Western Immunoblotting Technique Research Articles

The deleterious effects of cadmium on oxidative stress markers, drug-metabolizing, and antioxidant enzyme activities: Role of Silymarin and Garlic as Antioxidants.

Exposure to cadmium has been related to liver and kidney diseases such as polycystic and nephrotic syndrome. It is still unclear how cadmium contributes to these diseases. It is believed that the induction of oxidative stress resulting from the inhibition of antioxidant enzyme activities and changes in drug-metabolizing enzymes in the liver could explain the role of cadmium in the development of different diseases in the kidney and probably other organs. Changes in oxidative stress markers, antioxidant enzymes, and drug-metabolizing enzyme activities were assessed in the liver of male rats exposed to cadmium chloride. Additionally, the protective effects of silymarin and garlic extract against cadmium toxicosis were evaluated. Rats were randomly divided into eight groups as follows, groups 1, 2, 3, 4, and 5, received orally saline, CdCl2 (1mg/kg), garlic extract [800mg/kg], silymarin (25mg/kg) and silymarin plus garlic extract respectively for 28 consecutive days. Rats in groups 6, 7, and 8 were pretreated with the same doses of garlic, silymarin, and garlic plus silymarin, respectively for two hours before cadmium administration. The Western immunoblotting technique was used to investigate the protein expression of cytochrome P450 isozymes. Spectrophotometric methods were used to assess the activity of both antioxidant- and drug-metabolizing enzymes. Free radical levels [measured as thiobarbituric acid reactive substances (TBARS)], catalase, superoxide dismutase, and glutathione peroxidase activities increased whereas the levels of glutathione and the activities of glutathione S-transferase, glutathione reductase, and glutamyl transferase, cytochrome P450, aryl hydrocarbon dehydrogenase (AHH), dimethylnitrosamine-N-demethylase I (DMN-dI), 7-ethoxycoumarine-O-deethylase (ECOD), cytochrome b5 and NADPH-Cytochrome-c-reductase enzyme activities decreased after cadmium treatment. Furthermore, Western immunoblotting data revealed that glutathione peroxidase protein expression increased following cadmium exposure, but cytochrome P450 2E1 and 3A4 expressions were downregulated. However, pretreatment of rats with silymarin or garlic extract or both before cadmium administration was found to restore the protein expression of cytochrome P450 2E1 and 3A4, the level of free radicals, antioxidant enzymes, drug-metabolizing enzyme activities to their normal levels. Similarly, histological studies revealed that silymarin and/or garlic extract reduced the liver damage caused by cadmium. Silymarin and/or garlic extract reduced the adverse effects of cadmium on the activity of both drug-metabolizing and antioxidant enzymes activity. These antioxidants could be provided to those who work in cadmium-based sectors to help them cope with the adverse effects of cadmium on their kidneys. In addition, Inhibiting drug-metabolizing enzyme activity should be considered when administering therapeutic medications to persons exposed to cadmium because most therapeutic drugs and many endogenous substances are largely metabolized by these enzymes.

Trigonella stellata reduced the deleterious effects of diabetes mellitus through alleviation of oxidative stress, antioxidant- and drug-metabolizing enzymes activities

Ethnopharmacological relevanceGenus Trigonella has a history of folkloric medicinal uses in China, Japan, Egypt and India. There are a variety of therapeutic actions of Trigonella including hypocholesterolemia, hypoglycemia, antibacterial, antiviral, anti-inflammatory activities, antioxidants and appetite stimulant. Aim of the studyThe prevalence of diabetes mellitus is increasing annually. The present study aims at investigating the protective effects of Trigonella stellata against the adverse effects of diabetes mellitus through investigation of the changes in phase I & II drug-metabolizing enzyme activities, protein expression of cytochrome P450 isoenzymes [CYP2E1 & 3A4], oxidative stress, antioxidant enzymes as well as histopathology of both liver and kidney tissues. MethodsGC-MS and MALDI-TOF were used to analyze the main constituents of the aqueous and the ethanolic extract of Trigonella stellata. Western immunoblotting technique used to show the protein expression of CYP450 isozymes in different groups. Spectrophotometric- and fluorophotometric techniques were also used for assessment of different hepatic integrity enzymes. Histopathological techniques used to illustrate the changes in the tissues of both livers and kidneys after different treatments. ResultsTrigonelline was found to be the main constituent of both aqueous and ethanolic extract of Trigonella stellata. Administration of the aqueous and/or the ethanolic extracts of Trigonella stellata to the diabetic rats was found to decrease the blood glucose level, the biochemical markers of both liver (transaminases activities, Lactate dehydrogenase, gamma-glutamyl transferase) and the renal functions (urea, creatinine and bilirubin) which were increased in diabetic-treated rats relative to their normal levels. Diabetes mellitus potentially induced the oxidative stress, and also activities of dimethylnitrosamine N-demethylase I, cytochrome c-reductase, ethoxyresourfin O-deethylase, and the total hepatic content of cytochrome P450. On the other hand, the activity of catalase [CAT], superoxide dismutase [SOD], glutathione S-transferase [GST], glutathione reductase [GR], glutathione peroxidase [GPx] and levels of reduced glutathione [GSH] were potentially inhibited in diabetic rats compared to the control rats. However, treatments of diabetic rats with either aqueous and/or ethanolic extracts of Trigonella stellata restored such changes caused by diabetes almost nearly to their normal levels compared to the control group. Supporting the activity of dimethyl nitrosamine N-demethylase I activity, the protein expression of CYP2E1 was also induced in diabetic rats. However, the aqueous extract of Trigonella stellata was more effective than ethanolic extract in restoring the changes in the protein expression of CYP2E. On the other hand, the protein expression of CYP3A4 was markedly decreased in diabetic rats, and this decrease was partially restored to its normal level after treatment of diabetic rats with aqueous and/or ethanolic extracts. In addition, Trigonella stellata extracts alleviated the histopathological changes in livers and kidneys caused by diabetes mellitus. ConclusionIt is concluded that diabetes mellitus induced changes in oxidative stress, phase I & II drug-metabolizing enzymes, and antioxidant enzymes activities, whereas both extracts of Trigonella stellata alleviated such changes. Alterations in cytochrome P450 system should be considered when therapeutic drugs are administered to diabetic patients since most of xenobiotic are mainly metabolized by this system.

Alvocidib Potentiates the Activity of Venetoclax in Preclinical Models of Acute Myeloid Leukemia

IntroductionVenetoclax (ABT-199) is an approved BCL-2 inhibitor for the treatment of patients with chronic lymphocytic leukemia (CLL). Multiple clinical trials are underway to explore its efficacy in additional indications. While venetoclax demonstrated high remission rates in combination with azacitidine in early stage clinical trials, the question of durability of responses and primary and acquired resistance remain, especially given the modest activity and rapid development of resistance as a single agent. One reported mechanism of intrinsic resistance is high expression of other BCL-2 family proteins, including MCL-1. We and others have demonstrated that the CDK9 inhibitor, alvocidib, can mediate transcriptional repression of anti-apoptotic MCL-1. It has also been shown that alvocidib can increase pro-apoptotic BIM, a dual activator and sensitizer BH3-only protein that can directly induce apoptosis and simultaneously inactivate anti-apoptotic BCL-2 family proteins such as MCL-1 and BCL-2, thus having the same effect on mitochondria-associated apoptosis as MCL-1 down-regulation, with the potential to directly induce apoptosis. An alvocidib-containing cytotoxic chemotherapy regimen demonstrated favorable remission rates in high-risk AML patients over standard therapy in a randomized Phase 2 trial indicating its potential role and safety in AML. We hypothesized that alvocidib and venetoclax would synergize against AML cells by shifting the overall balance of pro- and anti-apoptotic BCL-2 proteins in favor of apoptosis and thus represent a novel active treatment regimen in AML.AimsThis study seeks to examine the efficacy of a treatment regimen containing alvocidib and venetoclax in multiple preclinical studies, including in vivo models of AML.MethodsCell viability assays interrogating alvocidib and venetoclax activity in cell lines were performed using CellTiter-Glo according to manufacturer's protocol. mRNA/miRNA expression changes were assessed using standard RT-PCR technique. Protein expression changes were assessed using standard western immunoblotting technique. To assess the efficacy of an alvocidib and venetoclax combination on tumor growth in an in vivo model, the OCI-AML3 xenograft mouse model and ex vivo studies with AML patient samples were performed.ResultsHerein we demonstrate that alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion in 3 out of 4 AML cell lines analyzed, while in cells where alvocidib did not reduce MCL-1 protein levels (i.e. MOLM-13) a dose-dependent decrease in miR17-92, and concomitant increase in BIM protein was observed after 24 hours of alvocidib treatment. The alvocidib-venetoclax combination resulted in very strong synergistic reductions of cell viability (with combination indices [CI] of 0.4 to 0.7), both in venetoclax-sensitive and resistant cells. The venetoclax-sensitive lines, MV4-11 and MOLM-13, exhibited 5- to 10-fold reduction of venetoclax EC50 values in the low nM range when combined with only 80 nM alvocidib. Importantly, venetoclax-resistant lines, OCI-AML3 and THP-1, exhibited at least 20-fold reduction of venetoclax EC50 values from near 1 µM to 10-50 nM, when combined with 80 nM alvocidib.In the venetoclax-resistant OCI-AML3 xenograft model, single agent alvocidib and venetoclax achieved tumor growth inhibition (TGI) of 9.7 and 31.5%, respectively, while the combination achieved 87.9% TGI at the same dose levels of individual drugs.ConclusionsTaken together, our data suggest that the combination of alvocidib with venetoclax is highly synergistic in vitro and in vivo, in both venetoclax-sensitive and -resistant AML across a heterogeneous genomic background. The particularly high level of synergy achieved in venetoclax-resistant cell lines highlights the central importance of both BCL-2 and MCL-1-mediated cell survival in AML. Importantly, the addition of alvocidib to venetoclax treatment reduced IC50s to clinically achievable concentrations. Therefore, we conclude that an alvocidib/venetoclax combination may be a novel approach for the treatment of AML and warrants further pre-clinical and clinical validation. DisclosuresWhatcott:Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Mesa:Celgene: Research Funding; Galena: Consultancy; Promedior: Research Funding; Ariad: Consultancy; Novartis: Consultancy; CTI: Research Funding; Incyte: Research Funding; Gilead: Research Funding. Peterson:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

Immunological diagnosis of human hydatid cyst using Western immunoblotting technique.

Background:Echinococcosis is a parasitic disease with worldwide distribution which is caused by the tapeworms Echinococcus granulosus. Diagnosis of the disease relies on imaging techniques, but the techniques are not able to differentiate the cyst from benign or malignant tumors; hence, appropriate serologic methods are required for the differential diagnosis of the infection.Materials and Methods:In this investigation, different sheep hydatid cyst antigens probed with thirty sera of patients with hydatid cyst and also thirty human normal sera using Western immunoblotting technique. Considering results of surgery as gold standard, sensitivity and specificity of Western blotting was estimated.Results:Sera of 29, 26, and 16 patients with hydatid cyst reacted with specific bands of hydatid cyst fluid (HCF), protoscolex crude antigen, and cyst wall crude antigen, respectively. However, none of the normal human sera reacted with those specific bands.Conclusion:A 20 kDa band of sheep HCF is an appropriate antigen for serodiagnosis of hydatid cyst infection.

A Nonglycosylated 27 KDa Molecule as Common Antigen between Human Breast Cancer and <i>Echinococcus granulosus</i> Hydatid Cyst Wall

Background: Hydatid cyst, which has anti-cancer activities, is outwardly covered with the cyst wall. It is in close contact with the host tissues and its molecules presented to the immune system. In this work immunological reaction of the sera of breast cancer patients with the hydatid cyst wall antigens has been investigated. Method: For this purpose, sera of patients with breast cancer, hydatid cyst and normal human sera were collected and their reaction with hydatid cyst wall antigens was tested using western immunoblotting technique. Results: All sera of patients with breast cancer, hydatid cyst and also human normal sera reacted with a band in western immunoblotting. However, sera of patients with breast cancer showed reaction with a 27 KDa band. Results of this work also revealed that this band was not glycosylated and may express only in some stages of breast cancer development. Conclusion: Sera of patients with breast cancer cross reacted with a nonglycosylated antigen of hydatid cyst wall. However, more work is recommended to find if this band involves in anticancer activity of the hydatid cyst.

Distribution of a pathological form of prion protein in the brainstem and cerebellum in classical and atypical cases of bovine spongiform encephalopathy

This study evaluated the distribution and signal intensity of a prion protein resistant to proteolysis (PrP(res)) in the brainstem and cerebellum of cattle affected with classical and atypical forms of bovine spongiform encephalopathy (BSE) using a Western immunoblotting technique. In both classical and atypical cases of BSE, a stronger signal was detected in the more rostral brainstem regions relative to the obex. In classical and H-type cases a significant decrease in the PrP(res) signal was found in the cerebellum when compared to that in the obex, whereas L-type BSE cases were characterised by signals of similar intensity in these regions. The uniform distribution of PrP(res) in the region rostral to the obex suggests that when autolysed samples are being tested for BSE, both classical and atypical forms are detectable, even when this target site is missing or cannot be clearly identified. The findings indicate that both the obex and rostral brainstem can be used for BSE diagnosis whereas use of the more caudal brainstem regions and cerebellum is not recommended.

Characterisation of Theileria parva isolates from Kiambu district, Kenya

Four Theileria parva isolates from Muguga area of Kiambu district, Kenya, were used to establish schizont-infected cell lines. Their protein antigens were then separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS page). The isolates were subsequently subjected to protein analysis and characterisation by the western immunoblotting technique. Probing for the polymorphic immunodominant molecule (PIM) was done using monoclonal antibody no. 4. SDS page detected up to 20 protein antigens of molecular mass 35,000–180,000 Da. The western blot analysis revealed a greater heterogeneity in the molecular mass ( M r) of PIM than previously thought. The M r of PIM varied between 80 and 90 kDa. The isolates further revealed different densities of surface epitopes with variable reaction to the monoclonal antibody. The implications of these findings to the epidemiology of east coast fever and immunisation programmes are discussed.

Evaluation of a Rapid Western Immunoblotting Procedure for the Diagnosis of Bovine Spongiform Encephalopathy (BSE) in the UK

Bovine brain tissue samples from 625 UK cattle, clinically suspected as bovine spongiform encephalopathy (BSE) cases, were used in a blind analysis to assess a rapid Western immunoblotting technique (Prionics Check; Prionics AG, Zurich), which detects bovine disease-specific protease-resistant prion protein (PrPSc). By means of statutory histopathological examination, 599 of the 625 cattle were confirmed as BSE cases by the demonstration of spongiform encephalopathy, the remaining 26 being classified as negative. Duplicate samples from the same animals were also examined by electron microscopy for the presence of abnormal brain fibrils (scrapie-associated fibrils; SAFs). The Prionics technique showed a high sensitivity, particularly when compared with the fibril detection test; the detection rates were 99·3% and 92·0% respectively, with histopathology being used as the “gold standard”. The false negative results by the Prionics test were possibly related to the sampling procedure. Analysis of 50 BSE-positive samples revealed similar glycoprofiles, the majority of PrPScisoforms being di-glycosylated protein. The Prionics test also detected PrPScin the four brain samples from the 26 histopathologically negative animals, apparently reducing the specificity of the test to 84·6%; however, confirmatory positive results in these samples were obtained by demonstrating SAF or by immunohistochemical examination, or both. It was concluded that the Prionics test detected PrPScin a small percentage (0·64%) of clinically suspected BSE cases showing no spongiform change. Since January 2000, the Prionics Western blot test has been introduced as one of the statutory tests for the diagnosis of clinically suspected BSE and scrapie cases in the UK.

Circulating autoantibodies to neuronal and glial filament proteins in autism

Automunumty may be a pathogenic factor in autism, a behavioral disorder of early childhood onset. Circulating autoantibodies are produced in organ-specific autoimmunity; therefore, we investigated them in the plasma of autistic subjects, mentally retarded (MR) subjects, and healthy controls. Autoantibodies (IgG isotype) to neuron-axon filament protein (anti-NAFP) and glial fibrillary acidic protein (anti-GFAP) were analyzed by the Western immunoblotting technique. We found a significant increase in incidence of anti-NAFP (P = .004) and anti-GFAP (P = .002) in autistic subjects, but not in MR subjects. Clinically, these autoantibodies may be related to autoimmune pathology in autism.

Analysis of two constitutive forms of microsomal heme oxygenase in different rat tissues.

To isolate and purify the heme oxygenase (HO) isoform in microsomal fractions of Sprague-Dawley rat liver and brain in order to understand the characteristics of the two constitutive forms and the mechanism of the occurrence of hyperbilirubinemia. After induction by hematin and phenylhydrazine, the rat liver and brain microsomal fractions were isolated and purified by DEAE-Sephacel and hydroxyapatite. Activity and the apparent molecular weight of the two isoforms [heme oxygenase 1 (HO-1) and heme oxygenase-2 (HO-2)] were measured. Kunming mice were used to prepare antiserum against purified liver HO-2. Rat liver HO-1 and brain HO-2 preparations were analyzed by the western immunoblotting technique. Two isoforms were purified and identified in the treated rat liver, and HO-1 was the predominant form with a ratio of 2:1. In the native state, HO-2 activity was detectable but HO-1 activity was increased in response to hematin and phenylhydrazine, while HO-2 activity was fully refractory to these agents. The apparent molecular weights of HO-1 and HO-2 were about Mr 30000 and Mr 36000 under reducing conditions, respectively. In the untreated liver and treated brain, only one peak of HO activity exhibiting an elution profile similar to that of HO-2 of the treated liver was detected. The presence of an activity peak was not found in the elution profile at the region where the inducible isoform of HO (HO-1) was expected. The apparent molecular weight in treated brain preparation was identical to that of the purified liver HO-2. Cross-reactivity of HO-2 in the brain microsomal preparation was established, but a reactivity of HO-1 in the liver was not observed by western immunoblotting analysis when antiserum to liver HO-2 was applied. Two constitutive forms of HO, designated as HO-1 and HO-2, exist in the treated rat liver. HO-1 is an inducible enzyme. In the treated rat brain only HO-2 exists and is a molecular entity similar to that found in liver. The two constitutive forms were different in molecular weight and in inducibility and immunochemical properties.

IgM antibody detection of ppUL80A and ppUL32 by immunoblotting: an early parameter for recurrent cytomegalovirus infection in renal transplant recipients.

The value of IgM detection for the early diagnosis of an active cytomegalovirus (CMV) infection in renal transplant recipients was evaluated prospectively. Sequential serum samples obtained from 22 allograft recipients with active CMV infection were tested for the presence of CMV-specific immunoglobulin M antibodies (IgM) by an enzyme-linked immunosorbent assay (ELISA) and a microparticle enzyme immunoassay (MEIA) and were compared with the Western-immunoblotting technique (IB). The time course of CMV IgM antibody detection was evaluated in relation to the shell vial assay (SVA), CMV disease, and immunosuppressive regimen. By IB, IgM antibodies against the capsid protein ppUL80a and the basic matrix phosphoprotein ppUL32 were detected in all 22 recipients with active CMV infection. Using the MEIA and the ELISA, the presence of CMV IgM antibodies was detected in 17 (77%) and ten (46%) of these 22 recipients, respectively. The SVA was the earliest parameter for detection of primary CMV infection in seven of nine (78%) recipients, in contrast to two of 13 (15%) patients with recurrent CMV infection (P < .05). The detection of IgM antibodies by IB was the earliest parameter for detection of recurrent CMV infection in seven out of 13 (54%) recipients in contrast to one out of nine (11%) patients with primary CMV infection (P < .05). During a primary CMV infection, the development of an abundant IgM antibody response was associated with recovery from CMV disease and the end of the viremic phase.

Pneumocystis cariniifrom pigs and humans are antigenically distinct

The antigens of Pneumocystis carinii cysts isolated from pigs and humans were compared by the Western immunoblotting technique. Convalescent pig serum reacted with two antigens (approximately 78 kDa and 32.5 kDa) of porcine P. carinii cysts, whereas convalescent serum from humans did not react with porcine P. carinii cyst antigens. The results indicate that porcine and human P. carinii cysts are antigenically distinct.

The effect of intravenous immunoglobulin on placental transfer of a platelet-specific antibody: anti-P1A1.

The isolated perfused lobule of human placenta was used as an in-vitro model to study the effect of intravenous immunoglobulin (IVGG) on the placental transfer of a human platelet-specific antibody (anti-P1A1). Normal human IgG was shown to transfer from the maternal to the fetal circulation of the placental model after a lag period of 2-3 h. IVGG also transferred across the placenta but only after a longer lag period (3-4 h) than normal human IgG at the same concentration, which suggests that IVGG may contain a factor that inhibits the transfer of its own component IgG. The sensitive Western immunoblotting technique was used to demonstrate progressive transfer of anti-P1A1 antibody to the fetal circulation after a 2-3 h lag period. When IVGG and anti-P1A1 antibody were added simultaneously to the maternal circulation, the transfer of platelet-specific antibody was strongly inhibited by IVGG. The inhibitory effect of IVGG on anti-P1A1 antibody transfer was consistent for three different batches of the same IVGG product (Sandoglobulin). These studies provide the first scientific data to support the use of IVGG to inhibit antiplatelet antibody transfer as part of the antenatal management of neonatal alloimmune thrombocytopenia.

Sensitivity of the standardized pseudorabies virus neutralization test varies with the test strain used.

The effect of altering the strain of the test virus used in the standardized pseudorabies virus neutralization (VN) test on the sensitivity of the assay was evaluated. Comparative VN tests were performed using 4 different strains: the avirulent Bartha parental, the avirulent recombinant Bartha gIIIKa, the moderately virulent Shope (currently used for the VN test at the National Veterinary Services Laboratory, Ames, IA), and the highly virulent P2208 (Funkhauser). A radioimmunoassay and a Western immunoblotting technique were employed to verify the presence of anti-pseudorabies virus (PrV) antibodies in sera. Statistical analysis indicated that replacement of the Shope strain by the Bartha gIIIKa or the P2208 strain resulted in VN titers that were 4.23- and 2.00-fold higher, respectively. Despite these differences, specificity with regard to PrV diagnosis was unaltered. This apparent enhancement of the sensitivity of the PrV VN test would be beneficial for the serologic identification of PrV-infected animals during an eradication effort.

Regulation of the glucocorticoid receptor in fetal rat lung during development

The effect of the synthetic glucocorticoid betamethasone on the regulation of the glucocorticoid receptor mRNA and on receptor protein was studied in fetal rat lung during development. Using a glucocorticoid receptor cRNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and by solution hybridization. A monoclonal antibody against the glucocorticoid receptor was used to study regulation of the receptor protein by the Western immunoblotting technique. In fetal rat lungs, of 16–21 days of gestation, as well as in adult lungs, betamethasone treatment resulted in a significant decrease of glucocorticoid receptor mRNA to 50–65% of the control level. In contrast, betamethasone treatment did not down-regulate the receptor protein in rat lungs of 16–19 days of gestation, whereas a decrease of glucocorticoid receptor protein to 40–60% of control was seen in lungs of 21 days of gestation, in postnatal and adult lung. These results provide data for a change in regulation in vivo of the glucocorticoid receptor by its homologous ligand in fetal rat lung during development.

Human pancreatic islet cell specific 38 kilodalton autoantigen identified by cytomegalovirus-induced monoclonal islet cell autoantibody

Our previous finding that about 15% of newly diagnosed patients with Type 1 (insulin-dependent) diabetes mellitus had human cytomegalovirus genome in their lymphocytes and islet cell autoantibodies in their sera, suggests that autoimmune Type 1 diabetes is associated with persistent cytomegalovirus infection under certain circumstances. This investigation was initiated to see if cytomegalovirus can induce islet cell autoantibodies and if the autoantibodies react with any specific islet protein(s). Monoclonal antibodies were generated after immunizing Balb/c mice with human cytomegalovirus. When these monoclonal antibodies were tested for the presence of islet cell antibodies were tested for the presence of islet cell antibodies, one (MCMVA-51) of 13 monoclonal antibodies reacted strongly with the islets. The titer of islet cell antibodies was 1:2000. When this monoclonal antibody was reacted with the proteins from the solubilized fraction of human pancreatic islets using the western immunoblotting technique, a band with a molecular weight of 38 kilodalton was detected. The 38 kilodalton band was not observed when the monoclonal antibody was reacted with the proteins prepared from pancreatic islet tissues of rats and mice or from other human organs including stomach, liver, spleen and brain, indicating that the 38 kilodalton protein is human islet cell-specific. It is concluded that human cytomegalovirus can induce islet cell antibodies that react with a 38 kilodalton human islet cell protein and that this protein component may represent islet cell-specific target antigens associated with persistent cytomegalovirus infection.

Identification et purification partielle d’antigènes solubles à partir de culture d’endozoïtes de &lt;em&gt;Besnoitia besnoiti&lt;/em&gt;

Soluble antigens from culture-grown Besnoitia besnoiti endozoites were identified following their partial purification by affinity chromatography. A specific eluate obtained after affinity chromatography on a column to which antibodies from serum of a naturally infected cow were bound exhibited seven polypeptide bands on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Five bands were observed in the eluate from an immunoadsorbent to which antibodies from an experimentally infected calf were coupled. Eluted antigens were reactive in the enzyme-linked immunosorbent assay (ELISA). Reactivity of electrophoretically resolved antigens with sera of endozoite-inoculated cattle and sera from field cases of besnoitiosis were studied using the Western immunoblotting technique.

Identification of the antigenic components of paramyxovirus-3, paramyxovirus-6 and Newcastle disease virus in turkeys

The aim of this study was to use the enzyme-linked immunosorbent assay (ELISA) and the Western immunoblotting as possible tools to differentiate infections in turkeys by different paramyxoviruses. Pooled hyperimmune sera of turkeys infected with either paramyxovirus-3 (PMV-3), paramyxovirus-6 (PMV-6), or Newcastle disease virus (NDV) were assayed for antibodies specific to the three viruses by the ELISA and Western immunoblotting. ELISA results showed cross reactions of turkey antibodies between PMV-3 and PMV-6 antigens, while turkey antibodies to NDV did not cross-react with any of the other paramyxoviruses. The immunoblots of sera from birds infected with PMV-3 (Minnesota turkeys and Iowa chickens) reacted to low molecular weight polypeptides of PMV-3 of 29, 32, and 34 kDa, and to a high molecular weight band of 200 kDa. The same Minnesota turkey sera had a cross reaction to the 200 kDa polypeptide of PMV-6, while the Iowa chicken sera did not. Both sera had no apparent reaction to NDV proteins. Western immunoblotting showed that the turkey PMV-3 sera had a specific reaction to a 220 kDa polypeptide present in PMV-3, but not in PMV-6, while the turkey PMV-6 sera had a specific reaction to a 130 kDa polypeptide present in PMV-6, but not in PMV-3. Immunoblots of pooled sera from turkeys infected with PMV-6 (Minnesota source) reacted to the 200 kDa protein present in both PMV-3 and PMV-6; however, no reaction occurred between this sera and NDV proteins. Sera from turkeys infected with NDV (Minnesota source) reacted to three major polypeptides in NDV, namely: the 58 kDa, 118 kDa, and 158 kDa. This sera had no detectable cross reactions to PMV-3 and PMV-6 in ELISA; however, it showed a reaction to a 75 kDa polypeptide in PMV-3, using the Western immunoblotting technique.

Evaluation of Western immunoblotting technique in the serological diagnosis of human syphilitic infections

Purified human syphilitic antibodies against both 15.5 Kd and 45 Kd treponemal antigens appear T. pallidum specific and do not cross react with antigens possessed by other treponemes (T. phagedenis, T. hyodysenteriae and a human intestinal treponeme). By using Western immunoblotting technique, 107 out of 110 syphilitic patients and 291 out of 294 subjects with serologically positive diagnostic tests for syphilis were found to have in their sera antibodies against a 15.5 Kd specific antigen of T. pallidum. These antibodies were present in 100% of the patient with secondary or early latent syphilis, both untreated and treated, in 98.24% of those with late latent treated syphilis and in 100% of patients with neurosyphilis. On the contrary, they were absent in 47 patients with false positive reactions for syphilis and in 121 healthy blood donors. For these reasons, the demonstration of these kind of antibodies in a patient's serum can be considered of high value in differentiating syphilitic patients from non infected individuals.

The antigen/receptor specificity of antigranulocyte antibodies in patients with SLE

The antigen/receptor specificity of antigranulocyte antibodies (AGAs) detected in the sera of patients with systemic lupus erythematosus (SLE) was investigated by inhibitory immunofluorescence test and Western immunoblotting technique. The interactions of AGAs with antigens of intact normal granulocytes were determined by inhibiting the binding of different myeloid monoclonal antibodies (mAbs). Seven of the studied 12 sera revealed binding to CD15 (X hapten) and/or to CD16 (FcR lo). The specificity investigation of AGAs was completed with Western immunoblotting technique. The binding of AGAs to bands with M r of about 50–60 kDa and at 30 kDa on unstimulated granulocyte plasma membrane preparation could be demonstrated from 4 out of 6 AGA positive SLE sera. The cause of the disappearance of bands on the phorbol-myristate-acetate (PMA) activated membrane except those of the 50–60 kDa bands is still to be discovered.