Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus

Background Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients’ sera.Methods Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. Results The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. Conclusion The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

Construction of Eukaryotic Expressing Plasmids Encoding HA and HA_1 of Influenza A Virus and Their Transient Expression in HEK293 Cells

In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus （A/PR/8/ 34） and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3. 1 （ ＋ ） to generate pcDNA3. 1 （ ＋ ）/HA and pcDNA3. 1 （ ＋ ）/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3. 1（＋）/HA and pcDNA3. 1（＋）/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3. 1 （＋）/HA or pcDNA3. 1 （＋）/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HAl , which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.

Expression of Recombinant Bovine Prion Protein PrP27-30 in CHO-K1 Cells

[Objective] To investigate the possibilities of expressing bovine PrP27-30 gene in CHO-K1 cells. [Method] The purified PCR products of PrP27-30 were digested and ligated to the pCI-neo vector to yield pCI-neo-PrP27-30 that was used as an expression vector. Then CHO-K1 cells were transfected by pCI-neo-PrP27-30, and stable expression clone cells were screened by methotrexate (MTX) at a concentration of 0.1 and 1.0 umol/L. The transient expression was detected by indirect immunofluorescence assay and western blot. [ Result] After drug selection with MTX, the expression of PrP27-30 gene was detected in CHO-K1 cells. [Conclusion] Recombinant protein PrP27-30 expressed in CHO-K1 cells has better immunoreactivity and can be used to study secondary structure and regulation mechanism of pathological isoform of prion protein (prpC).

Expression of Recombinant Human FADD,Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

The wild-type human Fas-associated death domain （FADD） protein was expressed as a His-tag fusion protein in Escherichia coll. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refolded and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD protein was allowed the production of high titre polyclonal antiserum. This new polyelonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofiuorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays. Cellular ＆ Molecular Immunology. 2008;5（6）：471-474.

Development and characterization of monoclonal antibodies against human aryl hydrocarbon receptor

Aryl hydrocarbon receptor（Ah R）, a ligand-dependent nuclear receptor, is involved in a diverse spectrum of biological and toxicological effects. Due to the lack of three dimensional（3D）crystal or nuclear magnetic resonance structure, the mechanisms of these complex effects of AhR remain to be unclear. Also, commercial monoclonal antibodies（mA bs） against human AhR protein（h Ah R）, as alternative immunological tools, are very limited. Thus, in order to provide more tools for further studies on h Ah R, we prepared two m Abs（1D6 and 4A6） against h Ah R. The two newly generated m Abs specifically bound to amino acids 484–508（located in transcription activation domain） and amino acids 201–215（located in Per-ARNT-Sim domain）of h Ah R, respectively. These epitopes were new as compared with those of commercial m Abs.The m Abs were also characterized by enzyme-linked immunosorbent assay, western blot,immunoprecipitation and indirect immunofluorescence assay in different cell lines. The results showed that the two m Abs could recognize the linearized AhR s in six different human cell lines and a rat hepatoma cell line, as well as the h Ah R with native conformations. We concluded that the newly generated m Abs could be employed in AhR-based bioassays for analysis of environmental contaminants, and held great potential for further revealing the spatial structure of AhR and its biological functions in future studies.