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Abstract: Hybrid hybridomas (quadromas) are derived by fusing at least two hybridomas, each producing a different antibody of predefined specificity. The resulting cell secretes not only the immunoglobulins of both parents but also hybrid molecules manifesting the binding characteristics of the individual fusion partners. Purification of the desired bispecific immunoprobe with high specific activity from a mixture of bispecific and monospecific monoclonal antibodies requires special strategies. Using a dual, sequential affinity chromatography (Protein-G chromatography followed by m-aminophenyleboronic acid agarose column), we have purified bispecific monoclonal antibodies (BsMAb) as a preformed HRPO (Horseradish Peroxidase) complex (BsMAb–HRPO). The quadroma culture supernatant was initially processed on a Protein-G column to isolate all the species of immunoglobulins. This pre-enriched fraction was subsequently passed through the aminophenyleboronic acid column super saturated with HRPO. The column matrix has the ability to bind to proteins such as HRPO with vicinal diols. The enzyme loaded column captures the desired bispecific anti-SARS-CoV×anti-HRPO species with the elimination of the monospecific anti-SARS-CoV MAb to result in a high specific activity diagnostic probe. The presence of anti-HRPO MAb is an acceptable impurity as it will not bind to the target SARS-CoV NP antigen and will get washed out during the ELISA procedure. [Copyright &y& Elsevier]

Abstract: Dengue and the more severe dengue hemorrhagic fever have been a very critical public health problem globally. Millions of people especially in the tropical areas get infected with dengue. An efficient diagnostic is very important for early screening of dengue infection. In dengue-infected patients, the nonstructural protein NS1 is present on the surface of infected cells and secreted in plasma. The NS1 antigen is an important target for developing a quick diagnostic largely due to its long presence in the blood. We have developed a simple-to-use immunoswab-based diagnostic procedure employing monoclonal antibodies and the second-generation quadromas. The detection limit for NS1 has been established to be in the subnanogram range. The assay is very sensitive, has a visual end point, and also being extremely inexpensive. With this assay, screening time for a dengue-infected person would be very rapid. [Copyright &y& Elsevier]

Abstract: The severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein is known to mediate receptor interaction and immune recognition and thus it is considered as a major target for vaccine design. The spike protein plays an important role in virus entry, virus receptor interactions, and virus tropism. Sensitive diagnosis of SARS is essential for the control of the disease in humans. Recombinant SARS-CoV S1 antigen was produced and purified for the development of monoclonal and bi-specific monoclonal antibodies. The hybridomas secreting anti-S1 antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. The sandwich ELISA was formed by using F26G18 as a coating antibody and biotinylated F26G18 as a detection antibody with a detection limit of 0.037μg/ml (p <0.02). The same detection limit was found with P136.8D12 as a coating antibody and biotinylated F26G18 as a detection antibody. The sensitivity was improved (detection limit of 0.019μg/ml), however, when using bi-specific monoclonal antibody (F157) as the detection antibody. In conclusion, the method described in this study allows sensitive detection of a recombinant SARS spike protein by sandwich ELISA with bi-specific monoclonal antibody and could be used for the diagnosis of patients suspected with SARS. [Copyright &y& Elsevier]

Abstract: Poxviruses are large DNA viruses that replicate in discrete locations in the cytoplasm of infected cells called viral factories. Because the host cell DNA replication machinery is located in the nucleus, poxviruses encode many of the proteins required for their own DNA replication, including a DNA polymerase. Although many if not all of the enzymes that are required for viral DNA replication have been identified, the actual mechanism of poxvirus DNA replication remains unclear. Two monoclonal antibodies and a polyclonal antibody against vaccinia virus DNA polymerase were produced and characterized for use as tools to investigate the mechanism of virus DNA replication. Although the monoclonal antibodies were not suitable for Western blotting, the polyclonal antibody was able to detect the protein in infected cell lysates using this method. In contrast, while the polyclonal antibody did not recognize the DNA polymerase when used for immunofluorescence microscopy, the monoclonal antibodies were able to detect the polymerase in vaccinia viral factories. In addition, one of these antibodies also stained viral factories produced by cowpox and ectromelia, two closely related viruses. Finally, all three antibodies were able to immunoprecipitate vaccinia DNA polymerase from infected cell lysates. These antibodies will be useful in experiments designed to describe more fully the role of the viral DNA polymerase in DNA replication of vaccinia virus. [Copyright &y& Elsevier]

Abstract: The emergence of severe acute respiratory syndrome (SARS) resulted in several outbreaks worldwide. Early tests for diagnosis were not always conclusive in identifying a SARS suspected patient. Nucleocapsid protein (NP) is the most predominant virus derived structural protein which is shed in high amounts in serum and nasopharyngeal aspirate during the first week of infection. As part of such efforts, a simple, easy to use immunoswab method was developed by generating a panel of monoclonal antibodies (MAbs), Bispecific MAbs and chicken polyclonal IgY antibody against the SARS-CoV nucleocapsid protein (NP). Employing the MAb-based immunoswab, an NP concentration of 200pg/mL in saline and pig nasopharyngeal aspirate, and 500pg/mL in rabbit serum were detected. BsMAb-based immunoswabs detected an NP concentration of 20pg/mL in saline, 500pg/mL in rabbit serum and 20–200pg/mL in pig nasopharyngeal aspirate. Polyclonal IgY-based immunoswabs detected an NP concentration of 10pg/mL in pig nasopharyngeal aspirate providing the most sensitive SARS point of care assay. Results show that the robust immunoswab method of detecting SARS-CoV NP antigen can be developed into an easy and effective way of identifying SARS suspected individuals during a future SARS epidemic, thereby reducing and containing the transmission. The key feature of this simple immunoswab diagnostic assay is its ability to detect the presence of the SARS-CoV antigen within 45–60min with the availability of the body fluid samples. [Copyright &y& Elsevier]

Abstract: Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP1,2 gene and subfragments of the GP1 gene were cloned in a bacterial expression vector as a C-terminal His6 fusion protein. Surprisingly, the full length EBOV GP1,2 gene could not be expressed in Escherichia coli. The subfragments of GP1 were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP1,2, GP1 subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP1 subfragment D protein was expressed exclusively as an insoluble ∼25kDa His6 fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP1 subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP1,2 protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment. [Copyright &y& Elsevier]

Abstract: Ebola virus (EBOV) causes hemorrhagic fever in humans and nonhuman primates with up to 90% mortality rate. In this study, Ebola virus like particles (EVLPs) and the aglycosyl subfragment of glycoprotein (GP1 subfragment D) were used to generate monoclonal antibodies (MAbs) against different epitopes of the viral antigens. Such MAbs could be useful in diagnostics and potential therapeutics of viral infection and its hemorrhagic symptoms. Hybridoma cell fusion technology was used for production of MAbs. The MAbs were characterized using ELISA and Western blot analysis. Furthermore, five recombinant sub-domains of GP1 subfragment D were produced, which were used as antigen in Western blot analysis for epitope mapping. Seventeen MAbs of different epitope specificities against EBOV antigens [virion protein (VP40), secreted glycoprotein (sGP), and GP1 subfragment D] were developed. Based on epitope mapping studies, the anti-GP MAbs were categorized into six groups. The binding of the three anti-sGP MAbs with different epitope specificities were mostly between aa 157 and 221. The two anti-VP40 MAbs with the same or overlapping epitopes are potentially good candidates for developing antigen detection assays for early diagnosis of EBOV infection. The anti-GP MAbs with different epitope specificities as an oligoclonal cocktail could be tested for therapy. [Copyright &y& Elsevier]

Abstract: We have developed a universal DC targeting vehicle that could be a convenient method to deliver any type of antigen to DC. P125, a quadroma (hybrid–hybridoma) secreting bispecific monoclonal antibodies (bsmAb), with one paratope specific for mouse DC DEC-205 and another paratope specific for biotin, was developed by PEG-fusion of the two parental hybridomas and selected by a fluorescence activated cell sorter. The bsmAb were purified using a biotin–Agarose column and the bsmAb activity was demonstrated using ELISA method employing mouse bone marrow DC and biotinylated BSA. Both confocal microscopy and ELISA studies have shown enhanced binding and internalization of biotinylated and FITC-labelled M13 to DC cell in the presence of bsmAb. In vivo studies in mice with biotinylated OVA has shown that in the presence of bsmAb and anti-CD40 mAb, both humoral and cell-mediated responses can be augmented. In addition, only a low concentration of antigen (500 fold less) is required using bsmAb to achieve a similar immune response in mice that were immunized using complete Freund''s adjuvant. In the absence of traditional adjuvants, bsmAb targeting of biotinylated antigens to DC could be an alternative, convenient method to deliver antigens to DC. Moreover, this method could be an alternative method to ex vivo stimulation of DC to overcome DC defects and for treatment of cancer. [Copyright &y& Elsevier]