ABSTRACT
Objective To obtain Chinese hamsterovary (CHO) cell line expressing human decay accelerating activity (hDAF) stably and to observe the protective effect of hDAF on heterologous cells under the circumstance of complement activation. Methods The eukaryotic expression vector DAF pcDNA3.1 was constructed and then transfected into CHO cells by lipofection. Monoclones of cells expressing hDAF stably were screened by the method of limiting dilution. hDAF expression was detected by flow cytometry. The decay accelerating activity of hDAF was determined by assay of C3 deposition and 51Cr release. Results The expression vector DAF pcDNA3.1 was successfully constructed, and monoclones of cells expressing hDAF were obtained. CHO cells expressing hDAF could decrease C3 deposition and attenuate the killing effect of activation of the complement system. Conclusion We have obtained CHO cell clones expressing hDAF stably, which is helpful for the further studies of the relationship of the structure with the functions of hDAF.
ABSTRACT
Objective To display efficient folding hDAF on the surface of yeast. Methods The hDAF open reading frame was cloned by PCR from DAF- pBluescript M13-（Amp＋）plasmid， then subcloned into the yeast surface displayed vector pYD1.The recombinant vector was transformed into yeast cells EBY100.Flow cytometric analysis was carried out to evaluate direct binding of anti-DAF mAbs onto the surface of yeast cells displaying DAF. Results Three mAbs against DAF different epitopes could bind onto DAF displayed on the surface of yeast.Conclusion Efficient folding DAF can be displayed on the surface of yeast potentially leading to the development of novel applications involving DAF yeast display.
ABSTRACT
Objective:To predict the secondary structure and B cell epitope of human decay accelerating factor Methods:The accuracy of two secondary structure prediction methods,Chou & Fasman and PHDsec,was compared,and then human DAF secondary structure was analyzed by PHDsec Hydrophilicity scores and solvent accessibility of DAF SCR1 4 were obtained by methods of Hopp & Woods and PHDacc respectively Combined with the secondary structure of DAF,the B cell epitopes in DAF were predicted Results:PHDsec which use multiple sequence alignment,achieve a better prediction accuracy There is no alpha helix in SCR1 4 of human DAF but only beta sheet and loop segments; The computer predicted most possible epitope in DAF localize within amino acid residues Pro73 Val79?Arg130 Leu139 and Glu156 Cys163 Conclusion:These results will be useful in estimate of the epitope and activity sites localized within human DAF
ABSTRACT
The technique of Clq-ELISA has been established in this laboratory for the detection of antihuman Clq monoclonal antibodies(McAb). This technique, as verified by displacement test, blocking test, and cross titration test, possesses high specificity. It is more sensitive than double immunodiffusion as verified by the tests of 15 specimens of mouse antihuman Clq serum and has better reproducibility. It is concluded that this technique is very useful for the detection of anti-Clq McAb in the supernatant of hybridoma culture.
ABSTRACT
This paper is to report a procedure to isolate C1q in human serum with anti-human Clq McAb affinity chromatography.This procedure is simple and rapid,which can be performed within 24 to 48 hr The yield of Clq was 7~10 mg per 100ml serum.The preparations were immunoglobulin free as judged by double diffusion and immunoelectrophoresis tassay and exhibited typical C1q subunit structure in SDS-PAGE.After biological activity evaluation with red cell agglutination and latex agglutination assay,this C1q preparation was found to be satisfactory in its activity.The anti-human Clq McAb sepharose 4B column can be used repeatedly and stored in 4℃ for approximately one year without loss of absorption activity.
ABSTRACT
Experimental acute serum sickness was produced in 20 rabbits By a sensitizing injection with bovine serum albumin (BSA) through the auricular vein. Five days after the sensitization, ten of the 20 animals were given a total body irradiation of 300 r from a 60Co sourse. No radiation was given to the other 10 animals which served as controls. Blood samples were taken from the rabbits of both groups before and 2 , 4 , 6 , 8 , 10, 12, 14, and 16 days after antigen injection. After the sera were seperated, the concentrations of the circulating immune complexes were determined with the PEG complement consumption test. It was found that the dynamic curves of the concentrations of the circulating immune complexes of the two groups were essentially similar. This result strongly suggests that total body irradiation of gamma rays given five days after the sensitization of an antigen exerts no influence on the formation of the circulating immune complexes though acute radiation sickness is well established.