Immunizing New Zealand Research Articles

Prokaryotic expression and antibody preparation of truncated glycoprotein of Crimean-Congo hemorrhagic fever virus

Objective To express and purify truncated glycoprotein(Gn, Gc1, Gc2) of Crimean-Congo hemorrhagic fever virus(CCHFV) strain 79121 from E. coli cells, and to prepare their polyclonal antibodies respectively. Methods The genes encoding Gn, Gc1 and Gc2 from CCHFV strain 79121 were amplified by RT-PCR, and cloned into prokaryotic expression vector pET-28a (+ ) to construct recombinant plasmids. The three recombinant plasmids were then transformed into E. coli BL21(DE3) to obtain the Gn-His, Gc1-His and Gc2-His fusion proteins through IPTG induction. The fusion proteins were purified by Ni-NTA purification system, and then analyzed by using SDS-PAGE. To prepare antisera, the purified Gn-His, Gc1-His and Gc2-His proteins were employed to immunize New Zealand White Rabbit (NZW), respectively. The titer and specificity of the antisera to each truncated glycoprotein were analyzed by using ELISA and Western blot, respectively. Results The recombinant expression vectors of pET-28a-Gn, pET-28a-Gc1 and pET-28a-Gc2 were constructed successfully by restriction enzyme analysis and DNA sequencing. The relative molecular mass(Mr) of Gn-His, Gc1-His and Gc2-His were approximately 25 000, 33 000 and 34 000, and the titers of the corresponding polyclonal antibodies were above 1∶25 600, 1∶6400 and 1∶25 600, respectively. The antisera were able to bind to the corresponding fusion protein specifically. Conclusion The recombinant expression of truncated glycoprotein (Gn, Gc1, Gc2) of CCHFV strain 79121 and the preparation of their high titer polyclonal antibodies laid foundation for further studying the biological function of glycoprotein and the rapid detection of CCHFV. Key words: Crimean-Congo hemorrhagic fever virus; Glycoprotein; Prokaryotic expression; Polyclonal antibody

Development and preliminary application of colloidal gold-based immunochromatographic test strips with colloidal gold for rapid detection of NJ001 specific antigen

Objective To develop a colloidal gold immunochromatographic assay for rapid detection of NJ001 specific antigen. Methods Polyclonal antibody was prepared by immunizing New Zealand rabbit with SPC-Al cells.Polyclonal antibody and Goat anti mouse IgG,as test line and control line respectively，were immobilized on nitrocellulose membrane,NJ001 was labeled by colloidal gold particles and then sprayed and fixed on fiberglass membrane as the combination pad.Together with a specimen pad ahead．The nitrocellulose membrane and the conbination pad were assembled into a test strip.The poisitive rate of NJ001 were examined from 52 lung adenocarcinoma patients and 40 healthy controls(HC).In the lung adenocarcinoma group, 49 were comfirmed by TNM, 15 stageⅠ/Ⅱ 34 stageⅢ/Ⅳ,and 3 cases without TNM staging data. 45 patients had lymphatic metastasis data,22 cases were of no lymphatic metastasis and 23 cases were of lymphatic metastasis.There were another 7 cases without the data of lymphatic metastasis. Results Positive rate of NJ001 in lung adenocarcinoma patients was 55.77%,significantly higher than that of HC(7.5%)（χ2=23.22，P＜0.001）.Positive rate of NJ001 specific antigen in patients at TNM stage Ⅲ/Ⅳ was higher than that at TNM stageⅠ/Ⅱ（64.17% vs 33.33%，χ2=4.141，P=0.042）; Positive rate in patients with lymph nodes metastasis was higher than that in patients without lymph nodes metastasis（69.57% vs 36.36%，χ2=4.98，P=0.026）. Conclusion This study successfully establishes a colloidal gold immunochromatographic assay that can be used to rapid detect NJ001 specific antigen in clinical serum samples.（Chin J Lab Med,2014,37:548-552） Key words: Lung neoplasms; Adenocarcinoma; Antibodies, monoclonal; Gold colloid; Immunochromatography

A preliminary study on serum protein SP70 as a novel biomarker for the detection of non-small cell lung cancer

Objective To evaluate whether the protein SP70 could be used as a serum biomarker for the diagnosis of non-small cell lung cancer （NSCLC）. Methods Polyclonal antibody was prepared by immunizing New Zealand rabbit with SPC-A1 cells.Sandwich ELISA was carried out by using newly-prepared polyclonal antibody（PcMb）coating assay plates，monoclonal antibody（McAb）NJ001 and HRP goat anti-mouse antibody as primary antibody and labeling antibody respectively.After optimizing the experiment conditions，serum from 175 lung cancer patients ［80 NSCLC adenocarcinoma，70 NSCLC squamous carcinoma and 25 small cell lung cancer（SCLC）］，25 benign lung disease（BLD）patients and 300 healthy controls （HC） were examined.CEA，NSE，CYFRA21-1 were measured by ECLIA for comparison. Results Positive rates of NSCLC adenocarcinoma，NSCLC squamous carcinoma，SCLC and BLD were 68.8%，51.4%，16.0% and 12.0% respectively，obviously higher than that of HC（7.3%）．NSCLC（adenocarcinoma，Squamous carcinoma） had significantly higher positive rate than SCLC（60.7% vs 16.0%，χ2=17.23，P<0.05）and BLD（60.7% vs 12.0%，χ2=20.41，P<0.05）.Among 68 NSCLC patients who had definite staging，positive rates at early stage（Ⅰ/Ⅱ，n=30） reached up to 76.7%.Meanwhile，positive rates of CEA，NSE and CYFRA21-1（32.7%，18.0% and 37.3%）were significantly lower than the targeting antigen to McAb NJ001 in NSCLC（60.7% vs 32.7%，χ2=23.63，P<0.05；60.7% vs 18.0%，χ2=57.22，P<0.05；60.7% vs 37.3%，χ2=16.34，P<0.05）. Conclusions It showed high positive rates of SP70 in the serum of NSCLC patients，which suggested that SP70 might be a potential valuable biomarker for the diagnosis of NSCLC.（Chin J Lab Med，2012,35：554-558） Key words: Lung neoplasms; Carcinoma; non-small-cell lung; HSP70 heat-shock proteins; Tumor markers，biological; Enzyme-linked immunosorbent assay