| Neutrophil Gelatinase-Associated Lipocalin (NGAL) involves in inflammation, embryonic development, immunity response, chemotaxis phenomena, signal transduction, developmentand progression of many cancers. It is a new biochemical marker for early renal damage. The aim of this study is to screen NGAL antibody by applying phage display technology and establish immune colloidal gold test technology to detect the NGAL change level which will help in the diagnose of the accute kidney injury.The immune Balb/C mice were used in this research. The total RNA was extracted from the spleen and reverse-transcribed into cDNA. Degenerateoligonucleotide primers of the light chain variable regions (VL) and heavy chain variable regions (VH) were designed and overlap extension PCR was conducted to connect these regions to obtain a single antibody gene (scFv). After the enzyme digestion by the sfiI and XbaI enzyme, the fragments were connected to phage carrier pCantab5E, and then transformed into Escherichia coli TGI to establish a 5 x107 capacity of rat source phage antibody library. The antibody library insertion rate was detected through PCR to 85%. Sequencing analysis of randomly picked monoclonal samples revealed differences in sequences of the antibody heavy chain and light chain variable region which means that the rat phage antibody library has better diversity.After four rounds of enrichment by applying recombinant protein NGAL as the target protein for the phage antibody library,30 monoclonal phage antibodies were picked for ELISA identification. The postive clones were built into CHO cell expression vectors, followed by environmental stress screening and recombinant expression into CHO cells. The purified expression antibodies were analyzed through SDS-PAGE, ELISA, and the affinity test was also conducted. The results showed that 11 monoclonal samples were strong positive clones. The titer of one of the monolonal antibodies was 1:107, and the affinity was 6.532×10-9 M. It demonstrates that we successfully got the NGAL genetic engineered antibody, and provided the foundation for the NGAL detection.By using combinant expression of antibodies and polyclonal antibodies prepared in our laboratory, NGAL immune colloidal gold test was established. The optimum conditions were investigated; the gold colloid antibody tag quantity was 12μg/mL,0.08% PEG-20000 was stabilizer, boric acid buffer (20 mM, pH 8.2, containing 0.01% Na2N3,0.08% PEG-20000) was gold colloid antibody preservation solution,20 mM, Tris-HC1 (containing 0.5% PVP-40,0.5% Trion) pH 9.0 was the treatment fluid for combination pad,0.01 mol/L, TBS (containing 1% BSA,0.05% Tween 20) pH 7.4 was the sample pad processing solution, the gold colloid antibody coating amount was 0.5μL/mL, the sheep-anti-NGAL polyclonal antibody coating amount was 1.2μg/mL, and sheep-anti-human IgG spraying quantity was 4μg/mL.The test strips were preserved from light at 37℃ and 4℃ respectively in the sealing and dryplace, the sensitivity and specificity were not affected after 20 weeks. The colloidal gold strip were also used to detect 40 clinical samples, the results were similar with commercial clinical test. In conclusion, the established immune colloidal gold test presented good stability, high specificity, and excellent viability as a clinical assay. |
PDF Full Text Request