
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D000915 Antibody Affinity A measure of the binding strength between antibody and a simple hapten or antigen determinant. It depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. It includes the concept of "avidity," which refers to the strength of the antigen-antibody bond after formation of reversible complexes. Affinity, Antibody,Antibody Avidity,Avidity, Antibody,Affinities, Antibody,Antibody Affinities,Antibody Avidities,Avidities, Antibody
D017434 Protein Structure, Tertiary The level of protein structure in which combinations of secondary protein structures (ALPHA HELICES; BETA SHEETS; loop regions, and AMINO ACID MOTIFS) pack together to form folded shapes. Disulfide bridges between cysteines in two different parts of the polypeptide chain along with other interactions between the chains play a role in the formation and stabilization of tertiary structure. Tertiary Protein Structure,Protein Structures, Tertiary,Tertiary Protein Structures
D055550 Protein Stability The ability of a protein to retain its structural conformation or its activity when subjected to physical or chemical manipulations. Protein Stabilities,Stabilities, Protein,Stability, Protein
D057127 Single-Chain Antibodies A form of antibodies consisting only of the variable regions of the heavy and light chains (FV FRAGMENTS), connected by a small linker peptide. They are less immunogenic than complete immunoglobulin and thus have potential therapeutic use. Fv Antibody Fragments, Single-Chain,ScFv Antibodies,Single-Chain Fv,Single-Chain Fv Antibody,Single-Chain Fv Antibody Fragments,Single-Chain Variable Fragment,Single-Chain Variable Fragment Antibodies,Single-Chain Variable Fragment Antibody,Single-Chain Variable Fragments,Antibodies, ScFv,Antibodies, Single-Chain,Antibody, Single-Chain Fv,Fragment, Single-Chain Variable,Fragments, Single-Chain Variable,Fv Antibody Fragments, Single Chain,Fv Antibody, Single-Chain,Fv, Single-Chain,Single Chain Antibodies,Single Chain Fv,Single Chain Fv Antibody,Single Chain Fv Antibody Fragments,Single Chain Variable Fragment,Single Chain Variable Fragment Antibodies,Single Chain Variable Fragment Antibody,Single Chain Variable Fragments,Variable Fragment, Single-Chain,Variable Fragments, Single-Chain

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