قراءة
عرض

• Immunological Techniques in Research and Clinical Medicine

• Philip L. Cohen, M.D. Chief of Rheumatology, LKSOM
• Presented by
• Dr. Minen Al-Kafajy
• December-18-2021

• Antibodies – Remarkable Tools for Research and Diagnosis

• You can make an antibody to practically any immunogen.
• Monoclonal antibodies have a single specificity, so the immunogen need not be pure (e.g., whole cells or lysates)
• Antibodies are stable (decades at ‐20C!)
• They can be covalently coupled to enzymes, chromophores, biotin, and many other things..


Immunologic Technoques

Albert Coons, M.D.

• 1912‐1978
Immunologic Technoques


• Coons’ Insight

• Antibodies can be “tagged” with small fluorescent molecules and still retain their binding specificity
• These “tagged” antibodies can be used as probes to visualize specific molecules in tissues, cells, or anywhere
Immunologic Technoques




Immunologic Technoques




Immunologic Technoques

SLE Systemic lupus erythrematosus

MGN membranous glomerulonephritis


Immunologic Technoques


• Multiple Color Immunofluorescence

للاطلاع

• Some Considerations for Fluorescence Staining

• Usually requires freshly snap‐frozen tissue
• Conjugated antibodies are less stable than native molecules, are light‐sensitive
• For fixed tissues, immunohistochemistry is preferred
• Can be quantitated using laser capture fluorescence microscopy
• Confocal and dual photon microscopy


Immunologic Technoques

• Methods for Diagnosis

• Immunodiffusion (Ouchterlony)
• Hemagglutination and latex agglutination
• Complement fixation
• ELISA
• Western Blot and other electrophoretic methods



Immunologic Technoques

• Antibodies as Tools for Quantitating Proteins and Almost Everything Else

• Monoclonal abs are exquisitely specific
• Essentially any molecule can be quantitated
• It helps to have two monoclonals with different specificity (sandwich assays)
• Examples of what’s measured: hormones, drugs, cytokines, tumor‐derived proteins (e.g. PSA)
• Sensitivity to ng levels at least


Immunologic Technoques

• Principle of the Sandwich ELISA

• ELISA Plate
Immunologic Technoques




• ELISA Plate Reader
Immunologic Technoques




Immunologic Technoques

• An Important ELISA Application

Immunologic Technoques

• Immunodiffusion Detection of Antibodies

Immunologic Technoques

• Hemagglutination on a Microtiter Plate

Immunologic Technoques





Immunologic Technoques

• Direct Coombs Test

Immunologic Technoques

• ELISA Principles

Immunologic Technoques

• Some Points About ELISAs

• Understand O.D.s (optical densities)
• How to make ELISAs quantitative
• Issues of Specificity and Stickiness

• Western Blot

Immunologic Technoques

• Typical Western Blot

Immunologic Technoques


• Uses of Western Blots

• For determining antibody specificity when the antigen is complex
• WBs are at best semi‐quantitative
• Some antibodies “don’t blot”
• Need for probity in data presentation

• Assays of Cellular Immunology

• DTH – “red bump in the skin”
• Most cellular assays depend on density separation of “PBM” – peripheral blood mononuclear cells

• Ficoll Hypaque Separation of PBM

Immunologic Technoques

• Using Isolated PBM to Study Immunity

• Counting cells via flow cytometry
• T‐cell functional assays: antigen, aCD3, or mitogen‐induced proliferation (CFSE preferred these days, formerly 3H thymidine incorporation Into DNA)
• Cytotoxicity
• Cytokine production
• Cell mixing and culture expts.


• Quantiferon Gold – a Cytokine Assay
Immunologic Technoques

• A Multiplex Cytokine Assay

Immunologic Technoques

• Proliferation Measured by CFSE Dilution

Immunologic Technoques

• Cytotoxic T Cells Destroying a Cancer Cell

Immunologic Technoques

• The End




رفعت المحاضرة من قبل: Ali Haider
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