A monoclonal antibody to scopolamine and its use for competitive enzyme-linked immunosorbent assay.

A hybridoma clone producing a monoclonal antibody (SC78.H81) against scopolamine was established. The monoclonal antibody was an IgG1 (k) antibody with high affinity (1.6 x 10(9) M-1 for methylscopolamine). The monoclonal antibody was cross-reactive with methylscopolamine and butylscopolamine, and showed weak cross-reactivity with 6 beta- and 7 beta-hydroxyhyoscyamine. The cross-reaction with L-hyoscyamine, atropine, scopine and DL-tropic acid was very weak. A competitive enzyme-linked immunosorbent assay using SC78.H81 was established to quantify scopolamine. The sensitivity of the assay allowed detection of 20 pg assay-1 (0.2 ng ml-1) of scopolamine. The assay was applied to the estimation of scopolamine content in hairy root cultures of a Duboisia hybrid.     

Conformational changes at the active site of adenylate kinase detected using a fluorescent probe and monoclonal antibody binding

A fluorescent probe, IAEDANS, was introduced into the active site of adenylate kinase (AK) by specifically modifying Cys-25. During modification, enzyme activity was greatly diminished. This probe allowed observation of conformational changes at the active site during denaturation that could not be detected directly in previous studies. The binding ability of modified AK with its monoclonal antibody (McAb3D3) was identical to that of native AK and the fluorescence of modified AK was quenched by interaction with McAb3D3. The relative fluorescence changes during the binding of modified AK with McAb3D3 in different concentrations of guanidine hydrochloride were monitored. The combination of this active site modification with the use of a conformation specific monoclonal antibody has potential for use in the study of the kinetics of folding of AK and in the detection of folding intermediates.     

Preparation and characterization of a human aurora-A kinase monoclonal antibody

We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an Elisa (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.     

MPM-12: a monoclonal antibody that predominantly stains mitotic cells and recognizes a protein kinase.

The monoclonal antibody MPM-12, raised by using partially purified extract of mitotic HeLa cells as the immunogen, preferentially stains the cytoplasm of mitotic cells by indirect immunofluorescence without exhibiting any species specificity. On immunoblots, MPM-12 recognizes three bands, of 155, 88, and 68 kDa, in mitotic HeLa cell extract but only the 68-kDa band in interphase cell extract. The 68-kDa band seems to be associated with chromatin while the other two are not. All three MPM-12 reactive peptides are phosphorylated, and the phosphorylation seems to be required for MPM-12 reactivity. The MPM-12 immunocomplexes exhibit autophosphorylating and histone H1 kinase activity.     

Characterization and diagnostic use of a monoclonal antibody for VP28 envelope protein of white spot syndrome virus

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21. After induction, the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production. It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection. Competitive PCR showed that the viral level was approximately 10⁴ copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay. Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.     

Development of a monoclonal antibody for use in radioimmunoassay of ethynylestradiol

Production of monoclonal antibodies (MABs) to a 17 alpha-ethynyl-1,3,5 (10)-estratriene-3,17 beta-diol (ethynylestradiol, EE2) hapten is described. Culture antibodies generated by hybridized cells tested in enzyme-linked immunosorbent assay (ELISA) bound the 6-oxoethynyl-estradiol-6-(0-carboxymethyl) oxime-bovine serum albumin conjugate (EE2-6-0 CMO-BSA) but not BSA. On radioimmunoassay (RIA) with tritiated ethynylestradiol (3H-EE2), some of the antibodies demonstrated high binding affinity (Ka = 2.8 X 10(10) M-1) to EE2. Estradiol-17 beta, estrone and estriol did not show any displacement of 3H-EE2 from the MABs even at high concentration (1 X 10(4) ng/mL). Among the widely used progestins, norethynodrel and norethisterone exhibited considerable cross-reactivity (25.7-100% and 0.3-54.1%, respectively) but not levonor-gestrel with the MABs. The high affinity demonstrated by the MABs to EE2 3-sulfate but not to EE2 17-sulfate and EE2 3,17-disulfate suggests the dominant role of the 17 beta-hydroxyl group in immunogenicity.     

A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities

Monoclonal antibody YC10 showed specificity for the phosphorylated form of human, bovine and porcine glial fibrillary acidic proteins (GFAPs) and negligible reactivity towards the dephosphorylated form of the GFAPs. Analysis of species specificity and of the epitope, determined using synthetic phosphopeptides, indicated that this antibody recognized the local phosphorylation-site sequence Thr-phosphoSer-Ala-Ala-Arg-Arg (residues 7-12 of GFAP). Making use of this antibody we developed a non-radioactive method to measure protein kinase activities. After incubation of a protein kinase with non-radioactive ATP in ninety-six wells coated with the synthetic peptide Arg-Arg-Arg-Val-Thr-Ser-Ala-Ala-Arg-Arg-Ser-Cys (residues 3-13 of GFAP), the phosphorylated product was detected by using this mouse antibody and peroxidase-labeled goat anti-mouse IgG. This method proved to be equally as sensitive as the radioactive method for the measurement of protein kinase activities and was less affected by concentrations of ATP present in the reaction mixture.     

Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope

The plasma cholesteryl ester transfer protein (CETP, Mr 74,000) has a binding site for neutral lipid which can readily equilibrate with lipoprotein cholesteryl esters or triglycerides. Recently, a monoclonal antibody (TP2) was obtained which neutralizes the cholesteryl ester (CE) and triglyceride (TG) transfer activities of the CETP. In this report, the epitope of the inhibitory monoclonal antibody has been localized to a hydrophobic 26-amino acid sequence at the COOH terminus of CETP. The Fab fragments of TP2 caused partial (50%) inhibition of CE transfer and complete inhibition of TG transfer by the CETP. Similarly, the Fab fragments inhibited (37%) the binding of CE to the CETP and abolished the binding of TG to the CETP. Surprisingly, the TP2 Fab was also found to enhance the binding of CETP to plasma lipoproteins and to phospholipid vesicles. In conclusion, the TP2 monoclonal antibody inhibits lipid transfer by blocking the uptake of lipid by CETP. The COOH-terminal epitope may be in or near the neutral lipid binding site. Occupancy of this site by TP2 Fab fragments or by neutral lipid may result in a conformational change of CETP causing enhanced binding to lipoproteins or vesicles.     

Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein

A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance.     

Effects of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils. Evidence for a role for proteolytically modified protein kinase C

A monoclonal antibody directed against the Ca2+-requiring proteinase (calpain) of human neutrophils was employed to assess the role of this proteinase in mediating the responses to stimuli such as phorbol 12-myristate 13-acetate or fMet-Leu-Phe. In the presence of either phorbol 12-myristate 13-acetate or fMet-Leu-Phe the antibody is taken up by the neutrophils, and a marked inhibition of intracellular calpain is observed. The decreased calpain activity is accompanied by (a) a significant decrease in the proteolytic conversion of native protein kinase C (Ca2+/phospholipid-dependent enzyme) to the soluble form that does not require Ca2+ or phospholipids for activity; (b) a marked increase in the production of superoxide anion; and (c) a decrease in the exocytosis of granule contents. The increase in superoxide production can be attributed to a more prolonged association of native protein kinase C with the plasma membrane, thus enhancing the phosphorylation of membrane proteins that precedes O(2-) production (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Sacco, O., and Horecker, B. L. (1986), Biochem. Biophys. Res. Commun. 140, 1121-1126). The decreased exocytosis can be attributed to a decreased phosphorylation of certain cytoskeletal proteins, catalyzed by the soluble form of protein kinase C (Pontremoli, S., Melloni, E., Michetti, M., Sparatore, B., Salamino, F., Sacco, O., and Horecker, B. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3604-3608); the subsequent reorganization of the cytoskeleton appears to be related to degranulation. These effects of the monoclonal anti-calpain provide direct evidence for an essential role for calpain in the activation of human neutrophils.     

Functional properties of a rat monoclonal IgE antibody specific for Schistosoma mansoni

A rat monoclonal antibody of IgE isotype (B48-14) raised against Schistosoma mansoni has been generated by the fusion of mesenteric lymph node cells from LOU/M rats immunized with a preparation of adult schistosome worms and IR973F nonsecreting rat myeloma cells. Investigation of the in vitro effector functions of this IgE antibody showed a high level of cytotoxicity against S. mansoni schistosomula in the presence of eosinophils, macrophages, and platelets. A significant level of protection (40 to 60%) against a challenge infection with S. mansoni cercariae was achieved by passive transfer experiment of B48-14 IgE to naive recipient rats. By immunoprecipitation, B48-14 IgE antibodies were shown to react with an antigen of 26 kDa present in excretion-secretion products of schistosomula, previously described as a potential immunogen eliciting a protective IgE response against schistosomiasis.     

Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification

Depth filtration has been widely used during process scale clarification of cell culture supernatants for the removal of cells and cell debris. However, in addition to their filtration capabilities, depth filters also possess the ability to adsorb soluble species. This aspect of depth filtration has largely not been exploited in process scale separations and is usually ignored during cell culture harvest development. Here, we report on the ability of depth filters to adsorptively remove host cell protein contaminants from a recombinant monoclonal antibody process stream and characterize some of the underlying interactions behind the binding phenomenon. Following centrifugation, filtration through a depth filter prior to Protein A chromatographic capture was shown to significantly reduce the level of turbidity observed in the Protein A column eluate of the monoclonal antibody. The Protein A eluate turbidity was shown to be linked to host cell protein contaminant levels in the Protein A column load and not to the DNA content. Analogous to flowthrough chromatography in which residence time/bed height and column loading are key parameters, both the number of passes through the depth filter and the amount of centrifuge centrate loaded on the filter were seen to be important operational parameters governing the adsorptive removal of host cell protein contaminants. Adsorption of proteins to the depth filter was shown to be due to a combination of electrostatic and hydrophobic adsorptive interactions. These results demonstrate the ability to employ depth filtration as an integrative unit operation combining filtration for particulate removal with adsorptive binding for contaminant removal.     

Techno-economic analysis of a transient plant-based platform for monoclonal antibody production

Plant-based biomanufacturing of therapeutic proteins is a relatively new platform with a small number of commercial-scale facilities, but offers advantages of linear scalability, reduced upstream complexity, reduced time to market, and potentially lower capital and operating costs. In this study we present a detailed process simulation model for a large-scale new “greenfield” biomanufacturing facility that uses transient agroinfiltration of Nicotiana benthamiana plants grown hydroponically indoors under light-emitting diode lighting for the production of a monoclonal antibody. The model was used to evaluate the total capital investment, annual operating cost, and cost of goods sold as a function of mAb expression level in the plant (g mAb/kg fresh weight of the plant) and production capacity (kg mAb/year). For the Base Case design scenario (300 kg mAb/year, 1 g mAb/kg fresh weight, and 65% recovery in downstream processing), the model predicts a total capital investment of $122 million dollars and cost of goods sold of $121/g including depreciation. Compared with traditional biomanufacturing platforms that use mammalian cells grown in bioreactors, the model predicts significant reductions in capital investment and >50% reduction in cost of goods compared with published values at similar production scales. The simulation model can be modified or adapted by others to assess the profitability of alternative designs, implement different process assumptions, and help guide process development and optimization.     

Characterization of monoclonal nonspecific suppressor factor (MNSF) with the use of a monoclonal antibody

The secretion of immunoglobulin (Ig) from cultured mononuclear cells by lipopolysaccharide (LPS) stimulation is inhibited by monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma. In an attempt to develop a murine monoclonal antibody (MAb) with specific reactivity against MNSF, a cell fusion technique that incorporated immune murine splenocytes and HAT-sensitive murine myeloma cells was used. Cross-reactivity experiments confirmed that the MAb (MO6) does not bind to unrelated proteins such as bovine serum albumin, mouse IgG, and murine interferon-gamma (IFN-gamma). There are no effects when anti-IFN-gamma antibodies are used with MNSF. As far as biological activity is concerned, MO6 inhibits in vitro the activity of MNSF in terms of the Ig secretion from cultured lymphocytes. By using MO6, affinity chromatography and immunoblotting were performed. The MNSF on the SDS-PAGE showed a band with m.w. of approximately 70,000, indicating the formation of an aggregate in saline; but after treatment with 0.4 M pyridine-acetic acid buffer, separate bands of 24,000 and 16,000 daltons were evident. Therefore MO6 recognizes 70,000 and both 24,000 and 16,000 daltons. Thus we confirmed by using this MAb and affinity chromatography, the existence of human counterpart, human nonspecific suppressor factor (hNSF), in supernatant from concanavalin A-stimulated T cells. When hNSF was fractionated by high pressure liquid chromatography (HPLC), the activity was found in a region corresponding to 70,000 daltons. However, when fractionated in pyridine-acetic acid buffer, hNSF activity was distributed in a slightly wider range of 15,000 to 30,000 daltons. Physicochemical analysis showed that the purified hNSF is resistant to either heating at 56 degrees C or to 2-mercaptoethanol treatment; however, it is labile to acidification at pH 2.0 and is also sensitive to protease treatment, the characteristics of which were similar to those of murine MNSF. Thus MO6 was confirmed to be a pertinent tool for isolation of hNSF, as well as for murine MNSF.