OBJECTIVETo identify a cost-efficient alternative antibody testing strategy for screening and confirmation of HIV infection by rapid simple tests (RSTs) and enzyme-linked immunosorbent assays (ELISAs).

METHODSFour RSTs (RST1, RST2, RST3, and RST4) and five ELISAs (ELISA1, ELISA2, ELISA3, ELISA4, and ELISA5) were evaluated in two phases by using banked and serum specimens prospectively collected at regional hospitals and voluntary counseling and testing (VCT) centers in Beijing. A total of 200 banked serum specimens were included in the first phase, including 62 HIV-positive, 127 HIV-negative and 11 indeterminate specimens. All specimens were tested by four RSTs and five ELISAs respectively. The second phase involved prospective testing of 389 routine specimens, including 92 HIV-positive, 287 HIV-negative, and 10 indeterminate specimens. All the specimens were tested by two RSTs (RST2 and RST4) and three ELISAs (ELISA1, ELISA3, and ELISA4), which were selected for their respective excellent sensitivity and/or specificity. Western blot (WB) was used as a gold standard for confirming the reactivity of all the specimens.

RESULTSSensitivity, specificity, and efficacy were calculated for each assay in two phases. In the first phase, four assays (ELISA4, RST2, RST3, and RST4) had a specificity of 100%. For the determination of efficacy, ELISA4, RST2, and RST4 were selected in the second phase. ELISA1 and ELISA3 which have a sensitivity of 95.9% and 93.2% respectively also entered this phase. In the second phase, all the five assays (ELISA1, ELISA3, ELISA4, RST2, and RST4) had a sensitivity and specifity of over 90%. ELISA1 had a sensitivity of 99% and ELISA4 a specificity of 99%.

CONCLUSIONThe sensitivity ELISA1 and the specificit of ELISA4 are comparable to ELISA/WB standard strategy. Application of this alternative testing strategy provides a cost-effective method for determining HIV prevalence in Beijing.

Blotting, Western ; methods ; China ; Enzyme-Linked Immunosorbent Assay ; methods ; HIV Infections ; diagnosis ; Humans ; Sensitivity and Specificity

OBJECTIVETo search for a new method of screening for molecular targets for androgen-dependent prostate cancer.

METHODSWe collected tissue samples and paired serum samples from 3 cases of androgen-dependent prostate cancer (ADPC) treated by surgical resection, and included another 3 samples of benign prostatic hyperplasia (BPH) tissue and normal human serum in the control group. The total proteins extracted were separated and transmembrane by two-dimensional gel electrophoresis, followed by hybridization with the sera of the patients with ADPC and those with hormone-independent prostate cancer (HIPC) as the primary antibodies. The differentially expressed proteins were compared by Western blot, analyzed by MALDI-TOF-MS mass spectrography, and verified by RT-PCR and Western blot following bioinformatic identification.

RESULTSThis modified method exhibited a significantly better effect in displaying differentially expressed proteins, by which 12 differentially expressed protein spots were identified, including Beclin1, glutathione S-transferase P (GSTP1-1), ZBTB7, dihydrodiol dehydrogenase 2 (DDH), enolase (ENO1), glucose-dependent insulin-releasing peptide receptor (GIPR), Mn-superoxide dismutase (MnSOD), phosphoglycerate mutase 1 (PGAM1), amino-peptidyl-prolyl cistrons isomerase (PPIA), and phospholipid-PE-binding protein (PEBP). The mRNA and protein expressions of Beclin1 were significantly down-regulated in androgen-dependent prostate cancer tissues.

CONCLUSIONThis modified serum-guided immunoblotting technique has provided a new method for clarifying the molecular mechanisms of the occurrence and progression of HIPC, in which Beclin1-mediated autophagy may play a key role.

Biomarkers, Tumor ; blood ; Blotting, Western ; Humans ; Immunoblotting ; methods ; Male ; Mass Spectrometry ; Prostatic Neoplasms ; genetics ; metabolism ; Proteomics

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.
Blotting, Western/methods ; Electrophoresis, Agar Gel/methods ; Human ; Korea ; Lymphokines ; Plants/immunology ; Pollen/analysis/*immunology ; Skin Tests/methods

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.
Blotting, Western/methods ; Electrophoresis, Agar Gel/methods ; Human ; Korea ; Lymphokines ; Plants/immunology ; Pollen/analysis/*immunology ; Skin Tests/methods

In Papua New Guinea, the laboratory diagnosis of HIV infection is based on proof of HIV antibody in the patient's serum. Under the government scheme, the testing is done in 30 laboratories, including the Papua New Guinea HIV Reference Laboratory (NRL), the Red Cross Blood Transfusion Service in Port Moresby, and 19 provincial and 9 district laboratories. An alternative testing strategy was adopted in 1993 based on a WHO recommendation, replacing the classical testing strategy (enzyme immunoassay + Western blot). The alternative testing strategy uses several EIA, rapid or simple HIV antibody assays for the detection and confirmation of the HIV antibody. This approach is faster and cheaper, with the same sensitivity and specificity as the classical testing algorithm. Except for the NRL, the Serodia Fujirebio HIV-1 gelatin particle agglutination assay is used throughout the country as the screening test. The PNG National HIV Reference Laboratory is the only laboratory authorized to perform confirmatory testing and to release positive results. Therefore, all serum samples reactive in the screening assay are sent to the NRL for confirmation by the battery of EIA, rapid or simple assays in accordance with the alternative testing strategy adopted. The paper explains the alternative testing strategy and highlights the principle of each individual test that is employed. PIP: In Papua New Guinea, HIV antibody testing is performed in 19 provincial and 9 district laboratories, the HIV National Reference Laboratory, and the Port Moresby Red Cross Blood Transfusion Service. Before 1993, enzyme immunoassay and Western blot were used for HIV serotesting and positive findings were sent to Australia for confirmation. Since 1993, the Serodia Fujirebio HIV gelatin particle agglutination assay has been used as the first screening test, followed by the enzyme-linked immunosorbent assay; the third test used for repeatedly reactive samples is generally the Immunocomb. All repeatedly positive results are forwarded to the reference laboratory for confirmation. Results are available within 7 days. In Papua New Guinea, the specificity of the Serodia Fujirebio test is consistently greater than 99%.
AIDS Serodiagnosis - methods ; Blotting, Western - methods ; Enzyme-Linked Immunosorbent Assay - methods ; Fluorescent Antibody Technique ; HIV Infections - diagnosis ; Papua New Guinea ; Polymerase Chain Reaction - methods ; Sensitivity and Specificity

BACKGROUND: In oxidative stress, reactive oxygen species (ROS) production contributes to cellular dysfunction and initiates the apoptotic cascade. Autophagy is considered the mechanism that decreases ROS concentration and oxidative damage. Propofol shows antioxidant properties, but the mechanisms underlying the effect of propofol preconditioning (PPC) on oxidative injury remain unclear. Therefore, we investigated whether PPC protects against cell damage from hydrogen peroxide (H₂O₂)-induced oxidative stress and influences cellular autophagy. METHOD: COS-7 cells were randomly divided into the following groups: control, cells were incubated in normoxia (5% CO₂, 21% O₂, and 74% N₂) for 24 h without propofol; H₂O₂, cells were exposed to H₂O₂ (400 µM) for 2 h; PPC + H₂O₂, cells pretreated with propofol were exposed to H₂O₂; and 3-methyladenine (3-MA) + PPC + H₂O₂, cells pretreated with 3-MA (1 mM) for 1 h and propofol were exposed to H₂O₂. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) reduction. Apoptosis was determined using Hoechst 33342 staining and fluorescence microscopy. The relationship between PPC and autophagy was detected using western blot analysis. RESULTS: Cell viability decreased more significantly in the H₂O₂ group than in the control group, but it was improved by PPC (100 µM). Pretreatment with propofol effectively decreased H₂O₂-induced COS-7 cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol during apoptosis. Western blot analysis showed that the level of autophagy-related proteins was higher in the PPC + H₂O₂ group than that in the H2O2 group. CONCLUSION: PPC has a protective effect on H₂O₂-induced COS-7 cell apoptosis, which is mediated by autophagy activation.
Animals ; Apoptosis* ; Autophagy* ; Blotting, Western ; Cell Survival ; COS Cells* ; Hydrogen Peroxide ; Methods ; Microscopy, Fluorescence ; Oxidative Stress ; Propofol* ; Reactive Oxygen Species

OBJECTIVETo study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure.

METHODSFP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index.

RESULTSCriteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

CONCLUSIONEstablishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China.

Blotting, Western ; methods ; Borrelia burgdorferi Group ; pathogenicity ; China ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Humans ; Lyme Disease ; diagnosis ; microbiology

OBJECTIVETo investigate the optimum proportion of Mosaicplasty and genes-enhanced tissue engineering for the repair of acute osteochondral defects.

METHODSWestern blot test was conducted to detect the expression of hTGF-beta1, Col II and Aggrecan in 3 groups, including hTGF-beta1, transduction group, Adv-betagal transduction group and control group without transduction. Eighteen 6-month-old Shanghai male goats (weight: 22 to 25 kg) were used. BMSCs were isolated from the autologous bone marrow, and were subcultured to get the cells at passage 3. Thirty-six medial femoral condyles were used and divided into 6 groups named AR, AL, BR, BL, CR, and CL. Acute cylindrical defects (9 mm in diameter and 3 mm in depth)were created in the weight bearing area of the medial femoral condyle of hind limbs. In the single group, the autologous osteochondral mosaicplasty was performed to repair the defect; in the combination group, besides the mosaicplasty, the dead space between the cylindrical grafts and the host cartilage were injected with the suspension of hTGF-beta1, gene enhanced autogenous BMSCs in sodium alginate, and CaCl2 was dropped into it to form calcium alginate gels. The autologous osteochondral transplantation cover rates of group AR was 44.44% single group, AL was 44.44% combination group, BR was 33.33% single group, BL was 33.33% combination group, CR was 22.22% single group, and CL was 22.22% combination group. The goats were killed 24 weeks after operation to receive gross and histology observation, which was evaluated by the histological grading scale of O'Driscoll, Keeley and Salter. Immunohistochemistry and TEM observation were also performed.

RESULTSWestern blot test showed the expression of the hTGF-beta1, Col II and the Aggrecan in the hTGF-beta1 transduction group were significantly higher than that of the Adv-betaga1 transduction and the blank control groups. The gross and histology observation revealed that each defects of six groups had different degrees of repairing. There was no significantly difference among the BL, AR, and AL groups. But the scores of the other three groups (BR, CR, and CL) were significantly poorer than the former three groups.

CONCLUSIONMosaicplasty associated with genes enhanced tissue engineering could repair the osteochondral defects effectively. With the autologous osteochondral transplantation coverage reducing, the advantage of the combination could have a better representation.

Animals ; Blotting, Western ; Bone Diseases ; metabolism ; pathology ; therapy ; Cell Line ; Goats ; Humans ; Immunoprecipitation ; Male ; Tissue Engineering ; methods

OBJECTIVETo study how the choices of the quick vs slow protein transfer, the blotting membranes and the visualization methods influence the performance of Western blotting.

METHODSThe cellular proteins were abstracted from human breast cell line MDA-MB-231 for analysis with Western blotting using quick (2 h) and slow (overnight) protein transfer, different blotting membranes (nitrocellulose, PVDF and nylon membranes) and different visualization methods (ECL and DAB).

RESULTSIn Western blotting with slow and quick protein transfer, the prestained marker presented more distinct bands on nitrocellulose membrane than on the nylon and PVDF membranes, and the latter also showed clear bands on the back of the membrane to very likely cause confusion, which did not occur with nitrocellulose membrane. PVDF membrane allowed slightly clearer visualization of the proteins with DAB method as compared with nitrocellulose and nylon membranes, and on the latter two membranes, quick protein transfer was likely to result in somehow irregular bands in comparison with slow protein transfer. With slow protein transfer and chemiluminescence for visualization, all the 3 membranes showed clear background, while with quick protein transfer, nylon membrane gave rise to obvious background noise but the other two membranes did not.

CONCLUSIONSDifferent membranes should be selected for immunoblotting according to the actual needs of the experiment. Slow transfer of the proteins onto the membranes often has better effect than quick transfer, and enhanced chemiluminescence is superior to DAB for protein visualization and allows highly specific and sensitive analysis of the protein expressions.

Blotting, Western ; instrumentation ; methods ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Humans ; Membranes, Artificial ; Proteins ; analysis

BACKGROUND: For donor samples showing reactive results in a human T-cell lymphotropic virus (HTLV) antibody test along with indeterminate results in Western blot assay, HTLV nucleic acid amplification test using laboratory-developed polymerase chain reaction (PCR) was performed. It is necessary to construct an adequate internal control (IC) to evaluate the accuracy of the results since we did not use an IC in the laboratory-developed PCR. METHODS: As a competitive IC, plasmid DNA containing the primer recognition sequence for amplification of the HTLV pX region was constructed. We determined the adequate concentration of the IC, which was added to the samples to evaluate the accuracy of the test results. RESULTS: When the plasmid DNA was added to the HTLV-positive samples, the amplified product of IC (400 bp) was detected with the HTLV gene (230 bp). The adequate concentration of plasmid DNA added as an IC was 1 pg. CONCLUSION: The construction of plasmid DNA as a competitive IC is an efficient method to evaluate accuracy of the test results. However, the production process for the competitive IC must be further developed. Therefore, it is necessary to compare with the performance of a non-competitive IC.
Blotting, Western ; DNA ; Humans ; Methods ; Nucleic Acid Amplification Techniques ; Plasmids ; Polymerase Chain Reaction* ; T-Lymphocytes ; Tissue Donors