Open sandwich ELISA with VH-/VL-alkaline phosphatase fusion proteins

Suzuki, Chikako; Ueda, Hiroshi; Tsumoto, Kouhei; Mahoney, Walt; Koyanagi, Izumi; Nagamune, Teruyuki

doi:10.1016/s0022-1759(99)00020-4

Cited by 43 publications

(38 citation statements)

References 31 publications

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“…However, mammalian APs differ from their E. coli counterpart in terms of Mg(II) secondary ligand (33), membrane anchoring (45), glycosylation (27), etc. APs represent a large research field as they are good models to study metal ion-dependent catalysis and are used in several application fields (molecular biology [3] and immunodetection [43]). …”

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confidence: 99%

Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Zappa

Rolland

Flament

et al. 2001

Appl Environ Microbiol

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This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiolreducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intraor interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

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confidence: 99%

Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Zappa

Rolland

Flament

et al. 2001

Appl Environ Microbiol

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“…The Asp residue at position 101 of the BAP protein was mutated to Ser because the D101S mutation was known to enhance the phosphatase activity with a higher Vmax than a wild-type enzyme. 10 To express the VL fragment, we chose Trx-tag as a fusion partner. Trx-tag has been used as a fusion tag for proper folding and soluble expression of many exogenous proteins.…”

Section: Design Of Os-elisa Assay System For Bz Detectionmentioning

confidence: 99%

Noncompetitive Immunodetection of Benzaldehyde by Open Sandwich ELISA

et al. 2009

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Benzaldehyde (Bz) is a typical fragrant compound for peach-flavored beverages. In the food and beverage industries there is great demand for a sensitive and easy detection system of Bz in order to ensure product quality control and to avoid contamination. For the noncompetitive detection of Bz, we applied an open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing an antigen-dependent reassociation of antibody variable region fragments, VH and VL. We cloned the VH and VL genes of an anti-Bz monoclonal antibody, and the fragments were individually expressed and purified as a bacterial alkaline phosphatase (BAP)-conjugated form for VH and as a thioredoxine (Trx)-fused form for VL, respectively. Using these VH and VL fragments, we successfully constructed the OS-ELISA system for Bz detection. The Bz-induced formation of a trimolecular complex composed of VH-BAP/Bz/Trx-VL was readily detected by a dosedependent increase in the BAP activity of the VH-fusion protein.

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“…The fragment was digested with EcoRV and HindIII, gel purified and ligated with pVH-PhoA(D101S), 17 which had been digested with the same. The ligation product was used to transform XL10-Gold and plated on LBAG plate, overnight at 37˚C.…”

Section: Expression and Purification Of Vh-ap Fusion Proteinmentioning

confidence: 99%

“…Also, the direct immobilization of MBP-VL was expected to result in an overall reduction in the assay time and steps. For detecting the bound VH, fusion protein with E. coli alkaline phosphatase mutant with higher Vmax than the wild-type enzyme (D101S) 17 was employed. The resultant dose-response curve using purified immobilized MBP-VL and VH-PhoA(D101S), together with detection with chemiluminescent substrate CDP star, is shown in Fig.…”

Section: Open Sandwich Elisa Using Mbp-vl and Vh-ap Proteinsmentioning

confidence: 99%

“…As a way to circumvent these limitations, we described a new immunoassay approach, called the open sandwich (OS)-ELISA. [17][18][19] Briefly, this assay exploits reassociation of the generally weak antibody variable region VH-VL complex by a bridging antigen. With the use of immobilized VL and enzyme-tagged VH fragments, one can measure less than 10 ng/mL of antigen in a shorter time period than using a conventional sandwich assay, due to the omission of an incubation/washing cycle.…”

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confidence: 99%

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