Immunological Techniques inResearch and Clinical MedicinePhilip L. Cohen, M.D.Chief of Rheumatology, LKSOM10 March 2016

Antibodies – Remarkable Tools forResearch and Diagnosis You can make an antibody to practicallyanything Monoclonal antibodies have a singlespecificity, so the immunogen need not bepure (e.g., whole cells or lysates) Antibodies are stable (decades at ‐20C!) They can be covalently coupled to enzymes,chromophores, biotin, and many otherthings.

Albert Coons, M.D.1912‐1978

Coons’ Insight Antibodies can be “tagged” with smallfluorescent molecules and still retain theirbinding specificity These “tagged” antibodies can be used asprobes to visualize specific molecules intissues, cells, or anywhere

Multiple Color Immunofluorescence

Some Considerations for FluorescenceStaining Usually requires freshly snap‐frozen tissue Conjugated antibodies are less stable thannative molecules, are light‐sensitive For fixed tissues, immunohistochemistry ispreferred Can be quantitated using laser capturefluorescence microscopy Confocal and dual photon microscopy

Antibodies as Tools for QuantitatingProteins and Almost Everything Else Monoclonal abs are exquisitely specific Essentially any molecule can be quantitated It helps to have two monoclonals withdifferent 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 Reader

An Important ELISA Application

Methods for Diagnosis Immunodiffusion (Ouchterlony)Hemagglutination and latex agglutinationComplement fixationELISAWestern Blot and other electrophoreticmethods

Immunodiffusion Detection ofAntibodies

Hemagglutination on a MicrotiterPlate

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 theantigen 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 densityseparation of “PBM” – peripheral bloodmononuclear cells

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Ficoll Hypaque Separation of PBM

Using Isolated PBM to Study Immunity Counting cells via flow cytometry T‐cell functional assays: antigen, aCD3, ormitogen‐induced proliferation (CFSE preferredthese days, formerly 3H thymidineincorporation Into DNA) Cytotoxicity Cytokine production Cell mixing and culture expts.

Quantiferon Gold – a Cytokine Assay

A Multiplex Cytokine Assay

Proliferation Measured by CFSEDilution

Cytotoxic T Cells Destroying a CancerCell

Transcription Factors and T‐cellSubsets FoxP3 for T regs, CD25 TH1 (T bet)TH2 (GATA 3)TH17 (RoR gamma T)

Macrophage Subsets Macrophages differentiate into functionalsubsets that can be recognized by surfacemarkers and by cytokine profiles Originally M1 and M2, getting more complex

Macrophages Differentiate into Distinct Phenotypes

Techniques to Study the ImmuneSystem Itself Is the patient immunodeficient? Does the patient have a malignancy of theimmune 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

Complement Assessment C3, C4 When to look at CH50?

Principle of Nephelometry

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

Lymphoma Stained with anti Kappa

The End