[Expression, purification, and characterization of an anti-human RBC ScFv-HIV gp160 fusion protein for hemagglutination-based rapid detection of antibodies to HIV in whole blood].

OBJECTIVE: To construct and express anti-human RBC and HIVgp160 fusion protein for rapid detection of antibody to HIV. METHODS: The gene of the anti human RBC ScFv and HIV antigen were constructed together into expression vector. The fusion protein was expressed in E. coli. RESULTS: The fusion protein was proved to be able to bind both anti-RBC and HIVgp160. It could cause agglutination of human RBC when HIVgp160 was present. CONCLUSION: The fusion protein has the potential in rapid detection of HIV.

[Identification and typing of human enteroviruses using an RT-PCR assay].

BACKGROUND: To establish a molecular detection and typing assay for identification and typing of human enteroviruses (HEV) which is suitable for clinical detection and epidemiologic research. METHODS: Using both primers specific for HEV genus and HEV typing primers and reverse transcription polymerase chain reaction (RT-PCR) the authors detected preliminarily HEV by agarose gel electrophoresis and then identified serotype through nucleotide sequence analysis of RT-PCR amplicons. The monospecific antisera neutralization was applied to validate the typing results. RESULTS: The serotype of 18 suspicious HEV samples was identified: 4 Coxsackievirus type A24 (CVA24), 3 CVB3, 1 CVB2, 1 CVA9, 1 CVA15, 1 Echovirus type 3 (E3), 1 E6, 1 E9, 1 E11, 1 E14, 1 E33 and 1 Rhinovirus type 9. The result was validated by monospecitive antisera neutralization. CONCLUSION: This RT-PCR assay for HEV detection and typing may be suitable for clinical detection and epidemic research since this method is sensitive and specific for direct identification and typing of HEV.

[Preliminary study on determination of specific cellular immunity of patients with hepatitis B].

BACKGROUND: To evaluate an enzyme linked immunospot (ELISPOT) method for testing the specific cellular immunity of patients with hepatitis B and preliminarily investigate into the difference of cellular immunity in patients with various types of hepatitis B. METHODS: The patients with acute hepatitis B, chronic hepatitis B liver cirrhosis, healthy persons with HBV vaccine immunization, healthy persons with past HBV infection and HBV naive persons were enrolled in this study. Their peripheral blood mononuclear cells were tested by ELISPOT to determine the number of gamma-interferon secreting cells. RESULTS: The number of gamma-interferon secreting cells was significantly different between the patients with acute hepatitis B and those with chronic hepatitis B, and between the patients with acute hepatitis B and those with liver cirrhosis (P=0.0209 and P=0.0211). CONCLUSION: The specific cellular immunity in the patients with hepatitis B could be evaluated by determining the number of gamma-interferon secreting cells with the method of testing their peripheral blood mononuclear cells by ELISPOT. The specific cellular immunity was stronger in the patients with acute hepatitis B than in those with chronic hepatitis B and liver cirrhosis.

[Prokaryotic expression and purification of human immunodeficiency virus p24 antigen].

OBJECTIVE: To express and purify the HIV p24 gene in E. coli cells, and identify p24 antigen activity. METHODS: The full length gene fragment of HIV p24 was amplified by PCR and inserted into the pRSET vector in order to construct the pRSET-p24 recombined vector. After transforming into E. coil, the purified p24 protein was prepared by metal-ligand affinity chromatography (IMAC). The accuracy of inserted gene and activity, specificity of HIV p24 proteins were detected by two enzymes digestion technology, SDS-PAGE, Western Blot (WB) and ELISA. RESULTS: The length of the HIV p24 gene fragment was 690 bp after digesting the recombinant plasmid T-p24 and pRSET-p24 with BamH I and Hind III. The expressed proteins had a single expected band of about 24 x 10(3) in SDS-PAGE. The specificity and activity of p24 protein were tested by WB and ELISA. CONCLUSION: The HIV p24 sequence from HIV-1 gene plasmid hasbeen expressed in E. coil. This protein possessed good specificity and activity.

[Preparation and characterization of the monoclonal antibody against HIV-1 p24 antigen].

AIM: To establish hybridoma cells secreting monoclonal antibodies (mAbs) against HIV-1 p24 antigen and characterize their properties. METHODS: BALB/c mice were immunized with purified p24 protein and then the from splenocytes immunized mice were fused with Sp2/0 cells. Monoclonal hybridoma cell lines were obtained by limiting dilution, HAT and HT-selective culture. The specificity of mAbs was identified by Dot blot and indirect ELISA. RESULTS: We had established two hybridoma cell lines secreting mAbs against HIV-1 p24 antigen. The titers of two mAbs in ascitic fluid were 1 x 10(-5) and 1.7 x 10(4)-1.8 x 10(4) mol/L, respectively. Both mAbs belonged to IgG1. Indirect ELISA detection demonstrated that both mAbs had no cross-reactivities with other viral antigens, such as HBcAg, HCV RNA positive sera and HIVgp41. CONCLUSION: Two hybridoma cell lines secreting mAbs against HIV-1 p24 antigen have been established successfully, which lays the foundation for detecting HIV-1 p24 antigen.