Measurement of scFv antibody affinity using non-competitive enzyme-linked immunosorbent assay

Objective: To measure scFv antibody af finity using non-competitive enzyme-linked immunosorbent assay (ELISA). Methods:Using serial dilutions of antigen (, ) and antibody (anti-MAGE-A1 scFv antibody), the affinity constant (K aff) was measured by non-competitive ELISA. Two sigmoid curves of optical density (OD) v ersus logarithms of antibody concentrations were estimated by SPSS 10.0 software . The maximum value of OD (OD-100) of each curve was computed respectively and OD-50, half of OD-100, was obtained. Then the concentrations of antibody corre sponding to OD-50 on the curves, named t and t were calculated . For =1/2 , K aff=1/{2 t- t}. Results: The affinity constant of scFv antibody was about 1.432×1 06 L/mol. Conclusion: Based on the Law of Mass Action, n on-competitive ELISA method for measurement of antibody-antigen affinity const ant is simple, rapid and reliable.

Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia co/i and its “oxidizing” mutant

AIM: To study the influence of redox environment of Escherichia coli (E(?) coli) cytoplasm on disulfide bond formation of recombinant proteins. METHODS: Bovine fibrobllast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E(?)coli strains BL21(DE3) and M15[pREP4] respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain,E(?)coli Origami(DE3). The 4 recombinant E(?)coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively. RESULTS: All recombinant E(?)coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv] was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from inclusion body in M15[pQE-HBscFv] was 30-35 mg/L and the affinity constant was 1.98×107 mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains. CONCLUSION: Modification of the redox environment of E(?)coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.

Experimental and modeling study of kinetics for methane hydrate formation in a crude oil-in-water emulsion

A low-viscosity emulsion of crude oil in water can be believed to be the bulk of a flow regime in a pipeline.To differentiate which crude oil would and which would not counter the blockage of flow due to gas hydrate formation in flow channels,varying amount of crude oil in water emulsion without any other extraneous additives has undergone methane gas hydrate formation in an autoclave cell.Crude oil was able to thermodynamically inhibit the gas hydrate formation as observed from its hydrate stability zone.The normalized rate of hydrate formation in the emulsion has been calculated from an illustrative chemical affinity model,which showed a decrease in the methane consumption（decreased normalized rate constant） with an increase in the oil content in the emulsion.Fourier transform infrared spectroscopy（FTIR） of the emulsion and characteristic properties of the crude oil have been used to find the chemical component that could be pivotal in selfinhibitory characteristic of the crude oil collected from Ankleshwar,India,against a situation of clogged flow due to formation of gas hydrate and establish flow assurance.

Effect of Tb(Ⅲ) on activity and stability of nattokinase

Nattokinase, is an effective fibrinolytic enzyme with the potential for fighting cardiovascular disease. The aim of study was to investigate the interaction of Tb（Ⅲ） with nattokinase and the impact of Tb（Ⅲ） on the enzyme activity and protein stability. The binding of Tb（Ⅲ） with nattokinase was studied by fluorescence spectrum in 100 mmol/L Tris-HCl（pH 8.0）. It could be seen that the protein bound one Tb（Ⅲ） with low affinity, and the binding constants K were 2.90×10~4 L/mol at 288 K. Although the activity of nattokinase determined by tetra-peptide substrate method at proper pH and temperature was not influenced for the binding of Tb（Ⅲ）, the transformation rate of substrate was increased to 113%. To better assess the stability of protease in the absence and presence of Tb（Ⅲ）, nattokinase was unfolded through continuous concentrations urea. Based on the model of structural element, the results showed that Tb（Ⅲ） could not change the average structural element free energy of nattokinase by the measurement of enzyme activity, but it could improve the stability of the global protein by the fluorescence spectral measurement.

Positron Characteristics in Cadmium and Zinc Chalcogenides

Electron energy levels and positron states have been calculated for cadmium and zinc chalcogenide compounds within the pseudo-potential approach and the independent particle model.Furthermore,the present contribution deals with the electron and positron chemical potentials allowing the calculation of the positron affinity to different materials of interest and hetero-structures formed by these materials.Besides,we here determine the positron diffusion constant by means of the positron deformation potential.An attempt has been made to scale positron affinity and diffusion constant with the lattice constant and the band gap energy,respectively.Such scaling is found to be not possible.The information gathered by the present study is of prime importance for a better understanding of positron trapping at interfaces and precipitates and should be useful in slow positron beam experiments.