A novel in vitro ELISA for estimation of glycoprotein content in human rabies vaccines

In vitro methods for quantification of immunodominant glycoprotein in the rabies vaccine formulations serve as good alternative to the cumbersome and variable mice potency assay as a batch release test for the vaccine. The present study presents the development of a sandwich ELISA with optimal concentrations of a high affinity recombinant diabody (D06) and a specific monoclonal antibody (M5B4) against rabies glycoprotein for its quantification in the vaccine formulations. The glycoprotein estimate correlated linearly (r² = 0.8) to the in vivo potency estimate for the vaccine formulations. This ELISA promises a good forecast of the mice potency values and thereby can serve as a simple, yet effective batch release test for the rabies vaccines replacing the in vivo assay.     

Genomewide mRNA profiling of esophageal squamous cell carcinoma for identification of cancer biomarkers

Cancer of the esophagus is of two main types, each with distinct etiological and pathological characteristics. Esophageal squamous cell carcinoma (ESCC) is predominant type of esophageal cancers worldwide comprising almost 95% of cases. While ESCC is prevalent in the developing world, esophageal adenocarcinoma is commonly seen in the developed country, usually in association with Barrett's esophagus. In spite of its higher prevalence, ESCC has not been studied as intensively as esophageal adenocarcinoma. ESCC and esophageal adenocarcinoma are common cancers worldwide with poor survival rate among patients mainly because both of these cancers lack early biomarkers of identification. Molecular mechanisms contributing to initiation and progression of esophageal squamous cell carcinoma are still poorly understood. Development of DNA microarray technology allows high-throughput identification of gene expression profiles in cancers. In order to identify molecules as candidates for early diagnosis and/or as therapeutic targets, we analyzed the mRNA expression profiles of 20 cases of ESCC using whole genome DNA microarrays. A total of 2,235 genes were differentially regulated in the tumors as compared to the corresponding adjacent normal epithelium of which 881 were significantly upregulated. We validated two molecules that were not previously reported to be overexpressed in ESCC, oral cancer overexpressed 2 (ORAOV2) and fibroblast activation protein (FAP), by immunohistochemical labeling of tissue microarrays and archival tissue sections and found that they were overexpressed in 98% (116/118) and 68% (79/116) of cases, respectively. By gene enrichment analysis, we identified significant downregulation of several genes in the arachidonic acid metabolic pathway. Overall, using this approach we have identified a number of promising novel candidates that can be validated further for their potential to serve as biomarkers for ESCC.     

NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules

Traditionally, the measurement of transport activity has employed radiolabeled compounds. The resulting experimental procedures do not measure transport in real time and are limited in temporal and spatial resolution. The use of epifluorescence microscopy provides the ability to measure transport activity in real time with high temporal and spatial resolution. Using epifluorescence microscopy we characterized the transport of the fluorescent organic cation, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD-TMA+, MW 266). NBD-TMA+ has structural characteristics common to other secreted organic cations and is fluorescent (lambda(ex)=458 nm; lambda(em)=530 nm). The excitation and emission spectra are insensitive to changes in [Cl-] and minimally sensitive to pH in the physiologically relevant range (pH 5.0-7.4). A microscope equipped with a photon-detection system was used to measure accumulation of NBD-TMA+ by isolated rabbit renal proximal tubules. Accumulation of NBD-TMA+ by proximal tubules was time dependent and saturable (Michaelis-Menten constant Km 12 microM). Proximal tubule accumulation of NBD-TMA+ was inhibited by the organic cations tetraethylammonium (TEA+) (apparent inhibitory constant K(app)TEA 134 microM), cimetidine, and N1-methylnicotinamide (NMN). Our experimental results provide strong evidence that NBD-TMA+ is transported by one or more of the basolateral organic cation transporters involved in the renal secretion of this chemical class of compound. This fluorescent substrate provides a sensitive means of investigating organic cation transport.     

Proteogenomic analysis reveals exosomes are more oncogenic than ectosomes

Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through tumor-derived EVs. Follow up integrated bioinformatics analysis highlighted the enrichment of oncogenic cargo in exosomes and ectosomes. Interestingly, exosomes induced significant cell proliferation and migration in recipient cells compared to ectosomes confirming the oncogenic nature of exosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes. The integrative proteogenomics approach utilized in this study has the potential to identify disease biomarker candidates which can be later assayed in liquid biopsies obtained from cancer patients.     

Timed pentylenetetrazol infusion test: a comparative analysis with s.c.PTZ and MES models of anticonvulsant screening in mice.

The intravenous pentylenetetrazol (i.v.PTZ) seizure test provides threshold dose for induction of seizures in individual animals. In the present study, the i.v. and s.c.PTZ seizure models in mice were compared for seizure pattern, intra- and interanimal variability. Anticonvulsant activities of several antiepileptic drugs (AEDs) at non-ataxic dose levels were evaluated in the PTZ and maximal electroshock (MES) seizure tests. In the i.v.PTZ test, at 0.5 ml/min rate of administration, the mean threshold PTZ doses for induction of clonus and tonic extensor were 44.17 and 99.59 mg/kg, respectively. The intra- and interanimal variabilities in the seizure response were low in the i.v.PTZ as compared to the s.c.PTZ model. Phenobarbital sodium, ethosuximide, sodium valproate, diazepam, tiagabine, oxcarbazepine and zonisamide enhanced threshold or onset latency for clonus in the i.v. and s.c.PTZ tests, respectively. Levetiracetam and pregabalin were active in the i.v.PTZ test, but had no effect in the s.c.PTZ test. Ability of AEDs to protect from tonic extensor was compared in the MES and i.v.PTZ tests. For this effect, phenobarbital sodium, phenytoin, carbamazepine, sodium valproate, gabapentin, oxcarbazepine, zonisamide and pregabalin were effective in the i.v.PTZ and MES tests. Ethosuximide, diazepam and levetiracetam were effective in the i.v.PTZ test, but not the MES test. On the contrary, lamotrigine and topiramate were active in the MES, but not the i.v.PTZ test. These results indicate that it is advantageous to use i.v.PTZ test as an acute seizure model for screening of antiepileptic drugs. This model can identify molecules with varied mechanism of action and broad clinical utility in the treatment of epilepsy.     

Recombinant Diabody-Based Immunocapture Enzyme-Linked Immunosorbent Assay for Quantification of Rabies Virus Glycoprotein

The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (V_H) and light chain (V_L) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human × mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r² value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P < 0.001), while the predicted potencies by the two tests did not differ significantly (P > 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.Rabies is a fatal viral infection of the nervous system affecting all mammals, including humans through bite wounds from a rabid animal, which can be prevented by vaccination coupled with administration of anti-rabies virus serum (6, 11). Rabies transmission from nonbite exposures is rare. Scratches, abrasions, open wounds, or mucous membranes contaminated with saliva or other potentially infectious material (such as brain tissue) from a rabid animal constitute nonbite exposures. Occasionally reports of nonbite exposure are such that postexposure prophylaxis is given. Inhalation of aerosolized rabies virus is also a potential nonbite route of exposure, but with the exception of laboratory workers, most people are unlikely to encounter an aerosol version of the rabies virus (5). Organ transplantations have also been credited with nonbite transmission of rabies from human to human (3). Despite significant scientific progress, rabies remains an important zoonotic disease globally. Annually, 20,000 deaths are reported in India, making rabies one of the major causes of human mortality (21). Vaccination is therefore considered one of the most viable and important methods for the prevention of rabies by way of preexposure prophylaxis in high-risk groups, postexposure prophylaxis in contact groups, and preexposure prophylaxis in pet animals that are at risk due to possible contacts with rabid animals. The most cost-effective means of prevention and control of rabies in humans is by eliminating rabies in dogs and other susceptible animals through vaccination.The NIH mouse protection test is an in vivo potency test that has been used widely by all manufacturers of rabies vaccines. The role of different immunological parameters and the presence of virus-neutralizing antibodies are not well established because of a weak correlation between the NIH potency test results and immunogenicity when vaccines containing different strains of rabies virus were tested (2). Furthermore, this method is time-consuming and expensive, requires a large number of animals, and involves the use of live rabies virus. As a result, there is increased exposure in human beings to live and virulent rabies strains. The NIH test also requires a secure biosafety level 3 (BSL-3) facility for housing and challenging the experimental animals. Therefore, for both practical and ethical reasons, replacement of this test by more rapid and reliable in vitro methods is highly desirable. Based on the fact that the rabies virus glycoprotein (RV GP) is the antigen responsible for inducing virus-neutralizing antibodies and conferring protection against a lethal intracerebral challenge, it has been suggested that the antigenicity of the rabies vaccines could be evaluated by titration of the RV GP (17).Though some laboratories have used enzyme-linked immunosorbent assay (ELISA) to assess RV GP content for determination of the potencies of inactivated vaccines, variable correlation between ELISA and the NIH test (7, 8, 9, 13, 14, 17, 18, 20) has been reported. Essentially, all these ELISAs incorporate the use of either polyclonal antibodies or hybridoma-derived monoclonal antibodies (MAbs). Although MAbs offer substantial advantages with respect to potency, reproducibility, and freedom from contaminants (4), they are difficult to prepare in a quality-assured manner.Recombinant DNA technology has been used to a great extent in the expression of antibodies/antibody fragments (12). Antibody fragments can be readily produced from the genes encoding antibody variable domains, which can be derived either from hybridomas (19) or from bacteriophage displaying antibody fragments (16). Diabodies are bivalent or bispecific antibody fragments generated by the dimerization of variable heavy (V_H)-light chain (V_L) fragments (10) as a result of reduction in the size of the linker between variable light and variable heavy chains (1), and these antibodies have many practical applications, including immunoassay and therapy.We describe for the first time the use of a recombinant diabody in the development of an ELISA for quantification of RV GP content in human rabies vaccines incorporating the PV strain of rabies virus and its comparison with the NIH mouse protection test.