浙大 高级免疫学技术 英文课件

Antibody conjugation
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Beads, sepharose – Target protein purification Fluorochrome – immunohistology, FACS Enzyme – Western blot, ELISA… Toxin – cancer immunotherapy Radioactive residues Gold Immunoassay
Each antigen may generate several antibodies for different sites (epitopes) on antigen.
170 suppliers in US
Polyclonal antibody
Antigen Immunization
Bleeding
Antibody conjugation
CD4
CD8
Western Blot
fluorescence microscope
FACS
Second Antibody
A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications.
Antibodies
FACS (Analysis and sorting) ELISA ELISPOT Tetramer
Technology which boost immunology
cell culture (in vitro culture system) Gene Profiling （parallel sequencing） Proteomics Transgenic and knockout animals
Specific secondary antibodies are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred.
Antibody conjugation toxin
Surface Antigen
Tumor Cell
Antibody for research
&
Antibody for clinic
Agglutination
It can be used to detect specific antibodies with known antigens or conversely specific antigens with known antibodies.
免疫学相关技术 （Technologies in Immunology）
王迪
浙江大学免疫学研究所 wangdi80@
Before
Years ago, immunologists typically spent the bulk of their time at the laboratory bench. Their research involved peering into a microscope and probably characterizing the different cells from a blood sample. And their understanding of the immune response was limited to what they could see and, based on that, what they could hypothesize.
Second Antibody
The primary antibody (in purple) binds to an antigen (in red). A labeled secondary antibody (in green), then binds to the primary antibody. The label is then used to indirectly detect the antigen.
Genomics
Proteomics
Transgenic and knockout animal models
Technology based on Immunology
Technology which boost immunology
Technology based on Immunology
Antibodies
ELISA ELISPOT
FACS (Analysis and sorting)
Tetramer Technology which boost immunology cell culture (cells and in vitro culture system) Gene Profiling Proteomics
Now
What is emerging is a more complex understanding of the science that involves many fields of science. Molecular Biology Biochemistry Structural Biology
1. Antibodies
Proteins secreted by B-lymphocytes (type of white blood cell), in vertebrates.
Recognise and bind to molecules (antigens) on foreign particles, marking them for destruction.
B
C
T
R
A
Gold nanoparticle labeled mouse anti-HCG antibody HGC Immobilized mouse anti-HCG antibody Immobilized anti-mouse IgG antibody
Technology based on Immunology
Protein G is from the cell wall of -hemolytic streptococci, G strain，MW 30~35 kDa. Protein A or Protein G can bind to the Fc domain of Ab.
Ab purification
Transgenic and knockout animals
Typical ELISA Procedure
ELISPOT
The Enzyme-linked immuno-sorbent spot (ELISPOT) assay is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983. The ELISPOT assay is based on, and was developed from a modified version of the ELISA immunoassay. Simply put, at appropriate conditions the ELISPOT assay allows visualization of the secretary product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information.
Direct :Human “ABO blood group” determination Indirect: Serum rheumatoid factors detection
Indirect agglutination:
Rheumatoid factors are a group of auto-antibodies produced by many individuals with rheumatoid arthritis. They can react with epitopes in the Fc region of IgG.
Monoclonal antibody
Freeze
Ab purification
*Affinity Purification: Protein A/G, Antigen peptide conjugated on sepharose or agarose beads. Protein A is from the cell wall of S. aureus，MW 42 kDa
APC
T cells
Antibody Purification
Affinity chromatography
Affinity chromatography uses antigen-antibody binding to purify antigens or antibodies To purify a specific antigen from a complex mixture of molecules, a monoclonal antibody is attached to an insoluble matrix, such as chromatography beads, and the mixture of molecules is passed over the matrix. The specific antibody binds the antigen of interest; other molecules are washed away. Specific antigen is then eluted by altering the pH, which can usually disrupt antibody-antigen bonds. Antibodies can be purified in the same way on beads coupled to antigen (not shown).