Western Blot Assay For Detection Research Articles

Downregulation of Cathepsin B Reduces Proliferation and Inflammatory Response and Facilitates Differentiation in Human HaCaT Keratinocytes, Ameliorating IL-17A and SAA-Induced Psoriasis-Like Lesion.

Psoriasis is a common inflammatory dermatology disease. Strongly expressed serum amyloid A (SAA) promotes psoriasis exacerbation through inducing IL-17 secretion. What's more, SAA can stimulate the release of cathepsin B. The current work was performed to demonstrate the specific effects of cathepsin B silencing on inflammatory response, proliferation, and differentiation of IL-17A and SAA-induced keratinocytes and to report the precise role of cathepsin B in psoriasis-like lesion. HaCaT keratinocytes received treatment with IL-17A (0, 10, 50, 100 ng/ml) or SAA (0, 1, 5, 10, 20 μg/ml) for 24 h to establish psoriasis-like keratinocytes model. HaCaT keratinocytes were transfected with small interfering RNA (siRNA)-cathepsin B for the functional experiments. Cathepsin B mRNA and protein levels were separately assessed by performing RT-qPCR and Western blot analysis. Then, CCK-8 for detection of cell proliferative capacity and Western blot assay for detection of Ki67 and PCNA expression were adopted to evaluate the influence of silenced cathepsin B on proliferation of IL-17A/SAA-induced HaCaT keratinocytes. Furthermore, IL-6, IL-1β, TNF-α, and p-NF-κB p65 were detected to assess the effects of cathepsin B knockdown on inflammatory response in IL-17A/SAA-induced HaCaT keratinocytes. In addition, assessment of KRT10, FLG, and LOR levels were applied to analyze the function of cathepsin B silencing on differentiation of IL-17A/SAA-induced HaCaT keratinocytes. Cathepsin B expression is distinctly elevated in IL-17A/SAA-induced HaCaT keratinocytes. IL-17A or SAA treatment enhanced proliferation, promoted the release of inflammatory factors, and arrested differentiation in HaCaT keratinocytes. Furthermore, downregulation of cathepsin B reduced proliferation, suppressed inflammatory response, and boosted differentiation in IL-17A/SAA-induced HaCaT keratinocytes. To sum up, cathepsin B silencing rescued excessive proliferation and inflammatory response and scarce differentiation in HaCaT keratinocytes induced by IL-17A and SAA. These findings prompted that cathepsin B might be a promising therapeutic target for psoriasis-like lesion, which helps to develop an anti-psoriatic agent.

Study on Leukemia Cell Differentiation and Drug Resistance By SATB1 Gene

Background and objective:In this article, four types of lymphoma cell lines U937, Raji, Hut-102, Akata, and three types of leukemia cell lines K562, HL-60, Jurkat were used the expression of SATB1 in leukemia cells, Through cell differentiation induced by all-TRANS Retinoic acid and DMSO, finds the SATB1 gene expression is reduced, suggest that the SATB1 gene may be involved in cell differentiation. By gene silencing, reduce the SATB1 gene expression, sensitivity to chemotherapy drugs is increased, suggest that the SATB1 gene may be associated with drug resistance.Methods:1. Western Blot Assay for detection of SATB1 gene expression in cell lines.2. DMSO and ATRA to HL-60, Jurkat cells for different times, SATB1 gene expression in the cells are detected by Western Blot.3. Building SATB1-shRNA, transfected Jurkat cells and to HL-60.4. Testing inhibition rate of chemotherapy drugs on HL-60, HL-60-SATB1-ctr, HL-60-SATB1-sh, Jurkat, Jurkat-SATB1-ctr, Jurkat-SATB1-sh cell, suggesting its relationship with the drug resistance.Results:1. SATB1 gene was expression in all the plant cells.2. The expression of SATB1 in U937, Raji, Hut-102, Akata four types of lymphoma cell line is low.3. It was highly expression in HL-60, Jurkat cells, but low in K562.4. DMSO and ATRA treat HL-60, Jurkat cells for 48 and 96 hours, HL-60 cells become larger, rounded, Jurkat cells into smaller, is more obvious for 96-hour.5. For HL-60, Jurkat cells, after DMSO and ATRA treated, the expression of SATB1 was decreased, more obvious for 96-hour.6. HL-60-SATB1-sh1, Jurkat-SATB1-sh1 cell compared with the control group, the SATB1 protein content decreased significantly, successfully building SATB1-shRNA HL-60 and Jurkat cell line.7. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity has improved significantly.Conclusion:1. The SATB1 gene expression in leukemia and lymphoma cells, and higher expression in leukemia cells.2. After DMSO and ATRA treatment, the SATB1 expression significantly reduced, suggest that SATB1 may be involved in cell differentiation.3. Successfully built SATB1-shRNA, and successfully transferred to HL-60 and Jurkat cell lines.4. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity increased significantly, prompting SATB1 may be related to drug-resistance. DisclosuresNo relevant conflicts of interest to declare.

Comparison of Surface-Plasmon-Resonance Biosensor Measurements and Western Blot Assay for Detection of GPE- Strain of Classical Swine Fever Virus by Using WH211 and WH303 Monoclonal Antibodies

Monoclonal antibodies WH211 and WH303 are in routine laboratory use for detecting Classical Swine Fever Virus (CSFV) or its major envelope E2 protein. While E2 is recognized as the most immunogenic protein, it is also the most variable and often not recognized by specific monoclonal against this protein. The aim of this study was to compare the detection sensitivity of WH211 and WH303 antibodies for CSFV GPE- strain via two well-known assays, Surface Plasmon Resonance Biosensor and Western Blot Assays. Both WH211 and WH303 Abs which specifically known to detect E2 gene (gp55) of CSFV at different recognition sites (epitope) were used as ligands to detect the GPE- strain (analytes). GPE− strain showed interaction with monoclonal antibodies at highest dilution of 1:1000 (v/v) in SPR assay. At lowest dilution (1:10), the interaction of GPE− strain with immobilized monoclonal antibody WH211 showed more than two-fold increase (163.5 RU) than the interaction with monoclonal antibody WH303 (60.0 RU). This study documented profound preference for WH211 as the target for CSFV E2 gene by having higher sensitivity towards GPE− strain but with lesser affinity. E2 epitope of GPE− strain was found undetectable when blotted with WH303 at the investigated dilutions as compared to WH211. The findings indicated a 2500-fold higher SPR sensitivity in detection in comparison to Western Blot and the limit of detection of GPE− strain by Western Blot could not be achieved beyond 1:10 dilution of monoclonal antibodies. Therefore, SPR approach could overcome the risk of GPE− vaccine strain from being invisible for identification. Although, WH303 was unable to recognize this strain by Western Blot, both WH211 and WH303 were applicable as a sensitive detection ligand for GPE- strain of CSFV using SPR analysis.

Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera

The present study describes the cloning and expression of ORF-2 region of TTV genome and the use of expressed peptide in developing immunoassay for detection of anti-TTV antibodies in serum. Presence of TTV-DNA in serum was detected by PCR amplifying N-22 region of ORF-1 of TTV genome. This was followed by genotyping of TTV by RFLP using N-22 amplicon. Using genotype-1 positive serum as the source of TTV, the ORF-2 region was amplified by PCR and subsequently cloned and expressed in pET-19b vector. The expressed protein, identified as 20 kDa protein on SDS-PAGE gel, was purified by affinity chromatography and then used as antigen to develop western blot assay for detection of anti-TTV antibodies in serum. Analysis of sera for anti-TTV antibodies and their comparison with presence of TTV-DNA, produced encouraging results. There was a good relation between presence of anti-TTV and TTV-DNA in these sera samples. Anti-TTV antibodies could be detected in all TTV-DNA positive sera irrespective of the presence of TTV-genotype. This investigation demonstrates that ORF-2 peptide may be used in developing immunoassay for identification of TTV infection.

Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43

The nucleocapsid (N) gene of human coronavirus strain OC43 (HCoV-OC43) was amplified by reverse transcriptase-polymerase chain reaction, and cloned in pENTR™/D-TOPO ® plasmid. This plasmid containing the N gene was recombined with in a BaculoDirect™ baculovirus DNA designed in order to express N protein in fusion with a C-terminal polyhistidine tag containing V5 epitope. Sf21 cells were transfected with recombinant baculovirus DNA. Recombinant N protein was extracted from infected cells, analysed by SDS-PAGE and Western blot, and purified by Ni 2+ affinity procedure. Sera from 100 healthcare workers and five 2–3-year-old children were tested in a Western blot assay using the purified recombinant N protein. All of the sera from adults and two of the sera from children have a positive result.

Assessment and Modification of a Western Blot Assay for Detection of Central Nervous System Tissue in Meat Products in the United States

Health hazards associated with meat contaminated by the bovine spongiform encephalopathy agent have led to the development of tests for the presence of this agent. The objective of this study was to optimize a neuron-specific enolase Western blot assay for use in the United States. We compared the original test with a modified protocol to evaluate the detection limit for the presence of central nervous system (CNS) tissue in experimentally inoculated samples and compared and evaluated the utility of these tests for detecting CNS tissue in retail sausages. Sensitivity and specificity of the original and modified protocols were evaluated using the kappa statistic to assess agreement between the results of the two protocols. The original protocol resulted in 100% specificity and 92% sensitivity for raw samples and 92% specificity and 72% sensitivity for cooked samples. The modified protocol resulted in 92% specificity and 89% sensitivity for raw samples and 83% specificity and 75% sensitivity for cooked samples. The kappa statistic for protocol comparison was 0.94 for raw samples and 0.74 for cooked samples. Both protocols correctly identified CNS tissue in positive controls for each replicate. Although the Western blot technique should be considered for screening for the presence of bovine CNS tissue in meat samples, the techniques should be further optimized to address problems of low sensitivity. A test with higher sensitivity is needed to protect consumers from food safety threats associated with bovine CNS tissue.

Characterization of monoclonal antibody against SARS coronavirus nucleocapsid antigen and development of an antigen capture ELISA

This report describes the production of several MAbs against N195 protein, a major immunodomain of SARS CoV nucleocapsid protein [He, Q., Chong, K.H., Chang, H.H., Leung, B., Ling, A.E., Wei, T., Chan, S.W., Ooi, E.E., Kwang, J., 2004. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11 (2) 417–422.]. One representative IgG1 monoclonal antibody (MAb), S-A5D5, was selected and characterized. S-A5D5 reacted specifically react with both recombinant and native nucleocapsid protein of SARS CoV. The reactivity of S-A5D5 with purified N195 protein and utilization of the MAb as a detector antibody to develop an antigen capture ELISA was assessed. As little as 37.5 pg of purified N protein and 50 TCID 50 of SARS CoV could be detected by the antigen capture ELISA. Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The N protein in N195-spike recombinant baculovirus-infected Sf-9 cells could also be identified. N protein was detected in 18 IFA IgM-positive serum samples collected from SARS confirmed patients, but not in nine samples collected from SARS recovery patient. No false positive results were given when 60 samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (IBV), chicken coronavirus, was tested. This monoclonal antibody-based antigen capture ELISA is thus a powerful tool for early diagnosis of SARS CoV infection.

Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.

A viral transmembrane recombinant protein-based enzyme-linked immunosorbent assay for the detection of bovine immunodeficiency virus infection

The expression of bovine immunodeficiency virus (BIV) truncated transmembrane envelope protein (designated hereafter tTM) in insect cells has been described previously (Abed, Y., St-Laurent, G., Zhang, H., Jacobs, R.M., Archambault, D., 1999. Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane proteins expressed from recombinant baculovirus. Clin. Diagn. Lab. Immunol. 6, 168–172). In this study, a tTM-based enzyme-linked immunosorbent assay (ELISA) was developed for the serodetection of BIV infection. A total of 109 bovine sera including 86 BIV-negative and 23 BIV-positive serum samples were tested. The ELISA results were compared with those of three Western blot assays using, as test antigens, cell culture-derived whole virus proteins (WB1), and the tTM (WB2) and p26 (WB3) fusion proteins expressed from recombinant baculovirus in insect cells, respectively. The concordances of the ELISA results with those of the WB1, WB2, and WB3 were 97.2, 100 and 97.2%, respectively. The tTM protein-based ELISA and Western blot permitted the detection of BIV infection in cattle whose sera failed to react with the p26 fusion protein and the whole virus protein preparation. The tTM recombinant protein was also used to study the kinetics of appearance of antibodies against BIV transmembrane envelope protein in rabbits infected experimentally with BIV. Antibodies to tTM were detected at 28 days post-infection and persisted through the entire 36–39.5 months experimental time period. The results of this study showed that the tTM-ELISA might be useful for the serodetection of BIV-infected animals, and for basic studies on BIV replication life cycle.

Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress.

The hepatotoxicity of acetaminophen is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the mechanism by which this metabolite produces the toxicity is unknown. The metabolite, which is both an electrophile and an oxidizing agent, may covalently bind to critical proteins, or it may initiate oxidative damage. We have previously developed a Western blot assay for detection of acetaminophen covalently bound to protein and have reported the relationship between covalent binding and the development of hepatotoxicity. Recently, we developed a Western blot assay for protein aldehyde formation, which may occur via the reactive oxygen species, the hydroxyl radical. In this paper, we have compared covalent binding to protein aldehyde formation. Toxic doses of acetaminophen (400 mg/kg) were administered to mice, and the mice were subsequently killed at 0, 1, 2, 4, and 6 h. Since the oxidizing agent FeSO4 has been reported to potentiate lipid peroxidation when administered with acetaminophen, other mice received FeSO4 (100 mg/kg) plus acetaminophen. Compared to saline-treated control mice, acetaminophen treatment significantly increased serum alanine aminotransferase levels, an index of hepatotoxicity, at 4 and 6 h, but not at 1 or 2 h. Acetaminophen plus FeSO4 treatment of mice significantly increased serum alanine aminotransferase levels at 2, 4, and 6 h compared to controls. Levels of alanine aminotransferase in serum of acetaminophen plus ferrous sulfate-treated mice were higher at 4 and 6 h than those of acetaminophen-treated mice, but not significantly different. FeSO4 alone did not increase alanine aminotransferase levels. Western blot assays revealed that acetaminophen did not cause an increase in protein aldehydes over control at any time, nor did acetaminophen plus FeSO4; however, FeSO4 alone increased the intensity of staining of the immunoblot for protein aldehydes over control at all times after 0 time. Acetaminophen-protein adducts were detected in acetaminophen- and acetaminophen plus FeSO4-treated mice. In vitro experiments indicated that FeSO4 plus tert-butyl hydroperoxide in the presence of bovine serum albumin increased protein aldehyde formation. Inclusion of acetaminophen in the incubation mixture inhibited protein oxidation of bovine serum albumin in a concentration dependent manner. The data indicate that acetaminophen quenches protein oxidation, presumably by reacting with the hydroxyl radical. These data are consistent with the theory that acetaminophen covalent binding is the primary mechanism of toxicity and argue against a role for protein oxidation in acetaminophen hepatotoxicity.

Analysis of the serologic relationship among San Miguel sea lion virus and vesicular exanthema of swine virus isolates. Application of the western blot assay for detection of antibodies in swine sera to these virus types.

Caliciviruses are positive-sense single-stranded RNA viruses with a single capsid protein. The serotypes of the marine mammal calicivirus, San Miguel sea lion virus (SMSV), are antigenically related to vesicular exanthema of swine virus (VESV) and are potentially hazardous to swine. Western blot assays using purified SMSV serotypes 1 and 4 were used to further examine the serologic relationship among SMSV and VESV isolates. With the exception of SMSV 8 and SMSV 12, rabbit polyclonal antisera generated against all the available SMSV and VESV isolates reacted positively, as assessed by western blot, with purified capsid protein from SMSV 1 and SMSV 4. Consequently, the SMSV 8 and SMSV 12 virus isolates may not be members of the SMSV/VESV calicivirus group. Using antisera from pigs experimentally inoculated with SMSV and VESV as positive controls, a western blot assay for these virus types was utilized to check for the presence of antibodies to calciviruses in swine sera. Sera from colostrum-deprived gnotobiotic pigs were used as a negative control in all experiments. Examination of sera from domestic and feral swine collected in Iowa, California, and Florida was completed using this technique. The presence of antibodies to these virus types was not detected in any of the porcine sera tested.