Laboratory techniques commonly used in immunology: Principles and examples Rebecca Jane Cox

1. Laboratory techniques commonly used in immunology: Principles and examples Rebecca Jane Cox Rebecca.cox@gades.uib.no

2. Antibody & antigen reaction basis of immunological assays Detection, purification and quantitation of antigens Majority antigens many epitopes heterogenous mixture of polyclonal antibodies Monoclonal antibodies derived from single clone to 1 epitope

3. (contains 4 MAbs) (MAb derived from 1 plasma cell)

4. Production of MAbs 1975 Georges Kohler and Cesar Milstein 1984 Nobel Prize in Physiology or Medicine Hybridoma Fusion of myeloma cell with normal Ab producing B cell Result immortal B cell producing MAb Hypoxanthine Aminopterin Thymidine medium) hypoxanthine-guanine phosphoribosyltransfera

5. Lag phase naïve B cells undergo clonal selection, clonal expansion & differentiation into memory & plasma cells Higher magnitude response Higher affinity (IgG) Shorter lag Large population memory cells, easily activated Naïve lymphocytes Memory lymphocytes

6. Immunoprecipitation in Gels (Ag & Ab form precipiate when in equivalence) • Antigen in well, antibody in gel. • Ring formed in area of equivalence • area of precipitation proportional to concentration of antigen • Antigen & antibody diffuse towards each other • Line of precipitation at equivalence

7. Agglutination Antibodies to sheep RBC agglutination Haemagglutination Inhibition Influenza virus agglutinates red blood cells Influenza specific antibodies inhibit agglutination

8. Radioimmunoassay • Introduced 1960 by SA Berson and Rosalyn Yalow to measure insulin in plasma (Nobel prize 1977) • I125 (emitting) also 3H ( emitting) • Competitive binding of radiolabeled antigen and unlabeled antigen • Solution or solid phase assays

9. Wells coated constant amount of antigen • Serum & labeled hepatitis surface antigen (HBsAg) added • Infected people less radiolabeled HBsAg bound • Standard curve used to calculate concentration of HbSAg in sample

10. Enzyme Linked Immunosorbent Assay (ELISA) • Qualitative or quantitative measurement of antigen or antibody • Enzyme linked to detector antibody • Soluble chromogenic substrate • Absorbance of product measured in an ELISA(spectrophotometer) • Chemiluminesence • Measurement of light instead of absorbance • Luxogenic substrate • Increased sensitivity (10-200 fold with enhancers)

12. Amplification of signal Biotin-avidin system • Amplification of signal • High affinity and specificity (avidin binds 4 molecules biotin) • Secondary antibody • Eg product detected MAB followed by enzyme labeled anti-mouse antibody

13. Multiplex Assays • Bead reader powered with Luminex xMAP technology • Reads 96-wells plates fully automated • Recognises multiple fluorescent signals, internal lasers • possible to measure up to 100 different biomolecules/well

14. Multiplex • Simultaneous quantitative analysis of up to 100 different biomolecules from a single drop of sample • Increase dynamic range in concentration for all analytes, because of the fluorescence and the superiour curve fitting modules • Increase of sensitivity of all analytes compared to ELISA • Decrease volume requirements for precious sample

15. Assay occurs on bead surface • 5.5µm bead diameter • Each bead contains different • concentration of RED and Infrared dyes • Up to 100 uniquely colored beads • Used for detection of cytokines Bead-Based Technology • BASED ONBEAD COLOUR

16. Purification of protein: Affinity chromatography Purification of antigen A from complex mixture

17. Purification of protein: Immunoprecipitates collected by magnetic beads

18. Production of antibodies by genetic engineering Phage display library

19. Western Blot • Protein mixture treated with detergent SDS • Separated by SDS PAGE - lower molecular weight migrate faster • Proteins blotted from gel to nitrocellulose membrane under electric current • Flood membrane with antibodies linked to enzyme used to visualise antigens • Developed insoluble substrate product deposited at site of reaction • Chemiluminsence use film (increased sensitivity)

23. Metabolic labeling • All actively synthesised cellular proteins labeled radioactive • amino acids • Cells lysed detergent • Precipitation specific proteins with MAbs on beads • Elute proteins in SDS • Run SDS PAGE

24. Immunohistology & Immunofluoresence • used to visualize subcellular distribution of biomolecules • Immuno-labeled tissue sections studied using a (fluorescence) microscope or by confocal microscopy • antibodies or antigens labeled with enzyme (immunohistology) or with fluorescent dyes (immunofluorescence)

25. Direct and Indirect Immunofluoresence Cells fixed to microsope slide

26. Collidal Immunogold staining in electron microscopy

27. Measuring lymphocyte responses

28. Isolation of lymphocytes Contains Sodium Diatrizoate & Polysaccharide aggregates erythrocytes (increasing sedimentation rate) lymphocyte suspensions contaminated (1-5% erythrocytes)

29. Separation of cells using magnetic beads Positive selection selects for cells containing surface marker Negative selection selects for cells other surface markers

30. Enzyme linked Immunospot Assay (ELISPOT) • Modification of ELISA • Quantification of number of cells producing antibody or • antigen e.g. Cytokine • Insoluble chromogenic or chemiluminescence substrate

31. Antigen coating (influenza proteins) Enzyme conjugated avidin Blocking (FCS) Colorless Substrate (enzymatic reaction) e S P

32. Sample (Lymphocytes) Colored Product (insoluble - spots) e S P Detector antibody (biotin conjugated) An ELISPOT well

34. What is Flow Cytometry? • Flow Cytometry is a powerful technique for cell counting and surface marker analysis • Flow Cytometry can provide quantitative information about cell surface markers • Based on immunofluorescence technique and computer-aided analysis • Allows simultaneous multiparametric analysis of cells

37. Neutrophils Lymphocytes Monocytes A flow cytometry scattergram Scatter Pattern of Human leukocytes Forward scatter (size) Side scatter (granularity)

38. Three Color Lymphocyte Patterns CD4 CD4 CD8 CD3 CD8 CD3

40. Fluorescence-activated cell sorter (FACS) • FACS is one version of Flow Cytometry, which can sort cells by their surface markers • Individual cell is positively or negatively charged based on their fluorescence color • When charged cells pass through an electric field, they are deflected and hence separated

41. Flow Cytometric uses of CFSE • CFSE or CFDA-SE: carboxyfluorescein diacetate, succinimidyl ester. • A fluorescein derivative which is cell permenant. • CFSE is partitioned equally among daughter cells with each division. • These properties allows simultaneous analysis of cell number, position, as well as division status. • Fluorochromes compatible with fluorescein can be used to probe other cellular properties. Dilution of CFSE with cell division Divisions: 3 2 1

42. Traditional methods CD4 proliferation after stimulation with specific antigen 3H-Thymidine incorporation into DNA CD8 cytotoxic activity after in vitro stimulation Release of 51Cr from labeled target cell killed by effector Measures response of bulk population Modern Methods Production of cytokines after in vitro stimulation with antigen Intracellular staining ELISPOT Staining with MHC/peptide complexes (tetramers) Measures single cell Quantification of T cell responses:

43. CD4 T cell proliferation assay

44. Polyclonal Activation • Activation of lymphocytes regardless of antigen specificity • Lectins (plant proteins) bind to TCR receptor stimulate T cells • E.g. Concanvalin A (Con A) Phytohaemagglutnin (PHA) • Useful positive control in assays

45. Proliferation assay: Setup 1. Harvest PBMC over Ficoll 2. Add killed virus or peptide antigen 3. Add PBMC Triplicate control wells: +ve control = PHA -ve control = no antigen ~1x105 cells/well 37OC 5% CO2 6 days 7. Obtain counts in automated rack β-counter 5. Add 1.0 μCi 3H-thymidine/well 37OC 5% CO2 18 hr 6. Automated harvest of cellular DNA onto glass filters

46. Cytotoxic T cell assay

47. CTL assay set-up: in vitro restimulation 1. Harvest PBMC over Ficoll Harvested live cells = Effector CTL for 51Cr release assay Infect PBMC with virus incubate 3-4 hr at 37OC Uninfect PBMC IL-2 50U/ml Mix in optimal ratio Incubate 37OC 5% CO2 7 days

48. CTL assay set-up: 51Cr release assay Harvest 100 μl supe for detection of γ-emission cpm Targets Effectors Triplicate wells: E:T ratio = 40:1, 20:1, 10:1, 5:1 3 days Weeks 4-5 hr at 37OC 5% CO2 OR Targets + 0.1% Triton =Total lysis medium = Spont. lysis PHA-stim’d autologous blast cells EBV-transf’d lymphoblastoid cell line Label with 51Cr; infect with virus or pulse with peptide

49. CD8 tetramer staining

50. MHC Tetramer staining of lymphocytes