Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B

Abstract

Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.

FIG. 1.

Agarose gels showing V_H, V_L, and ScFv gene amplicons. (A) Lane 1, 100-bp DNA ladder (Fermentas); lane 2, V_H gene amplicon (∼340 bp); lane 3, V_L gene amplicon (∼325 bp). (B) Lane 1, 100-bp DNA ladder (Fermentas); lane 2, ScFv gene amplicon (∼750 bp).

FIG. 2.

SDS-PAGE (A) and Western blot (B) profiles showing expression of anti-SEB ScFv in soluble form. (A) Lane 1, molecular mass markers (66, 45, 29, 20, and 14.2 kDa); lane 2, uninduced culture of 4PCL2-infected HB2151 cells; lanes 3 and 4, induced culture and periplasmic extract of 4PCL2-infected HB2151 cells, respectively, showing the anti-SEB ScFv band at ∼29 kDa. (B) Lane 1, molecular mass markers (97.4, 66.2, 45, 31, 26.6, and 14.4 kDa); lane 2, uninduced culture of 4PCL2-infected HB2151 cells; lanes 3 and 4, induced culture and periplasmic extract of 4PCL2-infected HB2151 cells, respectively, showing the anti-SEB ScFv band at 29 kDa.

FIG. 3.

ELISA (A) and Western blot (B) showing binding of anti-SEB ScFv with SEB antigen. ELISA was performed with different dilutions of soluble anti-SEB ScFv antibody. Periplasmic extract of untransformed HB2151 cells was used as a negative control (OD, 0.0945). (B) Lane 1, prestained molecular mass markers (70, 55, 35, 27, and 15 kDa); lane 2, r-SEB band at 30.5 kDa showing binding with anti-SEB ScFv. The bound anti-SEB ScFv antibody was detected using anti-E-tag HRP-conjugated antibody.

FIG. 4.

Homology of 4PCL2 ScFv heavy chain with mouse germ line genes. Nucleotide and amino acid sequences of 4PCL2 ScFv heavy-chain variable region aligned with the most homologous germ line V region (IGHV1-58*01, IMGT accession no. AC087166). In the case of germ line genes, only the differences in nucleotides and amino acids are shown. Amino acids are numbered according to the Kabat numbering scheme. The NCBI accession number for the sequence of 4PCL2 ScFv is GQ 465983.

FIG. 5.

Homology of the 4PCL2 ScFv light chain with mouse germ line genes. Nucleotide and amino acid sequences of 4PCL2 ScFv light-chain variable region aligned with the most homologous germ line V region (IGKV4-61*01, IMGT accession no. AJ231209). In the case of germ line genes, only the differences in nucleotides and amino acids are shown. Amino acids are numbered according to the Kabat numbering scheme. The NCBI accession number for the sequence of 4PCL2 ScFv is GQ465983.

FIG. 6.

IMGT collier de perle graphical 2-dimensional representation of 4PCL2 ScFv. (A) Variable heavy chain (V_H). (B) Variable light chain (V_L). Collier de perle representations are displayed according to IMGT unique numbering. Asterisks indicate differences between 4PCL2 ScFv and the mouse germ line genes most similar to 4PCL2 ScFv. Hydrophobic amino acids (those with positive hydropathy index values, i.e., I, V, L, F, C, M, and A) and tryptophan (W) are shown in blue circles. All proline (P) residues are shown in yellow circles. The complementarity-determining region (CDR-IMGT) sequences are delimited by amino acids shown in squares (anchor positions), which belong to the neighboring framework region (FR-IMGT). Hachured circles correspond to missing positions according to IMGT unique numbering. Colors of circle outlines indicate regions: in the V_H domain, red for CDR1-IMGT, orange for CDR2-IMGT, and purple for CDR3-IMGT; in the V_L domain, blue for CDR1-IMGT, green for CDR2-IMGT, and turquoise for CDR3-IMGT. Residues at positions 23 (first cysteine), 41 (tryptophan), 89 (hydrophobic amino acid), 104 (second cysteine), and 118 (hydrophobic amino acid) are conserved and shown in red letters.