Surface interactions between two of the main periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia

PURPOSE: Porphyromonas gingivalis and Tannerella forsythia have been implicated as the major etiologic agents of periodontal disease. These two bacteria are frequently isolated together from the periodontal lesion, and it has been suggested that their interaction may increase each one's virulence potential. The purpose of this study was to identify proteins on the surface of these organisms that are involved in interbacterial binding. METHODS: Biotin labeling of surface proteins of P. gingivalis and T. forsythia and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed to identify surface proteins involved in the coaggregating activity between P. gingivalis and T. forsythia. RESULTS: It was found that three major T. forsythia proteins sized 161, 100, and 62 kDa were involved in binding to P. gingivalis, and P. gingivalis proteins sized 35, 32, and 26 kDa were involved in binding to T. forsythia cells. CONCLUSIONS: LC-MS/MS analysis identified one T. forsythia surface protein (TonB-linked outer membrane protein) involved in interbacterial binding to P. gingivalis. However, the nature of other T. forsythia and P. gingivalis surface proteins identified by biotin labeling could not be determined. Further analysis of these proteins will help elucidate the molecular mechanisms that mediate coaggregation between P. gingivalis and T. forsythia.

Preparation and bioactivity evaluation of streptavidin-tagged human interferon- inducible T cell alpha chemoattractant bifunctional fusion protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare streptavidin-tagged human interferon-inducible T cell alpha chemoattractant bifunctional fusion proteins (SA/hI-TAC) and evaluate its biological activity.</p><p><b>METHODS</b>pET24a-SA-hI-TAC/pET21a-hI-TAC-SA plasmids were constructed and expressed in BL21. SA-hI-TAC and hI-TAC-SA fusion proteins were purified by Ni-NTA affinity chromatography, refolded by dialysis and identified by Western blotting. The bifunctionality of the fusion proteins (biotin-binding function and hI-TAC activity) was analyzed by flow cytometry and lymphocyte chemotaxis experiment, respectively.</p><p><b>RESULTS</b>SA-hI-TAC/hI-TAC-SA fusion proteins were expressed at about 12% and 25% of the total bacterial protein, respectively. The two fusion proteins had a purity of about 85% and 90% after purification, and their purity reached 98% after purification with S-100 gel filtration chromatography. Both of the fusion proteins were efficiently immobilized on the surface of biotinylated mouse bladder cancer MB49 cells (91.3% for SA-hI-TAC and 98.8% for hI-TAC-SA). SA/hI-TAC induced lymphocyte chemotaxis in a dose-dependent manner, and hI-TAC-SA showed a stronger chemotactic effect than SA-hI-TAC.</p><p><b>CONCLUSIONS</b>We successfully obtained SA/hI-TAC bifunctional fusion proteins, which may potentially be used in local treatment of tumor and as a tumor vaccine.</p>

Single-molecule detection and characterization of DNA replication based on DNA origami / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate single-molecule detection and characterization of DNA replication.</p><p><b>METHODS</b>Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication.</p><p><b>RESULTS</b>The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis.</p><p><b>CONCLUSIONS</b>The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.</p>

Construction of three HLA-A*0201-peptide tetramers and their preliminary application in detection of vitiligo-specific cytotoxic T lymphocytes / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct vitiligo-specific HLA-A*0201-peptide tetramers and to apply the constructed tetramers in detection of vitiligo-specific cytotoxic T lymphocytes (CTL).</p><p><b>METHODS</b>Proteins HLA-A0201*-BSP and β2M were obtained by effective prokaryotic expression. The purified proteins were refolded with vitiligo antigen peptides MelanA 26-35, gp100 209-217, and tyrosinase 1-9, respectively to form HLA-A*0201-peptide complex. The complex was biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with phycoerythrin (PE)-streptavidin at a ratio of 4∶1 and identified by Dot-blot assay. The capacity of tetramer to detect vitiligo-specific CTL was analyzed by flow cytometry.</p><p><b>RESULTS</b>The biotinylation of vitiligo-specific HLA-A*0201-peptide tetramers were successfully performed by Dot-blot. Flow cytometry analysis indicated that the tetramer effectively bound to specific CTL from peripheral blood of patients with vitiligo.</p><p><b>CONCLUSION</b>Three kinds of biotinylated vitiligo-specific HLA-A*0201-peptide tetramers have been constructed successfully. The tetramer can detect antigen specific CTL from patients with vitiligo.</p>

Functional Characterization of ABCB4 Mutations Found in Low Phospholipid-Associated Cholelithiasis (LPAC)

Multidrug resistance 3 (MDR3) is expressed on the canalicular membrane of the hepatocytes and plays an important role in protecting the liver from bile acids. Altered ABCB4 gene expression can lead to a rare hepatic disease, low phospholipid-associated cholelithiasis (LPAC). In this study, we characterized 3 ABCB4 mutations in LPAC patients using various in vitro assay systems. We first measured the ability of each mutant to transport paclitaxel and then the mechanisms by which these mutations might change MDR3 transport activity were determined using immunoblotting, cell surface protein biotinylation, and immunofluorescence. Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Both mutants showed significantly decreased total and cell surface MDR3 expression. These data suggest two missense mutations of ABCB4 may alter function of MDR3 and ultimately can be determined as LPAC-causing mutations.

Primary establishment of an alphaLISA assay for detection of HAV IgM / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop an AlphaLISA method for detection of antibody of Hepatitis A virus.</p><p><b>METHODS</b>After hepatitis A virus antigen was concentrated and biotinylated, optimal biotinylated HAV antigen, donor bead, acceptor bead concentration have been explored and determined by both dot-blotting and AlphaLISA methods. 97 samples including 23 serums from patients HAV infected and 70 serums from blood donors have been detected by new AlphaLISA method.</p><p><b>RESULTS</b>The sensitivity and specificity for anti-HAV IgM were 78% and 98.5%, the positive and negative predictive value were 95% and 93%.</p><p><b>CONCLUSION</b>A homogeneous and fast AlphaLISA assay for detection of HAV IgM has been established by preliminary verification on samples.</p>

Intravesical anchoring of streptavidin-tagged interleukin-4 fusion protein for immunotherapy of mouse superficial bladder cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the antitumor efficacy of streptavidin-tagged interleukin-4 (IL-4-SA) bifunctional fusion protein in the immunotherapy of mouse model of superficial bladder cancer.</p><p><b>METHODS</b>IL-4-SA fusion protein was prepared and its biological activity was determined. One day after MB49 cell implantation, 100 µl of 1 mg/ml NHS-PEO4-biotin was instilled into the bladder for 30 minutes, followed by intravesical instillation of 100 µl PBS, GFP-SA+IL-4 or IL-4-SA and incubation for 1 hour. The bladder irrigation was performed twice a week for three weeks. The CTL cytotoxicity and profile of CD8(+) tumor-infiltrating lymphocytes were analyzed.</p><p><b>RESULTS</b>The IL-4-SA fusion protein was durably anchored to the biotinylated mucosal surface of bladder wall for up to 5 days.On day 80 after the implantation of MB49 cells, all of PBS-treated mice died, and 8 out of 10 mice in the GFP-SA-treated group died from tumor burden.In contrast, 5 out of 10 mice in the IL-4-SA-treated group were tumor-free. The MB49 tumor-specific cytotoxicity from mice in the IL-4-SA group was (11.3 ± 1.2)%, (22.7 ± 1.5)% and (31.0 ± 3.0)% at the effector to target ratios of 1:1, 25:1 and 50:1, respectively. But the corresponding cytotoxicity was (4.3 ± 0.6)%, (9.0 ± 1.0)% and (14.3 ± 1.5)% in the GFP-SA+IL-4 group, and (3.3 ± 0.6)%, (7.3 ± 0.6)%, (12.7 ± 2.1)% in the PBS group. The tumor-specific cytotoxicity in the SA-CD40L group was significantly higher than that in the control groups (P < 0.05). The infiltrating CD8(+) T cells in tumors in the IL-4-SA-treated group were increased compare with those in other groups.</p><p><b>CONCLUSION</b>Intravesical anchoring of IL-4-SA elicites strong and long-lasting immunoprotection against superficial bladder cancer, and the novel immunotherapy may be an attractive therapeutic alternative in future.</p>

A novel immunotherapy for superficial bladder cancer by the immobilization of streptavidin-tagged bioactive IL-2 on the biotinylated mucosal surface of the bladder wall / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Intravesical administration of Bacillus Calmette-Guerin (BCG) after transurethral resection is by far the most effective local therapy for superficial bladder cancer, the fifth most common cancer in the world. However, approximately one-third of patients fail to respond and most patients eventually relapse. In addition, there are pronounced side effects of BCG therapy, such as BCG sepsis and a high frequency of BCG-induced cystitis. This study established a novel immunotherapy through immobilization of streptavidin-tagged human IL-2 (SA-hIL-2) on the biotinylated mucosal surface of bladder wall.</p><p><b>METHODS</b>A mouse orthotopic model of MB49 bladder cancer was established by perfusing MB49 cells into mouse bladders. The SA-hIL-2 fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall. Treatment began on day 1 after MB49 implantation, once every 3 days for 6 times. Immunohistochemical assay was performed to assess the persistence of SA-hIL-2 immobilized on the biotinylated mucosal surface of the bladder wall. The mice were monitored for tumor growth and survival. On day 60 after MB49 implantation, the SA-hIL-2-cured mice, which were found to have no hematuria or palpable tumors, were challenged with wild-type MB49 cells implanted into the pretreated bladder and monitored for survival.</p><p><b>RESULTS</b>SA-hIL-2 could be immobilized efficiently and durably on the bladder mucosal surface as long as 7 days. On day 60 after MB49 implantation, 9 out of 20 SA-hIL-2-treated mice survived, but all mice in PBS control group died. More importantly, 5 out of 9 tumor-free mice in the SA-hIL-2 group were protected against a second intravesical wild-type MB49 tumor challenge.</p><p><b>CONCLUSIONS</b>SA-hIL-2 fusion protein could significantly inhibit tumor growth and extend the survival time in the orthotopic model of MB49 bladder cancer.</p>

Immobilization of streptavidin-tagged bioactive hTNF-alpha on biotinylated mucosal surface of the bladder wall for treatment of superficial bladder cancer in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate a novel immunotherapy through immobilization of streptavidin-tagged hTNF-alpha on the biotinylated mucosal surface of the bladder wall for bladder cancer treatment in mice.</p><p><b>METHODS</b>A total of 120 female C57BL/6j mice were randomized into 5 equal groups, namely blank control, PBS, soluble hTNF-alpha, SA-GFP, and SA-hTNF-alpha treatment groups. Twenty-four hours after establishment of a mouse model of orthotopic superficial bladder cancer, SA-hTNF-alpha fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall, which was repeated every 4 days for a total of 6 sessions. Immunohistochemistry was performed to detect the retention time of SA-hTNF-alpha fusion protein in the biotinylated mouse bladder mucosa and the distribution of CD4(+) and CD8(+) lymphocytes in the mucosa and tumor tissues, with the tumor growth and mouse survival also observed. The cytotoxiciy of the tumor-specific lymphocytes was evaluated. The mice responding well to the treatment were re-challenged by MB49 and monitored for survival.</p><p><b>RESULTS</b>SA-hTNF-alpha could be efficiently and stably immobilized on the bladder mucosal surface for as long as 7 days. On day 60 after MB49 implantation, 18 out of 22 SA- hTNF-alpha-treated mice survived, with 9 appearing tumor-free, but all the mice in PBS control group died. Five out of 9 tumor-free mice in SA-hTNF-alpha group showed resistance to a re-challenge with intravesical MB49. The numbers of CD4(+) and CD8(+) lymphocytes were significantly greater in SA-hTNF-alpha group than in the other groups (P<0.05). The cytotoxicity of the tumor-specific lymphocytes was significantly stronger in SA-hTNF-alpha group than in the other groups (P<0.05).</p><p><b>CONCLUSION</b>SA-hTNF-alpha immobilized on the biotinylated mucosal surface of the bladder wall can significantly inhibit the tumor growth and promote the survival of the mice bearing orthotopic superficial bladder cancer.</p>

Computer-aided molecular modeling and activity estimation for ligand screening with specific phage clone as the target / 南方医科大学学报

To investigate the interaction between tumor necrosis factor alpha (TNF alpha) mimotopes and TNF alpha-binding peptides screened from random phage display peptide library with TNF alpha mimotopes displayed on phage clone as the target, the computational docking program AutoDock (with confirmation calculations using Discover) was used to predict and analyze the binding modes of LLT-18 (TNF alpha binding peptide, sequence EHMALTYPFRPP) with TNF alpha, after which LCS-7 (TNF alpha mimic phage clone, displayed positive sequence c-RRPAQSG-c) was docked to LLT-18 manually. The binding between LLT-18 and TNF alpha or LCS-7 was stabilized predominantly through electrostatic interaction and H-bond formation. The Arg residues in TNF alpha or LCS-7 were important for their interaction with LLT-18. For LLT-18, the key amino acid residues were Glu1, His2, Met3 and Tyr7. These results suggest the feasibility of screening ligand to single epitope with specific phage clone as the target, and of predicting the interaction between small peptides by computer-aided molecular modeling.

Preparation and characterization of biotinylated chitosan nanoparticles / 药学学报

Biotinylated chitosan nanoparticles (Bio-CS-NP) were prepared for the active delivery to cancer cells and its characterization was investigated in this study. The preparation process included two steps. First, biotinylated chitosan ( Bio-CS ) was obtained through a reaction between sulfosuccinimidobiotin and chitosan (CS). Second, Bio-CS-NP were prepared by the precipitation of Bio-CS with sodium chloride solution. With a biotin reagent box, the conjugation densities of biotin on the surface of Bio-CS-NP were determined. The morphology and diameter of the nanoparticles were assayed by transmission electron microscope (TEM) and laser light scattering particle analyzer, respectively. The uptake of nanoparticles by human hepotacarcinoma HepG2 cells, for example, Bio-CS-NP and chitosan nanoparticles (CS-NP) without any modification, was quantitatively examined. The results indicated that the conjugation densities of biotin on the surface of Bio-CS-NP were 2.2 biotin CS. Bio-CS-NP were spherical, smooth on the surface. The average diameter was 296.8 nm. The polydispersion index was 0.155. The uptake of Bio-CS-NP by HepG2 cells was much higher than that of CS-NP (P < 0.05). It demonstrated that Bio-CS-NP can be applied as a new vehicle to actively deliver anticancer drugs to tumor cells. The method for the determination of biotin was simple and practical.

Detection of HPV16 and 18 by in situ hybridization in precancerous and cancerous lesions of cervix.

Fifty cervical biopsies from women with preinvasive and invasive malignancies of uterine cervix and ten normal cervical biopsies were examined for the presence of human papilloma virus (HPV) 16 and 18 DNA sequences by in situ hybridization (ISH) method with biotinylated DNA probes. The overall positivity of HPV DNA was 48% (24/50). The positivity of HPV 16 DNA for low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) were 33.33%, 45.45%, 42.30% respectively. The positivity for HPV 18 DNA for LSIL, HSIL and SCC were 0%, 18.18%, 30.76% respectively. Two cases of cervical adenocarcinomas showed positivity for HPV 18 DNA only.

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.

Análisis histoquímico de células tumorales (cáncer de colon) tumor primario y metastásico mediante lectinas biotiniladas / Histochemical analysis on tumoral cells (colon cancer) primary and metastatic tumors by biotilinade lectines

De las etapas del desarrollo del cáncer la más amenazante es la metástasis, por lo que se han hecho esfuerzos en su estudio y comprensión. Nosotros estudiamos el glicocáliz de tumores primarios de colon y sus metástasis a sacro, ganglios linfáticos e hígado. Se utilizaron muestras tumorales de pacientes post quirúrgicos con cáncer de cólon. Los cortes fueron fijados e incubados en PSA, DBA, UEA1, jacalina y ELC. Como controles utilizamos azúcares inhibitorios específicos. La mayor intensidad correspondió a Jacalina en tumores primarios y metástasis. PSA,ECL, y UEA1 reaccionaron con intensidad menor. No se registraron diferencias entre los tumores primarios y metastáticos, y tampoco entre las diferentes metástasis. No hubo reacción con el DBA. Estos resultados no muestran diferencias en el Glicocáliz con la presencia de los carbohidratos estudiados entre el tumor primario y la metástasis. Es posible que se deban continuar los estudios con otras lectinas en la búsqueda de las moléculas que llevan a la selectividad por órgano de las metástasis tumorales...

Cloning and expression of a biotinylated multiple-epitope HCV fusion antigen gene / 中国实验血液学杂志

The aim was to develop a single multiple-epitope fusion antigen which incorporates all of the major immunodominant epitopes from the six functional regions of the HCV genome. A nucleic acid sequence consisting of viral core, E1, E2, NS3, NS4, and NS5 regions was constructed and inserted into the Promega Pinpoint Xa-1 T vector for inducing expression. The protein was expressed in JM109 (DE3) as a fusion protein with a 13 kD biotinlated tag to be used for detection and affinity purification. Immunogenicity and biotinylated tag of the fusion protein were detected by Western blot analysis with positive anti-HCV serum and streptavidin alkaline phosphatase. After purified by Promega SoftLink Soft Release Avidin Resin, the protein was pre-coated on microwell and detected with anti-core, anti-NS3, anti-NS4 and anti-NS5 positive sera by EIA, respectively. The results indicated that the recombinant soluble protein was expressed and labelled with biotin successfully, it reacted with anti-HCV positive serum, and exposed all of the major immunogenic epitopes chosen. In conclusion, this recombinant antigen may be used to design an double antigen sandwich anti-HCV immunoassay.

Produção de sonda de DNA diretamente biotinilada com N-hidroxisuccinimida biotina ester (BNHS) / Labeling DNA probes directly coupled with biotinyl-N -hydroxysuccinimide ester (BNHS)

A utilização de sondas de DNA para análise genômica em fase sólida tem se expandido e ultrapassado as áreas da pesquisa, estendendo-se ao terreno do diagnóstico de rotina nas áreas de medicina humana e animal, bem como nas ciências forenses. Estas sondas têm sido utilizadas desde o surgimento da primeira técnica de hibridização descrita com o southern blotting, que envolve a imobilização, em filtros de papel, de fragmentos de DNA separados eletroforeticamente. Outra técnica descrita como o dot-hibridização é menos complexa do que o southern convencional por não requerer o passo prévio de eletroforese e por ser menos exigente quanto à qualidade do DNA, já que dispensa a digestão com enzima de restrição. A marcação mais comum para estas sondas é o fósforo radioisótopo (32P), que, apesar de sua alta sensibilidade, apresenta os inconvenientes de risco físico, necessidade de autorização para manipulação de radioisótopos, alto custo e uma vida média muito curta. Uma série de alternativas para o 32P têm sido utilizadas, com marcas diretas ou secundárias, entre elas a biotina, que tem mostrado grandes vantagens e tem sido usada também em laboratórios especializados e bem equipados. Este trabalho descreve uma modificação do método clássico descrito para sondas de DNA acopladas por biotina hidrazida. Utilizamos a N-hidroxisuccinimida biotina (BNHS) em dimetilformamida, para marcar resíduos de citosina disponíveis em segmentos de fita simples de DNA extraído de Corynebacterium pseudotuberculosis. A variação da proporção de biotina e de DNA, bem como sua especificidade e sensibilidade, foram cuidadosamente testadas e criteriosamente analisadas através de ensaios de hibridização, adsorvendo-se DNA cromossômico do C. pseudotuberculosis e outros DNAs em papel de nitrocelulose, seguindo-se a incubação com as sondas biotiniladas descritas,avidina-peroxidase e revelação com 4-cloro-1-naftol. Os resultados mostraram que a sonda de DNA produzida tem as características favoráveis...

Relation between Beta-2-glycoprotein I and hepatitis B virus surface antigen / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To clarify the binding character between Beta-2-glycoprotein I (Beta-2-GPI) and HBsAg.</p><p><b>METHODS</b>Beta-2-GPI was purified from human plasma and labelled with biotin. Solid phase enzyme linked absorbance assay was used to investigate its binding with HBsAg.</p><p><b>RESULTS</b>Biotinylated Beta-2-GPI was found to bind HBsAg and the reaction could be inhibited by excess unlabelled Beta-2-GPI.</p><p><b>CONCLUSIONS</b>Beta-2-GPI may play a role in hepatitis B virus infection.</p>

Characterization of monoclonal antibodies recognizing alpha and beta subunits of human chorionic gonadotropin hormone.

Human chorionic gonadotropin (hCG) hormone is required for maintenance of early pregnancy and is a potential marker in the diagnosis and prognosis of both pregnancy and trophoblastic diseases. Murine hybridomas were generated against purified hCG. Seven hybrid clones secreting antibodies against hCG molecule with IgG1/kappa subclass were established. The indirect ELISA result demonstrated that six MAbs (BEL-1 to BEL-6) recognized hCG in both holo and free beta subunit form with negligible cross-reactivity against a closely related hormone, human luteinizing hormone (hLH). In this fusion, only one MAb (ALC-1) showed a cross-reaction with hLH, which designated an alpha subunit specific. The outcome of Western blot ascertained that ALC-1 recognized the conformational epitope on alpha subunit of hCG at Mr 23 kDa band in nonreducing condition (NR). In contrast, epitopes belonging to all MAbs recognized beta subunit of hCG were in linear peptide structure at Mr 34 kDa band (NR) and Mr 26 kDa band (R). These six MAbs were further characterized by using two beta subunit carboxy-terminal synthetic peptides (beta109-119 and beta109-145). Three of them (BEL-1, BEL-3, and BEL-4) recognized only epitope harboring in beta109-145 fragment, the others recognized both types of the synthetic peptide. In order to select the most suitable MAbs specific to beta subunit of hCG for exploiting with ALC-1 in the sandwich-type immunometric assay, competitive ELISA was employed. Six individual MAbs specific to beta subunit of hCG were used to compete with biotinylated ALC-1 to evaluate the proximity of their epitopes on the holo form of hCG molecule. Of all six MAbs, BEL-5 depicted the lowest percent inhibition result, which indicated the bottom-most steric hindrance effect. Consequently, MAb BEL-5 will be the most appropriate antibody to utilize in concert with ALC-1 in place of devising a sandwich-type immunometric assay for measuring holo-hCG level.

A simple and rapid non-radioactive oligonucleotide based hybridization assay for the detection of Wuchereria bancrofti.

Five biotin labeled oligonucleotides was designed based on a previously cloned and characterized repetitive DNA sequence specific for Wuchereria bancrofti. The oligonucleotide mix (containing five probes) when used in a hybridization assay, detected as little as 100 pg of purified W. bancrofti, microfilarial DNA, a single infective stage larva and a single microfilaria in 50 microl blood sample. A simple protocol was followed for the hybridization assay. Blood samples lysed with sterile distilled water and digested with proteinase K in the presence of a detergent were directly applied on to nylon membranes for dot blot assays. The DNA extract of mosquitos carrying infective stage larvae was eluted through sephadex G-50 minicolumns prior to blotting. The assay was also able to detect DNA extracted from microfilariae infected samples stored over five days at room temperature (28 degrees C). This simple and rapid oligonucleotide hybridization protocol with the highly sensitive chemiluminescent-based detection has significant potential for the development of a field kit to detect W. bancrofti infection.