蛋白质分析 protein assay
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• can be separated by electrophoresis
– Unique structure
• can be used to produce antibodies to that protein
• antibodies specific for a protein are the basis of immune assays
antiserum
Light chains
Ouchterlony immunodiffusion（免疫扩散）
Serum sample Ab
Countercurrent immunoelectrophoresis
（对流免疫电泳）
electric osmosis 电渗作用
？
Immunoelectrophoresis（免疫电泳）
– theoretically can detect all the proteins
Introduction
• The test purpose
– Deficiencies of specific proteins
• such as in immunoglobulin deficiency.
– Abnormal or inappropriate expression of proteins
Protein assay
Jim Liu Department of Clinical Laboratory The Second Affiliated Hospital of Nanchang University
Content
• Introduction • A short historical perspective • Qualitative techniques • Quantitative methods • Conclusion
– the concentration of the protein of interest will be crucial to the choice of test
Qualitative techniques
• Electrophoresis（电泳） • Ouchterlony immunodiffusion（Ouchterlony免疫扩
• e.g. in monoclonal gammopathies, autoimmune disease or atopy
Introduction plasma cells myeloma cells
• Principles of the protein analysis methods
– Different electrical charge
– polyclonal antibodies by immunizing animals with that human protein
– monoclonal antibodies by hybridoma technology
hybridoma cells
cell fusion
Produce Ab
Introduction
• Samples
– Blood(serum and plasma) – And other body fluids
• Urine • Pleural fluid • Cerebrospinal fluid
Introduction
• Interest proteins
– the immunoglobulins (antibodies)
IБайду номын сангаасmunoelectrophoresis（免疫电泳）
The immunoprecipitate arcs in the serum at bottom show a normal pattern; those for IgG, IgA and IgM are labelled. Note the absence of these immunoprecipitates in the upper sample, indicating that this patient was deficient in all three immunoglobulins.
散） • Countercurrent immunoelectrophoresis（对流免疫
电泳） • Immunoelectrophoresis（免疫电泳）
Electrophoresis（电泳）
• Electrophoresis equipment
cathode
5 microliters
negative charge
• Immunofixation electrophoresis 免疫固定电泳
– Permits the detection and typing of monoclonal antibodies or immunogloblins in serum or urine
Serum protein electrophores
Proliferate unlimitedly
hybridoma technology
Introduction
• The selection of an appropriate assay method
– whether a qualitative or quantitative result is required
• HBV, HCV, HIV antibodies
– the proteins of the complement system
• C3, C4, C1q
– Cytokines
• Growth factor, GF • Tumor necrosis factor, TNF • Interferon, IFN
Support Medium gels (such as agar, agarose and polyacrylamide)
isoelectric point
anode
Electrophoresis（电泳）
• Serum protein electrophoresis
anode
Density scan
cathode
The depth of the color
IgG/M/A
Electrophoresis（电泳）
• Serum protein electrophoresis
IgG/M/A？？？
multiple myeloma Bone marrow morphology
Myeloma cell
Electrophoresis（电泳）
– Unique structure
• can be used to produce antibodies to that protein
• antibodies specific for a protein are the basis of immune assays
antiserum
Light chains
Ouchterlony immunodiffusion（免疫扩散）
Serum sample Ab
Countercurrent immunoelectrophoresis
（对流免疫电泳）
electric osmosis 电渗作用
？
Immunoelectrophoresis（免疫电泳）
– theoretically can detect all the proteins
Introduction
• The test purpose
– Deficiencies of specific proteins
• such as in immunoglobulin deficiency.
– Abnormal or inappropriate expression of proteins
Protein assay
Jim Liu Department of Clinical Laboratory The Second Affiliated Hospital of Nanchang University
Content
• Introduction • A short historical perspective • Qualitative techniques • Quantitative methods • Conclusion
– the concentration of the protein of interest will be crucial to the choice of test
Qualitative techniques
• Electrophoresis（电泳） • Ouchterlony immunodiffusion（Ouchterlony免疫扩
• e.g. in monoclonal gammopathies, autoimmune disease or atopy
Introduction plasma cells myeloma cells
• Principles of the protein analysis methods
– Different electrical charge
– polyclonal antibodies by immunizing animals with that human protein
– monoclonal antibodies by hybridoma technology
hybridoma cells
cell fusion
Produce Ab
Introduction
• Samples
– Blood(serum and plasma) – And other body fluids
• Urine • Pleural fluid • Cerebrospinal fluid
Introduction
• Interest proteins
– the immunoglobulins (antibodies)
IБайду номын сангаасmunoelectrophoresis（免疫电泳）
The immunoprecipitate arcs in the serum at bottom show a normal pattern; those for IgG, IgA and IgM are labelled. Note the absence of these immunoprecipitates in the upper sample, indicating that this patient was deficient in all three immunoglobulins.
散） • Countercurrent immunoelectrophoresis（对流免疫
电泳） • Immunoelectrophoresis（免疫电泳）
Electrophoresis（电泳）
• Electrophoresis equipment
cathode
5 microliters
negative charge
• Immunofixation electrophoresis 免疫固定电泳
– Permits the detection and typing of monoclonal antibodies or immunogloblins in serum or urine
Serum protein electrophores
Proliferate unlimitedly
hybridoma technology
Introduction
• The selection of an appropriate assay method
– whether a qualitative or quantitative result is required
• HBV, HCV, HIV antibodies
– the proteins of the complement system
• C3, C4, C1q
– Cytokines
• Growth factor, GF • Tumor necrosis factor, TNF • Interferon, IFN
Support Medium gels (such as agar, agarose and polyacrylamide)
isoelectric point
anode
Electrophoresis（电泳）
• Serum protein electrophoresis
anode
Density scan
cathode
The depth of the color
IgG/M/A
Electrophoresis（电泳）
• Serum protein electrophoresis
IgG/M/A？？？
multiple myeloma Bone marrow morphology
Myeloma cell
Electrophoresis（电泳）