Four major immunologic methods
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Transcript of Four major immunologic methods
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Four Major Immunologic
Methods
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• ELISA (Enzyme-linked immunosorbant Assay
• Immunofluorescence- Flow cytometry (FACS) Lecture by Dr. Witte
• Western blot
• Immunoprecipitation
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Structure of AntibodyAntigen binding site
Five classes of antibody: IgM, IgG, IgA, IgE and IgD
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Ag + Ab Ag-Ab
k
1
-1
k
Ka = [AgAb]
[Ab] [Ag]
Association of antigen and antibody
• Ag-Ab reactions are reversible• Affinity of Ab for Ag can be measured
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EPITOPE = ANTIGEN-BINDING
SITE
POLYCLONAL vsMONOCLONAL ANTIBODIES
• Protein Antigens have many epitopes• B cells make Ab to a single epitope• Different clones of B cells make Ab
to different epitopes
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Polyclonal and MonoclonalAntibodies
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Antigen Excess Antibody Excess
Antigen-Antibody Interactions
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Haemagglutination
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ELISA- qualitative method
(a) Indirect ELIZADetection of Ab
Detection of Ag
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Structure of Antibody
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Competitive ELISA(quantitative method)
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Radioimmunoassay(quantitative method)
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Radioimmunoassay-Standard curve
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Immunoprecipitation - qualitative
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Figure A-18 part 1 of 2
Immunoprecipitation of Cell Lysates
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Figure A-18 part 2 of 2
Immunoprecipitation of Cell Lysates
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Figure A-20
Western Blot(qualitative and quantitative)
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1. Describe the difference between monoclonal and polyclonal antibodies
2. List 2 advantages of indirect reagents for antibody assays. 3. State the specificity of an indirect reagent. 4. Describe how to develop a "dip-stick" test for pregnancy using antibody to human
chorionic gonadotropin, a peptide hormone found in elevated levels in urine of pregnant women.
55. Design an ELISA for detection of antibody to HIV.
66. Design an immunologic assay to quantitate levels of insulin in a patient’s blood.
Problems
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