An investigation of the antigen P24 of 3,307 volunteer blood donors and 3,207 blood donors by kit of HIV Ag-1 monoclonal has shown that if there was weak possitive, it means that there was antigen P 24. However this has not been affirmed by quantified method. HIV antigen P24 expressed in the window period while there is no occurrence of HIV antibody. The identification of this antigen can shorten the window period in the diagnosis to help preventing from HIV infection via blood transfusion. The test of antigen P 24 helps also the physicians to determine the time for the chemotherapy and monitor the progress of disease during the treatment.
Blood Donors ; HIV Core Protein p24 ; HIV

OBJECTIVETo screen aptamers that can bind P24 antigen tightly and specificly, and verify its specificity and affinity.

METHODSPolycarbonate PCR plate was coated with P24 antigen and SELEX technology was used to screen aptamer on the PCR plate. The primary and secondary structure of these aptamers was analyzed by software. Through HRP labeled streptavidin and biotin labeled aptamers, the affinity and specificity of obtained aptamers were verified by ELISA.

RESULTSThe polycarbonate PCR plate could be coated with P24 antigen. Electrophoretic analysis showed the aptamers had been enriched. Sequence aligment analysis showed that these aptamers have consensus sequence and their apatial structure was multiple; ELISA verified that aptamers had high affinity with P24 antigen.

CONCLUSIONA simple method was established for screening aptamers that can bind HIV-1 P24 antigen specificly and tightly.

HIV Core Protein p24 ; analysis ; HIV-1 ; immunology ; Humans ; Polymerase Chain Reaction ; SELEX Aptamer Technique ; methods

BACKGROUND: In order to reduce the diagnostic window period between the time of human immunodeficiency virus (HIV) infection and serological diagnosis, new fourth generation screening assays which detect HIV p24 antigen and specific antibody simultaneously have been developed. In this study, we evaluated the performance of a new fourth generation assay. METHODS: We compared a new fourth generation assay, Architect HIV Ag/Ab combo, with another fourth generation assay AxSYM HIV Ag/Ab combo and a third generation assay, AxSYM HIV 1/2 gO for their performance. The assays were evaluated using 3 HIV seroconversion panels, 305 sera of healthy subjects and 100 sera of patients with HBsAg or anti-HCV antibodies. Within-run and total coefficient variations of the three screening assays were analyzed for the evaluation of precision. RESULTS: Architect HIV Ag/Ab combo shortened the window period by 8.7+/-2.1 days relative to AxSYM HIV 1/2 gO and 2.0+/-2.0 days relative to AxSYM HIV Ag/Ab combo in seroconversion panels. Architect HIV Ag/Ab combo presented the best performance in precision among the three reagents; total CV for positive control was 3.6%, 9.6% and 4.6% for Architect HIV Ag/Ab combo, AxSYM HIV Ag/Ab combo and AxSYM HIV 1/2 gO, respectively. Specificities of three assays were not different in this study. CONCLUSIONS: HIV Ag/Ab combined assays reduced the diagnostic window as compared to the third generation screening assays, enabling an earlier diagnosis of HIV infection. A new fourth generation assay, Architect HIV Ag/Ab combo presents a better performance than AxSYM HIV Ag/Ab combo, showing improved seroconversion sensitivity and precision.
Diagnosis ; Hepatitis B Surface Antigens ; Hepatitis C Antibodies ; HIV Core Protein p24 ; HIV Infections ; HIV Seropositivity ; HIV* ; Humans ; Indicators and Reagents ; Mass Screening

OBJECTIVETo establish and evaluate an Enzyme Immunoassay diagnostic kit combined with anti-HIV1/2 antibody and P24 antigen for shortening the examination window period of HIV infection in HIV laboratory diagnosis.

METHODSThe enzyme-linked reaction plates was coated by anti-HIV P24 monoclonal antibody and HIV 1/2 antigen. Labeling HIV1/2 antigen and anti-HIV P24 polyclonal antibody with horseradish peroxidase, setup an integrated ELISA kit for detecting anti-HIV-1/2 antibody and HIV P24 antigen, and evaluate the specificity and sensitivity of this kit.

RESULTSThe sensitivity of testing P24 antigen was up to 0.2 ng/ml. 78 serum samples of patients with AIDS, 85 serum samples of healthy people were compared with Abbott EIA kit, the coincidence was 100%. 12 051 sera from normal persons and patients were examined, the sensitivity of 100 %and specificity of 99.62 %, respectively.

CONCLUSIONThe anti-HIV1/2 antibody and HIV P24 antigen can be measured at the same time using this EIA kit, while the examination window period of HIV infection is shortened. Thus, the method is suitable for laboratory diagnosis and epidemiological investigation.

Enzyme-Linked Immunosorbent Assay ; HIV Antibodies ; blood ; HIV Core Protein p24 ; blood ; HIV-1 ; immunology ; HIV-2 ; immunology ; Humans ; Reagent Kits, Diagnostic

OBJECTIVETo study the pathological changes of the liver tissues of patients with HIV infection.

METHODS14 biopsy and 12 autopsy liver tissues were examined histologically. HIV-1 related antigen of outer membrane protein gp120 and capsid protein p24 were examined with their corresponding monoclonal antibodies by immunohistochemistry.

RESULTSIn the biopsy group, cytomegalic virus (CMV) infection was found in one (1/14) case, outer membrane protein gp120 and/or capsid protein p24 antigen were detected in Kupffer cells and in some of the lymphocytes in 11 cases. All the hepatocytes were negative for outer membrane protein gp120 and capsid protein p24 antigens. In the autopsy group, there were 5 (5/12) cases of liver tissues with CMV infection and 5 cases each with mycobacterium and Toxoplasma gondii infection. Capsid protein p24 was detected in liver tissues in 3 cases.

CONCLUSIONThere is HIV infection in liver tissue of patients with HIV. The rate of opportunistic infections in liver biopsy samples was lower than that in the autopsy liver tissues of patients with HIV.

Adult ; Female ; HIV Core Protein p24 ; biosynthesis ; genetics ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; HIV Infections ; pathology ; Humans ; Liver ; pathology ; Male ; Middle Aged

OBJECTIVETo express the Gag protein of HIV-1 strain CN54 in Pichia pastoris (P.pastoris), optimize fermentation parameters and purify Gag antigen.

METHODSThe Gag gene was subcloned into downstream of aox1 promoter of Pichia expression vector pPS1.0, an integrative vector which possesses an identical 5' untranslated region as the natural aox1 gene and employs both in vitro construction and in vivo selection for multi-copy integrants. The recombinant vector was introduced into P.pastoris strain GS115 by electroporation and selected with G418 for Gag gene integration. Super G418 resistant clones were selected and screened for Gag expression. The engineered P.pastoris was cultured to high cell density (>300 A600 Units/ml) in a 5L fermentor. Through methanol induction, the expression level of Gag reached 120 mg/L. Intracellularly expressed Gag was released by high-pressure homogenization and purified through Sepharose FF and DEAE Sepharose FF column chromatography, the purity of Gag reached up to 90%.

RESULTSWestern-blotting suggested that purified Gag expressed in P.pastoris could react specifically with serum from HIV infected individual.

CONCLUSIONGag antigen expressed in P.pastoris has provided a good basis for the development of a new generation of HIV vaccine candidates against some Chinese prevalent strains.

Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Fermentation ; Gene Expression ; HIV Core Protein p24 ; biosynthesis ; genetics ; isolation & purification ; HIV-1 ; genetics ; metabolism ; Humans ; Pichia ; genetics ; metabolism

New strategies to improve vaccine efficacy against human immunodeficiency virus type 1 (HIV-1) are still required. DNA vaccines, exhibiting potential advantages over conventional vaccines for their simplicity and versatility, can induce specific humoral and cellular immune responses. We developed a recombinant pVAX1 DNA vector carrying p24 gene of HIV-1. The results showed that pVAX1 mediated gene possessed the ability of effective expression in both transfected 293T cells and BALB/c mice. And pVAX1-p24 DNA prime and boost immunization can induce significant P24-specific humoral immune responses and cellular immune responses in BALB/c mice. Furthermore, immunization with pVAX1-p24 DNA prime and protein boost induced 7.3 to 8.0-fold greater p24-specific humoral responses than pVAX1-p24 DNA prime and boost, while the cellular immune responses induced by combined immunization was lower. The results suggested that pVAX1-p24 DNA and P24 protein vaccine is a promising HIV-1 vaccine, and the selections of the immunization strategies are important for the immunization results.
AIDS Vaccines ; genetics ; immunology ; Animals ; DNA ; genetics ; immunology ; HEK293 Cells ; HIV Core Protein p24 ; genetics ; immunology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; genetics ; immunology

OBJECTIVETo establish a specific and sensitive Enzyme-linked immunosorbent Assay (ELISA) kit for detection of HIV-1 antibody in urine using Escherichia coli expression products as coating antigen.

METHODSThe truncated HIV-1 gp41 gene fragment of major antigenic epitopes was inserted into the plasmid pET22b to obtain expression plasmid pET22b-mgp41. The recombinant antigen was expressed in BL21 (DE3) strains of Escherichia coli and was purified by immobilized metal chelation and gel filtration chromatography. Using this antigen as coating antigen, a HIV-1 urine antibody ELISA kit was developed. In order to examine the clinical utility of the kit, 5437 urine samples were assayed, which consisted of 641 urine samples from HIV infected patients and 4796 samples from normal subjects. Results The purity of purified antigen is up to 95%. Anti-HIV antibodies were detected in all the urine samples from HIV infected patients, and the diagnostic sensitivity for HIV-1 infection was 100%. In healthy control group, 71 cases showed false positive, the specificity was 98.52%.

CONCLUSIONThe HIV-1 urine antibody kit can be used in screening and diagnosing for HIV-1 infection.

Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; HIV Antibodies ; urine ; HIV Core Protein p24 ; genetics ; immunology ; isolation & purification ; HIV Infections ; immunology ; urine ; HIV-1 ; genetics ; immunology ; Humans ; Recombinant Proteins ; genetics ; immunology ; isolation & purification

BACKGROUND: Early diagnosis of HIV infection reduces morbidity and mortality. Fourth-generation HIV detection assays are more sensitive because they can detect p24 antigen as well as anti-HIV antibodies. In this study, we evaluated the performance of a new fourth-generation ADVIA Centaur HIV antigen/antibody combo (CHIV) assay (Siemens Healthcare Diagnostics Inc., USA) for early detection of HIV infection and reduction of false positive rate. METHODS: Four seroconversion panels were included. The third-generation ADVIA Centaur HIV 1/O/2 enhanced (EHIV) assay (Siemens Healthcare Diagnostics Inc., USA) and fourth-generation CHIV assay were used to test each panel for HIV infection. The presence of antigen was confirmed using HIV p24 antigen assay. To evaluate false-positivity and specificity, 54 HIV false-positive and HIV-negative serum samples from 100 hospitalized patients and 600 healthy subjects were included. RESULTS: Compared to the EHIV assay, the CHIV assay had a shorter window for three of the seroconversion panels: a difference of 10 days and two bleeds in one panel, and 4 days and one bleed in the other two panels. Only 34 of the 54 (63%) samples known to yield false-positive results by EHIV assay had repeatedly yielded reactive results in the CHIV assay. One of the 600 healthy subjects had a false-positive result with the CHIV assay; thus, the specificity was 99.85% (699/700). CHIV accurately determined the reactive results for the HIV-confirmed serum samples from known HIV patients and Korea Food & Drug Administration (KFDA) panels. CONCLUSIONS: The new fourth-generation ADVIA Centaur HIV assay is a sensitive and specific assay that shortens the serological window period and allows early diagnosis of HIV infection.
False Positive Reactions ; Female ; HIV Antibodies/*blood/immunology ; HIV Core Protein p24/*blood/immunology ; HIV Seropositivity/*diagnosis ; Humans ; Male ; Pregnancy ; Reagent Kits, Diagnostic ; Republic of Korea ; Sensitivity and Specificity ; Time Factors

OBJECTIVETo detect p24 antigen of human immunodeficiency virus (HIV)-1 in the liver biopsy specimens of patients with HIV infection.

METHODSLiver biopsy samples from 14 patients with HIV/AIDS (11 man, 3 women; age range 27-52 years; infection time range 8-13 years) were examined by immunohistochemistry prospectively.

RESULTSIntracellular expression of HIV-1 p24 antigen was detected in Kupffer cells, endothelial cells and hepatocytes. There were more HIV-positive liver cells in the patients with severer liver damage than those with milder liver damage (t=2.5189, P=0.0270).

CONCLUSIONThese findings indicate that HIV-1 could replicate in the liver of HIV-infected patients and might be related to the liver cells apoptosis.

Adult ; Female ; HIV Core Protein p24 ; biosynthesis ; HIV Infections ; metabolism ; virology ; HIV-1 ; physiology ; Hepatocytes ; metabolism ; pathology ; Host-Pathogen Interactions ; Humans ; Immunohistochemistry ; Liver ; metabolism ; pathology ; virology ; Male ; Middle Aged