BACKGROUND: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. METHODS: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize V(H) and V(L) fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. RESULTS: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of V(H) or V(L) domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of V(H) of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. CONCLUSION: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.
Alkaline Phosphatase ; Antibodies ; Clone Cells ; Cytoplasm ; DNA ; Escherichia ; Escherichia coli ; Immunoglobulin Fragments ; Indicators and Reagents ; Leucine Zippers ; Oxidoreductases ; Pathology, Molecular ; Pyruvic Acid ; Sprains and Strains

BACKGROUND: Although a skin test is the primary option for detecting allergen-specific IgE in clinics, the serum IgE immunoassay is also important because it allows for the diagnosis of allergy without any accompanying adverse effect on the patient. However, the low detection limit of IgE levels by immunoassay may restrict the use of the method in some occasions, and improving its sensitivity would thus have a significant implication in allergy-immunology clinics. METHODS: In this study, we attempted to detect specific serum IgE by using immuno-polymerase chain reaction (IPCR) which combines the antigen-antibody specificity of enzyme-linked immunosorbent assays (ELISAs) with the amplification power of PCR. RESULTS: Our results demonstrated that Blo t5-specific serum IgE can be detected by IPCR with a 100-fold higher sensitivity than ELISA, and cross-reactivity of serum IgE to other mite allergens is able to be analyzed by using only 0.3 microliter of serum sample. Use of real-time IPCR seemed to permit more convenient determination of specific serum IgE as well. CONCLUSION: We believe that IPCR can serve as a valuable tool in determining specific serum IgE, especially when the amount of serum sample is limited.
Allergens ; Antigens, Dermatophagoides ; Arthropod Proteins ; Cysteine Endopeptidases ; Enzyme-Linked Immunosorbent Assay ; Humans ; Hypersensitivity ; Immunoassay ; Immunoglobulin E ; Limit of Detection ; Mites ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Skin Tests