Application of Human Glycophosphatidylinositol (GPI) ELISA KIT_Industrial Additives

Background[1-3]

Human glycophosphatidylinositol (GPI) ELISA KIT uses a double-antibody sandwich method to determine the level of human glycophosphatidylinositol (GPI) in specimens. Use purified human glycophosphatidylinositol (GPI) antibody to coat the microwell plate to make a solid-phase antibody. Add human glycophosphatidylinositol (GPI) sequentially to the microwells coated with monoclonal antibody, and then mix it with HRP-labeled The human glycophosphatidylinositol (GPI) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme and into the final yellow under the action of acid. The depth of color is positively correlated with the human glycophosphatidylinositol (GPI) in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm, and calculate the concentration of human glycophosphatidylinositol (GPI) in the sample through the standard curve.

The core structure of GPI is composed of ethanolamine phosphate, three mannosides, glucosamine and inositol phospholipids. Generally speaking, the GPI anchors of any protein are not homogeneous, but are a mixture of a series of homologues. The C-terminus of the GPI-anchored protein is bridged to decorin through the pigment carbon black ethanolamine phosphate. This structure is highly conserved. There is also a phospholipid structure that connects the GPI anchor to the cell membrane. Decorin can be modified with a variety of side chains, such as ethanolamine phosphate groups, mannose, galactose, sialic acid or other sugar groups. Some complex side chain structures such as N-acetylgalactosamine are found in mammals and protozoa. In addition, lipid components such as 1,2-diacylglycerol, 1-alkyl-2-acylglycerol, monoacylglycerol and ceramide can be used as GPI anchor structures. Moreover, the acyl substituents of some GPI-anchored proteins may also be part of the functional structure. These acyl groups render the GPI anchor immune to PI-PLC (phosphat-idylinosito-l specific phospholipase C) by inhibiting phosphate formation.

Apply[4][5]

For research on the expression, purification and anti-tumor effect of GPI-anchored CD80-CD58 fusion gene in eukaryotic cells

The body’s anti-tumor immunity must rely on the antigens of tumor cells to be recognized by the body’s immune system before it can be activated. However, human tumors have weak immunogenicity, and there are multiple defects in antigen presentation, allowing tumors to evade immune recognition. Therefore, if immune-related genes can be introduced into tumor cells to prepare tumor vaccines, the antigenicity of tumor cells and the ability of antibodies to recognize and present tumor antigens can be enhanced, and the body’s anti-tumor immunity can be enhanced.

CD80 is a glycoprotein expressed on activated B cells, macrophages, dendritic cells, T cells, NK cells and other cells with antigen presentation functions. It binds to its ligand CD28 and plays a synergistic role in T cell activation. Stimulating effect. However, current research shows that, except for a few B lymphomas, CD80 molecules are not expressed on the surface of tumor cells. Even if tumor cells that do not express CD80 molecules are immunogenic, they still cannot effectively induce T cell activation and may even produce specific anergy. Cells expressing CD80 molecules become immunogenic. When recognized by CD8+T precursor CTL cells, the tumor antigen MHC-class I molecule complex provides the first signal, and the CD80 molecule provides the second signal by interacting with CD28 on the surface of T cells. .

As people’s understanding of Glycosyl phospharidyl inositol (GPI) anchoring signals continues to deepen, people have begun to use GPI anchors to anchor proteins on the cell surface to play the role of tumor vaccines. GPI-anchored proteins have no transmembrane region or intracellular part, do not span the cell membrane lipid bilayer, and are only anchored to the cell membrane through the GPI structure at its carboxyl terminus. When GPI-anchored proteins are incubated with cells, they can automatically integrate into the cell membrane surface and retain the ability to bind to their natural ligands. Taking advantage of the above structural and functional characteristics of GPI and the anti-tumor function of CD80, the carboxyl-terminal peptide chain coding sequence of CD58 and the extracellular coding sequence of CD80 were genetically recombined in vitro (the carboxyl-terminal peptide chain of CD58 contains an ω site, and its expression product is GPI-anchored protein), and then expressed in a eukaryotic expression system, and the GPI-anchored form of CD80 was obtained after purification.

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