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Separation of Monoclonal Antibodies from Cell-Culture Supernatants and Ascites Fluid Using Thiophilic Agarose

Thiophilic adsorption chromatography was first introduced by Porath and Belew (1) and was described as a one-step method for purification of monoclonal antibodies (MAbs) (2). As such, the method seems very attractive. Moreover, it operates with lyotropic salts, which are most agreeable r ...

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Detection, Purification, and Utilization of Murine Monoclonal IgM Antibodies

Antibodies of the IgM class are elaborated by many common murine hybridoma cell lines and often define important antigens. In contrast to IgG antibodies, however, IgM molecules exhibit little or no affinity for bacterial Ig binding proteins, such as protein A and protein G, which are almost univ ...

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Antigen Purification by Monoclonal Antibody Immunoaffinity Chromatography

In the past, isolation of a pure protein demanded many hours to develop a purification protocol, which included one or more cycles of time-consuming chromatography, only to have a final product that was enriched rather than purified. The development of a purification procedure had to be repeat ...

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Preparation and Use of Immunoaffinity Columns with Monoclonal Antibodies Without Purification from Ascites and Tissue-Culture Medium

Until recently, the primary methods available for preparing immunoaffinity columns with both polyclonal and monoclonal antibodies (MAbs) have required purification of the antibodies from serum, ascites, or tissue-culture medium prior to coupling to the affinity matrix beads or ...

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Coupling of Monoclonal Antibodies with Biotin

The avidin-biotin bond is the strongest known biological interaction between a ligand and a protein (K d = 1.3 � 10−15 M at pH 5) (1). The affinity is so high that the avidin-biotin complex is extremely resistant to any type of denaturing agent (2). Biotin (Fig. 1) is a small, hydrophobic molecule that functions as a c ...

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Coupling of Monoclonal Antibodies with Fluorophores

Immunofluorescence analysis has been greatly aided by the use of monoclonal antibodies (MAbs) modified by derivatization with fluorescent labels (1). Improvements of known fluorophores and development of new ones with a broader range of colors have paralleled the production of new d ...

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Coupling of Monoclonal Antibodies with Enzymes

As an outgrowth of the experience obtained from the chemical modification of proteins, a series of techniques to crosslink different proteins chemically has been developed in the last two decades. The conjugation of enzymes to antibodies, particularly to monoclonal antibodies (MAbs ...

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Use of Monoclonal Antibodies with Magnetic Particles to Separate Cell Subpopulations by Negative Selection

Sorting heterogeneous populations of cells into specific subpopulations has been greatly facilitated by the advent of monoclonal antibodies (MAbs) in combination with magnetic particles (1–35). MAbs provide the specificity necessary to distinguish one cellular subpopula ...

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Use of Monoclonal Antibodies with Magnetic Particles to Separate Cell Subpopulations by Positive Selection

The use of magnetic particles in combination with monoclonal antibodies (MAbs) has greatly simplified sorting heterogeneous populations of cells into specific subpopulations (1–35). MAbs which recognize particular cell surface markers provide the specificity necessary to ...

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Methods of Immunization to Enhance the Immune Response to Specific Antigens In Vitro

In vitro immunization involves the exposure of spleen cells to antigen in tissue culture rather than the antigenic stimulation of spleen cells via immunization of mice. The production of monoclonal antibodies (MAbs) to highly conserved molecules, such as enzymes (1,2), is possible using in ...

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Culture Conditions that Optimize Outgrowth of Hybridomas

Efforts to refine the methods of producing monoclonal antibodies (MAbs) of known specificity (1) have revealed there are many variables that affect the growth of hybridomas generated by the fusion of myeloma cell lines with spleen cells (reviewed in refs. 2 and 3). These include the cell-cycle st ...

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Production of Stable Heterohybridomas Producing Human Monoclonal Antibodies

In many circumstances, it is advantageous to have a continuous source of human antibody of a given specificity and immunoglobulin isotype. Reliance on human volunteers as a source of such antibody is problematic. Therefore, it has been a goal of investigators to establish immortal cell lines t ...

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Production of Monoclonal Antibodies in Swine

A major objective of this laboratory has been to improve the growth performance of swine with immunological manipulation. During the progress of the program, we demonstrated that a mouse monoclonal antibody (MAb) was able to enhance the somatogenic effect of porcine growth hormone (pGH) wh ...

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Production of Stable Bovine-Murine Interspecies Hybrids

An understanding of the processes involved in immunity to infection requires a clear knowledge of the different immunoglobulin (Ig) classes and subclasses, and their interaction with cells of the myeloid and lymphocytic lineages. Use has been made of Ig-secreting myelomas and plasmac ...

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Production of Monoclonal Antibodies in Horses

Monoclonal antibodies (MAbs) have been successfully used to evaluate immune responses in horses, and to target important antigens of equine infectious agents to which protective immune responses may be directed (1–5). Most of these studies are performed with murine MAb produced by fusi ...

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Use of Electric-Field-Mediated Cell Fusion to Produce Hybridomas Secreting Monoclonal Antibodies

The production of homogenous antibodies with a defined specificity is an optimal goal of antibody preparation. This goal was achieved by Kohler and Milstein with the development of the technology for hybridoma production in 1975 (1).

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Rapid Isotyping of Mouse Monoclonal Antibodies

Determination of class and subclass of monoclonal antibodies (MAbs) is important because several immunochemical and functional properties of antibodies depend on the isotype. Knowledge of the light-chain isotype (κ for 95% or λ for 5% of mouse antibodies) has limited practical value. In c ...

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Modified Microchemotaxis Assays

Chemotaxis is the process by which leukocytes are directed to sites of inflammation under the influence of a concentration gradient of the soluble chemotactic molecules. Upon encountering a chemotactic molecule, responding leukocytes begin to migrate directionally from regio ...

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Generation of Stable Cell Lines Expressing Chemokine Receptors

Most human and murine leukocytes express multiple chemokine receptors with overlapping ligand specificity. Therefore the creation of stable cell lines expressing specific chemokine receptors greatly facilitates chemokine receptor characterization, particularly ...

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Transwell Chemotaxis

Numerous and diverse biological functions are regulated by chemokines. In addition to the well characterized proinflammatory activities such as integrin activation, chemotaxis, lipid mediator biosynthesis, superoxide radical production, and granule enzyme release (r ...

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