Monoclonal antibodies beads

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

Figure 10.1 Home pregnancy testing devices, like the one seen here, utilize biotechnology techniques. This test uses paired monoclonal antibodies and colored beads to indicate increased human gonadotropin in urine. The second round well is a control, indicating the device is working properly by trapping a common urine protein.

Collm-Osdoby, P., Gursler, M J, Webber, D., and Osdoby, P (1991) Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts. J Bone Miner Res 6, 1353-1365... 

George F, Brisson C, Poncelet P, et al. Rapid Isolation of human endothelial cells from whole blood using S-Endol monoclonal antibody coupled to immuno-magnetic beads Demonstration of endothelial injury after angioplasty. Thromb Haemost 1992 67 147-163. 

Scale-up of cell cultures makes use of suspension cultures (erythropoietic cells or microcarriers) or, less often use of capillary beds (hollow fibre systems or glass bead columns), but these suffer from the same disadvantages seen with smaller scale cultures ( 3.4.4). In particular, nutrients are depleted as the medium flows through long columns or beds and high rates of flow coupled with recirculation are often employed. Nevertheless, Organon have used a hollow fibre dialysis system for production of monoclonal antibodies (Schonherr et al., 1985). Invitron s hollow fibre system has been used to produce cell conditioned media and the Cell-Pharm System (Jencons Ltd. Appendix 3) will produce up to 20 g cell secreted product per month. 

With peptide and oligonucleotide libraries prepared by split synthesis, the structure of a bound ligand on an individual bead can be determined by microsequencing [ 17,18]. This approach has been applied to testing libraries where the bead bound compounds are evaluated for their ability to bind to monoclonal antibodies or other recognition macromolecules. In one such approach, fluorescence-based methods are used to select the most... 

In the selected example by Lam et al. [101] many peptide libraries were prepared using the mix and split technique and tested in different on-bead screens. Incomplete libraries were tested (the population of most of them was more than a million compounds), and the positive structures were exploited through focused libraries. Some libraries were screened against an anti-insulin monoclonal antibody tagged with alkaline phosphatase, which allowed an enzyme-linked colorimetric detection. Only the beads bound to the murine MAb showed a tourquoise color, while the vast majority remained colorless (details of the technical realization of the assay can be found elsewhere [101, 102]). The chemical structure linked to the positive beads was then easily determined via Edman degradation of the peptide sequences. 

Immobilized histidine on agarose beads by means of aminohexyl spacer arm has also been utilized to purify polyclonal217 and monoclonal antibodies.218 Adsorption of IgG on histidine-agarose occurs in the presence of 25 mM Tris-HCl buffer, pH 7.4, and elution is achieved using a solution of 0.2 M sodium chloride in the same buffer. In described conditions the binding capacity of antibodies was about 11 mg/mL of resin and purity of separated antibody was estimated by the authors at around 98% when combined with ethanol precipitation. Dissociation constant was also determined and reported between 2.4 X 10-6 and 4.6 X 10-6 M. 

Aoyama, K., and Chiba, J. (1993). Separation of different molecular forms of mouse IgA and IgM monoclonal antibodies by high-performance liquid chromatography on spherical hydroxyapatite beads. J. Immunol. Methods 162, 201-210. 

Where sufficient antigen is available, e.g., a synthetic peptide, the monoclonal antibodies can be purified easily and quickly by affinity chromatography. Alternatively, purified preparations of monoclonal (e.g., MARK-1, ref. 5) or polyclonal antibodies to rat or mouse F(ab )2 can be used following their immobilization to crosslinked agarose or polyacrylamide bead supports. 

EL-4 cells (3 x 10 ) were lysed with N,N-dimethyl-N-(3-sulfopropyl)-3-[[(3a,5 P,7a, 12a)-3,7,12-trihydroxy-24-oxocholan-24-y 1]-amino]-l-propanaminium hydroxide (CHAPS). The nuclei and membranes were pelleted and the supematent lysate filtered to remove lipids. The lysate was sequentially passed over sepharose columns containing a) normal mouse serum b) Y-3 which is an anti-K monoclonal antibody. Both columns were washed with 45 coliunn volumes of progressively lower molarity salt solutions. The beads were then treated with acetic acid to release antigen-antibody complexes and the complex was denatured by boiling in 10% acetic acid. The mixture was filtered through a 3 kDa pore-size membrane and the filtrate containing MHC class I peptides subjected to reversed phase HPLC. 

The measurement of holotranscobalamin II is potentially useftil as a specific marker of biologically available vitamin Bi2, because only cobalamin bound to Tell is specifically available for uptake by aU cells. Other methods have been described for the measurement of holotranscobalamin in serum, one using an immobilized monoclonal antibody to human transcobalamin, followed by measurement of released cobalamin by CPB, This method is currently available as a commercial kit. The other method uses magnetic beads coated with cobalamin to precipitate apotranscobalamin followed by measurement of the holotranscobalamin in the supernatant by ELISA. Though these methods are claimed to be precise and simple to perform, there remains doubt over the interpretation of the measured concentrations, and over their sensitivity and specificity in the diagnosis of vitamin B deficiency. ... 

A nonisotopic ELISA method in which serum specimens are added to microtiter wells coated with human Tg is also available. In this method, antibody binding is assessed using a peroxidase-conjugated anti-IgG/o-phenylenedi-amine system. An automated two-step fluorescent enzyme immunoassay has been reported. In this assay, Tg is immobilized on magnetic beads, and anti-human IgG mouse monoclonal antibody is labeled with alkafine phosphatase 4-methyiumbelliferyl phosphate is used as the substrate. IRMA and ELISA both have similar detection limits (approximately 3 to 5 U/mL). A considerably more sensitive radioassay has been reported in which diluted serum is incubated with T-labeled Tg to allow formation of antigen-antibody complexes these complexes are then precipitated by adding solid-phase protein A. Its detection limit is reported to be approximately 0.2 U/mL. 

Antibodies to DNA RNA, DNA DNA and RNA RNA hybrids have been described (Section 7.4.2) (Stollar and Stollar, 1970 Rudkin and Stollar, 1976 Pisetsky and Coster, 1982). Monoclonal antibodies to unusual nucleic acids have also been reported (Lee et al., 1989) as well as against RNA DNA hybrids (Bogulawski et al., 1986). In capture assays, with DNA capture probe linked to nylon beads, hybrids resulting from annealing of the RNA target with the capture probe can be detected with the specific antibody. 

Cryptosporidium oocysts were selectively extracted (antibodies coupled to magnetic beads) from water concentrates using IMS. These oocysts were subsequently visualized using FITC conjugated monoclonal antibodies. However, dissociation from the immunomagnetic beads before labelling proved to be necessary (de Roubin et al. 2(X)2 Reynolds et al. 1999 Rushton et al. 2000). 

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