Immunologic Techniques in Medicine

• Immunological Techniques in Research and Clinical Medicine

• Philip L. Cohen, M.D. Chief of Rheumatology, LKSOM
• Presented by
• Dr. Minen Al-Kafajy
• Feb 16-21

• Antibodies – Remarkable Tools for Research and Diagnosis

• You can make an antibody to practically anything
• 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..

Albert Coons, M.D.

• 1912‐1978

• 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


















• Multiple Color Immunofluorescence

• 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

• 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

• Principle of the Sandwich ELISA

• ELISA Plate

• ELISA Plate Reader

• An Important ELISA Application

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

• Immunodiffusion Detection of Antibodies

• Hemagglutination on a Microtiter Plate

• Direct Coombs Test

• ELISA Principles

• Some Points About ELISAs

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

• Western Blot

• Typical Western Blot

• 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

• Cited ~14,000 times!

• Ficoll Hypaque Separation of PBM

• 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

• A Multiplex Cytokine Assay

• Proliferation Measured by CFSE Dilution

• Cytotoxic T Cells Destroying a Cancer Cell

• Transcription Factors and T‐cell Subsets

• FoxP3 for T regs, CD25+
• TH1 (T bet)
• TH2 (GATA 3)
• TH17 (RoR gamma T)


• Macrophage Subsets
• Macrophages differentiate into functional subsets that can be recognized by surface markers and by cytokine profiles
• Originally M1 and M2, getting more complex

• Macrophages Differentiate into Distinct Phenotypes

• Techniques to Study the Immune System Itself
• Is the patient immunodeficient?
• Does the patient have a malignancy of the immune system?
• Is the immune system causing injury?

• Assessing Humoral Immunity

• GLOBULIN levels (total protein minus albumin, or reported as globulin). Poor man’s test
• IgG and subclasses 1‐4
• IgA
• IgM
• Isoagglutinins
• Response to vaccination – pneumococcal, meningococcus

• Principle of Nephelometry

• Is it Cancer?
• Individual immunoglobulin molecules have either kappa or lambda light chains (about 60:40)
• In lymphoid tissues, cellular expression of kappa and lambda is likewise mixed
• So if lymphoid tissue has infiltrates of cells bearing (by IF or IHC) kappa or lambda only, the cells must be monoclonal and probably lymphoma
• Similar situation for T‐cell receptor V genes

• Lymphoma Stained with anti Kappa

• The End