OBJECTIVETo determine the contents of hirudin's hydrolysates in processed leeches, set up a new evaluating method of dot blotting to evaluate the qualities of those processed Chinese medicines as leeches.

METHODContents of hirudin's hydrolysates in processed leeches were determined by dot blotting with rat antibody of anti-hirudin as the first antibody. Blotting signal was analyzed by software of Quantity One.

RESULTContents of hirudin's hydrolysates in four batches of processed leeches were 296.51, 165.47, 95.58, and 298.05 microg g(-1), respectively.

CONCLUSIONDifference among four batches of processed leeches was significant in the content of hirudin's hydrolysates. Dot blotting, as a convenient and accurate method can be broadly used for evaluating processed products of Chinese crude drugs similar to leeches.

Animals ; Drugs, Chinese Herbal ; chemistry ; Hirudins ; immunology ; metabolism ; Immunoblotting ; methods ; Leeches ; chemistry ; Reproducibility of Results

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

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 x anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.
Animals ; Antibodies, Bispecific/*immunology ; HEK293 Cells ; Haptens/*immunology ; Humans ; Immunoblotting/*methods ; Immunoprecipitation/methods ; Rabbits ; Single-Chain Antibodies/immunology

OBJECTIVEUsing polymerase chain reaction-reverse blot dot (PCR-RDB) technique to establish a new method for hepatitis C virus (HCV) genotyping and to study the distribution of HCV genotypes in Foshan area.

METHODSHCV primers and probes were designed in 5'-untranslated region (nt-1-nt-299) of HCV. HCV RNA in serum was isolated and purified, and its cDNA was obtained by reversed transcription. Nested PCR using biotin-labelled primers, was done. PCR products were hybridized with immobilized specific probes (genotype 1a to 3b) on Biodyne C membrane to genotype HCV by color development while adding POD and TMB. A certain judgment could be made according to the position of color reaction. The reliability of this new method was verified by sequencing. HCV RNA levels in serum were determined by real time fluorescent quantitative (FQ)-PCR. 60 FQ-PCR-positive HCV sera from Foshan area were genotyped using this assay.

RESULTSAll 60 sera could be successfully genotyped by PCR-RBD. 50 (83.3%) cases were found to be genotype 1b, 2 (3.3%) as genotype 1a and 2 (3.3%) as genotype 2a while 5 (8.0%) to be mixture of genotype 1a and 1b, and 1 (1.7%) to be mixture of genotypes 1b and 2a. No genotypes 2b, 3a and 3b were found. The results of PCR-RDB genotyping methods coincided with sequence analysis.

CONCLUSIONNewly established HCV genotyping system was proved to be sensitive, specific, precise and economic, thus suitable for clinical and epidemiologic studies. The results of HCV genotyping showed that genotype 1b was the predominant genotype in Foshan area.

Genotype ; Hepacivirus ; classification ; genetics ; Hepatitis C ; virology ; Humans ; Immunoblotting ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Specific IgE against Acarus siro, Glycphagus domesticus, Tyrophagus putrescentiae, and Lepidoglyphus destructor have been investigated by ELISA in sera of 92 children. Of them, 41 were found to be specific IgE positive (> or = 0.35 IU/ml) against at least one of house dust mite species, Dermatophagoides pteronyssinus and Dermatophagoides farinae, by an immunoblot. In 65.9% of the dust mite-sensitized children, specific IgE against at least one of these mite species was found. Sensitization levels, including co-sensitization cases were found to be 35.7% against A. siro, 24.4% against T. putrescentiae, 31.7% against L. destructor, and 26.8% against G. domesticus. In non-sensitized children, dust mite sensitization level was found to be 25.5%. Breakdown of sensitization by individual species in this group was; against A. siro and T. putrescentiae at 7.8%, against L. destructor at 13.7%, and against G. domesticus at 9.8%. When all children were reckoned, 43.5% was found to be sensitized against at least one storage mite species, with sensitizations against A. siro at 18.5%, T. putrescentiae at 26.1%, L. destructor at 21.7%, and G. domesticus at 17.4%. In dust samples collected from the dwellings of children, distribution of species was found to be A. siro (17%), G. domesticus (23%), T. putrescentiae (29%), L. destructor (25%), and unidentified (6%). In Fisher's chi-square test on SPSS program, there was a relationship between dust mite sensitization and storage mite sensitization (P < 0.05), but no meaningful relationship was found on the basis of individual mite species.
Animals ; Antibodies/*blood ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay/methods ; Humans ; *Immunization ; Immunoblotting/methods ; Immunoglobulin E/*blood ; Mites/*immunology ; Prevalence ; Turkey

OBJECTIVEA PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis).

METHODS12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay.

RESULTSThe sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively.

CONCLUSIONOur findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

Antitubercular Agents ; pharmacology ; Drug Resistance, Bacterial ; Genotype ; Immunoblotting ; methods ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; Polymerase Chain Reaction ; methods ; Rifampin ; pharmacology ; Sensitivity and Specificity ; Time Factors

OBJECTIVETo research on the substantial foundation of the medical speciality of Chinese traditional medicines from immunogenicity.

METHODControl antigen with hot nature was prepared from the mixture of the aqueous extracts of three Chinese traditional medicines with three typical hot nature of Alpinia officinarum, Cinnamomum cassia and Curculigo orchioides, while that with cold nature prepared with Rheum palmatum, Anemarrhena asphodeloides, Coptis chinensis, and polyclonal antibody was prepared by immunizing rabbit with control antigen. Dot blotting was performed between the polyclonal antibody of control antigen and the aqueous extracts of nine Chinese traditional medicines on a piece of PVDF membrane, and the blotting signals were analyzed by the software of Quantity One.

RESULTBlotting signals with hot control antigen of nine Chinese traditional medicines in descending were Zingiber officinale, Aconitum carmichaeli, Eucommia ulmoides, Fraxinus rhynchophylla, Lonicera japonica, Anemarrhena asphodeloides, Coptis chinensis, Rheum palmatum and Phellodendron chinense, which degree of similarity to control antigen in peak value were 57.33%, 43.56 %, 34.16%, 30.2%, 28.81%, 26.53%, 21.68%, 17.62% and 14.85%, respectively. Blotting signals with cold control antigen were Rheum palmatum, Anemarrhena asphodeloides, Coptis chinensis, Phellodendron chinense, Zingiber officinale, Lonicera japonica, Fraxinus rhynchophylla, Eucommia ulmoides and Aconitum carmichaeli in descending, of which degree of similarity to cold control antigen in peak value were 55.22%, 54.23%, 46.72%, 34.08%, 30.3%, 24.48%, 24.33%, 20.35% and 15.17%, respectively. Results of cluster analysis with Wistar's method showed that nine medicines were classified into two groups, one group included Phellodendron chinense, Anemarrhena asphodeloides, Coptis chinensis, Rheum palmatum, another was Zingiber officinale, Aconitum carmichaeli, Eucommia ulmoides, Fraxinus rhynchophylla, Lonicera japonica.

CONCLUSIONBlotting signals of nine medicines with control antigen regularly varied with the alteration of medicine nature. The more similarity degree of the tested medicine to control antigen was smaller, the more distance of the tested medicine to control antigen was further. Dot immunoblotting was a practical and effective new method in researching the substantial foundation of the medical speciality of Chinese traditional medicines.

Animals ; Antibodies, Monoclonal ; immunology ; Antibody Formation ; Antigens ; immunology ; Blotting, Western ; Cold Temperature ; Drugs, Chinese Herbal ; analysis ; chemistry ; classification ; Hot Temperature ; Immunoblotting ; methods ; Male ; Medicine, Chinese Traditional ; methods ; Rabbits

OBJECTIVE: To investigate distribution specificity of human fucosyltransferase 5 (FUT5) as well as its expression and localization in spermatids. METHODS: Human semen, vaginal swab, saliva and venous blood from healthy individuals were collected. The spermatids were isolated and the spermatid membrane protein was then extracted. Expression levels of FUT5 from human spermatid membrane, seminal plasma, vaginal fluid, saliva and serum were detected by immunoblotting technique. The expression and localization of FUT5 in spermatids were analyzed by immunofluorescent method. RESULTS: Immunoblotting technique showed that FUT5 was expressed on spermatid membranes and in serum, but not in seminal plasma, vaginal fluid and saliva. The expressed FUT5 on spermatids was mostly localized on head of spermatids by fluorescent microscopy, suggesting that there was certain amount of FUT5 on human spermatid membrane, and the spermatids might be isolated from mixed stains with vaginal fluid by antigen-antibody reaction. CONCLUSION Human FUT5 shows a characteristic distribution specificity, and this feature may be used for identification of mixed stain involved in criminal sexual offence in future forensic practice.
Cell Membrane/metabolism* ; Female ; Fluorescent Antibody Technique/methods* ; Forensic Genetics/methods* ; Fucosyltransferases/metabolism* ; Humans ; Immunoblotting ; Male ; Saliva/metabolism* ; Semen/metabolism* ; Spermatids/metabolism* ; Vagina/metabolism*

After collecting calcareous corpuscles from plerocercoid of Spirometra mansoni (sparganum), we evaluated the antigenic values of calcareous corpuscles binding proteins obtained from the cyst fluid of Taenia solium metacestodes. Immunoblot analysis revealed that cysticercosis patient sera strongly recognized 10 and 95 kDa calcareous corpuscles binding proteins. This result demonstrated that calcareous corpuscles are bound with major secretory antigenic proteins, which is possibly involved in the secretory pathways of the 10 and 95 kDa proteins presenting in the cyst fluid of T. solium metacestodes.
Animals ; Antigens, Helminth/*analysis ; Carrier Proteins/*immunology ; Cysticercosis/diagnosis/immunology ; Helminth Proteins/*immunology ; Humans ; Immunoblotting/methods ; Molecular Weight ; Serologic Tests ; Sparganum ; Taenia solium/*chemistry/immunology

This study was aimed at investigating the possible effects of phytoceramide (Pcer) on learning and memory and their underlying mechanisms. Phytoceramide was orally administered to ICR mice for 7 days. Memory performances were assessed using the passive avoidance test and Y-maze task. The expressions of phosphorylated cAMP response element binding protein (pCREB), brain-derived neurotrophic factor (BDNF) were measured with immunoblot. The incorporation of 5-bromo-2-deoxyuridine (BrdU) in hippocampal regions was investigated by using immunohistochemical methods. Treatment of Pcer enhanced cognitive performances in the passive avoidance test and Y-maze task. Immunoblotting studies revealed that the phosphorylated CREB and BDNF were significantly increased on hippocampus in the Pcer-treated mice. Immunohistochemical studies showed that the number of immunopositive cells to BrdU was significantly increased in the hippocampal dentate gyrus regions after Pcer-treatment for 7 days. These results suggest that Pcer contribute to enhancing memory and BDNF expression and it could be secondary to the elevation of neurogenesis.
Animals ; Brain-Derived Neurotrophic Factor* ; Bromodeoxyuridine ; Cyclic AMP Response Element-Binding Protein ; Dentate Gyrus ; Hippocampus* ; Immunoblotting ; Learning ; Memory* ; Methods ; Mice* ; Mice, Inbred ICR ; Neurogenesis